LABORATORY STANDARD OPERATING

PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: COLLECTION, HANDLING, STORAGE SOP No: 006

AND SHIPMENT OF DBS (EID) FOR DNA-PCR
Version: Original
ASSAY.
Effective Date: January 2016 Page 1 of 9

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review:________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

1.0 PURPOSE / INTRODUCTION:
1.1 Major challenge in PMTCT is the presence of maternal antibodies in the
infant’s blood circulation and these trans-placental antibodies (IgG) from the
mother may persist for 15-18 months. If antibody based tests are performed on
such infants, chances of false positive result are high. The test is used for
children between 6 weeks up to 18 months.
1.2 Dried blood spots on filter paper (Schleicher and Schuell grade 903 or
Whatman BFC 180 paper) have been validated and it is very good technique
for handling and transporting specimen as compared to serum or plasma.
However, the accuracy of any laboratory procedure to detect such molecules is
as good as the manner in which the specimen is obtained and handled.
1.3 This SOP is meant to give guidelines in ensuring that DBS are collected,
stored and transported in accordance to the required laboratory standard to
safeguard on the quality of results received after analysis.

2.0 SCOPE / RESPONSIBILITY:

2.1 This SOP applies to all Health Care Workers involved in the collection of
DBS in facilities and during community activities.
2.2 It is the responsibility of the designated of the County Leads, Quality
Assurance and Quality Control (QC/QA) personnel, Laboratory Supervisors to
ensure that the current SOP is available to the Health Care Workers and the
procedure is followed as documented.

3.0 SAFETY/RISK ASSESSMENT:

3.1 Wear personal protective equipment.
3.2 Handle all samples as potential biohazards.
3.3 Avoid touching the circles with your finger (even when on gloves) or anything
else.

4.0 DEFINITIONS:
4.1 DBS: Dried Blood Spot.
4.2 DNA: Di-Nucleic Acid.
4.3 EDTA: Ethylene Diamine Tetra-acetic Acid.
4.4 EID: Early Infant Diagnosis.
 4.5 HIV: Human Immunodeficiency Virus.
4.6 MTC: Mother To Child.
4.7 PCR: Polymerase Chain Reaction.
4.8 PMTCT: Prevention of Mother to Child Transmission.
4.9 QA: Quality Assurance.
4.10 SOP: Standard Operating Procedure.

5.0 SPECIMEN:
5.1 Whole blood collected from a heel, finger prick using an EDTA capillary tube,
or EDTA anti-coagulated blood.
5.2 Infants age 1 – 4 months, less than 6kgs heel work best.
5.3 Infants age 5 – 10 months, less than 10kgs toes work best.
5.4 Larger infants use finger.

6.0 EQUIPMENT/ MATERIALS/ REAGENTS:

6.1 Equipment.
6.1.1 Not Applicable.
6.2 Materials.
6.2.1 EDTA capillary tubes.
6.2.2 Alcohol swab.
6.2.3 Desiccants.
6.2.4 Drying rack.
6.2.5 Gauze or cotton wool.
6.2.6 Glycine envelopes.
6.2.7 HIV diagnosis (DNA-PCR) laboratory requisition form.
6.2.8 Humidity indicator cards.
6.2.9 Sterile blood Lancet.
6.2.10 Powder free gloves.
6.2.11 S&S/Whatman filter paper.
6.2.12 Zip-lock bags.
6.3 Reagents.
Not applicable.

7.0 METHODOLOGY:
7.1 Principle.
DNA-PCR assay is a qualitative molecular based technique used for detection
of HIV viral nucleic acid in specimen. Application of correct specimen
collection technique, separation of plasma, storage and retrieval, packaging
and shipment assures of quality specimen for analysis and hence reliable
results for management of patients
7.2 Specimen Collection.
7.2.1 Confirm that the information on the request form is correct and
properly identify the patient.
7.2.2 Wear sterile powder free gloves.
7.2.3 Unpack the filter papers to be used from the batch and make sure that
no contact is made with the spot (circles) collection area.
7.2.4 Label the filter paper clearly using a biro pen with the correct patient
code and date of collection. Avoid using ink, pencil, felt pens and
placing on bench surface when writing.
7.2.5 Place the filter paper horizontally on a drying rack. Each filter paper
should be placed in a separate slot to avoid cross contamination.
7.2.6 Warm the heel, toe or finger then sterilize using an alcohol swab and
allow to air dry.
7.2.7 Press lancet into foot, toe or finger prick and wipe away the first drop
using a dry cotton wool/gauze.
7.2.8 Allow large drop (whole blood- 50µl) to collect. Collect using EDTA
capillary tube.
7.2.9 Add appropriately and directly to the circles on the filter paper under
sterile conditions. Make sure that each circle is completely filled or
otherwise fill at least 4 circles.
7.3 How to dry DBS.
7.3.1 Don’t touch or smear the blood spots.
7.3.2 Place drying rack horizontally on a bench for the filter paper to air dry
at room temperature (150C- 280C) overnight.
7.3.3 Keep drying filter paper away from direct sunlight, heat, water, dust
and bugs.
7.3.4 Do not allow DBS to touch anything during the drying process.
7.3.5 Dried DBS are:
 7.3.5.1 Chocolate brown in colour.
7.3.5.2 Dull and not shinning.
7.3.5.3 Tend to pull filter inwards and so appear curved.
7.4 Packaging DBS for storage.
7.4.1 Insert a dry DBS into sealable zip-lock bag.
7.4.2 Add 10 desiccant packets.
7.4.3 Add 1 humidity card.
7.4.4 Press air out of the zip-lock bag.
7.4.5 Seal and label the zip-lock bag.
7.4.6 Put into refrigerator at 40C for short-term storage or in a freezer at -
200C for long-term storage (over 90 days).
7.5 Packaging DBS for shipping.
7.5.1 Insert a dry DBS into a glycine envelope. Care should be taken not to
touch the area with blood spots.
7.5.2 Place the glycine envelope into a zip-lock bag (labelled area appearing
clearly) with a desiccant and a humidity indicator paper.
7.5.3 Expel as much air as possible from the bag and secure the zip-lock.

7.6 Sending DBS.

7.6.1 Ensure that each sample/batch of samples sent to the testing laboratory
is accompanied with an infant HIV diagnosis (DNA-PCR) laboratory
requisition form that is completely filled.
7.6.2 Place the packaged DBS in an envelope, address appropriately and
send through G4S courier.
7.7 DBS rejection criteria
7.7.1.1 Insufficient specimen collected
7.7.1.2 Scratched spots
7.7.1.3 Layer serum rings
7.7.1.4 Clotted blood
7.7.1.5 Improper drying
7.7.1.6 When the spots cannot elute.
7.7.1.7 Suspected contaminated specimen
7.7.1.8 Incorrect packaging of specimen
7.8 Results.
 Results of previous DBS samples will be received through G4S courier.
7.9 Limitations.
7.9.1 Limited detection of very low vireamia cases.
7.9.2 False positive or invalid results in case of contaminations

8.0 APPENDICES:
8.1 EID Request form.
9.0 REFERENCES:
9.1 Early infant diagnosis of HIV, standard operating procedure for DBS
collection for DNA-PCR testing, MOPHS/MOMS work-aid.
9.2 National public health laboratory services, strengthening laboratory services in
support of ART management (March, 2007), Laboratory standard operating
procedures, MOH publication, 1st edition
10.0 DOCUMENT CHANGE HISTORY:
10.1 Version Table:
Version 1: Collection, handling, storage Dated: SOP No.: No. Pages:
and shipping of DBS (EID) for DNA-PCR LGEN. 0022 08.
Assay.

Version 2: Dated: SOP No.: No. Pages:

.
Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log

Date of Changes made. Name of reviewer. Signature.
review.
11.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: Confirmatory test for HIV using First Response SOP No: 014
Version ORIGINAL
Effective Date: January 2016 Page 1 of 9

Signatures and Dates:

Author: _________________________________________________________________
Lab. Technologist Date

QA Review: ______________________________________________________________
QA Officer Date

Approving Authority: ______________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Responsibilities/ Applicability:
1.1 The designated Quality Assurance and Quality Control (QC/QA) personnel,
Laboratory Supervisors, SCMLTs, and CMLT.
1.2 It is the responsibility of all the laboratory technologist/technicians and students
(under supervision) to follow this SOP.
2.0 Purpose
2.1 HIV is recognised as the etiological agent of AIDS. The virus is transmitted by sexual
contact, exposure to infected body fluids or tissues and MTCT. The presence of HIV
elicits the production of antibodies specific to either HIV1 and or 2.Current available
HIV diagnostic testing principles include, HIV antibody detection, HIV antigen
detection, Combined HIV antibody/antigen detection, HIV viral nucleic acid
detection and HIV viral culture. Among the existing principles, HIV antibody
detection in whole blood, serum or plasma is the most commonly used.
2.2 This SOP is meant to give guidelines in ensuring that confirmatory test for HIV is
done in accordance to the required laboratory standard to safeguard on the quality
of results received.

1. Principle:
3.1 It’s based on immunochromatography principle in which nitrocellulose membrane is
pre-coated with HIV 1 antigen (gp41and p24) on test band 1 region and HIV 2 antigen
(gp36) on test band 2 region. When the test sample and assay diluent move through the
membrane, HIV antigens conjugated with colloidal gold particles binds to HIV1 and or
HIV 2 antibodies (s) present in the test sample. This conjugated antigen-antibody
complex moves through the nitrocellulose membrane and bind to the corresponding
immobilized HIV1 & 2 antigen (Test lines) leading to the formation of a visible colour
band. The control band will appear irrespective of reactive or non-reactive sample and it
servers for quality control.

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3.0 Specimen handling and preparation:
3.1 Collection:
3.2 Specimen handling.
3.3 Human serum, plasma or whole blood. Capillary whole blood should be tested
immediately.
3.4 Do not use haemolysed samples.
3.5 Samples stored at 2-80C and should be used within 3 days. For longer storage freeze
serum/plasma at -200C.

2. Quality control/Calibration:
4.1 The test is sensitive to humidity and temperature thus run test immediately after
removing from the foil pouch.
4.2 Run IQC with a known positive or negative sample.
4.3 An inbuilt procedural control. A coloured band should always appear at the control
window.
4.3 Store the card test and assay diluent at 4-300C until expiration date.

5.0 Equipment:
5.1 Timer

5.2 Materials
1. 70% methanol/Alcohol swabs.
2. Blood vacutainer/tube.
3. Cotton wool or gauze.
4. Fine tip sample pipette (enclosed in the test kit)
5. First Response® HIV 1-2.O card test kit.
6. PPEs e.g. Gloves and a Lab coat.
7. Sterile lancet

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6.0 PROCEDURE:
1. Confirm that the information on the request form is correct and properly identify the
patient.
2. Wear clean gloves.
3. Remove a test card and the sample pipette from the foil pouch and place it on a flat
surface.
4. Label each test with appropriate patient information.
5. Follow/see SOPs on how to collect venous/capillary blood.
6. Using a sample pipette, add 2 drops (20µl) of whole blood or one drop (10µl) of serum/
plasma to the sample well.
7. Add 1 drop (35µl) of assay diluent to the sample well.
8. Time and results can be seen within 5- 15 minutes but don’t interpret results after 15
minutes.

7.0 INTEPRETATION OF RESULTS

1. Presence of two coloured bands at HIV 1 (1) and control line (C) indicates a positive
result for HIV 1
2. Presence of two coloured bands at HIV 2 (2) and control line (C) indicates a positive
result for HIV 2.
3. Presence of three colour bands HIV 1 (1), HIV 2 (2) and control line (C) indicates a
positive result for HIV 1 and 2.
4. Presence of only 1 color band at control line (C) indicates a negative result.
5. If no color band appear at control line (C) OR appearance of 1 color band at the HIV 1 (1)
window only OR appearance of 1 color band at the HIV 2 (2) window only OR
appearance of 2 color bands at the HIV 1 (1) and HIV 2 (2) windows only, indicates
Invalid results.
6. Report results HTC positive or negative. Indicate the date and name of technical staff
reporting.

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7.0 LIMITATIONS OF THE PROCEDURE
1. Does not detect HIV1/2 antibodies in other body fluids.
2. Positive samples should be retested by a confirmatory HIV test.
3. A false negative result can occur in the following circumstances,
4. Low levels of antibodies (e.g. early sero-conversion specimen) are below the detection
limits of test.
5. Infection with variant of the virus that is less detectable by the assay configuration.
6. HIV antibodies in the patient that do not react with specific antigens utilized in the assay
configuration.
7. Specimen handling conditions, which result in the loss of HIV antibodies multivalency.

8.0 Explanation of Abbreviations and Terms:

AIDS: Acquired Immunodeficiency Syndrome.
HIV: Human Immunodeficiency Virus.
PPE: Personal Protective Equipment.
MTCT: Mother to Child Transmission.
QA: Quality Assurance.
IQC: Internal Quality Control.
HTC: HIV Testing and Counselling.
SOP: Standard Operating Procedure.

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10.0 References:

Reference Number or Authors Document Title

10.1 First Response® HIV 1-2.O card test, Rapid
immunochromatographic card test for the detection of
antibodies to HIV 1 & 2 in human whole blood/ serum/
plasma, Information sheet

10.2 HIV testing principle, KEMRI/CDC Presentation Part 3, Module

1.

11.0 Forms and Appendices:

Form or Appendix Number Title

11.1 SOP Copy Control and Updating Log
11.2 Training Documentation Log for SOP Files

12.0 Version Table:

Original: (Current) Dated: No.
Title: Confirmatory test for HIV using First June 2015 HIV RPD 002 Pages: 9
Response
Version 1 Dated: SOP No.: No.
Pages:
Version 2
Dated: SOP No.: No.
Pages:
Version 3:
Dated: SOP No.: No.
Pages:
Version 4: Dated: SOP No.: No. Pages:

Version 5: Dated: SOP No.: No. Pages:

Version 6: Dated: SOP No.: No. Pages:

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Appendix 11.1
SOP Copy Control and Updating Log

DOCUMENT COPY CONTROL

DATE PRINTED: Feb 2015 NUMBER OF COPIES:
SOP DISTRIBUTION
COPY 1 OF 1
LAB Section

By Initialing and dating below I understand and approve of the changes to the
attached SOP.
SOP CHANGES Changes Approval
Initials/Date
Date/Initials Nature of Change QA Approving
Authority

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 Appendix 11.2
Training Documentation Log for SOP Files
SOP NO : HIV RPD 00VERSION :
Laboratory Department
COPY 1 OF 3 ORIGINAL
Standard Operating Procedure
Effective Date: June 2015

Title: Confirmatory test for HIV using First Response

This SOP has been read and understood by:

Date: Printed Name OR Date: Printed Name OR

DD-MM-YY Initials Signature DD-MM-YY Initials Signature

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Supported by Centre for Health Solutions – Kenya (CHS)
 LABORATORY STANDARD OPERATING
PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: BLOOD GROUPING TILE METHOD SOP No: 002

Version: Original
Effective Date: January 2016 Page 1 of 7

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 PURPOSE / INTRODUCTION:
1.1 This SOP is meant to give guidelines in ensuring that blood grouping by tile or
slide method is done in accordance to the required laboratory standard to give
precise and accurate results.

2.0 SCOPE / RESPONSIBILITY:

2.1 This SOP applies to all laboratory technologists/technicians involved in the assay.
2.2 It is the responsibility of the designated Quality Assurance and Quality Control
(QC/QA) personnel, Laboratory Supervisors, SCMLTs, and CMLT to ensure that
the current SOP is available to the staff and the procedure is followed as
documented.

3.0 SAFETY/RISK ASSESSMENT:

3.1 Wear personal protective equipment. Handle all samples as potential biohazards.

4.0 DEFINITIONS:
4.1 EDTA: Ethylene Diamine Tetra-acetic Acid
4.2 PPE: Personal Protective Equipment.
4.3 QA: Quality Assurance.
4.4 Rh: Rhesus.
4.5 SOP: Standard Operating Procedure.

5.0 SPECIMEN:
5.1 Freshly prepared 20-30% red cell suspension from a clotted or EDTA anti-
coagulated blood sample.
5.2 Whole blood can also be used directly from a finger prick.

6.0 EQUIPMENT/ MATERIALS/ REAGENTS:

6.1 Equipment.
6.1.1 Electric centrifuge.
6.1.2 Weighing scale.

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 6.2 Materials.
6.2.1 Applicator sticks.
6.2.2 Disposal containers and bucket.
6.2.3 Grouping tiles or slides.
6.2.4 Pasteur pipettes.
6.2.5 PPEs e.g. Gloves and a Lab coat.
6.3 Reagent.
6.3.1 Antisera – anti-A, anti-B and anti-D.
6.3.2 Distilled water.
6.3.3 Sodium chloride powder

7.0 METHODOLOGY:
7.1 Principle.
7.1.1 Blood group antigens on the surface of red cells react specifically with
blood group antibodies to form agglutinates, which are detected using the
naked eye. The results are used to determine the major blood groups. Red
blood cells are mixed with antisera on a tile or slide and the presence or
absence of agglutination is noted.
7.2 Procedure.
7.2.1 Prepare Physiological saline as follows:
7.2.1.1 Weigh and dissolve 0.85g sodium chloride in 100ml distilled water
in a reagent bottle and label with the name of the reagent and date
of preparation. For daily use, transfer to a dropper bottle and label.
7.2.2 Prepare a 20-30% Red cell suspension as follows:
7.2.2.1 Transfer about 0.5 ml EDTA anti-coagulated blood or red cell
from a clotted sample into about 5ml physiological saline.
7.2.2.2 Wash the cells 3 times by adding 5-7 ml saline, centrifuging at
3000 rpm for 2-3 minutes and discarding the supernatant.
7.2.2.3 Make a 20-30% red cell suspension by mixing 5 drops of
sedimented cells in 20-25 drops of saline, holding the Pasteur
vertically.

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 7.2.3 Using a Pasteur pipette, place a drop of well-mixed red cell suspension/
whole blood to separate areas of the grouping tile/ slide. Label the areas A,
B and D.
7.2.4 Add a drop of anti-A, anti-B, anti-D to respective labelled areas.
7.2.5 Mix the antisera and red cell suspension thoroughly using a clean
applicator stick for each antiserum.
7.2.6 Gently tilt the slide continuously for 2 minutes.
7.2.7 Read and record the results.
7.2.8 Rinse the used tiles in running tap water and place in the bucket of 5%
Lysol® marked “SLIDES”. Place used applicator sticks in the bucket
marked “INCINERATION”.
7.3 Quality Control.
7.3.1 Check the reactions of the antisera using suspensions of known A, B and
D positive cells as follows:
7.3.1.1 Add 1 drop of known A, B and D positive cells to the drops of
anti-A, anti-B or anti-D, respectively, on the tile.
7.3.1.2 Mix with a clean applicator stick.
7.3.1.3 Gently tilt for 2 minutes. Strong agglutination indicates the antisera
are working.
7.4 Results.
7.4.1 Agglutination of red cells indicates a positive result.
7.4.2 A smooth suspension of red cells indicates a negative result.
7.4.3 Table 1: Results interpretation.
Anti-A Anti-B Anti-D Blood group
Negative Negative Positive O Rh D positive
Negative Negative Negative O Rh D negative
Positive Negative Positive A Rh D positive
Positive Negative Negative A Rh D negative
Negative Positive Positive B Rh D positive
Negative Positive Negative B Rh D negative
Positive Positive Positive AB Rh D positive

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 Positive Positive Negative AB Rh D negative

7.4.4 Report results as group A, B or AB, rhesus positive or negative.

Example: “Blood group A Rh D positive”
7.4.5 Indicate the date and name of technical staff reporting.
7.5 Limitations.
7.5.1 Samples that give weak or doubtful reactions should be re-tested using the
tube method.

8.0 APPENDICES:
8.1 Not Applicable.

9.0 REFERENCES:
9.1 AMREF (2008), Standard operating procedures for essential laboratory tests,
AMREF – MOH publication.
9.2 Monica Cheesbrough (2006), District laboratory practice in tropical countries,
Cambridge University press, Part 2.

10.0 DOCUMENT CHANGE HISTORY:

10.1 Version Table:

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 Version 1: Blood Grouping by Tile or Slide Dated: SOP No.: No. Pages:
Method. LBTS 0002 7.

Version 2: Dated: SOP No.: No. Pages:

Version 3: Dated: SOP No.: No. Pages:

1.1 SOP Review Log.

Date of Changes made. Name of reviewer. Signature.
review.

2.0 SOP AWARENESS LOG.

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I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

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LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………………………………………………………

COUNTY……………………………………………………………………………………………………………

SUB COUNTY………………………………………………….…………………………………………………

SOP Title: LABORATORY QUALITY CONTROL SOP No: 020

Version: Original
Effective Date: January 2016 Page 1 of 10

Signatures and Dates:

Author: _ ________________________________________________________________
Laboratory Technologist Date

QA Review: _______________________________________________________________
QA Officer Date

Approving Authority: _______________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 PURPOSE / INTRODUCTION:
1.1 Quality Control is the daily process designed to ensure that a test system
works properly. Quality control is a series of practices carried out for each
test on each day that subject tests are performed. It consists of analysing
specimens of known concentration, carefully recording and interpreting
these values before testing or releasing subject/patient results. It deals with
the analytical phase of testing.

1.2 Quality control is performed in the laboratory to ensure that accuracy and
precision are maintained in the performance of laboratory analysis each day.
There must be an on-going daily quality control program with established
internal review procedure. In addition, an external proficiency testing
program for each test performed should be implemented.

2.0 SCOPE / RESPONSIBILITY:

2.1 This SOP applies to all laboratory personnel and other departments that use
the laboratories services.
2.2 It is the responsibility of the designated of the County Leads, Quality
Assurance and Quality Control (QC/QA) personnel, Laboratory Supervisors to
ensure that the current SOP is available to the laboratory personnel and the
procedure is followed as documented.

3.0 SAFETY/RISK ASSESSMENT:

3.1 All specimens should be regarded as containing infectious agents and
universal precautions should be observed.

4.0 DEFINITIONS:

4.1 CA: Corrective Action

4.2 EQA: External Quality Assessment
4.3 QA: Quality Assurance
4.4 QC: Quality Control
4.5 SOP: Standard Operating Procedure

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5.0 SPECIMEN:
5.1 Quality Control material (in-house or commercial)

6.0 EQUIPMENT/ MATERIALS/ REAGENTS:

6.1 Equipment.
6.1.1 Respective laboratory equipment
6.2 Materials.
6.2.1 Control materials: Commercially prepared, assayed control specimens
will be incorporated in all analysis where applicable.
6.2.2 In assays where commercially prepared controls are not available,
laboratory prepared controls will be generated, quantitated and
validated before being placed in service.
6.2.3 Controls will be at least bi-level with normal and abnormal
parameters represented
6.3 Reagents.
6.3.1 Respective analytical reagents

7.0 METHODOLOGY:
7.1 Controls
7.1.1 Quality Control material from manufacturer or internally prepared
control material is used.
7.1.2 Assures the technician/technologist that the test system is
functioning properly.
7.1.3 Generates documentation for regulatory agencies that the laboratory
performs at a high level of competency.
7.1.4 Guarantees the suitability of results by monitoring test systems and
detecting variations in its performance.
7.1.5 Internal Quality Control procedures monitor analytical performance
relative to medical goals and alert analysts to unsatisfactory analytical
performance. The use of Quality Control procedures facilitates the
evaluation of laboratory analysis in progress to ascertain the
acceptability or unacceptability of subject or patient data.

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 7.1.6 External quality control and management information facilitates the
clinical interpretation of laboratory data by providing inter-laboratory
comparison of analytical and relative bias.

7.2 Control material incorporation

7.2.1 Quality control specimens will be incorporated into routine testing
each day subject or patient specimens are run where applicable and
performed by the same personnel.
7.2.2 Instrument maintenance is addressed in individual instrument
maintenance SOPs.

7.3 Reagent Quality Control

7.3.1 Change of reagent lot number will be documented on the appropriate
logs according to assay specific Quality Control and lot testing; refer
to assays Quality Control and Lot testing SOPs.
7.3.2 Controls will be run on new reagent lots prior to running subject
specimens.
7.3.3 Acceptable ranges will be determined by individual assays and kits
used but shall not be more than 3SD where applicable.
7.3.4 The use of expired reagents is not allowed.

7.4 Daily Operation

7.4.1 Run the control. Record the result on the control log sheet where
available.
7.4.2 Check the value obtained against the established reference values for
that control depending on the specific assays.
7.4.3 If the value is within the range of established values, run the next
control or proceed with client samples.
7.4.4 Refer to assay specific SOP where this is not applicable.

7.5 Out of Range Controls:

7.5.1 If the value is not within the range, plot and write “R” for rerun
adjacent to the point and repeat the test using the same control.

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 Record the result on the control log sheet and/or chart.
7.5.2 If the repeated value is within the range, run the next level of control
or proceed with client samples.
7.5.3 If the repeated value is still not within the range, plot and write “R2”
for second rerun adjacent to the point
7.5.4 Repeat the test using a new vial of control. If the value obtained is
within range, plot the value and write “New vial, control within range”
on comments.
7.5.5 Run the next control or proceed with client samples.
7.5.6 If the value is not within range, troubleshoot to establish the cause of
the problem before attempting to run more controls or client
samples.
7.5.7 Refer to manufacturer’s procedure manuals for troubleshooting
steps.
7.5.8 If the cause for Quality Control failure is established, e.g. instrument
related, QC material deterioration etc., document all the causes and
findings on the corrective action log; refer to SOP LGEN 0025 for CA
log.
7.5.9 After troubleshooting the instrument, repeat the control analysis.
7.5.10 If the value is within the range, plot the value and run the next level
control or proceed with client samples.
7.5.11 If the repeated value is consistently not within the range, contact the
Laboratory manager/designee. DO NOT run or report client samples
for that run.

7.6 Establishing Acceptable Limits.

7.6.1 Whenever a new lot of Quality Control material arrives, parallel
studies must be done before it is put into daily use.
7.6.2 The individual assay SOP should be followed for parallel studies and to
establish new ranges where applicable.

7.7 Sources of variation or error.

7.7.1 Random error

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 7.7.1.1 One control greater than 3SD or both controls greater than
2SD in same direction: May be due to instrument error
(voltage fluctuation, sampling error), sample error
(anticoagulant or drug interference), or human error (mixing
of control samples).
7.7.2 Systematic error
7.7.2.1 Both controls are greater than 2SD in the same direction or
one control is between 2 and 3SD on successive runs: May be
due to instrument error (dirty photometer, faulty ISE), or
decomposition of standards or reagents (evaporation,
crystallization, or microbial or chemical contamination).

7.8 Examination/Evaluation Procedure

7.8.1 Trend: 6 or more consecutive values that all increase or decrease.
7.8.1.1 A trend may be additive; when two controls are used, a trend
would be indicated when both controls have 3 or more
consecutive values all increasing or decreasing.
7.8.1.2 A trend is consistent with constant systematic error.
7.8.2 Shift: 6 or more consecutive values that fall on one side of the mean.
7.8.2.1 A shift may be additive.
7.8.2.2 A shift is consistent with constant systematic error.
7.8.3 Trends and shifts will be evaluated for cause. The procedure and
findings will be documented on the corrective action log for that
instrument. After completion of the evaluation, this information will
be forwarded to the Laboratory manager/designee for review and
appropriate action if required.

7.9 Quality Control Review

7.9.1 The Tech running the assay will review Quality Control reports daily
when run.
7.9.2 The section supervisor/designee will review QC reports at least
weekly as applicable and the Laboratory Manger/designee will review
QC reports at least monthly.

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 7.9.3 Initials of the reviewer and date will be annotated on QC sheet
summaries as applicable.
7.9.4 The monthly QC data has to be reviewed and signed by Laboratory
Manager/designee by the 15th day of each month. Document a CA for
late submission of QC data.
7.9.5 Retrospective control charts will be reviewed for outliers due to
clerical errors by Quality Assurance/Quality Control personnel and/or
section supervisors/designee.
7.9.6 Prospective control charts will be verified for adequacy of limits at the
various levels of reviews.
7.9.7 Preventive maintenance and function verification will be performed
and recorded in accordance with individual equipment maintenance
SOP.

7.10 Quality Control material and Specimen Rejection:

7.10.1 Quality control materials received from the vendor with broken
containers/vials, out of required temperature ranges, contaminated
and/or with short shelf life will be rejected and returned or destroyed
depending on the safety assessment.
7.10.2 Document any rejections and communications with the vendors.
7.10.3 Specimens which meet rejection criteria as defined in the Specimen
reception and rejection SOP and/or assay specific SOPs will be
rejected.
7.10.4 Rejection of specimens shall be documented in the Specimen
Rejection Form.

7.11 Specimens/reagents transfer:

7.11.1 Movement of specimens and/or reagents from one location to
another shall be authorized by the laboratory manager/designee and
will be dictated by the various reasons including cold storage
equipment malfunctions or inadequate storage space.
7.11.2 The Specimen/Reagent Transport Inventory Sheet (appendix 8.1)
must be completed and signed by all the relevant personnel

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 8.0 APPENDICES:

8.1 Specimens/Reagents Transport Inventory Sheet

Specimens/Reagents Transport Inventory Sheet

Date:

Laboratory site: 24hr contact:

Shipping personnel: Signature:

Courier: Contact:

Shipping date: Time: Shipping temp:

Receiving date: Time: Receiving temp condition:

Receiving technician: Signature

Specimens ID or Specimen/ Specimen/

Date Taken Reagents Lot# Reagent Reagent Sending Comment Receiving Comment
Volume (ml) Type

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Additional Comments: ___________________________________________________________________________________
______________________________________________________________________________________________________

Lab Manager/designee: (Printed):

Signature: Date:

9.0 REFERENCES:
9.1 NCCLS. Application of a Quality Management System Model for the
Laboratory Services; Approved Guideline – Third Edition. NCCLS document
GP26-A3 [ISBN 1-56238-553-4]. NCCLS, 940 West Valley Road, Suite 1400,
Wayne, Penneslyvania 19087 – 1898 USA, 2004.
9.2 NCCLS. A Quality Management System Model for Health Care; Approved
Guideline – Third Edition. NCCLS document HS1-A2 [ISBN 1-56238-554-2].
NCCLS, 940 West Valley Road, Suite 1400, Wayne, Penneslyvania 19087 –
1898 USA, 2004.

10.0 DOCUMENT CHANGE HISTORY:

10.1 Version Table:
Version 1: Laboratory Internal Quality Dated: SOP No. Pages:
Control No..LGEN. 10
0026
Version 2: Dated: SOP No.: No. Pages:

Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log.

Date of Changes made. Name of reviewer. Signature.
review.

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11.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

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 LABORATORY STANDARD OPERATING
PROCEDURES
FACILITY NAME…………………………………………………

COUNTY……………………………………………………………

SUB COUNTY………………………………………………………

SOP Title: BLEEDING TIME SOP No: 001

Version: Original
Effective Date: January 2016 Page 1 of 5

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Purpose & Scope
1.1Purpose
To outline the procedure for performing the Duke method Bleeding time test.

1.2 Scope
This procedure is applicable to Tabaka Mission Hospital Laboratory Haematology section.

1.3 Principle & Safety

1.3.1 Principle
Bleeding time test checks gross vascular status and platelet function. A controlled puncture
incision is made in the free hanging ear lobe and a piece of blotting paper is applied to the
bleeding drop. The length of time it takes for bleeding to stop is recorded.

1.3.2 Safety;
Handle all laboratory samples as if capable of transmitting infections; always use personal
protective equipment.

2.0 Abbreviation, Definitions and Terms

2.1 Abbreviations
N/A

2.2Definitions and Terms

N/A

3.0 Equipment and Materials/Reagents/Supplies

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Page 2 of 5
 3.1 Equipment
None

3.2 Materials/Reagents/Supplies
3.2.1 Sterile sharp blade
3.2.2 Timer
3.2.3 Blotting paper
3.2.4 Sterile alcohol swabs
3.2.5 Cotton wool

4.0 Responsibility
4.1 The Head of section is responsible for monitoring the effective implementation of this
procedure.
4.2 Technical staff are responsible for the implementation of this procedure.

5.0 Procedure
5.1 Procedural steps
5.1.1 Wipe the ear lobe with an alcohol swab.
5.1.2 Make an incision just deep enough to cause a tiny amount of bleeding on the free
hanging ear lobe
5.1.3 Apply piece of blotting paper to the bleeding drop every 30 seconds until the bleeding
stops, making sure that the paper does not touch the skin.
5.1.4 Wipe and dry the ear lobe with dry cotton wool.
5.1.5 Count the number of drops and multiply by 30 seconds.
5.1.6 Record the length of time it takes for bleeding to stop.

5.2 Reporting
5.2.1 Report in seconds
5.2.2 Normal ranges 60 seconds – 180 seconds.

5.3 Abnormal Values can be due to:

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Page 3 of 5
 5.3.1 Hodgkin’s disease
5.3.2 Acute Leukaemia
5.3.3 Disseminated intravascular coagulation.
5.3.4 Purpura
5.3.5 Haemolytic Disease of the newborn.

6.0 Related procedures

There are no related procedures

7.0 References
7.1 Nicholas J, Vardaxis, Pathology for the health sciences 2004, 7th Edition, Macmillan
Publishers Australia.

8.0 Appendix
N/A

Training log

All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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Page 4 of 5
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Page 5 of 5
 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME………………………………………………………………………………………………

COUNTY………………………………………………………………………………………………………..

SUB-COUNTY………………………………………………….……………………………………………..

SOP Title: DIFFERENTIAL COUNT SOP No: 009

Version: Original
Effective Date: January 2016 Page 1 of 8

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Purpose/Applicability

1.1. Purpose

To describe accurately the method for performing white blood cells differential count to
provide presumptive evidence of infections (bacterial, parasitic, viral), and screening for
leukaemia. The test is also performed to monitor treatment of these conditions.

1.2 Scope

This procedure applies to performing white blood cell differential count method at
Tabaka Mission Hospital laboratory

1.3 Principle/Background

There are five types of white cells in normal blood: neutrophils, lymphocytes,
monocytes, eosinophils and basophils. White cells are recognized by their appearance
(morphology) in a stained thin blood film. The differential white cell count is an
estimation of the percentage of each type of white cell in blood. The differential white
cell count is performed by examining a stained thin blood film. A platelet estimate and
the morphology of red cells and platelets is also examined.

The differential white cell count and blood cell morphology are performed for a variety
of reasons, including providing presumptive evidence of infections (bacterial, parasitic,
viral), determining the type of anaemia and screening for leukaemia. The test is also
performed to monitor treatment of these conditions.

2.0 Definitions /Abbreviations and Terms

TMHL: Tabaka Mission Hospital Laboratory

3.0 Equipment and Materials/Reagents/Supplies

1. Prepared Field Stain A and B, Giemsa or Leishman stains
2. Absolute methanol

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3. Grease pencil/pencil
4. Coverslips
5. Immersion oil
6. Clean glass slides
7. Differential blood cell counter
8. Staining rack
9. Timer clock/stopwatch
10. Microscope
11. Sharps box or container
12. Disposal bucket

4.0 Responsibility/Safety
4.1 Responsibility
All TMHL personnel working in haematology bench are responsible for the
implementation of this procedure.
4.2 Safety
Gloves must be worn at all times during the procedure

5.0 Procedure
5.1 Sample required
Capillary or venous blood anti-coagulated with EDTA. Prepare stained films within 4 hours
of collecting venous blood. Stored or haemolysed blood is not suitable for examination of
blood cell morphology. Do not use blood anti-coagulated with heparin because heparin
causes background staining, and poor staining and clumping of white cells and platelets.

5.2 Method
1. Prepare a thin blood film on a clean slide and label with the patient’s laboratory number
by writing with lead pencil across the thickest part of the film or on the frosted end of
the slide.
2. Fix by dipping in methanol for 2 seconds.

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3. Stain using the reverse Field stain, 10%Giemsa or Leishman stains and allow the film to
dry.
4. Place a drop of immersion oil on the film (towards the tail) and put on a coverslip. Place
the slide on the microscope stage.
5. Scan the whole film briefly using the lowest power objective available (x4 or x10) to
assess the quality of the film and staining technique, and to detect large parasites such
as microfilariae.
6. Swing the x10 objective into position and scan the "tail" (thin) area of the film. Assess
the distribution of the red cells for rouleaux formation. Estimate the total white cell
count and assess the predominant white cell type and the presence of patchy
abnormalities, e.g. eosinophilia or basophilia. With experience, white cells are easily
distinguished using the x10 objective. If you see a suspicious looking cell, briefly examine
it using the x40 objective and make a note of it.
7. Select a portion of the film where the red cells are evenly distributed and are just
overlapping. This is usually 2 or 3 fields inside the tail area. Swing the x100 objective into
position, examine the film systematically across its width and perform a differential
white cell count using a multi-key differential counter. Count each type of white cell in
every field to a total of 100 white cells.
8. Note and count abnormal or immature forms of white cells and include them in the
differential white cell count.
9. Note and count nucleated red cells per 100 white cells. Do not include nucleated red
cells in the differential white cell count. Now examine the red cells in at least 5 fields.
Determine size (by comparing with a small lymphocyte), shape, haemoglobin content
(whether central pallor is absent or occupies more than one third of the diameter of the
red cell), and presence of immature forms.
10. Now estimate the platelet count by comparing the number of platelets with the number
of red blood cells in a field. Normally the RBC: platelet ratio is about 10-15:1. Note any
marked variation in platelet size.
11. Use the xl00 oil immersion objective to examine inclusions, e.g. Howell-Jolly bodies,
basophilic stippling and malaria parasites. Record the results.

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 12. Remove the cover slip and place in the container of 5% Lysol marked "COVERSLIPS".
Place important or interesting slides face down on tissue paper for some hours or
overnight to remove the oil, and store for reference.

5.3 Procedural notes

• It is not possible to examine a thin blood film in a hurry. A good assessment takes about 15
minutes. A well-spread and well-stained film is essential. Accurate recognition of red cell
morphology improves with practice.
• In films prepared from capillary blood, platelets readily clump and are seen in masses at the
edges of the film. It is not be possible to estimate the platelet count in the presence of
marked platelet clumping.
• Large cells and parasitized cells are often "pushed" to the edges of the film during
spreading.

Summary of peripheral blood film examination

Objective
Assessment

X4 Quality of film and staining technique; presence of large parasites, e.g.

microfilariae.
X10 Arrangement of red cells, e.g. presence of rouleaux; estimation of total
white cell count; predominant white cell type and presence of patchy
abnormalities.
X40 Differential white cell count including abnormal or mature forms; red cell
size, shape, haemoglobinisation, immature forms; estimation of platelet
count and size.
X100 Cell inclusions; detailed examination of individual cells.
(Oil
immersion)

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6. Quality control

Refer to a book, atlas or bench aids if you find cells that are not easily identified.
Abnormal findings must be counter checked by a responsible experienced person.
Store stained slides for checking by a visiting supervisor.
Send stained slides to a reference laboratory for an expert opinion.

7. Interpretation
• Clinically useful red cell forms include hypochromic microcytic cells, oval macrocytic cells,
spherocytic cells, fragmented red cells, sickle shaped cells, nucleated red cells, red cells with
inclusions.
• Clinically useful white cell forms include toxic granulations in neutrophils, "left shift" of
neutrophils (immature neutrophils), atypical lymphocytes, hyper-segmented neutrophils
and blast cells.

Reference values for differential white cell counts [Dacie, Lewis. 1993]
Infants at Birth Children (aged 6 yrs)
Adults

% of total Absolute % of total Absolute % of total WBC count

WBC count WBC count
count (x109/1) count (x109/1)
Neutrophils 40 – 75 2.0 - 7.5 40 – 75 5 – 13 20 - 60
Lymphocytes 20 – 50 1.5 – 4 29 – 47 3.5 - 8.5 55 - 85
Monocytes 2 - 10 0.2 - 0.8 4–8 0.5 - 1.5 7 - 15
Eosinophils 1–6 0.04 - 0.4 1 – 13 0.1 - 2.5 3-8
Basophils 0–1 0.01 - 0.1 0–1 0.01 – 0.1 0.1 – 1

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8. Reporting results

Report percentage of different types of white cells and indicate the reference range. Report
normal and any abnormal white cells, red cells or platelets.
Example: WBC (differential count) Neutrophils 62%
Lymphocytes 32%
Monocytes 4%
Eosinophils 2%
Basophils 0%

Comments:
RBC morphology: Normocytic normochromic
WBC: Normal in number and appearance and distribution
Platelets: Adequate in numbers, normal size.

Indicate date and name of technical staff reporting.

9. References

• F. Baker and S. Silverton (1980). Introduction to medical laboratory technology. 5th

Edition.

• AMREF (2008). Standard operating procedures, Essential Laboratory Tests

• Dacie and Lewis (1993). Practical haematology

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Training Log
All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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 LABORATORY STANDARD OPERATING
PROCEDURES
FACILITY NAME……………………………………………………………..

COUNTY……………………………………………………………………….

SUB COUNTY…………………………………………………………………

SOP Title: CAPILLARY BLOOD COLLECTION SOP No: 004

Version: Original
Effective Date: January 2016 Page 1 of 16

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review:
__________________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

1.0 PURPOSE / INTRODUCTION:
1.1 Blood specimens are obtained by either venous or capillary punctures. The
source of the specimen is determined chiefly by the quantity of blood required
to perform the laboratory procedures, age and condition of the subject/patient.
1.2 Sample volume/test requirements will be as specified in the protocol and must
be in line with the clients safety standards.
1.3 The purpose of this Standard Operating Procedure is to provide guidelines
proper specimen collection in accordance to the required laboratory standards
and to safeguard on the quality of results received after analysis.
1.4 It will also outline the order of drawing, care of hematomas, color-coded
system for easy identification of collection tubes, emergency action to take if
the subject becomes ill during the phlebotomy procedure and Quality
Assurance/Quality Control procedures.
1.5 Strict adherence to SOP requirement ensures quality laboratory results.

2.0 SCOPE / RESPONSIBILITY:

2.1 This SOP applies to all Health Care Workers involved in the collection of
Capillary/Venous blood in facilities and during community activities.
2.2 It is the responsibility of the designated of the County Leads, Quality
Assurance and Quality Control (QC/QA) personnel, Laboratory Supervisors to
ensure that the current SOP is available to the Health Care Workers and the
procedure is followed as documented.

3.0 SAFETY/RISK ASSESSMENT:

3.1 Wear personal protective equipment.
3.2 Handle all samples as potential biohazards.
3.3 Never recap needles.

4.0 DEFINITIONS:
4.1 CPR: Cardiopulmonary Resuscitation.
4.2 EDTA: Ethylene diamine tetra-acetic acid
4.3 I.V.: Intravenous.
4.4 QA: Quality Assurance.
 4.5 QC: Quality Control.
4.6 SOP: Standard operating procedure.
4.7 SST: Serum Separation Tube

5.0 SPECIMEN:
5.1 Use capillary blood from the side of the finger in adults and children.
5.2 In infants, collect capillary blood from the side of the heel or big toe.
5.3 Volume of venous blood collected will depend with the individual tests
requested. Please see individual tests SOPs for the required blood volumes.

6.0 EQUIPMENT/ MATERIALS/ REAGENTS:

6.1 Equipment.
6.1.1 Not applicable.
6.2 Materials.
6.2.1 Vacutainer Holder
6.2.2 Syringes, Needles/ butterfly
6.2.3 Tourniquet
6.2.4 Isopropyl alcohol (70%)
6.2.5 Phlebotomy trays
6.2.6 Sharps container
6.2.7 Swabs
6.2.8 Gauze
6.2.9 Gloves
6.2.10 Lab coat
6.2.11 Rockers/Mixers/Rollers
6.2.12 Sample racks
6.2.13 Lancets
6.2.14 Hand paper towels
6.2.15 BD Vacutainer Tubes or equivalent
6.2.15.1 Red Top - Tubes do not contain anticoagulant and are
used to obtain clotted or serum specimens. Serum is required
for serology tests, most chemistries, compatibility tests, and
blood typing and cold agglutinins.
6.2.15.2 Serum Separation Tube (SST),{Marble top/gold
 topped} - Is a serum separation tube containing “Thixotropic
Gel” in the bottom of the tube. During centrifugation, the gel
temporarily becomes fluid and moves to the dividing point
between the serum and cells. These tubes are known as “Quick
Clot” tubes and are used for STAT chemistry testing.
6.2.15.3 Grey Top - Tubes that can contain anticoagulant
potassium/sodium oxalate or sodium fluoride or sodium
iodoacetate and are used to obtain whole blood or plasma. The
glucose tolerance test, fasting blood sugar, lipase, lactate,
alcohol test, protein, urea, bilirubin and bicarbonate are drawn
in these tubes. For lactate testing, tubes of blood should be kept
closed at all times in a vertical, stopper-up position. Keep
samples on ice. Plasma should be physically separated from
contact with cells within 15 minutes of sample collection, and
analysed without delay
6.2.15.4 Green Top - Tubes contain sodium or lithium heparin
anticoagulant. The most common test drawn is for whole blood
studies, chemistries, immunophenotyping and heavy metals.
6.2.15.5 Purple Top - Tubes contain the anticoagulant ethylene
diamine tetra-acetic acid (EDTA) and are used to obtain whole
blood or plasma. Most common tests drawn for are CBC
differential, CD4 T-cell enumeration, viral load, glycosylated
Hb analysis. Erythrocyte sedimentation rate, reticulocyte count,
red blood cell cholinesterase, sickle cell test and lead.
6.2.15.6 Yellow Top - Tubes contain Acid Citrate Dextrose
(ACD) anticoagulant and used to obtain whole blood for
PBMC separation and plasma for storage
6.2.15.7 Blue Top - Tubes contain Sodium Citrate and used to
obtain whole blood for coagulation determinations and
erythrocyte sedimentation rate.
6.3 Reagents.
6.3.1 Not applicable.

7.0 METHODOLOGY:
7.1 Procedure.
7.1.1 Sequence of blood specimen draws
7.1.1.1 Whole blood
7.1.1.2 First draw- blood culture tubes, sterile tubes
7.1.1.3 Second draw – tubes with no additives (i.e. red)
7.1.1.4 Third draw – coagulation tubes (i.e. light blue)
7.1.1.5 Last draw – tubes with additives.
7.1.2 Quality assurance/quality control
7.1.2.1 Procedures to be followed to ensure as little trauma to the
clients as possible include the following:
7.1.2.1.1 Label tubes IMMEDIATELY after phlebotomy.
7.1.2.1.2 If you notice the venepuncture area beginning to
swell while drawing the blood, immediately release
the tourniquet, remove the needle, and apply
pressure with gauze or equivalent.
7.1.2.1.3 Remove tourniquet after drawing the subject’s
blood.
7.1.2.2 If an accidental needle stick occurs, contact your co-
worker/immediate supervisor, safety officer, post exposure
prophylaxis designate and the site director. Wash the area with
soap and running water and follow up with medical treatment.
7.1.2.3 Do not collect venous blood from the arm of recent or with an
I.V infusion.
7.1.2.4 Collect venous blood from upper stream if both arms are on I.V
infusions or in case of disability situations.
7.1.2.5 Samples should be delivered to the lab within one hour of
collection unless specified otherwise.
7.1.2.6 Sample collection devices must be used within their expiration
dates.
7.1.3 Preparation of clients.
7.1.3.1 Properly identify the subject; this should be done in accordance
with the protocol requirement or method of patient/participant
identification. This should include but not limited to:
7.1.3.2 Call out the participant’s names.
 7.1.3.3 Sit the subject comfortably in the chair. Introduce yourself.
7.1.3.4 Explain the procedure to the patient/client.
7.1.3.5 Assemble all materials to ensure easy access, check tests
requested and select proper tubes.
7.1.3.6 If tubes are unknown, ask laboratory supervisor/designee for
correct tube and volume. Tubes that contain additives should be
gently tapped to dislodge any additive that may be trapped
around the stopper.
7.1.3.7 Wear the appropriate personal protective equipment (PPE)
7.1.3.8 Have the subject/patient roll up sleeve, place tourniquet above
elbow and tighten enough to find appropriate vein.
7.1.3.9 Palpate the antecubital fossa area and locate the desired vein.
Loosen tourniquet where necessary.
7.1.3.10 Starting from the centre and working outward, clean
area with 70% alcohol/ equivalent in a circular direction.
7.1.4 Vacutainer technique.
7.1.4.1 Open needle or butterfly package but do not remove the needle
shield.
7.1.4.2 Thread the needle into the holder until secure.
7.1.4.3 Do not re-palpate disinfected site. Have the subject make a fist
and straighten arm.
7.1.4.4 Remove the needle cover and inspect the needle to ensure that
it is not damaged.
7.1.4.5 Position needle with bevel up, parallel to and over the top of
the vein. Insert the needle quickly under the skin and then into
the vein.
7.1.4.6 After entry into the vein, loosen/release the tourniquet, then
push the tube all the way into the holder and allow the blood to
fill the tube.
7.1.4.7 To fill other tubes, remove the full tube and insert new tubes
until all required tube are filled.
7.1.4.8 Label the samples immediately after collection.
7.1.4.9 Countercheck client’s identification available on the request
and tube labels. Labels should include subject’s identification
 number, date and time of specimen collection
7.1.4.10 Place tubes in an appropriate rack/rocker/roller (where
applicable) for laboratory testing.
7.1.4.11 If no blood flows into the tube or blood ceases to flow
before an adequate specimen is collected, the following steps
are suggested to complete satisfactory collection:
7.1.4.11.1 Push tube forward until tube stopper has been
penetrated. If necessary, hold in place to ensure
complete vacuum draw.
7.1.4.11.2 Confirm correct position of needle/cannula in the
vein.
7.1.4.11.3 If the tube has a poor vacuum, remove the tube and
replace with a new tube.
7.1.4.11.4 If second tube does not draw, release and remove
tourniquet then remove needle and discard. Repeat
procedure.
7.1.4.12 Upon completion of the venepuncture, remove the
needle from the subject’s arm and apply pressure to the site
using a sterile piece of gauze/equivalent. Instruct the subject to
continue applying pressure for 2-3 minutes. Folding the arm is
not recommended.
7.1.4.13 Immediately dispose of needles into a sharps container.
7.1.4.14 When the venepuncture site has stopped bleeding, place
a Band-Aid over the site and escort the subject out of the
phlebotomy area.
7.1.4.15 Precautions
7.1.4.15.1 The only areas that are authorized for the
phlebotomist to draw blood from are arms and
hands.
7.1.4.15.2 Arterial sticks are strictly prohibited. In the event
that an accidental arterial stick occurs, immediately
loosen and remove tourniquet then remove needle
and place direct pressure on the site.
7.1.4.15.3 Maintain pressure on the site for minimum of three
 minutes.
7.1.4.15.4 After bleeding has clearly stopped, place a pressure
dressing on the site and have the subject apply more
direct pressure to the site for another 15 minutes.
7.1.4.15.5 If bleeding does not stop, a clinician should be
notified immediately.
7.1.4.15.6 Specimen collected for coagulation from
intravenous line should be flushed with saline
before drawing sample.
7.1.4.15.7 The first 5ml of blood should be drawn off and
discarded before the coagulation tube is filled.
7.1.4.15.8 The correct concentration of the anticoagulant is
fundamentally important to the precision of the
results for coagulation.
7.1.4.15.9 Do not under fill or overfill the tube for coagulation
studies.
7.1.4.15.10Specimens for coagulation studies should be
collected in 3.2% buffered sodium citrate
anticoagulant.
7.1.5 Syringe Technique:
7.1.5.1 Break seal in the syringe by removing the plunger up and down
in the barrel. Expel all air from the syringe.
7.1.5.2 Insert the needle into the syringe. Twist the needle on the
syringe and make sure it fits securely. Some syringes come
with the needle already in place.
7.1.5.3 Do not re-palpate disinfected site. Have the subject make a fist
and straighten arm.
7.1.5.4 The syringe should be placed below the venepuncture site to
prevent backflow, and the arm placed in the downward
position.
7.1.5.5 Hold the subject’s arm firmly 1 to 2 inches below the puncture
site pulling the skin tight with your thumb.
7.1.5.6 Hold the syringe with the opposite hand between the thumb and
the last three fingers. Rest the index fingers against the hub of
 the needle to serve as a guide.
7.1.5.7 The needle should be in the bevel up position, pointing in the
same direction as the vein, and should make an approximate
15-degree angle with the arm.
7.1.5.8 The vein should be entered slightly below the area where it can
be seen. In this way, there is tissue available to serve as an
anchor for the needle.
7.1.5.9 As the needle enters the vein slightly less resistance should be
felt.
7.1.5.10 A small amount of blood will flow into the neck of the
syringe as the needle enters the vein.
7.1.5.11 When using a syringe, care must be taken not to pull on
the plunger too rapidly or forcefully. This may cause the blood
to haemolyse, pull the wall of the vein down on the bevel of the
needle causing the blood flow to stop, or cause the needle to
inadvertently be pulled out of the vein.
7.1.5.12 When blood enters the syringe, release the tourniquet.
7.1.5.13 After the desired amount of blood is obtained, remove
the needle. A gauze pad should be placed lightly over the
venepuncture site and slight pressure applied to the pad as the
needle is slowly removed. The bevel should still be in the
upward position.
7.1.5.14 Instruct the subject to continue applying pressure for 2-
3 minutes.
7.1.5.15 Fill the required tubes by placing the needle into the top
of the tube and allowing the vacuum to draw blood into the
tube until the blood flow stops. This process should be
accomplished quickly before the blood begins to clot.
7.1.5.16 Label the samples immediately after collection.
7.1.5.17 Countercheck client’s identification available on the
request and tube labels. Labels should include subject’s
identification number, date and time of specimen collection
7.1.5.18 Place tubes in an appropriate rack/rocker/roller (where
applicable) for laboratory testing.
 7.1.5.19 Immediately place the syringe in a sharps container
with the unsheathed needle attached.
7.1.5.20 If an accidental needle stick occurs, contact your co-
worker/immediate supervisor/safety officer/post exposure
prophylaxis designate and the site director.
7.1.5.21 Wash the area with soap and running water and follow
up with medical treatment. (See needle stick injury protocol).
7.1.5.22 When the venepuncture site has stopped bleeding, place
a bandage over the site and escort the subject out of the
phlebotomy area.
7.1.5.23 Note: In case of difficult draw by this method, a
clinician can use the femoral vein to obtain blood.

7.2 Finger stick Procedure

7.2.1 The best locations for finger sticks are the 3rd (middle) and 4th
(ring) fingers of the hand. Do not use the tip of the finger as it
is very sensitive or the centre of the finger. Avoid the side of
the finger where there is less soft tissue, where vessels and
nerves are located, and where the bone is closer to the surface.
The 2nd (index) finger tends to have thicker, skin. The fifth
finger tends to have less soft tissue overlying the bone. Avoid
puncturing a finger that is cold, swollen, scarred, or covered
with a rash.
7.2.2 Massage finger gently 5-6 times from base to tip to aid blood
flow.
7.2.3 Have the client hold their hand in a defendant position to help
increase blood supply to the hand.
7.2.4 Clean the site with saturated 70% isopropyl alcohol pad. Allow
the site to dry completely so as to provide effective disinfection
and to prevent possible haemolysis by residual alcohol.
7.2.5 Using a sterile lancet or equivalent, make a skin puncture just
off the centre of the finger pad.
7.2.6 The puncture should be made perpendicular to the ridges of the
fingerprint so that the drop of blood does not run down the
 ridges.
7.2.7 Wipe away the first drop of blood, which tends to contain
excess tissue fluid.
7.2.8 Collect drops of blood into the collection device by gently
massaging the finger. Avoid excessive pressure that may
squeeze tissue fluid into the drop of blood.
7.2.9 Have the patient hold a small gauze pad over the puncture site
for a couple of minutes to stop the bleeding.
7.2.10 Dispose of contaminated materials/supplies in designated
containers.

7.2.11 Continued Bleeding:

7.2.11.1 Apply pressure to the site with a gauze pad until
the bleeding stops.
7.2.11.2 Wrap a gauze bandage tightly around the arm
over the pad.
7.2.11.3 Tell the subjects to leave the bandage on the site
for at least 15 minutes.
7.2.11.4 Excess Bleeding:
7.2.11.5 If the bleeding persists longer than 5 minutes,
contact the clinician in charge.
7.2.11.6 Continue applying pressure on the site as long as
necessary to control the bleeding.

7.3 Emergency Phlebotomy Procedure

7.3.1 In the event a subject starts feeling sick or appears to be
passing out, follow these steps:
7.3.1.1 Remove the tourniquet and withdraw the needle from
the arm at the first sign of reaction during the
phlebotomy.
7.3.1.2 If possible, clear the phlebotomy room area so that the
subject experiencing the adverse reaction can be
attended to in privacy.
7.3.1.3 If the subject does not respond rapidly to the measures
 listed below, contact the on-site supervisor or clinician
immediately.
7.3.2 Subject Feels Faint:
7.3.2.1 Talk to the subject to ensure that they are conscious and
able to follow simple instructions.
7.3.2.2 Call for assistance.
7.3.2.3 Administer small amounts of aromatic spirits of
ammonia by inhalation. Utilise an ice pack or a cold
compress for head and or/neck.
7.3.2.4 If subject is sitting, lower his/her head and arms. If
possible, assist the subject to the floor and elevate feet
above the level of the heart.
7.3.2.5 Allow the subject to drink water.
7.3.2.6 Continue to monitor the subject until he/she feels fully
recovered.
7.3.2.7 Notify the site supervisor or clinician of the situation.
7.3.3 Fainting Subject:
7.3.3.1 Seek assistance in the phlebotomy area.
7.3.3.2 Never leave the subject unattended.
7.3.3.3 Place the subject on their back and raise the subject’s
feet.
7.3.3.4 Be sure the subject has an adequate airway, is breathing,
and has a pulse.
7.3.3.5 Administer aromatic spirits of ammonia by inhalation.
The subject should respond by coughing.
7.3.3.6 Loosen tight clothing.
7.3.3.7 Apply cold compresses to the subject’s head and /or
neck.
7.3.3.8 Continue to monitor the subject’s breathing and pulse
until assistance arrives from a clinician.
7.3.4 Nauseated subject:
7.3.4.1 Make the subject as comfortable as possible.
7.3.4.2 Instruct the subject to breathe deeply and slowly.
7.3.4.3 Apply cold compresses on the subject’s forehead and
 back of neck.
7.3.5 Vomiting subject:
7.3.5.1 Give the subject an emesis basin/equivalent, and have
tissue/paper towel/equivalent ready.
7.3.5.2 Give the subject water to rinse out his/her mouth.
7.3.5.3 Report the incident to the site supervisor or clinician.
7.3.6 Convulsing subject:
7.3.6.1 Call for help and notify a physician immediately.
7.3.6.2 Prevent the subject from injury by placing him/her on
the floor.
7.3.6.3 Be sure the subject has an adequate airway, is breathing
and has a pulse.
7.3.6.4 Be cautious because some people exhibit great muscular
strength and are difficult to handle during severe
seizures.
7.3.6.5 Continue to monitor the subject’s breathing and pulse
until the clinician arrives.
7.3.7 Cardiac or Pulmonary Arrest subject:
7.3.7.1 Call for help and notify the clinician immediately.
7.3.7.2 If the subject is in cardiac arrest, begin CPR
immediately if you are trained, and continue until the
clinician arrives.
7.3.8 Care of Hematomas:
7.3.8.1 A hematoma is a small collection of blood under the
skin. Initially, it may appear as a small lump. This may
occur following a phlebotomy procedure when blood is
taken for laboratory testing. There might be slight pain
or discomfort at the site of the hematoma. The skin will
reabsorb this blood within several days. During the
reabsorption process the skin will appear “black and
blue”. Any extension of this area of discoloration
represents your body’s way of removing the blood from
a place it should not be. This extension of discoloration
DOES NOT mean that you are continuing to bleed. As
 the blood is reabsorbed, skin colours of brown and
yellow will be seen.
7.3.8.2 First and most importantly, do not engage in activity
that requires strenuous use of the arm. For the first 24
hours after blood collection, place crushed ice in a
plastic bag, wrap it in cloth, and hold on the hematoma
for approximately 15 minutes. Repeat at intervals
during the first 12 hours. If you secure the cold pack to
the site of the hematoma with a pressure dressing, make
sure the dressing is not applied too tightly. If the
pressure dressing becomes too tight as evidenced by
tingling or cold fingers, discoloration of the hand or
lower arm, or discomfort, unwrap the dressing and
rewrap with less pressure. Do not keep dressing on
longer than 15 minutes at any one time. Do not go to
bed with the dressing on.
7.3.8.3 After 24 hours, warm compresses are advised
periodically. A warm washcloth may be used for this
purpose. The warm compress should be applied to the
hematoma for 15 minutes. Warm soaks will quicken the
reabsorption process of blood and relieve any
tenderness that may be present.
7.3.8.4 Remember, the best way to prevent a hematoma from
occurring is by applying DIRECT PRESSURE to the
site of the blood collecting area. Direct pressure will
inhibit blood from seeping out of the vein; allow for the
needle punctured site in the vein to close, and therefore
decreases the chance of a hematoma developing.
Maintain direct pressure to the site for 3 to 5 minutes
after blood collection.
7.3.8.5 Any further questions you may have can be directed to
the site supervisor or clinician.
8.0 APPENDICES:
8.1 Not applicable.
9.0 REFERENCES:
9.1 AMREF (2008), Standard operating procedures for essential laboratory tests,
AMREF – MOH publication.

9.2 Monica Cheesbrough (1998), District laboratory practice in tropical

countries, Cambridge University press, Part 1.

9.3 Monica Cheesbrough (2006), District laboratory practice in tropical

countries, Cambridge University press, Part 2.

10.0 DOCUMENT CHANGE HISTORY:

10.1 Version Table:
Version 1: Collection of Capillary/Venous Dated: SOP No. No. Pages:
blood LGEN 0020 17.

Version 2: Dated: SOP No.: No. Pages:

Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log.

Date of Changes made. Name of reviewer. Signature.
review.
11.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

 LABORATORY STANDARD OPERATING
PROCEDURES
FACILITY NAME………………………………………………………………

COUNTY…………………………………………………………………………

SUB COUNTY……………………………………………………………………

SOP Title: CD4 TESTING SOP No: 005

Version: Original
Effective Date: January 2016 Page 1 of 10

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Purpose & Scope

1.1 Purpose
This SOP describes the procedure for performing CD4, CD3 count for adults, It is a simple, cost-
effective and reliable tool available in modern Immunology to monitor the immune status of HIV
infected adults

1.3 Principle & Safety

A single test requires one convenient, ready-to-use reagent tube pair: one tube determines the
absolute number of helper/inducer T lymphocytes (CD4/CD3), the other tube determines the
absolute number of suppressor/cytotoxic T lymphocytes (CD8/CD3).

Both tubes measure the absolute number of total T lymphocytes (CD3). Simply follow the whole
blood staining procedure to prepare the samples, and then run the samples on the FACSCount
instrument. A built-in screen displays instructions for operation. The procedure requires minimal
sample handling.

When whole blood is added to the reagents, fluorochrome-labelled antibodies in the reagents bind
specifically to lymphocyte surface antigens. After a fixative solution is added to the reagent tubes,
the sample is run on the instrument. Here, the cells come into contact with the laser light, which
causes the fluorochrome-labelled cells to fluoresce. This fluorescent light provides the information
necessary for the instrument to count the cells.

In addition to containing the antibody reagent, the reagent tubes also contain a known number of
fluorochrome-integrated reference beads. These beads function as fluorescence standard for locating
the lymphocytes and also as quantification standard for enumerating the cells.

Analysis is automatic. The software identifies the T-lymphocyte populations and calculates the
absolute counts. Results print immediately after samples are run and absolute cell counts for
CD4 (helper/inducer T lymphocytes), CD8 (suppressor/cytotoxic T lymphocytes) and CD3 (total
T lymphocytes), as well as CD4/CD8 (helper/suppressor T-lymphocyte ratio) are reported

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1.3.2 Safety;
Lab coats
Latex gloves

2.0 Abbreviation, Definitions and Terms

2.1 Abbreviations

2.2Definitions and Terms

If applicable

3.0 Equipment and Materials/Reagents/Supplies

3.1 Equipment

3.11BD Facscount machine

3.124ml EDTA tube
3.13BD Microtainer (K2E)
3.14Electronic pipette
3.15Coring station
3.16Reagent tube lids
3.17Gloves
3.18 Waste bucket with 2% sodium hypochlorite (bleach)

3.2 Reagents

3.21. FACS Count Reagent kit

3.22FACS Count Control kit
3.23FACS Flow
3.24FACS Clean

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3.25 FACS Rinse.

4.0 Responsibility
All medical laboratory technicians, technologists working in the immunology laboratory CD4
bench Quality assurance officer and Laboratory in charge are responsible for understanding and
implementing this SOP

5.0 Procedure
5.1 ENTERING CONTROL & REAGENT INFORMATION

The FACSCount reagents and control beads are each assigned specific lot codes and specific bead
counts. Carefully enter the lot codes and bead counts prior to running controls or samples. This
information is stored and does not need to be changed between runs UNLESS a new lot of controls
or a new lot of reagents is used.

5.11 Press [CONTROL] from the FACSCount screen.

The CONTROL BEADS screen is displayed.
5.12Enter the eight-digit control bead lot code. This information is found on the control bead tray.
5.13 Enter the bead counts for the Low, Medium and High controls. This information is found on
the control bead tray.
5.14 Press [CONFIRM] to save this information to the protocol disk. The REAGENTS screen is
displayed.
5.15Enter the eight-digit reagent lot code. This information is found on the foil bag and on the
reagent pair tab. Press [ENTER] to move to the next field.
5.16Enter the CD4 and CD8 reference bead counts for the reagent lot. This information is found in
the foil bag label.
5.17Press [CONFIRM] to save this information to the protocol disk.
5.18Enter the normal control ID (up to 15 characters).

5.2 PREPARING & RUNNING PATIENTS SAMPLES

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5.21Identify the patient samples adequately with the laboratory request form and collect blood in
the microtainer tubes or the EDTA vacutainer tube
5.22In the laboratory, the technician/Technologist responsible removes the required number of
reagents (CD4, CD8) tubes from the refrigerator and vortex for 6
Seconds upside down and then for another 6 seconds upright.
5.23Place the vortexed reagent tubes onto the supplied workstation.
5.24Label each reagent tube with the relevant Patient ID number and site for ease of identification
and to eliminate errors.
5.25Use the supplied coring station to remove the lids off the reagent tubes.
5.26Add 50µl blood to each tube using the electronic pipette (use reverse pippeting) cover with
lids supplied and incubate for 30 minutes at room temperature in the workstation provided,
ensuring the lid is closed at all times to prevent exposure to light
5.27Prior to turning on the FACS Count machine, ensure that the FACS Flow supply tank (White
cap) is full and that the Waste tank (Red cap) is empty.
5.28Turn on the FACS Count machine ensure that the diskette
Protocol is in the drive at all times.
5.29After 60 minutes incubation, add 50µl fixative solution to each tube and vortex prior to
reading on the machine.
5.29.1 Place the reagent tube onto the holder beneath the probe and allow the machine to read
the CD4+ sample. The tube will automatically come down once reading is complete. 5
5.29.2 A printout is automatically issued from the FACS Count machine upon completion of
sample analysis.
5.29.3 Perform daily clean on the FACS COUNT machine
5.29.4 Shut down the machine and empty the Waste tank into the designated sink.
5.29.5 Remove the CD4,CD3 result sheet and record the results in the laboratory records and
attach the original onto the patients request form.

5.3 ENTERING PATIENTS NUMBER & REAGENT INFORMATION

Patients ID number for each sample is entered before running the sample on the FACS
Count instrument.

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 5.30On the FACS Count screen or the CONTROL results screen, press [SAMPLE].
5.31Enter or verify the reagent lot code and reference bead counts and press [CONFIRM].
5.32The SAMPLE screen is displayed.
5.33Enter the patients ID number (up to 15 characters). If the patient ID number from a previous
run exists, press [ClrAcc#] to remove it.
5.34Click enter on the FACS COUNT instrument and follow the instruction to proceed

5.3 CALIBRATION OF FACS COUNT MACHINE

5.31Procedure running CD4,CD3 controls

FREQUENCY

This procedure is performed DAILY to ensure that the machine is properly calibrated and
is issuing reliable results.

Controls are prepared by adding normal blood, then fixative solution, to the CD4, CD3 reagent
tubes. Before reagent tubes are run on the instrument, control beads are added.

One Reagent tube Low (red top) Approximately 50 beads/50ul

One Reagent tube Medium (blue top) Approximately 250 beads/50ul

One Reagent tube High (purple top) Approximately 1000 beads/50ul

5.31Take three of Reagent vials and label them: low, Medium, and High
5.32Vortex these reagents vial (upside down for 6 seconds and upright for 6 seconds) and open
using the Coring station.
5.33Use blood sample from a single control donor and pipette 50ul of blood into these reagent vials.
Remember to change pipette tips between tubes. Cap the tubes and vortex upright for 6

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 seconds.
5.34Incubate these vials for 60 minutes at room temperature in the work station (protect from light).
5.35Prior to turning on the FACS Count machine, ensure that the FACS Flow supply tank (White
cap) is full and that the Waste tank (Red cap) is empty.
5.36Turn on the FACS Count Machine (15 minutes prior to use) ensure that the protocol diskette is
in the drive at all times.
5.37After 60 minutes incubation, add 50µl fixative solution to each tube and vortex upright for 6
seconds.

5.4 (Run the samples on the BD FACSCount instrument within 2hours of adding controls
beads to reagent tubes)
5.41Place the Low, Medium, and High tubes in the control area of the workstation and Uncap the
Reagent tubes
5.42Vortex the Zero/Low control bead pair and pipette 50ul of Low control beads (red top) into the
CD4 reagent tube labelled Low (blue top). Before opening the Zero/Low control bead pair with
the coring station, vortex the pair upside down for 6 seconds, then upright for 6 seconds. Cap
the controls, after use and store upright. For subsequent uses of the control bead pair, vortex
upright for 6 seconds.
5.43Vertex the medium/High control bead for 6seconds.Pipette 50ul of the Medium control beads
(green top) into the CD4,CD3 reagent tube labelled Medium (blue top).
5.44Vortex the Medium/High control bead pair and pipette 50ul of the Medium control beads (blue
top) into the CD4CD3 reagent tube labelled Medium (green top).
5.45Pipette 50ul of the High control beads (purple top) into the CD4,CD3 reagent tube labelled
High (blue top).
5.46Recap the reagent tubes with new caps. There should now be three Reagent containing the
following control beads:
Reagent Tube Control
One reagent tube CD4 Percentage Tube Low

One reagent tube CD4 Percentage Tube Medium

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 One reagent tube CD4 Percentage Tube High
5.47Run the tubes on the FACSCount instrument immediately or within 2 hours of adding control
beads to reagent tubes. Store Samples at room temperature in the workstation until they are run
on the instrument. Vortex upright for 6 seconds immediately before running.
5.48If the calibration procedure was successful the printout will read PASSED.
5.49Continue with rest of FACSCount testing as per usual or shut down the machine and empty the
waste tank.

6.0 Related Procedures

Blood is collected in a 4ml EDTA vacutainer tube or BD Microtainer (K2E Tubes).Normal

blood is used to run controls and must be collected from a normal donor stored no longer than 24
hours at room temperature(20-25C)

7.0 References
BD Biosciences Instrument User’s Guide

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TRAINING LOG:
By signing this log is an acknowledgement that I have read and understood the attached
document
DATE NAME SIGNATURE

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME……………………………………………………………………………………………………….

COUNTY…………………………………………………………………………………………………………………..

SUB-COUNTY………………………………………………….………………………………………………………..

SOP Title: EXTERNAL QUALITY ASSESMENT SOP No: 012

Version: Original
Effective Date: January 2016 Page 1 of 8

Signatures and Dates:

Author: _ ________________________________________________________________
Laboratory Technologist Date

QA Review: _______________________________________________________________
QA Officer Date

Approving Authority: _______________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 PURPOSE / INTRODUCTION:
1.1 External Quality Assessment is a system for objectively checking the
laboratory’s performance using an external agency or facility.
1.2 EQA provides:
1.2.1 comparison among different test sites
1.2.2 early warning for systemic problems
1.2.3 objective evidence of testing quality
1.2.4 areas that need improvement
1.2.5 training needs
2.0 SCOPE / RESPONSIBILITY:
2.1 This SOP applies to all laboratory personnel and other departments that use
the laboratories services.
2.2 It is the responsibility of the designated of the County Leads, Quality
Assurance and Quality Control (QC/QA) personnel, Laboratory Supervisors to
ensure that the current SOP is available to the laboratory personnel and the
procedure is followed as documented.

3.0 SAFETY/RISK ASSESSMENT:

3.1 Wear personal protective equipment.
3.2 All survey samples should be treated as potentially infectious and should be
handled as if they are capable of transmitting diseases.
3.3 Gloves should be put on before opening the container and should be kept on
and replaced if contaminated throughout the period samples are handled.
3.4 All high altitude samples should be opened in a hood or biological safety
cabinet.
3.5 Survey samples, reagents and disposable equipment used in testing should
be autoclaved or incinerated and disposed of as hazardous waste.
3.6 Some of the specimens in this survey are classified as toxic, corrosive, irritant
and dangerous to the environment.
3.7 In case of any accident seek medical attention

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4.0 DEFINITIONS:

5.0 SPECIMEN:
5.1 Proficiency testing samples
6.0 EQUIPMENT/ MATERIALS/ REAGENTS:
6.1 Equipment.
6.1.1 Instruments specific for assays.
6.2 Materials.
6.2.1 Assay related laboratory consumables
6.3 Reagents.
Assay specific reagents

7.0 METHODOLOGY:
7.1 Reception
7.1.1 On receiving the proficiency panel kit, the technician/ technologist
should inspect the package for any damage and proper packaging
depending on the specific panel requirements.
7.1.2 If the kit is damaged or incomplete, a Corrective action log should be
completed and the laboratory manager/ designee notified.
7.1.3 If packaging is okay, go ahead and open to inspect the kit content.
7.1.4 Confirm the content with the package insert or packing list
accompanying the samples.
7.1.5 Should the kit have a problem and/or replacement samples required,
the Lab manager /designee will request for new samples.
7.1.6 Record the kit content package in the reagents inventory book
7.1.7 Report to the manager/designee and the relevant section staff the
arrival of the proficiency panel kit and hand over the panel to the
section where possible.
7.2 Storage and Stability
7.2.1 The kit samples must be stored in accordance with the kit instruction
storage information

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7.3 Specimen analysis
7.3.1 The specimen must be brought to recommended analytical
temperature prior to use as applicable
7.3.2 The specimen must be examined or tested with the laboratory’s
regular patient workload by personnel who routinely perform the
testing in the laboratory and they should rotate in the section.
7.3.3 Sharing of proficiency test results and communication with other
laboratories prior to evaluation is not permitted.
7.3.4 Proficiency testing specimens/materials SHALL NOT be referred to be
analysed in another laboratory.
7.3.5 If there is more equipment in a section, the PT samples/material shall
be run with the second equipment and the results filed to be graded
when external evaluation report is received where applicable.
7.3.6 The technician/technologist shall analyse the proficiency sample
according to the kit instructions
7.4 Submitting Results
7.4.1 Follow the kit instructions manual to submit results by the preferred
method.
7.4.2 Results are filled in the reporting form by the operator where
applicable.
7.4.3 A second person reviews these transcribed reports against the raw
data
7.4.4 If submitting results online, the sections technical staffs enters on the
online report form.
7.4.5 These are counterchecked and confirmed with the raw data by
another technical staff
7.4.6 Laboratory manager/designee, Section head/designee and Quality
Assurance unit personnel counterchecks all reports entered on to the
forms against the raw data.
7.4.7 If results are faxed, a copy of the fax delivery confirmation should be
filed with the results report and if by mail a copy of the courier send-
out sheet should be made available.

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 7.4.8 Note: In case results submission may be delayed due to lack of
kits/controls, equipment breakdown, the provider will be requested
for an extension of the submission deadline.
7.5 Residual Samples Handling
7.5.1 PT specimen/material should be discarded once acceptable results
have been received and no more corrective action is required.
7.5.2 PT specimen/material may be used for training or validation
purposes.
7.6 Results Review
7.6.1 On receiving back the report of results, the Lab manager/designee
should go through them with the respective section personnel, make
relevant comments including biases or trends where necessary, and
append a signature while dating.
7.6.2 All documentation should be completed within two weeks on
reception of the PT panel test results.
7.6.3 Biases and trends should be investigated as soon as possible
7.6.4 A corrective action/variance shall be filled for all undesirable results.
7.6.5 Any un-graded PT analytes received should be graded using the
participants’ summary reports, documented in the results form signed
and dated.
7.6.6 Un-grading could be due but not limited to:
7.6.6.1 Lack of peer group to compare with, so the results should be
compared with all methods in the laboratory.
7.6.6.2 The laboratory submitting results after the cut-off/due date
7.6.6.3 The lab not submitting results at all
7.6.6.4 The laboratory made an error in completing the result form
7.6.6.5 Educational challenge
7.6.7 Grading for the second/ backup equipment shall be done once results
are received.
7.6.8 Results of the grading should be documented and reviewed as
appropriate.

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 7.6.9 Results and any other related documents are filed in the sections EQA
folders as permanent laboratory records and may be retrieved on
request.
8.0 APPENDICES:
NA

9.0 REFERENCES:
9.1 NCCLS. Application of a Quality Management System Model for the
Laboratory Services; Approved Guideline – Third Edition. NCCLS document
GP26-A3 [ISBN 1-56238-553-4]. NCCLS, 940 West Valley Road, Suite 1400,
Wayne, Pennsylvania 19087 – 1898 USA, 2004.
9.2 NCCLS. A Quality Management System Model for Health Care; Approved
Guideline – Third Edition. NCCLS document HS1-A2 [ISBN 1-56238-554-2].
NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087 – 1898
USA, 2004.

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10.0 DOCUMENT CHANGE HISTORY:
10.1 Version Table:
Version 1: External Quality Assessment - Dated: SOP No.: No. Pages:
Proficiency Testing LGEN. 0007 8.

Version 2: Dated: SOP No.: No. Pages:

.
Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log

Date of Changes made. Name of reviewer. Signature.
review.

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11.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME……………………………………………………………………………………………………………….

COUNTY………………………………………………………………………………………………………………………….

SUB COUNTY………………………………………………….……………………………………………………………….

SOP Title: FIELD STAINING SOP No: 013

Version: Original
Effective Date: January 2016 Page 1 of 5

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.1 Purpose

To accurately define a method of staining of thick blood films using field stain

1.2 Scope

This procedure applies to staining of thick blood films using field stain at Tabaka Mission
Hospital Laboratory

1.3 Definitions, Acronyms, symbols

AMREF: African medical and research foundation

TMHL: Tabaka Mission Hospital Laboratory

1.4 Principle

Field stain is an aqueous (water based) Romanowsky stain used for staining blood films.
Romanowsky stains contain Eosin Y, an anionic acidic dye, and Azure B, an ionic basic thiazine
dye obtained by oxidation of methylene blue. The anionic dye stains the basic components
(cytoplasm) of cells red; the ionic dye stains the acid components (nucleus) of cells blue.
When these dyes are combined in an aqueous solution, the dye complex precipitates.
Therefore Field stain is prepared as two separate aqueous solutions. Field stain is the stain of
choice for malaria diagnosis in small laboratories with low to moderate workload

2.0 Safety precautions

Gloves must be worn at all times during the procedure

3.0 Procedures

3.1 Equipment, reagents and materials

1. Field Stain A and B powders – commercially available

2. Sodium azide powder – commercially available
3. Distilled water
4. Filter paper (Whatman No 1)

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 5. Measuring cylinder
6. Conical flasks 250 ml
7. Reagent bottles
8. Staining jars
9. Slide drying rack
10. Cotton wool or gauze
11. Weighing scales

3.2 Preparation of reagent

1. Field stain A solution: Dissolve 12.5 g of Field stain A powder in 500 ml of warm
distilled water. Add 0.5 g sodium azide.
2. Field stain B solution: Dissolve 12.5 g of Field stain B powder in 500 ml of warm
distilled water. Add 0.5 g sodium azide.
3. Filter the stains into separate reagent bottles and label with the name of the reagent
and the date of preparation. For daily use, transfer 100ml into separate staining jars
and label.

3.3 Sample required

Use capillary or venous blood anti-coagulated with EDTA. For parasite examination,
preferably collect blood at times of fever. Prepare and stain thick blood films within 1
hour of blood collection. Stored blood is not suitable for parasite examination.

3.4 Procedure

1. Prepare a thick blood film and leave to dry in air.

2. Stain the thick blood film using the Field stain technique by dipping the slide as
follows:
Field stain A – 4 seconds
Tap water – 5 seconds
Field stain B – 4 seconds
Tap water – 5 seconds.

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 3. Clean the back of the slide and place the slide upright on the drying rack. Allow the
film to dry at room temperature away from direct sunlight and hot objects.

4.0 Procedural notes

• Timing is critical because the staining process is so rapid.
• Keep the Field stain jars covered to avoid dust and evaporation. Filter the stains
regularly to remove dirt and scum.
• Sodium azide is used as preservative.

5.0 Quality control

Prepare two thick blood films:
1. Stain with old stain.
2. Stain with the newly prepared stain.
3. Compare the staining reaction.

References

• F. Baker and S. Silverton (1980). Introduction to medical laboratory technology. 5th

Edition.

• AMREF (2008). Standard operating procedures, Essential Laboratory Tests

Associated records and forms

Not applicable

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Training log

All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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 LABORATORY STANDARD OPERATING
PROCEDURES
FACILITY NAME…………………………………………………………………

COUNTY……………………………………………………………………………

SUB COUNTY………………………………………………………………………

SOP Title: Collection and processing of Sputum for AFB SOP No: 007
Version ORIGINAL
Effective Date: January 2016 Page 1 of 9

Signatures and Dates:

Author: ___________________________________________________________________
Lab. Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: _________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Responsibilities/ Applicability:
1.1 Technical staff is responsible for the preparation, review and updating of the SOP.
1.2 It is the responsibility of all Lab staff to follow and implement this SOP.
1.3 The laboratory in charge or designee to is responsible for ensuring that the SOP is
followed and implemented.
1.4 This SOP applies to all qualified Laboratory personnel.

2.0 Purpose/ Principle:

2.1 This SOP describes the procedures for processing and staining of sputum samples.
2.2 Sputum is the collected secretion from the mucous membrane lining the trachea, bronchi,
and bronchioles of the respiratory tract.
2.3 ZN staining is a technique used to stain Mycobacterium species for example M.
tuberculosis, M. leprae, and M. ulcerans. Mycobacterium unlike other bacteria does not
stain well with gram stain technique.
2.4 Principle: the cell wall of some bacteria e.g. Mycobacterium tuberculosis is made up of a
waxy lipid substance mainly mycolic acid that can only be penetrated by hot carbol
fuchsin stain which stains the protoplasm of the bacteria cell red in such a way that the
red colour cannot be easily removed by acid alcohol. Mycobacterium tuberculosis when
it retains the red colour of carbol fuchsin after treatment with acid- alcohol are referred to
as acid fast bacilli (AFB). Those bacteria that are non acid fast are easily decolorized and
can be stained with methylene blue or malachite green (counter stains) and takes up the
colour of the counter stain.

3.0Specimen handling and preparation:

3.1 Collection:
3.1.1 Sputum should be collected in an open air as far away as possible from other
people. Sputum specimen should never be collected in a laboratory or in an
enclosed area.
3.1.2 Health worker should wear protective clothing that is; lab coats, masks and
gloves when handling sputum.
3.1.3 Clearly demonstrate and instruct the patient on how to collect sputum.

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 3.1.4 An ideal container for sputum collection should be wide mouthed, has a screw cap
and is leak proof.
3.1.5 Instruct the patient on how to open and close the container, the ideal time of
sputum collection and the importance of collecting sputum and not saliva.

3.2 Specimen handling in the processing area.

3.2.1 Verify the patient detail that is; name, IP/OP number, date, residence, new or
follow-up, age on both request form and specimen match.
3.2.2 Check whether specimen container is tightly closed.
3.2.3 If the specimen meets the above criteria start processing, if not reject and follow
the specimen rejection criteria.
3.2.4 Record in the specimen register.

4.0 Quality control/Calibration:

4.1 Use a known AFB positive smear preferably one whose quantification of positivity is
(+), also a known negative smear is used to QC the reagents.
4.2 Should be done either daily or weekly whenever sputum smears are stained and results
documented in the logbook or sputum AFB register.

5.0 Materials and Equipment:

5.1 Carbol fuchsin 5.9 Methylated spirit or 70% alcohol
5.2 3%Acid-alcohol 5.10 Staining rack
5.3 Wide-mouthed poly-pots 5.11 Microscope slides
5.4 Applicator sticks 5.12 Burner flame
5.5 5% phenol 5.13 Oil immersion
5.6 Grease pencil/pencil 5.14 Gauze
5.7 Gloves 5.15 Microscope
5.8 Masks 5.16 Biosafety cabinet

6.0 PROCEDURES:
6.1 Preparation of Carbol fuchsin reagent (1 litre)
Requirements;

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 6.1.1 Basic fuchsin -------10 grams
6.1.2 Phenol Crystals----50 grams
6.1.3 Absolute ethanol---100 mls
6.1.4 Distilled water -----900 mls
Procedure;
6.1.5 Dissolve phenol in distilled water
6.1.6 Dissolve basic fuchsin in absolute ethanol with aid of gentle heat.
6.1.7 Combine the two solutions. Mix well and filter.
6.1.8 Label with stain name, date prepared and name of person who prepared.
6.1.9 The stain is stable indefinitely.

6.2 Preparation of 3% acid alcohol

6.2.1 20% Sulphuric acid.
6.2.2 Absolute ethanol/methanol------------970 mls
6.2.3 Measure 970 mls of absolute methanol/ethanol and top up with 30 mls of
hydrochloric acid.
6.2.4 Label as 6.1.8 above.
Note: 3% acid alcohol is used for tubercle bacilli, 1% acid alcohol is used for
Leprae bacilli as this bacilli is weakly acid fast. Use of acid alcohol is
recommended and has more advantages over 20% sulphuric acid used alone.
6.2.5. Advantages of acid alcohol.
6.2.5.1 Decolourization is completed quickly and the margins and underside of the slide
are more completely cleaned and freed from deposits of stain.
6.2.5.2 Certain other acid fast bacilli which may be encountered in pathological specimen
and may otherwise be confused with tubercle bacilli are decolorized by acid
alcohol.
6.2.5.3 It does not corrode the bench surface.
6.3 Macroscopic examination.
6.3.1 Observe and report the appearance of the sputum either as muco-purulent, blood
stained or salivary.

6.4 PREPARATION AND STAINING OF SMEARS FOR AFB.

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 6.4.1 Preparation of sputum smears should be done in a biosafety cabinet.
6.4.2 Using an applicator stick/wire loop, pick a portion of the sputum specimen and
spread on the slide.
6.4.3 Spread sputum material evenly in appropriate area of 2cm by 1 cm so that
newsprint are readable on drying.
6.4.4 Air dry smear completely and then fix smear in flame.
6.4.5 Put slide in staining rack and apply filtered carbol fuchsin stain and heat until
steam rises. Keep the stain hot for 5 minutes. Do not allow the stain to boil or dry
on the slide.
6.4.6 Wash with water.
6.4.7 Decolorize with;
6.4.7.1 3% acid alcohol for tubercle bacilli
6.4.7.2 1% acid alcohol for Leprae bacilli until the smear appears pale pink.
6.4.8 Wash with several changes of water.
6.4.9 Counter stain with malachite green or methylene blue for 1-2 minutes using
longer time if the smear is thin.
6.4.10 Wash with water and stand in a draining rack to dry. Do not blot dry.
6.4.11 Examine under oil immersion objective.

7.0 Results reporting / Reference ranges.

7.1 For Mycobacterium Tuberculosis and M. ulcerans acid-fast bacilli appear as; red, straight
or slightly curved rods occurring singly or in small groups.
7.2 Cells appear as green or blue depending on the counter stain used.
7.3 Background material id either green or blue.
Note: if any definite bacilli are seen, report the smear as AFB positive.

7.4 Report semi-quantitation of the bacilli as follows;

No AFB in at least 100 HPF’s No AFB seen (0)

1-9 AFB/100HPF’s Report the exact number
10-99 AFB/100 HPF’s +

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 1-10 FB /HPF in at least 50 fields ++
More than 10 AFB/HPF in at least 20 fields +++

7.5 Record the results in the patient request form, microbiology register or enter into
computer system where applicable and also indicate the date, time and name of technical
staff reporting
7.6 Ready results are taken to the reception area for dispatch or entered to computer where
applicable.

8.0 Explanation of Abbreviations and Terms:

8.1 MICRO-Microbiology 8.6 QC-Quality Control
8.2 SOP - Standard Operating 8.7 QA - Quality Assurance
Procedure. 8.8 IP/OP-In Patient/Out Patient
8.3 AFB-Acid Fast Bacilli 8.9 ZN- Ziehl Neelsen
8.4 HPF-High Power Field 8.10 Mls - Millilitres
8.5 NA- Not Applicable

9.0 References:
Reference Number or Authors Document Title
9.1. AMREF SOP’S Essential laboratory Tests ©2008

9.0 Forms and Appendices:

Form or Appendix Number Title

10.1 SOP Copy Control and Updating Log

10. Training Documentation Log for SOP Files

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 10.0 Version Table:
Original: (Current) Dated: COUNTYAFB 001 No.
Title: Collection and processing of Sputum May 2015 Pages:07
for AFB
Version 1 Dated: SOP No.: No.
Pages:
Version 2
Dated: SOP No.: No.
Pages:
Version 3:
Dated: SOP No.: No.
Pages:
Version 4: Dated: SOP No.: No. Pages:

Version 5: Dated: SOP No.: No. Pages:

Version 6: Dated: SOP No.: No. Pages:

Appendix 10.1
SOP Copy Control and Updating Log

DOCUMENT COPY CONTROL

DATE PRINTED: Feb 2015 NUMBER OF COPIES:
SOP DISTRIBUTION
COPY 1 OF 1
LAB Section

By initialling and dating below I understand and approve of the changes to the
attached SOP.
SOP CHANGES Changes Approval
Initials/Date
Date/Initials Nature of Change QA Approving

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 Authority

\
Appendix 10.2
Training Documentation Log for SOP Files
SOP NO: COUNTYAFB
Laboratory Department
COPY 1 OF 3 001VERSION: ORIGINAL
Standard Operating Procedure
Effective Date: May 2015

Title: Sputum Processing and staining by Ziehl Neelsen Method

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This SOP has been read and understood by:

Date: Printed Name OR Date: Printed Name OR

DD-MM-YY Initials Signature DD-MM-YY Initials Signature

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………………………………………………………

COUNTY…………………………………………………………………………………………………………

SUB COUNTY………………………………………………….………………………………………………

SOP Title: RHEUMATOID FACTORS SOP No: 026

Version: Original
Effective Date: January 2016 Page 1 of 6

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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 1. PURPOSE/APPLICABILITY

PURPOSE: To provide guidelines for the proper procedure of determination of Rheumatoid

factors to ensure accurate and reliable results.
1.1 Applicability: TMH Lab technologists/technicians and students.

1.2 Principle:

Rheumatoid Factor is based upon the agglutination reaction between Rheumatoid factors
(R.F) of a patient specimen or control serum and human immunoglobulin G (IgG) coated onto
polystyrene latex particles. The positive reaction is indicated by a distinctly visible agglutination
of the latex particles in the test cell of the slide.

2. Abbreviations and Terms

2.1. R.F.: Rheumatoid Factor

2.2. TMH: Tabaka Mission Hospital
2.3 Lab: Laboratory
2.4 Tech: Technologist/Technician
2.5 IAW: In accordance with

3. Equipment and materials

3.1 Equipment
3.1.1. Centrifuge
3.1.2. Shaker
3.1.3. Pipette and pipette tips

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 3.1.4. Slide or tile (black background)
3.1.5. Applicator sticks
3.1.6. Latex gloves

3.2 Reagents

3.2.1. R.F Latex Reagent (White cap)

3.2.2. Control serum positive (red cap)
3.2.3. Control serum negative (green cap)
3.2.4. Stability : Reagents are stable up to expiry date when stored at 2 - 8◦c
3.2.5. Specimen - Serum stability: Up to 24 hours at 2 - 8◦c
Up to 4 weeks at -20◦c

4. Responsibilities
4.1 The laboratory in-charge is responsible for ensuring the implementation of this SOP
with the assistance of Quality Officer to ensure strict adherence to this procedure.
4.2 All personnel involved in the execution of this SOP must ensure that they are
properly trained.
4.3 Quality Assurance: SOPs are to be reviewed/approved by the in-charge office of
quality, or designee, prior to the responsible approving authority.

5. PROCEDURES

5.1. Qualitative determination (screening test)

a) Bring the reagents and serum sample to room temperature

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 b) Mix the Latex Reagent carefully prior to use to suspend the Latex
particles completely
c) Pipette/drop onto separate cells of the slide:

Serum sample 40 ul (1 drop)

Red cap (positive control) 1 drop
Green cap (negative control) 1 drop

d) Add Latex Reagent (white cap) onto all sample and control cells (1 drop each).
e) Mix both separate sticks and spread the fluid over the entire area of the
particular cell.
f) Tilt the slide back and forth for 2 minutes so that the mixture rotates inside the
cells or place the slide on the shaker at 1000 r.p.m.
g) At the end of the 2 minutes read the results under bright artificial light.

5.2 Interpretation of Results

5.2.1 Distinct agglutination indicates R F content of more than 20 iu/ml in
the non diluted serum specimen.
5.2.2 Quality control - Positive and negative controls are to be used in each
series. Their results should be compared with the unknown specimen
to distinguish possible granularity from agglutination.
Positive control - distinct agglutination within 2 minutes
Negative control - Smooth suspension with no visible agglutination
after 2 minutes.

NOTES

1. Contaminated and markedly lipemic sera may cause non-specific reactions and should
therefore not be tested.

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 2. A reaction time longer than 2 minutes may lead to false positive results due to a drying
effect.
3. The final diagnosis should not be made using the result of a single test but should be in
correlation with other clinical findings.
4. During dispensing hold the dropper vertically.
5. All reagents contain sodium oxide. Do not swallow. Avoid contact with skin and mucus
membranes.

6. REFEFENCES

AUTHOR DOCUMENT TITLE

Humatex RF Insert

Multer W. The serology of Rheumatoid Arthritis

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Training log
All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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 LABORATORY STANDARD OPERATING
PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: Blood Grouping By Tile or Slide Method SOP No: 003
Version Original
Effective Date: January 2016 Page 1 of 9

Signatures and Dates:

Author: ____________________________________________________________________
Lab. Technologist Date

QA Review: ________________________________________________________________
QA person Date

Approving Authority: _________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author/Reviewer QA Review Approving Authority

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1.0 Responsibilities/ Applicability:
1.1 Technical staffs are responsible for preparation, review and updating of the SOP.
1.2 It is the responsibility of all Lab staff to follow and implement this SOP.
1.3 The laboratory in-charge or designee is responsible for ensuring that this SOP is followed
and implemented.
1.4 This SOP applies to Laboratory personnel, designated Quality Assurance unit personnel
and the laboratory in-charges, Sub County Medical laboratory Coordinator, County Medical
Laboratory Coordinator.

2.0 Purpose/ Principle

2.1 This SOP describes the procedures for blood grouping using slide or tile method.
2.2 Principle – Blood group antigens on the surface of red cells react specifically with blood
group antibodies to form agglutinates, which are detected using the naked eye. The results
are used to determine the major blood groups. Red blood cells are mixed with antisera on a
tile or slide and the presence or absence of agglutination is noted.

3.0 Specimen handling and preparation:

3.1 EDTA anti-coagulated blood sample or clotted sample or 20-30% red cell suspension.
3.2 A drop of blood may also be used directly from finger prick.
3.3 Grouping is done immediately the sample is received. After grouping samples are kept until
evening before being disposed.
3.4 Examined slides are put in a bucket of 10% sodium hypochlorite after reporting of patient
results
3.5 Always wear gloves and lab coat when handling specimen. All spillages are wiped
immediately with 10% sodium hypochlorite.

4.0 Quality control/Calibration:

4.1 Check the reactions of the antisera using suspensions of known A, B and O positive cells.
This should be done daily.

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4.2 Add 1 drop 4% washed cells of known A, B and O positive cells to the drops of anti-A, anti-
B and anti-D, respectively, on the tile.
4.3 Mix with a clean applicator stick. Gently tilt for 10-15 seconds. Strong agglutination
indicates the antisera are working.
4.4 Documentation the results of QC in blood grouping quality control chart.
4.5 Calibration: NA

5.0 Materials and Equipment:

5.1 Weighing scales
5.2 Applicator sticks
5.3 Glass Pasteur pipettes
5.4 Tile or slide
5.5 Antisera-Anti A, anti B, anti-D
5.6 Sodium chloride crystals or commercially available normal saline.
5.7 Distilled water
5.8 Disposal containers and bucket with 10% sodium hypochlorite
5.9 Grease pencil/marker pen

6.0 PROCEDURES:
6.1 Preparation of Physiological saline
6.1.1 Weigh 0.85grams of sodium chloride powder,
6.1.2 Put in a reagent bottle and add 100mls of distilled water to dissolve
6.1.3 Label the reagent bottle with the name of the reagent, date of preparation and initials of
person who prepared.
6.1.4 For daily use, transfer into a dropper bottle and label.

6.2 Preparation of 20-30% Red cell Suspension.

6.2.1 Transfer about 0.5 ml EDTA anti-coagulated blood or red cells from a clotted sample
into a test tube containing about 5ml physiological saline.
6.2.2 Wash the cells by adding 5-7 ml physiological saline, centrifuge at 3,000 rpm for 2-3
minutes and discard the supernatant. Repeat this step 3 times.

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6.2.3 Make a 20-30% red blood cell suspension by mixing 5 drops of sediment washed cells in
20-25 drops of physiological saline, holding the Pasteur pipette vertically.

6.3 Blood grouping

6.3.1 Label the areas of slide or tile as A, B and D with a grease pencil.
6.3.2 Place a drop of anti-A, anti-B, anti-D on respective labelled areas of the ruled tile or
slide.
6.3.3 Add drop of well-mixed red cell suspension to each drop of anti-serum.
6.3.4 Mix the antisera and red cell suspension thoroughly using a clean applicator stick for
each antiserum.
6.3.5 Gently tilt the slide /tile continuously for 2 minutes.
6.3.6 Read and observe for agglutination. Record the results.
6.3.7 Rinse used tiles in running tap water and place in the bucket of 10% sodium hypochlorite.
6.3.8 Place used applicator sticks in the safety box

7.0 Results reporting / Reference ranges

7.1 A strong agglutination of red blood cells in the presence of any of the grouping antisera
indicates a positive result.
7.2 A smooth suspension or no agglutination of red blood cells at the end of 2 minutes indicates
a negative result.
7.3 Report results as of blood group as either A, B, O or AB, rhesus positive or negative.
Example: “Blood group A RhD positive”.
7.4 Record the results in the patient request form, BTS register or enter to computer where
applicable and also indicate the date and name of technical staff reporting
7.5 Ready results are taken to the reception area for dispatch or entered to computer where
applicable.

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8.0 Explanation of Abbreviations and Terms:
8.1 NKR: Nakuru County Medical Laboratory Department.
8.2 BTS: Blood Transfusion Section.
8.3 SOP: Standard Operating Procedure.
8.4 RPM: Revolution per Minute.
8.5 Rh: Rhesus.
8.6 QC: Quality Control
8.7 QA: Quality Assurance
8.8 EDTA: Ethylene Diamine Tetra Acetic acid.
8.9 AABB: Association of American Blood Banks.
8.10 ISBT: International Society for Blood Transfusion.
8.11 AMREF: African Medical Research Foundation.
8.12 NA: Not applicable.

9.0 References:
Reference Number or Authors Document Title
9.1. AMREF SOPs Essential laboratory tests 2008
9.2 AABB 14th Edition 2003
9.3 ISBT Volume 3 No.2 June 2008

10.0 Forms and Appendices:

Form or Appendix Number Title
10.1 SOP Copy Control and Updating Log
10.2 BTS Reagent Quality control Chart
10.3 Training Documentation Log for SOP Files

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11.0 Version Table:
Original Dated: SOP No.: 004 No.
Title: May Pages:10
Blood Grouping By Tile or Slide Method 2015
Version 1: Dated: No.
Pages:
Version 2 No.
Dated: SOP No.: Pages:
Version 3: No.
Dated: SOP No.: Pages:
Version 4: Dated: SOP No.: No. Pages:

Version 5: Dated: SOP No.: No. Pages:

Appendix 10.1
SOP Copy Control and Updating Log

DOCUMENT COPY CONTROL

DATE PRINTED: May. 2015 NUMBER OF COPIES:
SOP DISTRIBUTION
COPY 1 OF 3 COPY 2 OF 3 COPY 3 OF 3
LAB Section

By initialling and dating below I understand and approve of the changes to the
attached SOP.

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 SOP CHANGES Changes Approval
Initials/Date
Date/Initials Nature of Change QA Approving
Authority

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Appendix 10.2
BLOOD TRANSFUSION - REAGENT QUALITY CONTROL CHART.

Date

Reage Manufactu Appearan Performa Manufact Appearan Performa Manufact Appearan Performa
nt rer Lot No. Exp. Date ce nce urer Lot No. Exp. Date ce nce urer Lot No. Exp. Date ce nce

Anti -
A

Anti B

Anti D
Album
in

AHG
Origin
al A1
Cells
Origin
al B
Cells

O pooled
antibody
screen
cells
tech.
Initials

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 Appendix 10.3
Training Documentation Log for SOP Files
Nakuru County Medical SOP No: 001
Laboratory Department
COPY 1 OF 3 VERSION: Original
Standard Operating Procedure
Effective Date: May. 2015

Title: Blood Grouping By Tile or Slide Method

This SOP has been read and understood by:

Date: Printed Name OR Date: Printed Name OR
DD-MM-YY Initials Signature DD-MM-YY Initials Signature

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: RESULT VALIDION SOP No: 022

Version: Original
Effective Date: January 2016 Page 1 of 5

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

1.0 PURPOSE / INTRODUCTION:
1.1 This procedure describes steps to ensure that laboratory results are accurate
and reliable prior to reporting. This procedure has been developed to explain
how to validate the results of laboratory tests before sending them to the
requestor
2.0 SCOPE / RESPONSIBILITY:
2.1 This SOP applies to all laboratory personnel and other departments that use
the laboratories services.
2.2 It is the responsibility of the designated of the County Leads, Quality
Assurance and Quality Control (QC/QA) personnel, Laboratory Supervisors to
ensure that the current SOP is available to the laboratory personnel and the
procedure is followed as documented
3.0 SAFETY/RISK ASSESSMENT:
NA
4.0 DEFINITIONS:
NA

5.0 SPECIMEN:
NA
6.0 EQUIPMENT/ MATERIALS/ REAGENTS:
NA
7.0 METHODOLOGY
7.1 Procedure
7.1.1 Result validation consists of verifying that the internal quality controls
are acceptable, the quality management system functions correctly,
and the results make sense.
7.1.2 The laboratory management or authorized person will verify that:
7.1.2.1 The internal quality controls are acceptable. If this is not the
case, the technologist will discuss this with the laboratory
management. If appropriate, the equipment will be calibrated,
controls run and evaluated, and the test will be repeated.
 7.1.2.2 Any required calibration of the equipment was done correctly.
If this is not the case, the technologist will discuss this with the
laboratory management. If appropriate, the test will be
repeated.

7.1.2.3 There have been no recent reports of a problem in the

processing workflow.

7.1.2.4 The results are in concordance with the clinical details stated
on the request form.

7.1.2.5 Critical results are discussed with the technologist and these
results are reported as indicated in the Critical Results
Reporting SOP.LGEN. 0006.

7.1.2.6 Abnormal laboratory’s results are verified by repeating the

test. The laboratory management may request (at no cost for
the patient) that the specimen be sent to a reference
laboratory for confirmation. In that case, follow the
Laboratory Specimen Referral SOP. LGEN. 0017

7.1.2.7 Appropriate commentary accompanies the test result to

facilitate the requestor's comprehension of the result.

7.1.3 When results indicate a complementary test should be performed, it

will be discussed with the requestor. If the requestor agrees and if
conditions allow, the additional test may be carried out. The
laboratory management will then register the new test to be
performed.

7.1.4 The test reports are handwritten or printed, reviewed and initialled or
signed by the performing technologist.

7.1.5 The test report is then reviewed and initialled or signed by

management or designee. By this the laboratory management
acknowledges review of the report and accepts responsibility for
initiating any necessary follow-up.
 7.1.6 The charts and original laboratory’s report are stored in the
appropriate location.

8.0 APPENDICES:

9.0 REFERENCES:
NA

10.0 DOCUMENT CHANGE HISTORY:

10.1 Version Table:
Version 1: Dated: SOP No. No. Pages: 5
LGEN. 0021

Version 2: Dated: SOP No.: No. Pages:

Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log.

Date of Changes made. Name of reviewer. Signature.
review.
11.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to contact
my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: CRPTOCCOCCAL ANTIGEN SCREENING TEST SOP No: 023

Version: Original
Effective Date: January 2016 Page 1 of 8

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 PURPOSE / INTRODUCTION:
1.1 The CrAg lateral flow Assay is an immunochromatographic test system for the
qualitative and semi-quantitative detection of the capsular polysaccharide
antigens of Cryptococcus species complex (Cryptococcus neoformans and
Cryptococcus gattii) in serum, plasma and CSF. The CrAg Lateral Flow Assay is
a prescription use laboratory assay which can aid in diagnosis of
cryptococcosis.
1.2 Cryptococcosis is caused by both species of the Cryptococcus species
complex (Cryptococcus neoformans and Cryptococcus gattii). Individuals with
impaired cell mediated immunity are at greater risk of infection.
Cryptococcosis is one of the most common opportunistic infections in AIDS
patients. Detection of CrAg in serum and CSF has been extensively utilized
with very high sensitivity and specificity.

2.0 SCOPE / RESPONSIBILITY:

2.1 This SOP applies to all laboratory technologists/technicians involved in the
assay.
2.2 It is the responsibility of the designated Quality Assurance and Quality
Control (QC/QA) personnel, Laboratory Supervisors, SCMLTs, and CMLT to
ensure that the current SOP is available to the staff and the procedure is
followed as documented.

3.0 SAFETY/RISK ASSESSMENT:

3.1 Wear personal protective equipment. Handle all samples as potential
biohazards.
3.2 Always wear gloves when handling reagents which are preserved with
0.095% (w/w) sodium azide. Sodium azide should never be flushed down the
drain as this chemical may react with lead or copper plumbing to form
potentially explosive metal azides.
3.3 Excess reagents should be discarded in an appropriate waste receptacle.

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4.0 DEFINITIONS:
4.1 AIDS: Acquired Immuno-Deficiency Syndrome.
4.2 C.S.F: Celebral Spinal Fluid.
4.3 CrAg: Cryptococcal Antigen.
4.4 HIV: Human Immuno-Deficiency Virus.
4.5 LF: Lateral Flow.
4.6 PPE: Personal Protective Equipment.
4.7 QA: Quality Assurance.
4.8 SOP: Standard Operating Procedure.

5.0 SPECIMEN:
5.1 Non-haemolysed serum/plasma and CSF.
5.2 If a delay is encountered in specimen processing, storage at 2-80C up to 72hrs
is permissible.
5.3 Specimens may be stored for longer periods at less than -200C provided they
are not repeatedly thawed and refrozen.
5.4 Specimen in transit should be maintained at less than -200C or 2-80C.

6.0 EQUIPMENT/ MATERIALS/ REAGENTS:

6.1 Equipment.
6.1.1 Adjustable pipette (40 - 80µl).
6.1.2 Timer.
6.2 Materials.
6.2.1 Disposable micro-centrifuge tubes, test tubes, or a micro-titer plate.
6.2.2 Pipette tips.
6.2.3 PPEs e.g. Gloves and a Lab coat.
6.3 Reagents.
6.3.1 CrAg LF tests strips (50 strips in desiccant vial).
6.3.2 CrAg positive control 1ml: Glycine-buffered saline spiked with
cryptococcal antigen.

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 6.3.3 LF Specimen diluent 2.5ml: Glycine-buffered saline containing
blocking agents and a preservative.
6.3.4 LF titration diluent 6.0ml: Glycine-buffered saline containing a
preservative.

7.0 METHODOLOGY:
7.1 Principle.
7.1.1 The CrAg lateral flow assay is a dipstick sandwich
immunochromatographic assay. Specimens and specimen diluents are
added into an appropriate reservoir, such as a test tube, and the
lateral flow device is placed into the reservoir. The test uses specimen
wicking to capture gold-conjugated, anti-CrAg monoclonal antibodies
and gold-conjugated control antibodies deposited on the test
membrane. If CrAg is present in the specimen, then it binds to the
gold-conjugated, anti-CrAg antibodies. The gold labelled antibody-
antigen complex continues to wick up the membrane where it will
interact with the test line, which has immobilized anti-CrAg
monoclonal antibodies. The gold-labelled antibodies antigen complex
forms a sandwich at the test line causing a visible line to form.
7.1.2 With proper flow and reagent reactivity, the wicking of any specimen,
positive or negative, will cause the gold-conjugated control antibody
to move to the control line. Immobilized antibodies at the control line
will bind the gold-conjugated control antibody and form a visible
control line.
7.1.3 Positive test results create two lines (test and control). Negative test
results form only one line control. If a control line fails to develop the
test is not valid.
7.2 Qualitative Test Procedure.
7.2.1 Add 1 drop of LF specimen diluent into an appropriate reservoir
(disposable micro-centrifuge tube, test tubes, or micro-titer plate).
7.2.2 Add 40µL of specimen to the container and mix.
7.2.3 Submerge the white end of a cryptococcal antigen lateral flow test

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 strip into the specimen.
7.2.4 Wait 10 minutes.
7.2.5 Read and record the results.
7.3 Semi-Quantitative Titration Test Procedure.
7.3.1 Prepare dilutions starting with an initial dilution of 1:5, followed by
1:2 serial dilutions to 1:2560.
7.3.2 Place 10 micro-centrifuge or test tubes in an appropriate rack and
label them 1 to 10 (1:5 through 1:2560). Addition dilutions may be
necessary if the specimen is positive at 1:2560.
7.3.3 Add 4 drops of LF specimen diluent to tube number 1.
7.3.4 Add 2 drops of LF titration diluent to each of the tubes labelled
number 2 to10.
7.3.5 Add 40µl of specimen to tube number 1 and mix well.
7.3.6 Transfer 80µl of specimen from tube number 1 to tube number 2 and
mix well. Continue this dilution procedure through tube number 10.
Discard 80µl from tube number 10 for a final tube volume of 80µl.
7.3.7 Submerge the white end of a cryptococcal antigen lateral flow test
strip into the specimen in each of the 10 tubes.
7.3.8 Wait 10 minutes.
7.3.9 Read and record the results.
7.4 Quality Control.
7.4.1 CrAg positive control can be evaluated by adding 1 drop of LF
Specimen diluent followed by 1 drop of CrAg positive control to a
tube.
7.4.2 A negative control can be evaluated by adding 2 drops of LF Specimen
diluent to a tube.
7.4.3 Insert a test strip into the tubes and read after 10 minutes. Two (2)
lines (test and control) indicate a positive result and one line (control)
indicates a negative result.
7.5 Results.
7.5.1 Read the reactions, the presence of two lines (a line in the test zone
and a control line), regardless of the intensity of the test line,

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 indicates a positive result.
7.5.2 For the semi-quantitative titration procedure, the patient’s titer
should be reported as the highest dilution that yields a positive result.
7.5.3 A single control line indicates a negative result. Negative results do
not rule out the diagnosis of disease. The specimen may be drawn
before detectable antigen is present.
7.5.4 If the control line does not appear, the results are invalid and the test
should be repeated.
7.6 Limitations.
7.6.1 The assay performance characteristics have not been established for
matrices other than serum, plasma and CSF only.
7.6.2 Depending on the disease and organism prevalence, testing should
not be performed as a screening procedure for the general
population. The predictive value of a positive or negative serologic
result depends on the pretest likelihood of Cryptococcal disease being
present. Testing should only be done when clinical evidence suggests
the diagnosis of Cryptococcal disease.
7.6.3 Testing haemolysed serum samples could lead to false negatives due
to the high background colour of the strip.
7.6.4 This assay was not evaluated for potential interference related to
specimen pre-treatment with 2-mercaptoethanol or with specimens
including the following substances: Vaginal cream, caffeine, ascorbic
acid, itraconazole, amphotericin B, acetaminophen, or acetylsalicyclic
acid.

8.0 APPENDICES:
8.1 Not Applicable.

9.0 REFERENCES:
9.1 CrAg lateral flow assay information sheet, for the detection of cryptococcal
antigen, immuno-Mycologics,Inc. - 2700 technology PI.
9.2 Internet sources.

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10.0 DOCUMENT CHANGE HISTORY:
10.1 Version Table:
Version 1: CrAg Testing Using CrAg LF Test Dated: SOP No.: No. Pages:
Strips. . LSERO 0004. 08.

Version 2: Dated: SOP No.: No. Pages:

Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log.

Date of Changes made. Name of reviewer. Signature.
review.

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11.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………………………………………………………………….

COUNTY……………………………………………………………………………………………………………………….

SUB COUNTY………………………………………………….…………………………………………………………….

SOP Title: GENE EXPERT TESTING SOP No: 015

Version: Original
Effective Date: January 2016 Page 1 of 11

Signatures and Dates:

Author: _ ________________________________________________________________
Laboratory Technologist Date

QA Review: _______________________________________________________________
QA Officer Date

Approving Authority: _______________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 PURPOSE / INTRODUCTION
1.1 GeneXpert is a molecular diagnostic, Real Time PCR test (ability to monitor or
visualize accumulation of PCR products using fluorescence) that detects
pulmonary and extra pulmonary MTB DNA. It uses the principal of PCR where
the machine breaks and replicates DNA for analyses. It also detects the MTB
strain, which is RIFAMPCIN sensitive or resistant. GeneXpert is more sensitive
than ZN and FM.

1.2 Criteria for GeneXpert includes, All retreatment cases (failures, relapses,
return after default), DR TB contacts, HIV positive smear negatives, Smear
positive refugees, to diagnose TB in Children under 5 years and HCW with
symptoms.

2.0 SCOPE / RESPONSIBILITY

2.1 This SOP applies to all laboratory technologists/technicians involved in the
assay.
2.2 It is the responsibility of the designated Quality Assurance and Quality
Control (QC/QA) personnel, Laboratory Supervisors, SCMLTs, and CMLT to
ensure that the current SOP is available to the staff and the procedure is
followed as documented.

3.0 SAFETY/RISK ASSESSMENT

3.1 Wear personal protective equipment. Handle all samples as potential
biohazards.
3.2 The Buffer used is poisonous and care should be observed. In case it sprinkles
on eyes or skin, wash with plenty of water and seek medical assistance
immediately.
3.3 Sputum is High infectious and care should be observed while processing.
Always process sputum in Microbiology safety cabinet in a well-ventilated
room.

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4.0 DEFINITIONS

4.1 MTB: Mycobacterium tuberculosis

4.2 NTRL: National TB Reference Lab.
4.3 DNA: Deoxyribonucleic Acid.
4.4 PCR: Polymerase Chain Reaction.
4.5 UPS: Uninterrupted Power Supply.
4.6 QA: Quality Assurance.
4.7 RIF-resistant: Rifampicin Resistant.
4.8 SOP: Standard Operating Procedure.

5.0 SPECIMEN
5.1 At-least 2ml good quality sputum in a graduated falcon tube.
5.2 Saliva, Blood stained or sputum with food particles should not be used.
5.3 Storage of sputum, <35°C maximum 3 days or 4°C maximum 10 days.

6.0 EQUIPMENT/ MATERIALS/ REAGENTS

6.1 Equipment.
6.1.1 GeneXpert machine.
6.1.1.1 Operating Environment Temperature range 18–30 ºC with an
upper humidity limit of 90% non-condensing.
6.1.2 A Computer (Pentium IV, 2GB RAM).
6.1.3 A printer.
6.1.4 Adjustable pipette.
6.1.5 GeneXpert software
6.1.6 Software barcode scanner.
6.1.7 UPS.

6.2 Materials.
6.2.1 Pasteur pipettes.
6.2.2 Pipette tips
6.2.3 PPEs e.g. Gloves and a Lab coat.

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 6.2.4 Timer.
6.3 Reagent.
6.3.1 Disposable GeneXpert cartridge.
6.3.2 Sample reagent/buffer.

7.0 METHODOLOGY
7.1 Principle.
7.1.1 PCR is a common method of creating copies of specific fragments of
DNA. Can produce sufficient copies to be detectable. PCR is
sometimes called molecular photocopying. Chain reaction relays on
DNA replication process by repeated cycles. Qualitative method
(presence or absence of sequences). Quantitative method (direct or
gene expression).
7.1.2 The technology involves Patient Sample Washing → DNA/RNA
extraction → DNA or RNA purification → Amplification → Detection →
Analysis (Positive/Negative Results).
7.2 Sample processing.
7.2.1 Confirm that the information on the request form is correct and
properly identify the specimen.
7.2.2 Enter the patient’s details in GeneXpert Register.
7.2.3 Assemble all materials required to ensure easy access and confirm
tests requested.
7.2.4 Wear the appropriate personal protective equipment (PPE).
7.2.5 Bring the reagents, controls and patients’ sample to room
temperature (18 - 25oC) if applicable.
7.2.6 Label the patient’s request form and sample (falcon tube) with lab No’
from the Register.
7.2.7 Arrange the sample on a rack.
7.2.8 Unscrew to open the falcon tube, Pipette and add sample
reagent/buffer into the falcon tube with the sputum, ration of (buffer)
2:1 (sputum) i.e.

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 Sputum /Control 2ml 4ml
Buffer 4ml 8ml

7.2.9 Screw tight the falcon tube. Shake vigorously.

7.2.10 Incubate the mixture at room temperature (18 - 25oC) for 10minutes.
7.2.11 Shake again and incubate further at room temperature (18 - 25oC) for
5 minutes.
7.2.12 Unwrap and open the cartridge.
7.2.13 While holding the Pasteur pipette vertically dispense 2ml of the
mixture into the specimen hole of the cartridge.
7.3 Xpert Procedure.
7.3.1 Switch on Genexpert Machine.
7.3.2 Switch on Computer.
7.3.3 Log on to Windows as the Cepheid user to operate the system.
7.3.3.1 User name: Cepheid
7.3.3.2 Password: cphd
7.3.4 Double click on Xpert-Program shortcut
7.3.5 Log-in with user account
7.3.6 Click “CREATE TEST”
7.3.7 A window opens requesting to scan the cartridge barcode
7.3.8 Take the barcode scanner and scan the cartridge barcode while
holding the yellow button pressed.
7.3.9 After barcode scanning, this window with all details appears.
7.3.10 Enter Patient ID (Name) and Sample ID (Lab Serial No)
7.3.11 The software automatically assigns a module, which should be used
for the test.
7.3.12 Click on “START TEST”
7.3.13 Completely open lid of the assigned module. The green light will be on
above the module you’re supposed to use.
7.3.14 Enter cartridge carefully with the barcode being in front.
7.3.15 Closing lid automatically starts test (the door should “click” before
test starts).

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 7.3.16 Wait the test to run for 2 hours.
7.3.17 Read and record the results.
7.3.18 Open the module lid and discard the cartridge in a biohazard bucket.
7.4 Quality Control.
7.4.1 Each GeneXpert cartridge is a self-contained unit test device (to
minimize risk of contamination).
7.4.2 The use of external controls will have limited use in assuring proper
assay performance.
7.4.3 Cepheid designed specific molecular methods to include 3 kinds of
internal controls that enable the system to detect specific failure
modes that could result in a false result.
7.4.3.1 Probe check control.
7.4.3.2 Specimen processing controls (SPC)/Endogenous control (for
Xpert BCR/ABL)
7.4.3.3 Sample Adequacy Control (SAC) (for Xpert CT/NG).
7.5 Viewing Specific Test Results.
7.5.1 Click on “VIEW RESULTS”.
7.5.2 Click on “VIEW TEST” to see a list of all results.
7.5.3 Double click the test, which you would like to see. (Alternatively:
highlight & click “OK”.)
7.5.4 The result window opens.
7.5.5 Positive results always highlighted in RED
7.5.6 Negative results always highlighted in GREEN
7.5.7 Reporting.
7.5.7.1 MTB detected or MTB not detected. MTB detected can be low,
medium or high.
7.5.7.2 RIF resistance detected or RIF resistance not detected or RIF
resistance indeterminate.
7.5.7.3 Error/Invalid = repeat test.
7.5.8 Report results as displayed on the screen.
7.5.9 Indicate the date and name of technical staff reporting.
7.6 Editing a Printable PDF Report.

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 7.6.1 Click on “VIEW RESULT”.
7.6.2 Click on “REPORT”.
7.6.3 Select the test(s) that you’d like to be in the report.
7.6.4 Click on “Preview PDF” or “Generate report file”.
7.6.5 A test report opens (PDF file), which can be printed and/or saved.
7.7 Daily Archival of Results.
7.7.1 Click on “Data Management”.
7.7.2 Click on “Archive test”.
7.7.3 Select the test(s) you want to archive.
7.7.4 Click on “Delete archived tests”. That means the tests that you
archived will not show in the software anymore.
7.7.5 For monthly archival of results, Click on “Proceed” in the next
window.
7.7.6 The files will be saved in the “Export” folder. The filename contains
the date of archiving.
7.7.7 Click SAVE.
7.7.8 Click OK.
7.7.9 The archived files have to be burned to a CD every month! (Refer to
Computer Guidelines for SOP).
7.7.10 Store the CD in the laboratory/store CD off site.
7.8 How to Retrieve Results from Archive.
7.8.1 Click on “Data Management”
7.8.2 Click on “Retrieve test”
7.8.3 Select the file that you want to retrieve from the archive. Note: Only
in the next window you will see, which tests are in this file.
7.8.4 Click on “Open”
7.8.5 Select the test(s) that you would like to retrieve from the archive.
7.8.6 Click OK
7.8.7 Click “Proceed”
7.8.8 Click OK.
7.8.9 To view results, continue with “How to view results”.
1.1.1 Print result where applicable.

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 1.1.2
1.2 Limitations.
1.2.1 Do not use a cartridge that has been dropped or shaken after you
have added the treated sample.
1.2.2 Do not use a cartridge if it appears wet or if the lid seal appears to
have been broken.
1.2.3 Do not use a cartridge having a damaged reaction tube/ handle
cartridge by PCR reaction tube.
1.2.4 Do not use cartridges beyond expiry date
1.2.5 Store Cartridge between 2 - 28°C
1.2.6 Use the cartridge within 30 minutes after opening the cartridge lid
(open only when adding sample).
1.2.7 The cartridge is stable up to 7 days after opening the package.
1.2.8 Avoid pipetting possible particles from the sample tube to the
cartridge.
1.2.9 Avoid creating bubbles when pipetting the sample into the cartridge.
1.3 Calibration.
1.3.1 Check the calibration status of each module.
1.3.2 Click on the Maintenance screen choose Maintenance > Module
Reporters.
1.3.3 The Module Reporters window appears which indicates the last date
of calibration for every module.
1.3.4 Calibration interval: 1 year or every 2000 runs (whichever comes
earlier)
1.3.5 When the calibration is due, a reminder will appear (but instrument
can be still used), i.e. from 1800 runs onwards
1.3.6 Contact DLTLD
1.3.7 The serial number of the instrument is necessary (found at the back of
the instrument & on the Certificate of calibration paper).
1.3.8 Fill the given forms & fax to Cepheid prior to sending the modules.
1.3.9 Cepheid will send replacement modules to you.
1.3.10 Once you received those, replace your own modules.

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 1.3.11 Disinfect the cartridge bay of your own modules.
1.3.12 Take out the modules and install the new modules
1.3.13 NOTE: The modules may be only taken out by staff of the supervising
laboratory!!
1.3.14 Run the calibration cartridges on the instrument & return results to
Cepheid
1.3.15 Cepheid issues a code if results are ok.
1.3.16 Enter code in your software & calibration is done.
1.4 Maintainance
1.4.1 Cleaning Xpert and surroundings.
1.4.1.1 Wipe with 10% Bleach – prepared within one week.
1.4.1.2 Wait 5 minutes.
1.4.1.3 Wipe with 70% ethanol.
1.4.1.4 Let dry.
1.4.1.5 Repeat wiping with 70% ethanol two times using every time a
fresh tissue.
1.4.2 Disinfect plunger monthly.
1.4.2.1 Remove cartridges from the instrument.
1.4.2.2 In the GeneXpert window, click “MAINTENANCE/Plunger
maintenance” on the menu bar.
1.4.2.3 The Plunger Maintenance window opens.
1.4.2.4 Select the module you want to clean or click on “CLEAN ALL”.
1.4.2.5 Follow the instructions in the dialog box, then click “OK”.
1.4.2.6 In the instrument, the plunger rod in the selected module
lowers into the cartridge bay.
1.4.2.7 Dip a number of swabs in the 10% bleach solution.
1.4.2.8 Wipe the plunger rods with the swabs. Use a fresh swab for
each plunger rod. Wait 5 minutes.
1.4.2.9 Dip a number of swabs into the 70% alcohol solution.
1.4.2.10 Wipe the plunger rods with the swabs. Use a fresh
swab for each plunger rod.
1.4.2.11 Repeat steps 1.4.2.5. and 1.4.2.6. two times

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 1.4.2.12 In the Plunger Maintenance dialog box, click Move Up
(or Move Up All). The plunger rod moves back up to its resting
position.
1.4.2.13 Click Close to dismiss the Plunger Maintenance dialog
box

2.0 APPENDICES
2.1 DLTLD Contact: email: [email protected], [email protected],
2.2 Cepheid representative, Kenya hotline number: 0720258323 or 0720649130
Email: [email protected]

3.0 REFERENCES
3.1 www.cepheid.com
3.2 GenXpert Operator Manual.

4.0 DOCUMENT CHANGE HISTORY

10.1 Version Table:
Version 1: MTB Diagnosis by GeneXpert Dated: SOP No. No. Pages:
Machine. LMIC 0001 12.

Version 2: Dated: SOP No.: No. Pages:

Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log.

Date of Changes made. Name of reviewer. Signature.
review.

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11.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: SICKLING TEST SOP No: 024

Version: Original
Effective Date: January 2016 Page 1 of 5

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Purpose & Scope
1.1Purpose
To outline the procedure to be followed when performing the sickling test.

1.2 Scope
This procedure is applicable to Tabaka Mission Hospital Laboratory Haematology section.

1.3 Principle & Safety

1.3.1 Principle
Sodium metabisulfate is a reducing agent and it draws oxygen from the red blood cells
depriving the blood of the oxygen. In the deoxy form, hemoglobin S starts to polymerize
and precipitate out in solution. This distorts the RBC making it rigid and produces the
sickle shaped cell.

1.3.2 Safety;
Always wear personal protective equipment when handling potentially infectious material.

2.0 Abbreviation, Definitions and Terms

2.1 Abbreviations
2.1.1 SOP: Standard Operating Procedure
2.1.2 PMHL: Princess MARINA HOSPITAL LABORATORY

2.2Definitions and Terms

N/A
3.0 Equipment and Materials/Reagents/Supplies

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 3.1 Equipment
3.1.1 Microscope

3.2 Materials/Reagents/Supplies
3.2.1 Slides
3.2.2 Cover slip
3.2.3 Paraffin wax
3.2.4 Sodium metabisulfate
3.2.5 Pipette
3.2.6 Pencil
3.2.7 4ml EDTA blood sample
3.2.8 Petroleum jelly
3.2.9 Test tube

4.0 Responsibility
4.1 The Head of section is responsible for monitoring the effective implementation of this
procedure.
4.2 Technical staff is responsible for the implementation of this procedure.

5.0 Procedure
5.1 Procedural steps
5.1.1 Label the slide with the patient number and one of the patient names
5.1.2 Put 2 drops of fresh 2% sodium metabisulfate in the test tube and mix with 1 drop of
well mixed EDTA patient blood.
5.1.3 Gently mix and place a drop onto the slide
5.1.4 Apply a cover slip avoiding creating air bubbles
5.1.5 Create an air tight seal around the cover slip with petroleum jelly
5.1.5.1 Put a small amount of petroleum jelly onto a clean slide
5.1.5.2 Light a burner

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 5.1.5.4 Melt the petroleum jelly and quickly drip it onto the edges of the cover slip
creating the airtight seal.
5.1.5.5 Repeat the process until all the four corners of the cover slip are sealed
5.1.6 Check the sealed slide under the microscope. Cells should be in a single layer
5.1.7 Allow to stand for 15-30minuts at room temperature in a wet chamber
5.1.8 Examine the slide under x10 the x40 objectives for sickle cells
5.1.9 Incubate the slide in a wet chamber at 37OC for 6- 24hours
5.1.10 Examine again under the microscope X10 or X 40
5.1.11 Report the results and record in the Peripheral blood smears report book

5.2 Quality control

5.2.1 Use EDTA blood sample
5.2.2 Avoid air bubbles when putting the cover slip
5.2.3 Use freshly prepared 2% sodium metabisulfate
5.2.4 Use a known sickle cell slide as a quality control

5.3 Reporting of results

5.3.1 Report as positive where sickle cells are seen under the microscope x40 objective
5.3.2 Report as negative where red blood cells appear normal at x40 objective

6.0 Related procedures

There are no related procedures

7.0 References
7.1 Heilmeyer L. and Begemann H. Atlas of Clinical Haematology 6th Edition 2005 Library
of Congress Cataloging-in-publication Data.
7.2 Dacie and Lewis Practical Heamatology, 2006 10th Edition, Churchill Livingstone.

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Training log

All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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LABORATORY STANDARD OPERATING PROCEDURES

FACILITY NAME………………………………………………………………………………………………………

COUNTY…………………………………………………………………………………………………………………

SUB-COUNTY………………………………………………….……………………………………………………..

SOP Title: DIRECT COOMBS ANTIGLOBULIN TEST SOP No: 010

Version: Original
Effective Date: January 2016 Page 1 of 8

Signatures and Dates:

Author: __________________________________________________________________
Laboratory Technologist Date

QA Review: _______________________________________________________________
QA Officer Date

Approving Authority: _______________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

1.0 PURPOSE / INTRODUCTION:
1.1 DCT/DAT detects in vivo coating of patient red cells - either IgG antibodies,
C3, or both. Within the patient's blood stream antibodies attach to their
specific antigens on the red blood cells. This happens in HDN, incompatible
transfusion reactions, and in autoimmune haemolytic anaemia acquired
haemolytic anaemias. Certain drugs are also known to activate complement
and it can also coat the cells in vivo. When the blood is drawn the antibodies
and/or complement have already attached to the red cells. Thus, red should
be washed 3 or more times.

1.2 A small proportion of normal individuals have a positive DAT without

evidence of decreased red cell survival. Thus, a positive DAT, by itself, does
not mean that the patient has an immune haemolytic anaemia. A positive
DAT due to IgG is seen most frequently in patients with warm
autoantibodies. Approximately 1/3 of these patients also have C3 on the red
cell membrane. A positive DAT due to C3 alone is seen in patients with cold
autoantibodies, paroxysmal cold hemoglobinuria, and in some drug induced
haemolytic anaemias. The offending antibodies are typically of the IgM
isotypes that efficiently bind C3. IgM antibodies are not directly detected by
the DAT, but are detected indirectly by the presence of C3 on the red cell
surface.

2.0 SCOPE / RESPONSIBILITY:

2.1 This SOP applies to all laboratory technologists/technicians involved in the
assay.
2.2 It is the responsibility of the designated Quality Assurance and Quality
Control (QC/QA) personnel, Laboratory Supervisors, DCMLTs, and CMLT to
ensure that the current SOP is available to the staff and the procedure is
followed as documented.

3.0 SAFETY/RISK ASSESSMENT:

3.1 Wear personal protective equipment. Handle all samples as potential
biohazards.
4.0 DEFINITIONS:
4.1 AHG: Anti- human Globulin.
4.2 IgG: Immunoglobulin G.
4.3 IgA: Immunoglobulin A.
4.4 IgM: Immunoglobulin M.
4.5 C3: Complement.
4.6 HDN: Haemolytic Disease of the New born.
4.7 DAT: Direct Anti-globulin Test.
4.8 DCT: Direct Coombs Test.
4.9 Mm: Millimetres.
4.10 PPE: Personal Protective Equipment.
4.11 QA: Quality Assurance.
4.12 RBC: Red Blood Cell.
4.13 SOP: Standard Operating Procedure.

5.0 SPECIMEN:
5.1 Freshly prepared 3-5% red cell suspension from a clotted blood sample.

6.0 EQUIPMENT/ MATERIALS/ REAGENTS:

6.1 Equipment
6.1.1 Electric centrifuge
6.1.2 Microscope.
6.2 Materials
6.2.1 PPEs e.g. Gloves and a Lab coat.
6.2.2 Small glass test tubes (75x12mm).
6.2.3 Pasteur pipettes.
6.2.4 Microscope slide.
6.3 Reagent
6.3.1 Normal saline.
6.3.2 Coombs serum/ AHG.
6.3.3 IgG anti-D

7.0 METHODOLOGY:
7.1 Principle
7.1.1 The anti-human serum when added to sensitized cells forms bridges
between the antibodies and the end result is firm agglutination of red
cells. The same principle also works for complement sensitized red
cells.
7.1.2 The most important thing is that the unattached serum globulins
should be removed from around a sensitized cell before AHG is
added. If the unattached globulins aren't removed they will combine
and neutralize the anti-human serum hence give a false negative test
result.
.
7.2 Procedure
7.2.1 Prepare a 3-5% red cell suspension as follows:
7.2.1.1 Transfer about 0.5 ml red cell from a clotted sample into about
5 ml normal saline.
7.2.1.2 Wash the cells 3 times by adding 5-7 ml saline, centrifuging at
3000 rpm for 2-3 minutes and discarding the supernatant.
7.2.1.3 Make a 3-5% red cell suspension by mixing 1 drop of
sedimented cells in 20-25 drops of saline, holding the Pasteur
pipette vertically.

7.2.2 Label a test tube DCT.

7.2.3 Using the Pasteur pipette, put 2 drops of the 3-5% red cell suspension
into the test tube.
7.2.4 Add 2 drops of coombs serum/AHG.
7.2.5 Mix and centrifuge at 1500rpm for 1 minute.
7.2.6 Re-suspend the contents and examine for agglutination both
macroscopically and microscopically.

7.3 Quality Control

7.3.1 Use cells sensitized with IgG to ensure that the AHG reagent is
working.
7.3.2 Prepare sensitized cells as follows:
 7.3.2.1 Place 4 drops of O positive blood in a 12 x 75 mm test tube.
7.3.2.2 Add 2 drops of IgG anti-D
7.3.2.3 Incubate at 37oc for 60 minutes in water bath.
7.3.2.4 Wash the cells 3 times by adding 5-7mls saline, centrifuging at
3000 rpm for 2-3 minutes and discarding the supernatant.
7.3.2.5 Make 3-5% cell suspension (see 7.2.1.1 to 7.2.1.3)
7.3.2.6 Mix 1 drop of 3-5% cell sensitised cell suspension and 1 drop
AHG in a test tube.
7.3.2.7 Centrifuge at 1000 rpm for 1 minute.
7.3.2.8 Examine for agglutination macroscopically.
7.3.2.9 Agglutination indicates that cells have been sensitized and
demonstrates that AHG reagent is working properly.

7.4 Results.
7.4.1 Agglutination of red cells indicates a positive result.
7.4.2 A smooth suspension of red cells indicates a negative result.
7.4.3 Report results DCT/DAT positive or negative. Indicate the date and
name of technical staff reporting.

7.5 Limitations.
7.5.1 False negative results may occur due to improper washing of the red
cells to remove the unattached globulins.
7.5.2 False positive results may occur due to extreme reticulocytosis, lead
poisoning, drug induced haemolysis and certain viral diseases on rare
occasions.
7.5.3 Rarely, patients with immune haemolysis may have a negative DAT.
Many of these patients have IgG, IgM, or IgA antibodies detectable on
the red cell only with more sensitive techniques. (MicroCoomb’s test)

8.0 APPENDICES:
8.1 Not Applicable.
9.0 REFERENCES:
9.1 Immunohaematology/ Blood Transfusion Science, Jomo Kenyatta University
of Agriculture and Technology Notes, Prof. Mwanda. O.
9.2 Monica Cheesbrough (2006), District laboratory practice in tropical countries,
Cambridge University press, Part 2.
9.3 Internet sources.

10.0 DOCUMENT CHANGE HISTORY:

10.1 Version Table:
Version 1: Direct Coombs/ Anti-globulin Dated: SOP No.: No. Pages:
test. LBTS. 0001 08.
 Version 2: Dated: SOP No.: No. Pages:

Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log

Date of Changes made. Name of reviewer. Signature.
review.

11.0 SOP AWARENESS LOG

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………………………………………………………………..

COUNTY………………………………………………………………………………………………………………………

SUB COUNTY………………………………………………….……………………………………………………………

SOP Title: ERYTHROCYTE SEDEMENTATION RATE SOP No: 011

Version: Original
Effective Date: January 2016 Page 1 of 5

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Purpose & Scope
1.1 Purpose
To outline the procedure to be followed when performing ESR.

1.2 Scope
This procedure is applicable to Tabaka Mission Hospital Laboratory Haematology section.

1.3 Principle & Safety

1.3.1 Principle
When anti-coagulated blood is allowed to stand undisturbed for a period of time,
the RBCs gradually settle out of the plasma. RBCs have a net negative surface
charge and these repulsive forces are counteracted if there are increased
quantities of positively charged plasma proteins the RBCs settle more rapidly due
to rouleaux formation. The ESR is directly proportional to the RBC mass and
inversely proportional to the plasma viscosity. The rate at which the RBCs fall in
one hour is measured in mm/hour.

1.3.2 Safety
Always wear personal protective equipment when handling potentially infectious
material.

2.0 Abbreviation, Definitions and Terms

2.1 Abbreviations
1. ESR: Erythrocyte sedimentation rate
2. PMH: Princess marina hospital
3. EDTA: Ethylene diamine tetra acetic acid
4. RBC: Red blood cell
5. PTB: Pulmonary tuberculosis
6. Mm: Millimetres

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 7. Ml: Millilitres

2.2Definitions and Terms

N/A

3.0 Equipment and Materials/Reagents/Supplies

3.1 Equipment
i. Timer

3.2 Materials/Reagents/Supplies
i. 4ml EDTA sample
ii. Westergren tube
iii. Westergren rack
iv. ESR cups (contain 0.5 ml of 3.8% sodium citrate)

4.0 Responsibility
4.1 The Head of section is responsible for monitoring the effective implementation of this
procedure.
4.2 Technical staff is responsible for the implementation of this procedure.

5.0 Procedure
5.1 Procedural steps
5.1.1 Label the ESR cups with the sample number e.g. H333
5.1.2 Remove the cap from the ESR cup
5.1.3 Pour well-mixed EDTA blood up to the mark, ensuring that there are no air
bubbles (air bubbles invalidates the test result)
5.1.4 Replace the cap and invert 8 times to mix the blood and sodium citrate already
inside the cup

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 5.1.5 carefully insert the Westergren tube into the plungeable vial cap of the blood
mixture twisting as you push down.
5.1.6 Ensure the blood is up to the zero mark before placing on Westergren rack
5.1.7 Place the tube in the Westergren rack
5.1.8 Set the timer on from one hour
5.1.9 Read from the top to the point where RBC are separated from the plasma in mm
at the end of one hour.
5.1.10 Record results and enter your initials under READ BY

5.2 Reporting
5.2.1 Report in mm/hour
5.2.2 Normal ranges,
Females: 0mm to 20mm/hour
Males: 0 to 15mm/hour

5.3 An increased ESR can be due to:

5.3.1 Pregnancy
5.3.2 Rheumatoid arthritis
5.3.3 Polycythemia
5.3.4 Sickle cell anemia
5.3.5 Congestive heart failure

6.0 Related procedures

There are no related procedures

7.0 References
7.1 Rodak B.F. Haematology Clinical Principles and Applications 2002, 2nd edition
Saunders

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8.0 Appendix
Training log
All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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 LABORATORY BIOSAFETY STANDARD OPERATING
PROCEDURES
FACILITY NAME…………………………………………………………………………………………………

COUNTY……………………………………………………………………………………………………………

SUB COUNTY………………………………………………….…………………………………………………

SOP Title: General Biosafety Procedure SOP No: 016

Version: Original
Effective Date: January 2016 Page 1 of 38

Signatures and Dates:

Author: _ ________________________________________________________________
Laboratory Technologist Date

QA Review: _______________________________________________________________
QA Officer Date

Approving Authority: _______________________________________________________

Laboratory In-charge Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1. Purpose Applicability:
1.1. The establishment of standard policies and procedures are necessary for safe
laboratory conduct, handling laboratory hazards, and contingency planning for safety
issues as part of the department safety program.
1.2. Laboratory personnel must have knowledge of safe laboratory procedures and an
awareness of potential hazards. Adherence to appropriate safety practices will
prevent serious laboratory accidents.

2.0. Responsibilities
2.1 This SOP applies to all laboratory personnel and other departments that use the
laboratory services.
2.2 It is the responsibility of the designated of the County Leads, Quality Assurance and
Quality Control (QC/QA) personnel, Laboratory Supervisors to ensure that the current
SOP is available to the laboratory personnel and the procedure is followed as
documented.

3.0 Safety /Risk Assessment:

3.1 Refer to procedure

4. METHODOLOGY:

4.1 General Laboratory Safety Standards, Practices and General Requirements for
Personnel on Laboratory Safety:
4.1.1Become familiar with the location and proper use of emergency exits,
fire equipment and first aid stations.
4.1.2 Follow all outlined safety instructions/policies.
4.1.3 Read all labels carefully.
4.1.4 Post appropriate warning signs within the laboratory.

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4.1.5 Read all posted signs.
4.1.6 Label reagents, materials, and storage containers legibly and
according to regulations.
4.1.7 Inform co-workers of any potential health hazard associated with the
work performed.
4.1.8 Report all unsafe conditions or equipment to the laboratory
manager/designee and/or safety officer/designee.
4.1.9 Treat all materials as toxic until they are adequately identified or
characterized.
4.1.10 Do not use laboratory equipment for non-laboratory procedures.
4.1.11 Place “not in use” signs on equipment awaiting repair or installation
and commissioning.
4.1.12 Cover and store all cutting or capping instruments used in the
laboratory when they are not in use.
4.1.13 Label defective laboratory equipment and remove it from the
laboratory area.
4.1.14 To improve laboratory safety and efficiency, store materials and
equipment in their proper places.
4.1.15 Store bottles of caustic materials, i.e., bleach in a secondary container
that will contain any possible leakage.
4.1.16 Store no more than 500mL of ethanol or isopropyl alcohol on the
bench top. Additional amounts kept in the laboratory must be kept in
a locked "flammable liquids" cabinet.
4.1.17 Treat carefully all systems under vacuum or positive pressure.
4.1.18 Do not move equipment between laboratories unless absolutely
necessary.
4.1.19 Never smell or taste any substance used in the laboratory.
4.1.20 Do not eat or drink in laboratory areas.
4.1.21 Do not smoke anywhere in the building.
4.1.22 freezers.
4.1.23 Do not put laboratory materials, including pencils and pens into your
mouth.

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 4.1.24 Keep hands away from mouth and face.
4.1.25 Never mouth pipette anything in any area of the laboratory.
4.1.26 Wash hands frequently when in the laboratory and always before
leaving the laboratory, this is essential to avoid becoming exposed to
chemical irritants and infectious agents.
4.1.27 Cover all cuts or abrasions on the hands with adhesive bandages
before entering the laboratory area.
4.1.28 Do not apply cosmetics or lip balm while in the laboratory area.
4.1.29 Do not manipulate contact lenses while in the laboratory area.
4.1.30 If possible avoid wearing contact lenses in areas where dust,
abrasives, corrosives, acids, caustics, chemical vapours or fumes are
present. These materials can get under the lenses and damage the
eyes.
4.1.31 All personnel - wear hair and/or beards such that they will not come
in contact with working surfaces, specimens or laboratory equipment.
4.1.32 Do not wear loose or dangling jewellery in the laboratory if there is a
chance that it may come in contact with work surfaces, specimens, or
moving parts of equipment.
4.1.33 Do not wear sandals or open-toed shoes or go barefoot in the
laboratory to avoid injuries from falling objects, broken glass or toxic
spills.
4.1.34 Use universal precautions while in the laboratory. Handle all
laboratory specimens as if they are potentially infectious.
4.1.35 Decontaminate work surfaces with bleach and/or alcohol before and
after use and immediately after any spill.
4.1.36 Keep laboratories clean and free of material not pertinent to the
procedures being performed.
4.1.37 Always keep laboratory doors closed when performing procedures.
4.1.38 Remove personal protective equipment when leaving the laboratory
area.
4.2 Personal protective equipment (PPE)
4.2.2 Wear full-length solid front disposable gowns or full-length laboratory

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 coats completely fastened or buttoned while working in the
laboratory area.
4.2.3 Do not wear laboratory coats or disposable gowns outside the
laboratory.
Wear fitting gloves while processing specimens or performing
laboratory tests.
4.2.4 Change gloves immediately they become contaminated with blood,
serum, plasma or any other body fluids or if they tear.
4.2.5 Use safety goggles or other eye or face protection when handling
specimens or performing procedures where there is an eye-impact
hazard.
4.2.6 Use full-face shields (worn or mounted) if there is a possibility of
being exposed to aerosols, splashes from blood products, corrosives,
acids, caustics or other chemicals.
4.2.7 Become familiar with the use of the emergency eye rinsing facilities
available at designated sinks in the laboratory. Become familiar with
the use of emergency shower
4.2.8 Personal protective equipment are provided for the protection of
laboratory personnel; PLEASE USE THEM.
4.3 General Policies
4.3.2 The Laboratory director/designee appoints safety officer.
4.3.3 The Ministry of Health provides safe working conditions as follows:
4.3.3.1 Employees who work in biohazard areas should have adequate
protective equipment and clothing.
4.3.3.2 Accident prevention as an integral part to safe laboratory
functions.
4.3.3.3 No job shall be initiated without implementing appropriate
safety measures.
4.3.3.4 Access to laboratories limited to authorized laboratory
personnel only.
4.3.3.5 Review of safe work practices and monthly documentation
and annotated in the Quality Improvement program minutes.

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4.4 Procedures for Reporting and Documenting Incident/Accident
4.4.2 Supervisors at all levels review accident prevention measures, analyse
any accident that occur and take appropriate corrective actions.
These reviews are documented and the documentation is placed in
the laboratory safety file.
4.4.3 The laboratory manager or designee reviews and signs off on all
incident reports as part of the laboratory Quality Improvement
Program.
The laboratory director or designee conducts an Annual Review of all
safety policies, records and work practices and document such
reviews in
4.4.4 the QI annual appraisal reports
4.4.5 All guest shall be briefed on laboratory safety standards before being
allowed to work in any of the laboratory areas.
4.4.6 Accidents resulting in fatalities or in hospitalisation of any
employee(s) will be reported to the lab director or designee
immediately where possible or within 24 hours.
4.4.7 A Safety Checklist will be completed at the end of each week.

4.5 Reporting Incident/Accident

4.5.2 Contact the first line supervisor/designee immediately.
4.5.3 Within one hour, if possible
4.5.4 Report the exposure event to the PEP designate; if he is not available
inform one of the coordinators. Phone numbers are posted on the lab
notice board.

4.6 Guideline for an exposure event

Refer to SOP LGEN 0023 –Post Exposure Prophylaxis Procedure

4.7 Biohazard Control Procedures

4.7.2 Use universal precautions standards as required at all times.
4.7.3 Decontaminate laboratory work surfaces when work is finished and

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 after any splash or spill of biological material using 10% bleach
solution or 70% alcohol.
4.7.4 Place biohazard waste in appropriately marked containers for
biohazard waste.
4.7.5 Liquid biohazard waste may be decontaminated by dispensing them
into a 10% bleach solution or 70% alcohol as applicable.
4.7.6 Decontaminate reusable supplies such as trays, racks and pipettes by
soaking in a 10% bleach solution or wiping surface with 10% bleach,
or by wiping with 70% alcohol.
4.7.7 Replace gloves if they become contaminated, punctured, or torn.
4.7.8 Remove gloves when using the laboratory telephones/general
computers to avoid contaminating the person who might be using the
phone/computer.
4.7.9 Perform all procedures with potential for aerosol formation in
biological safety cabinet, e.g. Opening of containers of whole blood,
serum, plasma, performing aliquoting procedures.
4.7.10 Use the appropriate safety cabinet or chemical hood when working
with materials that are infectious, toxic, corrosive, dusty, or irritating
to skin or eyes.
4.7.11 Dispose of all sharps, e.g. pipette tips and pipettes, into appropriate
sharps containers.
4.7.12 Wash hands thoroughly before leaving the laboratory.
4.8 Biological Safety Cabinets
4.8.2 Determine the biological safety level of the laboratory, type of
infectious agents or biochemical hazard that may be present, and the
nature of the work performed.
4.8.3 Use total exhaust safety cabinets for operations that utilize hazardous
chemicals and volatile toxins.
4.8.4 Use a biological safety cabinet when performing procedures that may
create an inhalation or aerosol hazard.
4.8.5 Use biological safety cabinet when working with infectious agents
classified as Biosafety Level-2 (BSL-2) or greater

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 4.8.6 Biological Safety Cabinets MUST be inspected and certified when
installed or relocated and annually thereafter.
4.8.7 Documentation of certification consists of a sticker with the
certification date affixed to each BSC.

4.9 Use of the Biological Safety Cabinet

4.9.2 Place safety cabinets in an area of low traffic and low draft if possible.
4.9.3 Wear protective clothing (PPE).
4.9.4 Minimize the amount of supplies and equipment inside the hood.
4.9.5 Do not place objects over the ventilation grills in the front of the hood
or block the grills in the back of the hood. This will interfere with the
laminar airflow in the hood.
4.9.6 Keep to a minimum the amount of traffic past the face of the hood.
4.9.7 DO NOT use heating devices or Bunsen burners inside the cabinet
unless absolutely necessary.

4.10 Procedures for Handling Hazardous Spills

4.10.2 Infectious Spills
4.10.3 STOP work immediately and ASSESS the extent of the danger.
4.10.4 ALERT other personnel in the immediate area.
4.10.5 Remove contaminated gloves, discard, and replace with clean
gloves.
4.10.6 Immediately DECONTAMINATE all body areas that may have
been exposed to infectious material as follows (Ask for the help of a
co-worker if necessary):
4.10.6.1.1 Initially soak clothing or exposed body areas with
70% alcohol.
4.10.6.1.2 Wash thoroughly with soap and water - a minimum
of three minutes. Do not abrade skin during the
washing process.
4.10.6.1.3 Use the eyewash if eyes were exposed.
4.10.7 Change gloves again and take any FIRST AID measures

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 necessary.
4.10.8 RESTRICT ACCESS to the area, cover the spill with absorbent
material and thoroughly soak with freshly prepared 10% household
bleach.
4.10.9 Allow the spilled material to stay in contact with the
disinfectant for 30 minutes and then clean up the area.
4.10.10 Do not pick up any containers until they have been
decontaminated
4.10.11 Remove protective clothing and discard.
4.10.12 NOTIFY the Safety officer and supervisor as soon as possible.
Do not leave the area until this is done.
4.10.13 Prepare a REPORT of Incident or Accident with the help of the
supervisor.
4.10.14 Chemical Spills
4.10.14.1 Immediately FLUSH any exposed body areas and
clothing with large volumes of water.
4.10.14.2 Request the help of a co-worker, if necessary.
4.10.14.3 Use emergency body shower or eye showers as
needed.
4.10.14.4 Put on protective clothing (PPE) - Laboratory coat,
gloves, Safety goggles, glasses, or face shield.
4.10.14.5 Retrieve the Chemical Spill clean-up materials
4.10.14.6 Place absorbent NEUTRALIZING material over the spill
area. Use enough material to absorb the entire spill.
Place the used absorbent material in the CLEAR plastic bags provided
and close securely with the fastener.
4.10.14.7 REFER to the MSDS for proper DISPOSAL or contact the
Safety officer for directions.
4.10.14.8 NOTIFY the Safety Officer or Laboratory
manager/designee of the incident within one hour.
4.10.14.9 Prepare a REPORT of Incident or Accident with the help
of the supervisor/designee.

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4.11 Hazardous Waste Disposal
4.11.2 Infectious or Pathological Waste (Soft Waste)
4.11.2.1 Includes any solid or liquid biological material that may
contain or be contaminated with potentially communicable
disease agents.
4.11.2.2 Locate red infectious/biohazard waste disposal
containers in each area of the laboratory where infectious
waste may accumulate.
4.11.2.3 Line containers with disposable bright orange/red
biohazard bags before placing infectious waste into them to
prevent leakage.
4.11.2.4 Included in this category are:
4.11.2.4.1 Used gloves
4.11.2.4.2 Transfer pipettes (In burn up bins)
4.11.2.4.3 Test tubes
4.11.2.4.4 Specimen containers
4.11.2.5 Fill infectious waste bag/containers no more than
three-quarters full.
4.11.2.6 Remove biohazard bags from the hoods after each
day's use or as needed.
4.11.2.7 Transport bags and containers to the biohazard storage
room.
4.11.2.8 Biohazard wastes bags are incinerated
4.11.3 Sharps Disposal
4.11.3.1 Sharps are pipette tips, pipettes, needles, syringes,
broken glassware, blood collection tubes, microwell plates or
any object that can puncture the skin.
4.11.3.2 All sharps must be prepackaged in puncture resistant
containers.
4.11.3.3 Dispose of sharps in appropriate biohazard sharps
containers provided for this purpose.

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 4.11.3.4 Do not dispose of any other trash into these
containers.
4.11.3.5 Do not overfill sharps disposal containers. Fill to 3/4
capacity.
4.11.3.6 Needles should not be recapped, bent, or broken prior
to disposal.
4.11.3.7 Do not remove needles from disposable syringes.
4.11.3.8 Expel excess liquid from syringes or pipettes.
4.11.3.9 Dispense infectious liquid into a 10% bleach solution
prior to disposal.
4.11.3.10 Lock filled containers.
4.11.3.11 Dispose off sharps containers in biohazard containers.
4.11.4 Chemical Waste
4.11.4.1 All chemical containers must be labelled with the
name of the chemical, name and address of the
manufacturer and specific hazard warnings.
4.11.4.2 Material Safety Data Sheets (MSDS) must be readily
available for each chemical or reagent used in the laboratory.
4.11.4.3 Non-hazardous chemical waste can be disposed of by
pouring down the sink:
4.11.4.4 Hazardous chemical waste includes toxic, corrosive,
ignitable, reactive, and explosive materials.
4.11.4.5 Store corrosive chemicals in an approved corrosive
container.
4.11.4.6 Store flammables in a flammable cabinet separate
from corrosives and oxidizers.
4.11.4.7 Store oxidizers separately.
4.11.4.8 Store organic acids separate from mineral acids.
4.11.4.9 Store concentrated acids and bases in a fume hood
with a barrier between them.
4.11.4.10 Do not store hazardous chemicals on shelves above
eye level (approx. five feet).

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 4.11.4.11 Label chemical storage areas according to National Fire
Protection Association (NFPA) standards.
4.11.4.12 Handle any material that is not infectious or
radioactive and whose risk to public safety is in doubt, as
hazardous chemical waste until clarification can be obtained.
Dispose of chemical waste according to Safety Guidelines Also refer to MSDSs.
4.11.4.13Do not dispose of hazardous chemicals by pouring them
down the sink.
4.11.4.14If in doubt as on how to dispose of hazardous chemicals
consult with the Safety Officer, laboratory manager/designee and
MSDS.
4.11.4.15NOTE: All laboratory-generated hazardous wastes will be
disposed of according to the local and state regulations.

5.0 Infection Control/Blood borne Pathogens Exposure Control Plan

5.1 The Blood borne Pathogens Standard was issued to reduce the
occupational transmission of infections caused by microorganisms sometimes
found in human blood, body fluids, or other materials.
5.2 Hepatitis B Virus (HBV) and Human Immunodeficiency Virus (HIV), as well
as a variety of other infectious agents, have been shown to be responsible for
infecting workers handling/exposed to human blood and other body fluids
containing these viruses.
5.3 Routes of infection have been by needle-stick injuries and by direct
contact of mucous membranes and non-intact skin with contaminated blood
or body fluids and other materials.
5.4 Occupational transmission of Hepatitis B Virus occurs much more often
than transmission of Human Immunodeficiency Virus. However, the lethal
nature of HIV requires that all possible measures be used to prevent the
exposure of workers to the HIV virus.
6.0 Scope and Procedures
6.1The Ministry of Health has established an infection control plan to
minimize and prevent, when possible, employee exposure to disease causing

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 microorganisms transmitted through human blood, body fluids and other
potentially infectious materials.
6.2The plan involves all employees who are or may be exposed to blood and
other potentially infectious materials as part of their duties.
6.3Review the plan at least annually and update as necessary.

A copy of the laboratory infection control plan is available to be read and as for reference by
employees, as part of the SOP/policy. Keep the copy in the main laboratory areas accessible
6.4 Basic components of this exposure control plan include:
6.4.1 Exposure determination.
6.4.2 Methods of compliance.
6.4.3 Procedures for evaluation and follow-up of exposure incident.
6.4.4 Employee training.
6.4.5 Record keeping procedures
6.4.5.1 Exposure Determination.
6.4.6 The exposure control plan includes all job categories in which
there is a possibility that an employee will have skin, eye,
mucous membrane, or potential contact with blood or other
potentially infectious materials.
6.4.7 Job categories with a definite risk of exposure
6.4.7.1 Phlebotomists
6.4.7.2 Laboratory supervisors and managers
6.4.7.3 Laboratory technologists and technicians
6.4.7.4 QA/QC technologists and technician
6.4.8 Job categories with a possible risk of exposure
6.4.8.1 Clerical staff - accidental spills in sample receiving
areas.
6.4.8.2 Department Directors, supervisors, QA Officers -
accidental spills or aerosols while observing testing.
6.4.9 Potentially infectious materials include:
6.4.9.1 Blood and blood products
6.4.9.2 Cerebrospinal fluid

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 6.4.9.3 Semen
6.4.9.4 Vaginal secretions
6.4.9.5 Pleural fluid
6.4.9.6 Pericardial fluid
6.4.9.7 Peritoneal fluid
6.4.9.8 Any body fluid visibly contaminated with blood
6.4.9.9 Unfixed tissues or organs (other than intact skin)
from human, living or dead.
6.4.9.10 Blood, organs, or other tissues from
experimental animals infected with HIV or HBV.
6.4.9.11 Cell or tissue cultures, organ cultures, and
culture media containing HIV or HBV virus.

6.4.10 Methods of Compliance (Universal precautions)

6.4.10.1 Always handle all blood or other body fluids as
potentially infectious materials capable of
transmitting an infectious disease.
6.4.10.2 Use engineering and work practice controls to
minimize or eliminate employee exposure by:
6.4.10.2.1 Maintain or replace faulty
parts/instruments on a regular basis:
6.4.10.2.2 Hand washing facilities
6.4.10.2.3 Sharps containers
6.4.10.2.4 Personal protective equipment
6.4.10.2.5 Waste removal
6.4.10.2.6 Absorbent material for spills (Spill
Removal Kits)
6.4.10.2.7 Laundry/housekeeping procedures
6.4.10.2.8 Biohazard labels
6.4.10.3 General hygiene measures
6.4.10.3.1 Practice appropriate hand washing,
it is the primary protective infection

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 control measure.
6.4.10.4 Sharps management
6.4.10.4.1 Do not bend, recap, reuse, remove
or share contaminated needles or
other contaminated sharps.
6.4.10.4.2 Throw them without manipulation
into sharps’ container while
maintaining their integrity.
6.4.11 Precautions when handling, procuring, and transporting
specimens
6.4.11.1 Wear PPE as needed at all times. Follow
Universal Precautions.
6.4.11.2 Place blood specimens or other potentially
infectious material in a container that prevents
leakage during collection, handling, processing,
storage, transport, or shipping.
6.4.11.3 Seal specimen containers before storing,
transporting or shipping.
6.4.11.4 Use the following containers in the laboratory:
6.4.11.4.1 Tubes with screw caps that is leak-
proof.
6.4.11.4.2 Test tube racks of a size to hold the
types of tubes used in the
laboratory.
6.4.11.4.3 Red-orange or red plastic biohazard
bags with seals.
Seal the primary specimen container and place it in a
6.4.11.5 Secondary container if specimens are to be
transported or shipped.
6.4.11.6 Follow all state and local regulations for
shipping potentially infectious materials.
6.4.11.7 Consult with supervisors and carefully handle

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 shipments that are not submitted or received in
appropriately labelled sturdy containers that
prevent leaks/spills or exposures.
6.4.11.8 Reject and discard shipments that show obvious
signs of leaks/spills. Document and ensure the
director or designee is notified of the occurrence.
The manager or designee will notify the submitting
facility of the incident.
6.4.12 Management of contaminated equipment
6.4.12.1 Assess equipment and decontaminate before
servicing or shipping.
6.4.12.2 If equipment cannot be fully decontaminated
attach a label stating which parts remain
contaminated.
6.4.12.3 Decontaminate with 10% bleach solution or
70% alcohol unless otherwise stipulated by the
manufacturer.
6.4.13 Personal protective equipment
6.4.13.1 The employer will provide personal sanitary and
reliable protective equipment for the employees to
use when exposure is likely to occur. Employees
will be trained in and know where this equipment is
located so that it is used by them in technical areas
in situations where exposure is likely to occur.
6.4.13.2 Employees will be trained to recognize these
instances.
6.4.13.3 Gloves, laboratory coats, hood, hood sleeves,
safety glasses, and full safety face shields must be
available.
6.4.13.4 Wear protective equipment whenever there is a
possibility of exposure of the skin, eyes, mouth, or
other mucous membranes to infectious, bio

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 hazardous or chemical materials.
6.4.13.5 Wear properly fitting gloves. Small, medium,
large and extra-large sizes are available. Let the lab
manager/designee know when your particular size
is running low.
6.4.13.6 Hypoallergenic gloves and cotton glove liners
are available for employees who have allergies to
latex gloves or the powder in gloves.
6.4.13.7 Remove and dispose of coats, gowns, or gloves
as soon as possible whenever they become
contaminated with potentially infectious material
or they tear as the employee is working with
potentially infectious material.
6.4.13.8 Place disposable lab coats, gowns, etc. in
biohazard waste container after use. Use a
disposable lab coat for no longer than two weeks.
6.4.13.9 Remove all personal protective equipment
before leaving the laboratory area.
6.4.13.10 Remove filled biohazard bags from hoods each
day when work is completed or as soon as they are
filled up.
6.4.13.11 Disposable gloves:
6.4.13.11.1 Replace immediately when they
are contaminated or their ability to
function as a barrier is
compromised, i.e. torn or punctured.
6.4.13.11.2 Do not wash or decontaminate
single-use gloves for re-use.
6.4.13.11.3 Discard all gloves into a
biohazard container.
6.4.13.11.4 Always decontaminate and/or
wash your hands whenever you

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 remove gloves
6.4.13.12 Utility gloves
6.4.13.12.1 Decontaminate and reuse utility
gloves from spill removal kits if they
are in good condition.
6.4.13.12.2 Discard gloves that show signs of
deterioration such as cracking or
peeling or whenever their ability to
act as a barrier is compromised.
6.4.13.12.3 Discard torn or punctured gloves.
6.4.13.13 Use full safety face shields (worn or mounted) if
there is a chance that aerosols of blood or other
potentially infectious materials may contaminate
eyes, nose or mouth.
6.4.13.14 Wear safety goggles when the chance of
exposure is to the eyes but not to the mouth or
nose.
6.4.13.15 The use of regular eyeglasses in lieu or safety
glasses is permitted if they have side protectors.
The wearing of contact lenses is not considered a
form of protection against exposures.
6.4.13.16 Wear laboratory coats, disposable or reusable
gowns
6.4.13.16.1 While performing assays.
6.4.13.16.2 While performing quality control
duties.
6.4.13.16.3 When opening tubes of blood,
serum, plasma, or other fluids.
6.4.13.16.4 While operating or cleaning
laboratory instruments.
6.4.13.16.5 When decontaminating and
cleaning up chemical or biological

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 spills.

6.5 Housekeeping
6.5.4 Maintain the workplace in a clean and sanitary condition.
6.5.5 Clean equipment and environmental working surfaces with
appropriate disinfectants:
6.5.5.1 After completing any procedure in the laboratory
6.5.5.2 Immediately following any spill of blood or other
potentially infectious material.
6.5.5.3 At the end of each work shift if a work surface has
been used during the day.
6.5.6 Replace protective coverings, e.g. plastic wrap, aluminium foil
from equipment or environmental surfaces if they become
contaminated or if there is a possibility that they may have
become contaminated.
6.5.7 Decontaminate pails, cans, and other receptacles that may
become contaminated with potentially infectious materials. If
they become visibly contaminated, decontaminate
immediately.
6.5.8 Do not reuse “disposable” containers.

6.6 Regulated Medical Waste

They includes:
6.6.4 Liquid or semi-liquid blood or other potentially infectious
materials.
6.6.5 Contaminated items that, if compressed, would release blood
or other potentially infectious materials in a liquid or semi-
liquid state.
6.6.6 Items caked with dried blood or other potentially infectious
materials that would release these materials during handling.
6.6.7 Contaminated sharps (properly stored in sharps containers).
6.6.8 Pathological and microbiological waste containing blood or

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 other potentially infectious materials.
6.6.9 Cultures, stocks, and vaccines.
6.6.10 Animal waste.

6.7 Waste Containers

6.7.4 Place all regulated waste materials into containers that are:
6.7.4.1 Closable
6.7.4.2 Constructed to contain all contents and prevent
leakage of fluids during handling, storage, transport
or shipping.
6.7.5 Use red or red-orange primary containers with a biohazard
label.
6.7.6 Use rigid secondary containers with biohazard labels.
6.7.7 Seal containers prior to moving or removal from the
laboratory to prevent spillage or protrusion of contents during
handling, storage, transport, or shipping.
6.7.8 Place containers that have become contaminated on the
outside into a second container with the same characteristics
as the initial container.

6.8 Labels
6.8.4 Place warning labels on the following:
6.8.4.1 Containers of regulated medical waste.
6.8.4.2 Refrigerators and freezers containing blood or
other potentially infectious material.
6.8.4.3 Containers used to store, transport, or ship blood
or other potentially infectious materials.
6.8.4.4 Contaminated equipment-include on the label
parts that cannot be cleaned and so remain
contaminated.
6.8.5 Labels must have the biohazard symbol and the text
“Biohazard”.

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 6.8.6 Use fluorescent orange or red-orange labels with lettering and
symbols in a contrasting colour.
6.8.7 Labels must be an integral part of the container or affixed as
close as feasible to the container. Place them on opposite
sides of the containers.
6.8.8 Affix labels so that they cannot be lost or unintentionally
removed.
6.8.9 No label is required on individual containers of blood or other
potentially infectious material if they are placed in labelled
containers during storage, transport, shipment, or disposal.

6.9 Hepatitis B Vaccination

6.9.4 All staff who have risk of exposure to blood borne pathogens
should receive Hepatitis B vaccine.
6.9.5 Any staffs who routinely works in the lab (non-administrative
personnel) is identified as having a risk for exposure to blood
borne pathogens.
6.9.6 Post exposure evaluation and follow-up are offered if there is
an exposure incident on the job.
6.9.7 Medical evaluations and vaccination series are performed by,
or under the supervision of, a licensed physician, physician’s
assistant, or nurse practitioner as per recommendations by
ministry of health guidelines.
6.9.8 Hepatitis B vaccination will be administered as recommended
by the manufacturer of the vaccine being administered.
6.9.9 Currently no routine booster dose is recommended
6.9.10 Vaccinations are not available to the following:
6.9.10.1 Employees who have previously received the
complete Hepatitis B vaccination series.
6.9.10.2 Employees for whom the vaccine is medically
contraindicated.
6.9.10.3 Employees with potential exposure risk who

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 choose not to take the Hepatitis B vaccination.
Those employees must sign a declination statement

6.10 Employee Training

6.10.4 All employees will be trained regarding blood borne pathogens
and all other laboratory safety issues.
6.10.5 Train annually during working hours.
6.10.6 Provide additional training whenever there are changes in
tasks or procedures that affect occupational exposure. Limit
this training to the new exposure situation.
6.10.7 Tailor the training approach to the educational level, literacy,
and language of the employees.
6.10.8 Training is given by the Safety Officer on the Procedures to use
in an emergency involving exposure to blood or other
potentially infectious materials.

6.11 Emergency First Aid Procedures

6.11.4 First aid is the treatment of a sick or injured person before
regular medical or surgical attention can be obtained.
6.11.5 Procedures should never supersede nor take the place of
proper medical attention.
6.11.6 Notify emergency personnel promptly.
6.11.7 Never administer first aid beyond one’s training/qualifications.
6.11.8 Restrict procedures to approved methods of artificial
respiration, oxygen administration, control of bleeding, and
treatment for shock or burns
6.11.9 Keep calm and use common sense.
6.11.10 Under most circumstances, no more than two persons
are needed to attend to an injured person.
6.11.11 Give the injured person room to breathe.
6.11.12 Do not move an injured person unless fumes, fire, or
other hazards exist. Moving may cause additional harm in case

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 of broken bones or head, spinal column, or internal injuries.
6.11.13 Artificial Respiration/Cardiopulmonary Resuscitation
procedures are the methods of choice. Only perform these
procedures if certified to do so!
6.11.14 Minor Injuries
6.11.14.1 Provide a first aid kit for treatment.
6.11.14.2 Notify the Safety Officer, QA Officer or
Laboratory manager if materials are used from the
kit so that the kit can be stocked with used
materials.
6.11.15 Burns
6.11.15.1 Smother flames on a person’s clothing by rolling
the victim in a coat or blanket.
6.11.15.2 Do not attempt to remove clothing that
adheres to the skin.
6.11.15.3 Submerge a localized burn in cold/ ice water.
6.11.15.4 Do not open blisters.
6.11.15.5 For chemical burns flush contaminated skin
areas with copious amounts of water for at least
fifteen (15) minutes.
6.11.16 Bleeding
6.11.16.1 Stop arterial or other copious bleeding before
giving any other treatment.
6.11.16.2 Hold a clean compress directly on the wound
and apply hand pressure.
6.11.16.3 If the wound is on an extremity, apply pressure
to the points as well.
6.11.16.4 Tourniquets are not recommended, except as a
final resort.
6.11.16.5 If bleeding is not profuse, carefully wash any
foreign material from the wound with soap and
water.

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 6.11.16.6 Apply antiseptic to all parts of the wound.
6.11.16.7 Cover lightly with a clean dressing such as
sterile gauze.
6.11.16.8 Puncture wounds must always receive prompt
medical attention since antiseptic may not reach
the bottom of the wound.
6.11.17 Fractures
6.11.17.1 Do not move the victim unless there is an
immediate hazard.
6.11.17.2 Leave splinting of broken bones to a physician.
6.11.17.3 Treat for shock and bleeding.
6.11.18 Shock
6.11.18.1 Shock is present in all injuries to some degree.
6.11.18.2 Shock may kill a victim even when other injuries
have been treated.
6.11.18.3 Shock Symptoms.
6.11.18.3.1 Paleness
6.11.18.3.2 Cold, moist skin with or without
sweating
6.11.18.3.3 Nausea
6.11.18.3.4 Shallow breathing
6.11.18.3.5 Trembling
6.11.18.4 Treatment
6.11.18.4.1 Keep the victim warm.
6.11.18.4.2 Lower the head or raise the feet.
6.11.18.4.3 Keep the airway open.
6.11.18.4.4 Give liquid stimulants such as hot
coffee or tea, if possible. (This is not
possible in the laboratory)
6.11.18.4.5 Do not attempt to give liquids if
the victim is unconscious.
6.11.18.4.6 Never give victims alcoholic

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 liquids.
6.11.19 Electrical Shock
6.11.19.1 Shut off the current, if possible.
6.11.19.2 Using heavy insulated or rubber gloves,
carefully remove the victim from the source.
6.11.19.3 Give artificial respiration if necessary.
6.11.19.4 Keep the victim warm.
6.11.20 Poisons
6.11.20.1 Attempt to determine the kind of poison
involved.
6.11.20.2 Observe the victim for shock.
6.11.20.3 Refer for immediate medical attention.
6.11.21 Industrial Safety
6.11.21.1 Use and storage of flammable/combustible
liquids.
6.11.21.2 Store/place only enough of a flammable
chemical to meet daily requirements at the
workbench.
6.11.21.3 Use approved safety containers for storage of
flammable materials.
6.11.21.4 Store no more than five litres of flammable
liquids on open shelves.
6.11.21.5 Store flammable materials in excess of five
litres in metal safety cabinets and mark the
cabinets accordingly.
6.11.21.6 Do not place flammable liquid storage cabinets
in corridors, stairways, or hallways.
6.11.21.7 Use only explosion-proof refrigerators for
storing flammable materials that require
refrigeration. Ensure that the refrigerator is
labelled.
6.11.21.8 Keep flammable liquids, acids, and corrosives

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 segregated from one another and display
appropriate signs for each.
6.11.21.9 Keep flammable liquids stored inside buildings
at least eighteen (18) inches away from electric
lights, steam pipes, and ceilings.
6.11.21.10 Maintain at least an 18-inch clearance between
stored flammable materials and combustible
interior partition walls of buildings, except where
otherwise approved by the Primary Fire
coordinator.
6.11.21.11 Do not use gasoline, naphthalene, benzene or
other highly flammable volatile liquids for cleaning
purposes.
6.11.22 Chemical Hygiene Plan
6.11.22.1 The Laboratory Director is responsible for the
overall implementation of the chemical hygiene
plan.
6.11.22.2 The Laboratory Director will review the annual
Chemical Hygiene Plan effectiveness.
6.11.22.3 Plan will describe policies and procedures for all
operations that involve hazardous chemicals.
6.11.22.4 The Designee for the Department under the
scope of the lab manager will ensure the use of
personal protective equipment.
6.11.22.5 The Lab manager and designee will assure
exposure monitoring when permissible levels are
exceeded.
6.11.22.6 The Laboratory Safety Officer is responsible for
implementing the chemical hygiene plan.
6.11.22.7 After the initial Safety Orientation, employees
are responsible for using PPE when handling
corrosive or flammable materials.

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 6.11.22.8 Thick gloves or double gloving, in addition to
protective eyewear and lab coats should be used
when handling corrosives.
6.11.22.9 Gloves, eyewear and a lab coat should be used
when handling flammable materials.
6.11.22.10 The role of supervisors is to enforce the use of
PPE, monitor for chemical use safety and provide
recommendations to the Chemical Hygiene Plan
officer.

6.12 Duties of the Safety Officer.

6.12.4 Oversight of lab safety policies and procedures.
6.12.5 Continuing staff education of these policies.
6.12.6 Management of employee injuries and exposures. This would
include following up with occurrences, looking for trends and
making engineering or work practice control changes if
necessary.
6.12.7 Regular safety audits, which include fire and electrical safety,
ergonomics, chemical hygiene, housekeeping, waste
management, PPE, and infection prevention.
6.12.8 Act as the laboratory Chemical Hygiene Officer.
6.12.9 Maintenance of an inventory of all hazardous materials and all
hazardous waste.
6.12.10 Ensure that there are precautionary labels posted on
the containers of all hazardous chemicals indicating type of
hazard (for example corrosive, carcinogen) and immediate
reminders if what to do if accidental contact occurs.
6.12.11 Ensuring that the Department’s hazardous waste is
kept at a minimum.
6.12.12 Ensuring that all personnel are properly trained in
hazard communications, use of proper protective equipment,
and storage and waste disposal, and maintain records of,

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 laboratory safety trainings
6.12.13 Ensuring that appropriate Material Safety Data Sheets
(MSDS) are available in each work area or in a central area and
that MSDS guideline for exposure monitoring and use of
protective equipment are followed.
6.12.14 Investigating all hazardous material spills and
exposures.
6.12.15 Ensuring that chemical fume hood, laminar flow hoods,
and other protective equipment as needed (respirators if
used) are inspected and certified as functioning correctly on
an annual basis.

6.13 Evacuation Plan and Procedures

6.13.4 Responsibilities for the primary evacuation of the laboratory in
case of an emergency lie with the laboratory personnel.
6.13.5 Evacuation route maps are posted next to each fire exit route.
6.13.6 To prevent injury and limit the spread of the emergency
situation the following steps take precedence over firefighting
or damage control:
6.13.7 Alert personnel in the immediate vicinity of the emergency
incident, of the nature and extent of the emergency.
6.13.8 Activate the fire alarm.
On sound of alarm, all activities should be stopped immediately
irrespective of their nature and the building evacuated!
6.13.9 Summon the fire department.
6.13.10 Shut all doors to the laboratory as personnel evacuate
the building.
6.13.11 The last personnel to leave are the lab supervisors,
making sure all have evacuated/shut doors.
6.13.12 Inspect all labs to make sure no one is inside.
6.13.13 Be available to direct the fire department and
emergency vehicles to the scene of the emergency.

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6.14 Safety drills
6.14.4 Conduct evacuation drills at least once a year to evaluate
disciplined and orderly evacuations.
6.14.5 Procedures to follow:
6.14.5.1 Stop working, leave the working stations, and
calmly exit the building as you shut the doors.
6.14.5.2 Congregate approximately 100 feet from the building until the
drill is concluded.

6.15 Electrical Safety

6.15.4 Yearly, facilities maintenance will check each circuit supporting
fixed, electrical receptacles for polarity and ground integrity.
6.15.5 Facilities maintenance will be informed of the arrival of new
instruments and appliances.
6.15.6 They will check for adequate grounding and current leakage
before initial use.
6.15.7 They will also be informed when repair, modification or a
problem is suspected, so this check can also be done.
6.15.8 Facilities will document these checks on their database and/or
tags on the equipment.
6.15.9 Replace damaged or deteriorated cords. Do not attempt to
repair them.
6.15.10 Inspect open equipment such as motors for dust and
grease accumulation.
6.15.11 Do not overload sockets. Insist that maintenance
personnel survey loads on individual circuits regularly and
distribute them so that none has an excessive load.
6.15.12 Call the safety officer and maintenance officer
immediately to examine circuits with blown fuses or opened a
circuit breaker.
6.15.13 Do not store flammable materials in biological safety

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 hoods, incubators, or refrigerators that have not been fitted
with explosion-proof motors or equivalent shielding.
6.15.14 The facilities manager and/or laboratory supervisor will
maintain documentation of inspections and repairs for each
piece of equipment.

6.16 Chemical Toxin Hazard Control (including carcinogens).

6.16.4 Notify all personnel before using any hazardous chemical toxin
in the laboratory.
6.16.5 Label containers of any chemical toxin used in the laboratory.
6.16.6 Identify the chemical toxin by type of hazard (poison, caustic,
skin or eye irritant),
6.16.7 Precautions to be taken when using the substance, and
instructions to follow in case of accidental exposure or
contamination.
6.16.8 Conduct an annual inventory of all chemical toxin materials
used in the laboratory.
6.16.9 Exposure to carcinogens is of particular concern for the
laboratory worker. Classification of carcinogens includes:
6.16.9.1 Class I - chemicals considered to be
carcinogenic
6.16.9.2 Class II - chemicals suspected of being
carcinogenic
6.16.10 Keep the use of carcinogenic material at the lowest
practical volume to reduce potential hazard.
6.16.11 Take the following safeguards when handling chemical
toxins or Class I carcinogens to avoid accidents:
6.16.11.1 Wear personal protective equipment (PPE),
including a fully fastened laboratory coat or smock
and protective gloves.
6.16.11.2 Discard gloves after each use and after overt
contact with any chemical toxin hazard.

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 6.16.11.3 Do not wear PPE outside the laboratory area.
6.16.11.4 Conduct all procedures involving weighing or
working with large amounts of carcinogenic
material in a chemical fume hood.
6.16.11.5 Close the protective glass front as much as
possible.
6.16.11.6 Do not eat, drink, smoke, remove or insert
contact lenses, or apply cosmetics in areas where
chemical toxin or carcinogenic material is used or
stored.
6.16.11.7 Always use mechanical pipetting aids for all
pipetting procedures. Do not pipette any chemical
toxin or carcinogen by mouth under any
circumstances.
6.16.11.8 Wash hands thoroughly immediately after
completion of a procedure in which chemical toxin
or carcinogenic substances have been used.
6.16.11.9 Wash hands thoroughly before leaving the
laboratory.
6.16.11.10 Cover work surfaces on which chemical toxins
are used with steel or plastic trays, dry absorbent
paper, or other impervious material.
6.16.11.11 Thoroughly decontaminate or dispose of
protective work surfaces upon completion of
procedures involving chemical toxins or
carcinogens.
6.16.11.12 Cover analytical instruments used to measure
or weigh carcinogens with a disposable weight
sheet, if possible. If no covering is used thoroughly
decontaminate the instrument after use.
6.16.11.13 Decontaminate or dispose of contaminated
materials in labeled containers for disposal of these

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 types of materials. Always refer to MSDS. If
necessary consult with the Safety Officers.

6.17 Radiation Safety

6.17.4 Refer to the Radiation Protection Act, chapter 243, (Laws of
Kenya).
6.17.5

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7 APPENDICES:
DAILY SAFETY CHECKLIST.
Section/Bench Name Month/Year
DATE OF MONTH
DAILY ACTIVITY 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Avail gloves.

Decontaminate &
organise bench.

Avail sharp box.

All bins with liners
in place.

Refill jik jar.

Ensure proper
ventilation.

No obstructions to
exits/corridors.

Correct harzard
labels to hazardous
reagents/chemicals.
TECH’S INITIALS.

COMMENTS

1) ……………………………………………………………………………………………………………………… KEY: √- Done, X - Not done

2) ……………………………………………………………………………………………………………………… NA- Not Applicable, H- Holiday, W- Weekend

Safety Officer Review _______________________________________________________________

Date_________________________

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 7.0 Explanation of Abbreviations and Terms:
7.1 BSC: Biosafety Cabinet.
7.2 MSDS: Material Safety Data Sheet.
7.3 PEP: Post Exposure Prophylaxis
7.4 PPE: Personal Protective Equipment.
7.5 QA: Quality Assurance.
7.6 QC: Quality Control.
7.7 QI : Quality Improvement.
7.8 SOP: Standard Operating Procedure

8.0 References:
Reference Number or Authors Document Title
8.1
8.2.

9.0 Forms and Appendices:

Form or Appendix Number Title

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 10.0 Version Table:

Original: (Current) Dated: SOP No.: No.

Title Pages:

SOP Copy Control and Updating Log

DOCUMENT COPY CONTROL

DATE PRINTED: May’ 2012 NUMBER OF COPIES: 5
SOP DISTRIBUTION
COPY 1 OF 1
Main Lab

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By initialling and dating below I understand and approve of the changes to the
attached SOP.
SOP CHANGES Changes Approval
Initials/Date
Date/Initials Nature of Change QA Approving
Authority

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 Appendix 7.2
Training Documentation Log for SOP Files
St.Marys Mission Hospital SOP No:
Laboratory Department COPY 1 OF 5 Supersedes: None
Standard Operating Procedure Effective Date: October
2015
Title:
This SOP has been read and understood by:
Printed Name
Date: Printed Name OR Date: OR
DD-MM-YY Initials Signature DD-MM-YY Initials Signature

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITYNAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: POST EXPOSURE PROPHYLAXIS SOP No: 021

Version: Original
Effective Date: January 2016 Page 1 of 9

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 PURPOSE / INTRODUCTION:
1.1 Health Care Workers (HCWs) are at risk of exposure to potentially infectious
materials through contact with contaminated blood and other body fluids
through needle stick injuries, injuries by other sharp objects and non-intact skin
and mucous membranes. This means that all specimen should be treated as if
contaminated with HIV, hepatitis B & C blood borne viruses and other infectious
pathogens. To avoid exposure to these pathogens precautions should be taken
when handling possibly contaminated specimen by including the use of PPEs,
proper handling and disposal of sharps, safe disposal of contaminated waste,
safe handling of soiled linen, adequate disinfection procedures and universal
Hepatitis B vaccination of laboratory personnel.
1.2 This SOP defines the procedures to be followed by a HCW in case of exposure to
a potentially infectious material in the laboratory.

2.0 SCOPE / RESPONSIBILITY:

2.1 This SOP applies to all laboratory personnel and other departments that use the
laboratories services.
2.2 It is the responsibility of the designated of the County Leads, Quality Assurance
and Quality Control (QC/QA) personnel, Laboratory Supervisors to ensure that
the current SOP is available to the laboratory personnel and the procedure is
followed as documented
3.0 SAFETY/RISK ASSESSMENT:
3.1 Wear personal protective equipment. Handle all samples as potential
biohazards.
3.2 All contaminated wastes should be safely disposed off.
3.3 Surfaces contaminated with HIV infected products should be immediately
disinfected.

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4.0 DEFINITIONS:
4.1 ART: Antiretroviral Therapy.
4.2 CSF: Cerebral Spinal Fluid.
4.3 HbsAg: Hepatitis B Surface Antigen.
4.4 HBV: Hepatitis B Virus.
4.5 HCV: Hepatitis C Virus.
4.6 HIV: Human Immunodeficiency Virus.
4.7 MLTS: Medical Laboratory Technicians/ Technologists.
4.8 PEP: Post Exposure Prophylaxis.
4.9 PPE: Personal Protective Equipment.
4.10 QA: Quality Assurance.
4.11 SOP: Standard Operating Procedure.

5.0 SPECIMEN:
5.1 Not applicable.

6.0 EQUIPMENT/ MATERIALS/ REAGENTS:

6.1 Equipment.
6.1.1 Not applicable.
6.2 Materials.
6.2.1 Not applicable.
6.3 Reagents.
6.3.1 Not applicable.

7.0 METHODOLOGY:
7.1 Procedure for Post-exposure management in occupational exposure
7.1.1 Encourage bleeding from the site but do not scrub or cut the site,
washing it with soap and water

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7.1.2 Do not apply caustic agents, including antiseptics or disinfectants to the
exposed area.
7.1.3 Inform the department manager of the exposure.
7.1.4 Record in incident/accident register. The following information must be
provided:
7.1.4.1 Date and time of exposure.
7.1.4.2 Exposure site (s).
7.1.4.3 Where and how the exposure occurred.
7.1.4.4 If a sharp object was involved, type and brand of device.
7.1.4.5 Type and amount of fluid exposed to.
7.1.4.6 Severity of exposure (e.g. depth of sharp puncture, intact skin,
eyes
7.1.4.7 Patient history (if HIV patient, check stage of disease, viral load,
history of ART etc.)
7.1.5 The department manager will refer the exposed staff for PEP to the
medical officer in-charge/designee who will evaluate the exposed staff on
potential infectious hazard based on;
7.1.5.1 Type an amount of body fluid/ tissue.
7.1.5.2 Type of exposure, whether needle stick injury, non-intact skin
exposure or mucous membrane exposure.
7.1.5.3 Status of the source and the infectious material - HIV, HbsAg and
HCV antibody tests.
7.1.5.4 HIV, HBV and HCV immune status of exposed laboratory staff.
7.1.5.5 Assess the potential risk of infection
7.1.5.6 Both the source and exposed person need to be counselled for
HIV-testing.
7.1.6 A known source should be tested for HIV; if the source person is not
willing to be tested, he/she should not be coerced into having the test.
7.1.7 Discarded sharps/needles should not be tested

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 7.1.8 The exposed person should not receive ARV drugs without being tested.
However, where immediate testing is not feasible, treatment should not
be delayed since HIV testing can be carried out the following day or soon
thereafter.
7.1.9 Counselling and support should be provided to the exposed and for those
who decline to be tested, they should be offered further appropriate
support. HIV test should be done at baseline, at 3 months and at 6
months for person exposed.
7.1.10 Other baseline tests to be carried out where feasible include: FBC, LFT
and renal function.
7.2 Risk Assessment

Low risk High risk.

Type of Intact skin. Mucus membrane/ non-intact skin,

exposure. Percutaneous injury.

Source. HIV-negative HIV status unknown; clinically

well/unwell.
Material. Saliva, tears, sweat, faeces, Semen, vaginal secretions, synovial,
urine, sputum, vomit pleural, pericardial,
peritoneal, amniotic fluids
Blood and bloody bodily fluids; CSF;
viral cultures.

7.3 Summary of medical management of medical PEP

Considerations Recommended Action

Eligibility • High risk exposure
• Exposure within 72 hours
• Exposed individual is not HIV infected
• Source individual HIV positive or of
unknown HIV status

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Counselling and testing • Offer information on risks and benefits
the exposed individual • Verbal consent adequate
• Base-line HIV test in HIV exposed person
• Voluntary testing for both exposed and
source individuals

ARV agents for PEP • Adult: Preferred : TDF + 3TC + ATV/r

x 1 month
• Children: Preferred : ABC + 3TC + LPV/r

Time of initiation • As soon as possible after exposure, but no

later than after 72 hours

Duration of therapy • 28 days

Dose of PEP • Same as indicated for ART; use dosing wheel

for children
Follow-up • Follow up client at 7 days, 14 days and 28
days
• Follow-up HIV testing at 3 and 6 months
after exposure
• Pregnancy testing
• Hb (if AZT-containing regimen used for PEP)
• Hepatitis B vaccination if not previously
immunized Management of side effects due
to PEP
• Adherence counselling, risk reduction,
Counselling trauma and mental
• health counselling, social support and safety
Other services for • STI prophylactic treatment to all
sexual assault • Emergency contraceptive for non-pregnant
women Tetanus Toxoid for any physical
injury of skin or mucous membranes
• Documentation of clinical evidence of
assault and collection
• of forensic evidence
• NOTE: Refer to National Guidelines on
management of Sexual Violence in Kenya for
comprehensive management

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 7.4 NOTE: Counselling on behaviour change and risk reduction should be offered to
clients with repeated exposure risk

8.0 APPENDICES:
Not applicable.

9.0 REFERENCES:
9.1 Coast provincial general hospital ART Programme (March 1, 2005), SOP for
laboratory services, MSH/MoH.
9.2 Ministry of Health; National AIDS and STI Control Program (NASCOP). Guidelines
on Use of Antiretroviral Drugs for Treating and Preventing HIV Infection: A rapid
advice, 2014
9.3 National public health laboratory services, strengthening laboratory services in
support of ART management (March, 2007), Laboratory standard operating
procedures, MOH publication, 1st edition.

10.0 DOCUMENT CHANGE HISTORY:

10.1 Version Table:
Version 1: Post - Exposure Prophylaxis. Dated: SOP No.: No. Pages:
LGEN. 0023 9.

Version 2: Dated: SOP No. No. Pages:

Version 3: Dated: SOP No.: No. Pages:

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10.2 SOP Review Log
Date of Changes made. Name of reviewer. Signature.
review.

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1.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………………………………………………………….

COUNTY……………………………………………………………………………………………………………….

SUB COUNTY………………………………………………….…………………………………………………….

SOP Title: GRAM STAINING SOP No: 018

Version: Original
Effective Date: January 2016 Page 1 of 8

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Purpose & Scope
1.1Purpose
This procedure outlines the protocol for performing the Gram stain.
1.2 Scope
This procedure is to be used in the bacteriology for the identification of organisms based
on their Gram reactivity.
1.3 Principle & Safety
1.3.1 Principle
Bacteria can be recognized as Gram positive (blue-black/purple) if they retain the primary
dye complex of crystal violet and iodine in the face of attempted decolourization, or as
Gram negative (pink) if decolourization occurs as shown by the cell accepting the counter
stain Safranin. Generally the mechanism of the Gram stain is: The fixed bacteria are
stained with the triphenylmethane dye, crystal violet. Next the smear is flooded with
Gram’s iodine solution, which oxidatively forms an insoluble complex with the crystal
violet. The smear is then flooded with the organic solvent, acetone-alcohol. Depending on
cell permeability the crystal violet-iodine complex will be washed from Gram negative
bacteria in solvent but not from Gram positive bacteria. Upon counterstaining with
Safranin, organisms which had been decolorised by the ethanol (Gram negative) will stain
pink. Gram positive organisms which retained the crystal violet will appear blue-
black/purple microscopically.

1.3.2 Safety
Handle all laboratory samples as if capable of transmitting infections; always use personal
protective equipment

2.0 Abbreviation, Definitions and Terms

2.1 Abbreviations
2.1.1 CSF: Cerebrospinal fluid
2.1.2 FRM: Form

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 2.1.3 MB: Microbiology
2.1.4 TMHL: Tabaka Mission Hospital Laboratory
2.1.5 QC: Quality control
2.1.6 SOP: Standard Operating Procedure
2.1.7 v/v: volume for volume

2.2 Definitions and Terms

2.2.1 None

3.0 Equipment and Materials/Reagents/Supplies

3.1 Equipment
3.1.1 Microscope
3.1.2 Bunsen burner

3.2 Materials/Reagents/Supplies
3.2.1 Crystal violet stain
3.2.2 Lugol’s Iodine
3.2.3 Acetone alcohol
3.2.4 Safranin or Neutral red stain
3.2.5 Frosted end glass slides
3.2.6 Immersion oil
3.2.7 Staining rack
3.2.8 70% v/v methanol/ethanol

4.0 Responsibility
4.1 The Head of section is responsible for monitoring the effective implementation of this
procedure.
4.2 Technical staff is responsible for the implementation of this procedure.

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5.0 Procedure
5.1 Preparation of smears
5.1.1 Label the frosted end of the slide using a lead pencil with the specimen number and
the type of specimen, e.g. pus, CSF, fluid etc.
Specimens received on swabs
5.1.2 Gently touch the swab to the slide and make a thin smear of the specimen by
carefully rolling the swab along the surface of the slide.
5.1.3 Do not use a back-and-forth scrubbing motion.
Liquid specimens
5.1.4 Spread a loopful of the material over an appropriate area of the slide to make a thin
film
Tissue specimens
5.1.5 Prepare a thin smear of the homogenated/ground tissue material
5.1.6 Avoid large particles of the tissue.
Isolated colonies on media
5.1.7 Make a barely turbid suspension of the material in a small drop of sterile distilled
water or saline
5.1.8 The film should be homogenous and the material should be mixed and spread
gently

5.2 Slide fixing

You can use either heat or alcohol to fix slides for Gram stain, but not both.

Heat fixing
5.2.1 Allow the smears to air dry prior to staining. Do not heat the slide dry
5.2.2 Fix the specimens or organisms by passing the underside of the slide quickly through
a flame 3 times. The slide should be just warm when applied to the back of the hand.
5.2.3 Note: Do not overheat
5.2.4 Allow the slide to cool - this will happen quickly - in just a few seconds

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5.2.5 Note: Do not add stain to hot slide

Alcohol fixing
5.2.6 Allow the smears to air dry prior to staining
5.2.7 Cover the smear with 70% v/v methanol or ethanol, absolute alcohol is also
adequate
5.2.8 Leave the alcohol on the smear for a minimum of 2 minutes or until the alcohol dries
on the smear. Another way is to put slide in 70% v/v alcohol for 1 minute and the take out
to dry.

5.3 Staining
5.3.1 Flood slide with crystal violet solution and let stand for one full minute.
5.3.2 Drain off excess stain by tilting the slide and briefly wash gently with a stream of
water.
5.3.3 Quickly drain off excess water by tilting the slide
5.3.4 Flood the slide with Lugol’s Iodine solution and allow it to remain on the slide for
one full minute
5.3.5 Wash off the iodine gently with water
5.3.6 Decolorize rapidly with acetone-alcohol until no more colour comes from the film -
usually 10 seconds or less.
5.3.7 Wash gently with water
5.3.8 Flood the slide with Safranin or Neutral red and leave for 2 minute.
5.3.9 Wash gently with water.
5.3.10 Wipe the back of the slide clean with tissue and place slide in a rack to air dry

5.4 Microscopy
5.4.1 Examine the smear microscopically, first with the 40X objective lens to check the
staining and to see the distribution of material.

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5.4.2 Put a drop of immersion oil in the centre of the smear and examine using the oil
immersion objective lens (100X) to look for the bacteria and cells.
5.4.3 Open fully the condenser iris when using the oil immersion lens.

5.5 Results Interpretation

5.5.1 Gram positive bacteria - dark purple
5.5.2 Gram negative bacteria - pale to dark red
5.5.3 Yeast cells - dark purple
5.5.4 Nuclei of pus cells – red
5.5.5 Epithelial cells – pale red

5.6 Reporting of Gram Smears

The report should include the following information:
5.6.1 The numbers of bacteria present, whether many, moderate, few or scanty
document.
5.6.2 The morphology of the bacteria, whether cocci, diplococci, rods or coccobacilli
5.6.3 Report intracellular organisms when they are present inside pus cells.
5.6.4 The presence and number of pus cells
5.6.5 Presence of yeast and epithelial cells.

5.7 Quality Control

5.7.1 Controls should be run on a daily basis with known smears containing Gram positive
and Gram negative bacteria
5.7.2 Gram positive control: Staphylococcus aureus
5.7.3 Gram negative control: Escherichia coli
5.7.4 Record the QC results on the appropriate book.

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6.0 References
7.1 Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H.1999. Manual of
Clinical Microbiology, 7th Edition. ASM Press.
7.2 Winn, W., Allen, S., Janda, W., Koneman, E., Procop, G., Schreckenberger, P. and
Woods, G. 2006. Color Atlas and Textbook of Diagnostic Microbiology, 6th Edition.
Lippincott Williams and Wilkins.
7.3 Cheesbrough, M.1984.Medical Laboratory Manual for Tropical Countries, Volume 2
Microbiology. 1st Edition. ELBS, Cambridge.

8.0 Appendix

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Training log
All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………………………………………………………

COUNTY……………………………………………………………………………………………………………

SUB COUNTY………………………………………………….…………………………………………………

SOP Title: Specimen referral procedure SOP No: 028

Version ORIGINAL
Effective Date: January 2016 Page 1 of 8

Signatures and Dates:

Author: ______________________________________________________________
Lab. Technologist Date

QA Review: ___________________________________________________________
QA Officer Date

Approving Authority: ____________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 Responsibilities/ Applicability:
1.1 This SOP applies to all laboratory personnel and other departments that use the
laboratories services.
1.2 It is the responsibility of the designated of the County Leads, Quality Assurance
and Quality Control (QC/QA) personnel, Laboratory Supervisors to ensure that
the current SOP is available to the Health Care Workers and the procedure is
followed as documented.

2.0Purpose/ Principle
2.1 Referral system is a mechanism to enable clients’ health needs to be
comprehensively managed using resources beyond those available where they
access care.” National Referral Strategy and Investment Plan for Health Services:
2010 – 2015
2.2 Effective referral system ensures accessible and affordable services, timely
diagnosis, appropriate clinical management, and support to public health
interventions.
2.3 Batched specimens should be stored at the appropriate temperature and
conditions that maintain specimen integrity
2.4 Shipment day(s) for batched specimen should be documented and
communicated to the clinical team

3.0Specimen handling and preparation:

3.1Procedure
3.1.1 Specimen collection
3.1.2Collect and handle appropriately all specimen according to the tests
requested (refer to specific assay SOP)
3.1.2 Staff should be trained on phlebotomy and other specimen collection
procedures
3.1.3 Proper instructions should be given to patients
3.1.4 Use appropriate containers
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 3.1.5 Specimen for Bacteriological analysis should be collected in transport
media where applicable

3.2 Packaging and Transportation from satellites facilities

3.2.1 Confirm that the specimen are properly labelled with the recommended
identifiers and all request forms are duly filled
3.2.2 All specimen should be properly packaged and packaging must follow the
International Air Traffic Association (IATA) regulation i.e. Triple packaging
3.2.3 All specimen should be packed according to their SOPs with due attention
to temperature requirements
3.2.4 All specimen should be logged in facility specimen referral logs before
dispatch
3.2.5 Specimen shipment log should be duly filled and accompany the specimen
to the nodal facility.
3.2.6 All specimen should be transported in a cool box
3.2.7 Staff transporting specimen should be trained and certified in proper
procedures for packaging and shipping dangerous goods
3.2.8 Specimen referral form should be duly be duly filled

4.0 Specimen reception at the nodal facility (County referral Laboratory)

4.1 All specimen shipped to the referral should be received by a qualified laboratory
personnel.
4.1.1.1 The Laboratory staff at the nodal facility should counter check
that all specimen delivered are accompanied by the respective
request forms and shipment manifest form.
4.1.1.2 All specimen should be documented in the Laboratory Reception
book
4.1.1.3 Specimen should be rejected and communicated to the courier
using the following criteria:
4.1.1.3.1 Missing/soiled/incompletely filled request forms

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 4.1.1.3.2 Evidence of spillage or possible contaminations
4.1.1.3.3 Inadequate volume of specimen collected per test
requested
4.1.1.3.4 Specimen mislabelling
4.1.1.3.5 Incorrect packaging of specimen
4.1.1.3.6 Unsuitable specimen collected
4.1.1.4 Specimen Rejection Form should be dully filled, signed by the
supervisor and filed in respective folder
4.1.1.5 Rejected specimen should be disposed of appropriately
4.1.1.6 Specimen Referral Form should be filed in the Specimen Referral
Form folder and produced for transport refund where applicable
4.1.2 Specimen handling at the nodal laboratory
4.1.2.1 Specimen that qualify for acceptance should be accessioned into
the respective laboratory reception register and shipped to
appropriate working bench for analysis.
4.1.2.2 However, specimen for further referral should be stored
appropriately with attention to specific storage requirements till
the time of shipment
4.1.2.3 On the date of shipment to the Regional or National referral
laboratories, all specimen should be packaged appropriately and
well labelled, documented in the respective Specimen dispatch
register, shipped within the specified conditions to ensure
specimen integrity.
4.1.2.4 A copy of Specimen Referral Form should be completely filled and
accompany the specimen to the referral laboratory.
4.1.2.5 The laboratory courier should ensure that the recipient facility
countersigns, dates, and stamp a copy of the Specimen Referral
Form as an acknowledgement of receipt of specimen
4.1.2.6 In case of any specimen rejections, the courier should
communicate immediately to the concerned facility clinical staff

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 for possible re-collection of specimen.
4.1.2.7 All rejections should be documented with reasons for rejection
and action taken and the individual communicated to.
4.1.2.8 Specimen Referral Form refer should be filed in the Specimen
Referral Form folder and produced for transport refund where
applicable.
4.1.3 Sending Laboratory Reports and Results to Satellite Laboratory
4.1.3.1 Laboratory reports may be send through the following means:
4.1.3.1.1 Providing hard copy of the reports. These should be
collected when specimen are delivered to the nodal
site
4.1.3.1.2 Using electronic communication system. Individual
facilities should access the results online or through
access portals, print and file the results as
appropriate.
4.1.3.1.3 Using smartphones and Short Message Service system.
Used when results for urgent tests are ready for
collection, and to provide preliminary reports to the
designated facility focal point person.
4.1.4 Communication between Satellite and Nodal Sites
4.1.4.1 Nodal sites should provide information to satellite facilities on
the following:
4.1.4.1.1 Rejected specimens and corrective measures to
prevent recurrences
4.1.4.1.2 Analytical service interruptions or delays ( breakdown
of service) and resumption
4.1.4.1.3 Alteration in the analytical schedules for the specific
tests , changes of analytical method, or changes for
reference values
4.1.4.1.4 New tests added to menu at the nodal site. This will

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 include indications for performing the test, specimen
requirements, and the testing schedule if batch
analysis is applied.

9.0 References:
Reference Number or Authors Document Title
9.1. Specimen referral procedure

9.0 Forms and Appendices:

Form or Appendix Number Title
10.1 SOP Copy Control and Updating Log
10. Training Documentation Log for SOP Files

10.0 Version Table:

Original: (Current) Dated: AFB 001 No.
Title: Specimen referral procedure. June 2015 Pages:07
Version 1 Dated: SOP No.: No.
Pages:
Version 2
Dated: SOP No.: No.
Pages:
Version 3:
Dated: SOP No.: No.
Pages:
Version 4: Dated: SOP No.: No. Pages:

Version 5: Dated: SOP No.: No. Pages:

Version 6: Dated: SOP No.: No. Pages:

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Appendix 10.1
SOP Copy Control and Updating Log

DOCUMENT COPY CONTROL

DATE PRINTED: Feb 2015 NUMBER OF COPIES:
SOP DISTRIBUTION
COPY 1 OF 1
LAB Section

By initialling and dating below I understand and approve of the changes to the
attached SOP.
SOP CHANGES Changes Approval
Initials/Date
Date/Initials Nature of Change QA Approving
Authority

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 Appendix 10.2
Training Documentation Log for SOP Files
SOP AFB 001VERSION : ORIGINAL
Laboratory Department COPY 1 OF 3
Effective Date: June 2015
Standard Operating Procedure
Title: Specimen referral procedure.

This SOP has been read and understood by:

Date: Printed Name OR Date: Printed Name OR
DD-MM-YY Initials Signature DD-MM-YY Initials Signature

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LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME……………………………………………………………………………………………………………

COUNTY………………………………………………………………………………………………………………………

SUB COUNTY………………………………………………….…………………………………………………………..

SOP Title: GIEMSA STAINING SOP No: 017

Version: Original
Effective Date : January 2016 Page 1 of 5

Signatures and Dates:

Author: __________________________________________________________________
Laboratory Technologist Date

QA Review: _______________________________________________________________
QA Officer Date

Approving Authority: _______________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1 Purpose

To accurately define a method of staining of thick blood films using giemsa stain

1.1 Scope

This procedure applies to staining of thick blood films using giemsa stain at Tabaka mission
hospital laboratory

1.2 Applicable Personnel

All cadres of qualified medical laboratory technical staff and specially trained staff at all
health facility levels.

1.3 Definitions, Acronyms

TMHL- Tabaka mission Hospital Laboratory

EDTA Ethylene diamine tetra-acetate

2 principal

Giemsa is a Romanowsky stain used for staining blood films. Romanowsky stains contain
Eosin Y, an anionic acidic dye, and Azure B, an ionic basic thiazine dye obtained by oxidation
of methylene blue. When the dyes are diluted in buffer, the anionic dye stains the basic
components (cytoplasm) of cells red, and the ionic dye stains the acid components (nucleus)
of cells blue. Giemsa stain may be used in 3%, 5% or 10% dilutions with different staining
times.

2.1 Safety precautions

Gloves must be worn at all times during the procedure

3.Procedures

Equipment, reagents and materials

1. Giemsa powder – commercially available or ready made Giemsa stock solution
2. Methanol
3. Glycerol

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 4. Filter paper (Whatman No 1)
5. Measuring cylinder
6. Glass rod
7. Conical flask 200-250 ml
8. Brown reagent bottle
9. Buffered water pH 7.2
10. Staining jar or staining rack
11. Slide drying rack
12. Cotton wool or gauze
13. Weighing scales
14. Timer clock/stop watch

3.1 Preparation of reagent

1. Weigh 3.8 g Giemsa powder and transfer into a dry brown bottle.
2. Measure 250 ml methanol and add to the stain. Mix well using a glass rod.
3. Measure 250 ml glycerol and add to the stain. Mix well using a glass rod.
4. Place the bottle of stain in a water bath at 50-60 C or if not available, at 37 C for up to
° °

2 hours, to help the stain to dissolve. Mix well at intervals.

5. Label with the name of the reagent and date of preparation, and mark
“inflammable”. Store at room temperature in the dark.

3.2 Sample required

Use capillary blood or venous blood anti-coagulated with EDTA. Prepare stained slides
within 1 hour of blood collection. Stored blood is not suitable for parasite examination.

4 Procedure
1. For a 10% solution, dilute 10ml of the stock Giemsa stain with 90 ml (1:10) buffered
water at pH 7.2.
2. Prepare a thick blood film and leave to air dry.
3. Place the slide on a staining rack and flood with diluted Giemsa stain. If staining several
slides, place the slides in a staining jar and cover with diluted Giemsa stain. Stain for 10
minutes.

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5. Wash in buffered water at pH 7.2.
6. Clean the back of each slide with cotton wool or gauze and place the slide upright on the
drying rack. Allow films to dry at room temperature away from direct sunlight and hot
objects.

4.1 Procedural notes

Use only glycerol supplied for laboratory use. Glycerol supplied for general use may be
contaminated with water which will cause the stain to precipitate.

4.2 Quality control

Prepare two thick blood films:
1. Stain with old stock.
2. Stain with the newly prepared stock.
3. Compare the staining reaction and adjust the staining time as necessary.

5. References

• F. Baker and S. Silverton (1980). Introduction to medical laboratory technology. 5th

Edition.

• AMREF (2008). Standard operating procedures, Essential Laboratory Tests

Associated records and forms

Not applicable

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Training log

All members of staff who use this document must date and sign in the table below to
indicate that they have read, understood and are going to implement the document in their
work.

Print Name Date Signature

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Supported by Centre for Health Solutions – Kenya (CHS)
 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: SYPHILLIS TEST SOP No: 025

Version: Original
Effective Date: January 2016 Page 1 of 7

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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 1. PURPOSE/APPLICABILITY

1.1 PURPOSE: To provide guidelines for the proper procedure of testing for syphilis
antibodies so as to ensure accurate and reliable results.

Applicability: TMH Lab Technologist/Technicians and students

Principle: During the testing procedure, the specimen, serum or plasma is mixed with the
carbon antigen reagent and allowed to react for eight minutes. If anti lipoidal antibodies are
present in the specimen, they will react with the reagent forming visible black floccules. If anti
lipoidal antibodies are not present in the specimen there will be no flocculation.

2. Abbreviations and Terms

2.1 TMH: Tabaka Mission Hospital

2.2 LAB: Laboratory
2.3 TECH: Technologist/Technician
2.4 VDRL: Venereal Disease Research Laboratory

3. Equipment and Materials

3.1 Equipment
3.1.1 Centrifuge
3.1.2 Shaker/mechanical rotor
3.1.3 Slide/Tile
3.1.4 Applicator sticks/stirrer

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 3.1.5 Rubber teats
3.1.6 Needle dropper
3.1.7 Pipettes
3.1.8 Test tubes

3.2 Reagents

3.2.1. Carbon antigen suspension

3.2.2. Positive control
3.2.3. Negative control
3.2.4. Isotonic saline

Reagent storage and stability - Store the reagents at 2-8◦c. Do not freeze. The
shelf life of the reagent is as per the expiry date mentioned on the reagent vial
label. Avoid exposure to elevated temperatures and air as the reagent is highly
sensitive to denaturation and drying.

4. Responsibilities

4.1 The Lab manager is responsible for ensuring the implementation of this SOP with
the assistance of quality officer to ensure strict adherence to this procedure.
4.2 All personnel involved in the execution of this SOP must ensure that they are
properly trained.
4.3 Quality Assurance: SOPs are to be reviewed/approved by the in charge, office of
quality or designee, prior to the responsible approving authority.

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5. Procedures

5.1 Bring all reagents and samples to be tested to room temperature before testing.
Thoroughly mix the carbon antigen reagent suspension by gentle agitation before
testing.
5.2 Qualitative method
1) Place one drop of the test sample positive and negative controls onto
separate reaction circles of the slide using sample dispensing pipette.
2) Add one drop of well-mixed carbon antigen reagent next to the test
sample, positive control and negative control by using a needle dropper
which dispenses 6-18ul of the reagent. Do not let the dropper tip touch
the liquid on the slide.
3) Using a mixing stick, mix the test sample and carbon antigen reagent
thoroughly spreading uniformly over the entire reaction circle.
4) Immediately rotate the slide gently and continuously either manually of on
a mechanical rotor at 1000 r.p.m.
5) Observe fir flocculation macroscopically at 8 minutes.

5.3. Quantitative Method

1) Using isotonic saline prepare serial dilutions of the test sample 1:2, 1:4,
1:8, 1:16, 1:32, 1:62. 1:128 and so on.
2) Perform the qualitative test procedure using each dilution as test
specimen.
3) The titre is reported as the reciprocal of the highest dilution, which shows
a positive test result.

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 5.4 INTERPRETATION OF TEST RESULTS

5.4.1 Qualitative method

a) Large and medium black floccules against white background- Reactive

b) Small black floccules against white background – weekly reactive
c) No floccules - non reactive

• Flocculation is a positive test result and indicates the presence of

anti lipoidal antibodies in the test samples.
• No flocculation is a negative test result and indicates absence of
anti lipoidal antibodies in the test sample.

5.4.2 Quantitative Method- The titre of anti lipoidal antibodies is the highest
dilution of test sample giving a positive test result.

NOTES

1) Samples not tested immediately may be stored at 2-8◦c for up to 48 hours.

2) The reagent and sample dispensing device should dispense precisely 16-18µl and 50µl
respectively.
3) It is strongly recommended that results of the test should be correlated with clinical
findings to arrive at the final diagnosis.
4) Dispose all used and contaminated material as per standard Biohazard safety guidelines.
5) Very slight roughness should be interpreted as a negative test results.
6) Carbon antigen reagent should be gently but thoroughly mixed before testing to dispose
the carbon particles uniformly and improve test readability.

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 7) The reagent contains sodium oxide 0.1% as preservative. Avoid contact with skin and
mucus. On disposal flush with large quantities of water.

6. REFERENCES

AUTHOR TITLE
M.C. Grew B.E. American Journal of Clinical Pathology
Pang Boarn, Mary C. Isolation and purification of serological
active phospholid from breef heart
Monica Cheesbrough Medical Laboratory manual for tropical
countries
Eurocarb Insert

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7. Forms and Appendices

Training log

All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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 LABORATORY STANDARD OPERATING
PROCEDURES
FACILITY NAME…………………………………………………

COUNTY……………………………………………………………

SUB COUNTY……………………………………………………..

SOP Title: INDIRECT COOMBS TEST SOP No: 019

Version: Original
Effective Date: January 2016 Page 1 of 8

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.0 PURPOSE / INTRODUCTION:
1.1 ICT is a technique used to detect red cell antibodies in patient serum that will
react with RBCs carrying the corresponding antigen. It detects RBCs, which have
been sensitized in-vitro. In clinical practice this is referred to as the "antibody
screen" and is part of the type and screen procedure. Approximately 5% of
patients have a positive ICT due to IgG antibodies, IgM antibodies, or both. Most
clinically significant alloantibodies are IgG antibodies that react best at 37°C and
are formed as a result of previous exposure via transfusion or pregnancy, e.g.
antibodies to Rh, Kell, Kidd, and Duffy red cell antigens. IgM antibodies are
usually not clinically significant (except for ABO antibodies) but are a source of
in-vitro serologic difficulty that may delay transfusion. Examples include
antibodies to the Lewis, I, P, M, and N red cell antigens. IgM antibodies react
best at cold temperatures (4°C) and are usually naturally occurring in that they do
not require a sensitizing event.

1.2 ICT applications includes; to detect antigens on RBCs using antiserum containing
antibodies directed against specific antigens to detect weaker variants of Du, to
detect an unknown antibody in serum using a reagent RBCs that are known to
contain most common RBC antigen, to identify antibodies using a panel of
selected group O cells known to posses this common antigen in different
combinations, used to titrate antibodies in which the relative strength of a known
antibody is determined by testing various dilutions of serum with cells known to
contain the corresponding antigen and it's also used to determine the serologic
compatibility of donors' blood with recipient serum.

2.0 SCOPE / RESPONSIBILITY:

2.1 This SOP applies to all laboratory technologists/technicians involved in the assay.

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 2.2 It is the responsibility of the designated Quality Assurance and Quality Control
(QC/QA) personnel, Laboratory Supervisors, SCMLTs, and CMLT to ensure that
the current SOP is available to the staff and the procedure is followed as
documented.
3.0 SAFETY/RISK ASSESSMENT:
3.1 Wear personal protective equipment. Handle all samples as potential biohazards.

4.0 DEFINITIONS:
4.1 AHG: Anti- human Globulin
4.2 ICT: Indirect Coombs Test
4.3 IgG: Immunoglobulin G
4.4 IgM: Immunoglobulin M
4.5 mm: Millimeters
4.6 PPE: Personal Protective Equipment
4.7 QA: Quality Assurance
4.8 RBC: Red Blood Cell
4.9 SOP: Standard Operating Procedure

5.0 SPECIMEN:
5.1 Freshly prepared 3-5% red cell suspension from pooled Blood Group O donor
blood.
5.2 Patient Serum/ test serum.

6.0 EQUIPMENT/ MATERIALS/ REAGENTS:

6.1 Equipment.
6.1.1 Electric centrifuge
6.1.2 Microscope.
6.2 Materials.
6.2.1 PPEs e.g. Gloves and a Lab coat.
6.2.2 Small glass test tubes (75x12mm).
6.2.3 Pasteur pipettes.

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 6.2.4 Microscope slide.
6.3 Reagent.
6.3.1 Normal saline.
6.3.2 Coombs serum/ AHG.
6.3.3 IgG anti-D.

7.0 METHODOLOGY:
7.1 Principle
7.1.1 The purpose of the indirect antiglobulin test is to detect in vitro
sensitization of red cells. This is done when sensitization does not lead to
direct agglutination. This occurs when there are too few antigens on the
red cell, too few antibodies in the serum and those antibodies are in the
IgG class.
7.2 Procedure
7.2.1 Prepare a 3-5% O pooled cell suspension as follows:
7.2.1.1 Transfer about 0.5 ml O cells each from different (3 to 4) donor
cells into about 5 ml normal saline.
7.2.1.2 Wash the cells 3 times by adding 5-7 ml saline, centrifuging at
3000 rpm for 2-3 minutes and discarding the supernatant.
7.2.1.3 Make a 3-5% red cell suspension by mixing 1 drop of sedimented
cells in 20-25 drops of saline, holding the Pasteur pipette
vertically.

7.2.2 Label a test tube ICT.

7.2.3 Using the Pasteur pipette, put 2 drops of the 3-5% red cell suspension into
the test tube.
7.2.4 Add 2 drops of patient serum to be tested.
7.2.5 Mix well and centrifuge at 1000rpm for 1 minute. This immediate
centrifugation may detect an antibody which otherwise may show prozone
characteristic.
7.2.6 Dislodge gently and observe for macroscopic agglutination. If
agglutination is absent, weak or questionable, re-suspend the contents and

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 incubate for 30-60 minutes at 370C. A 60-minute incubation is
recommended rather than a shorter period when testing for serum
antibodies of unknown strength.
7.2.7 Centrifuge at 1000rpm for 1 minute.
7.2.8 Dislodge gently and observe for macroscopic agglutination. If reaction is
still negative, weak or questionable proceed as follows,
7.2.9 Wash the cells four times in normal saline pouring off all the supernatant
saline and re-suspend the cells in the last drop of saline remaining.
7.2.10 Add 1 drop of AHG.
7.2.11 Mix well and centrifuge at 1000rpm for 1 minute.
7.2.12 Dislodge gently and observe for macroscopic and microscopic
agglutination.
7.3 Quality Control
7.3.1 Use cells sensitized with IgG to ensure that the AHG reagent is working.
7.3.2 Prepare sensitized cells as follows:
7.3.2.1 Place 4 drops of O positive blood in a 12 x 75 mm test tube.
7.3.2.2 Add 2 drops of IgG anti-D
7.3.2.3 Incubate at 37oc for 60 minutes in water bath.
7.3.2.4 Wash the cells 3 times by adding 5-7mls saline, centrifuging at
3000 rpm for 2-3 minutes and discarding the supernatant.
7.3.2.5 Make 3-5% cell suspension.
7.3.2.6 Mix 1 drop of 3-5% cell sensitized cell suspension and 1 drop
AHG in a test tube.
7.3.2.7 Centrifuge at 1000 rpm for 1 minute.
7.3.2.8 Examine for agglutination macroscopically.
7.3.2.9 Agglutination indicates that cells have been sensitized and
demonstrates that AHG reagent is working properly.
7.4 Results.
7.4.1 Agglutination in step 7.2.6 or 7.8.2 indicates presence of a saline
agglutinin in patient's serum - ICT positive result.
7.4.2 Agglutination or hemolysis in step 7.2.6, 7.8.2 and 7.2.12 indicates iso-

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 immunization - ICT positive result.
7.4.3 Agglutination in step 7.2.12 indicates that an incomplete antibody is
present - ICT positive result.
7.4.4 A smooth suspension of red cells in all steps indicates - ICT negative
result.
7.4.5 Report results ICT positive or negative. Indicate the date and name of
technical staff reporting.
7.5 Limitations.
7.5.1 If the ICT (antibody screen) is positive, additional testing is required to
determine the specificity of the antibody.

8.0 APPENDICES:
8.1 Not Applicable.

9.0 REFERENCES:
9.1 Immunohaematology/ Blood Transfusion Science, Jomo Kenyatta University of
Agriculture and Technology Notes, Prof. Mwanda Otieno.
9.2 Internet sources.
9.3 Monica Cheesbrough (2006), District laboratory practice in tropical countries,
Cambridge University press, Part 2.

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10.0 DOCUMENT CHANGE HISTORY:
10.1 Version Table:
Version 1: Indirect Coombs Test. Dated: SOP No.: No. Pages:
LBTS 0004 08.

Version 2: Dated: SOP No.: No. Pages:

Version 3: Dated: SOP No.: No. Pages:

10.2 SOP Review Log.

Date of Changes made. Name of reviewer. Signature.
review.

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11.0 SOP AWARENESS LOG.

I, the under named, have read and understand the contents of this SOP. I agree to
contact my supervisor/ designee if I have any query.

NO. DATE NAME SIGNATURE

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 LABORATORY STANDARD OPERATING
PROCEDURES
FACILITY NAME…………………………………………………

COUNTY…………………………………………………………

SUB COUNTY………………………………………………….

SOP Title: MONITORING AND MAINTENANCE SOP No: 008

OF LABORATORY REFRIGERATORS
Version: Original
Effective Date: January 2016 Page 1 of 11

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

1. Purpose Applicability:
1.1.1The Laboratory keeps quality control (QC) records for each freezers/refrigerators
provides documentation of all instrument parameters and the initials of the individual
completing the monitoring task.
1.1.2 These refrigerators are used for storage of samples, test kits/reagents/media etc.
1.1.3 To ensure the quality assurance of all data for clinical work, the refrigerators are
monitored for performance, and preventive maintenance
Conducted so that freezers/refrigerators are maintained within appropriate parameters
2. Responsibilities
2.1. It is the responsibility of all staff to follow the SOPs that impact clinical activities
performed.
2.2. Technical staff are responsible for the preparation, review and updating all SOPs relative
to their daily operations.
2.3. The County Medical Laboratory Coordinator/designee and QA/QC personnel are
responsible for ensuring that all SOPs are updated annually to meet the standards outlined
within this SOP.
2.4. The County Medical laboratory Coordinator/designee is responsible for reviewing,
signing, dating and approving all procedural standards and policies as well as any changes
that are made.

3. Equipment & Reagents

3.1. Equipment, Reagents and Materials
3.2. Thermometer
3.3. Gloves
3.4. 70% ethanol/alcohol.
3.5. Absorbent material (e.g. sponge, Gauze).
3.6. Refrigerators (2-8°C)
.

4. PROCEDURES:
4.4. Safety
4.4.39. Standard Laboratory Safety Training.
4.4.40. Wear proper (Personnel Protective Equipment’s) PPE; lab coat and
gloves/Cryogen where applicable.
4.4.41. Do not stick your hand into the fan behind the compressor while cleaning the
unit.

4.5. Daily Temperature Check/ Monitoring

4.5.39. Check temperature within the refrigerator(s) using standardized thermometers
located inside and on the LED display (where applicable) every morning and
afternoon including holidays and weekends.
4.5.40. Check that all the doors are properly closed and the door seals are in good
condition.
 4.6. Check the cleanliness of the refrigerator(s).
4.7. At the end of each month, daily temperature monitoring records are reviewed.
4.8. Measurements and Records
4.8.39. Record the findings on the temperature chart and initial (appendices 7.1)
4.8.40. If temperature is out of range, troubleshoot and notify the Maintenance officer
and note the problem in the Corrective Action Log.
4.8.41. Transfer the contents into another refrigerator(s) and (backup) until the problem is
fixed.
4.8.42. A new temperature recording chart is generated by the lab in charge.
4.8.43. The records hard copies are filed in the equipment maintenance charts folders in
the Lab.
4.9. Maintenance
4.9.39. Quarterly/ scheduled Maintenance
4.9.39.4. Refrigerator - Cleaning shelves and cabinet interior.
Note: Cleanness can be done as need be.
4.9.40. Semi-Annual / Annual Maintenance
This can be done by the maintenance team.

5.0 Procedural Note:

5.1.1. Refrigerators are used for storing samples/reagents some are connected to will be
provided with access control logs that are filled out by the personnel indicating the date,
time of accession, temperature just before entry, purpose of entry, box number and slot
number when applicable, time returned (if applicable), the temperature after the purpose
of entry, comments and personnel’s initials.

5.1 Quality Assurance

5.1.2 Ensuring the maintenances and thermometer verifications are done as scheduled
and documentations filed in the equipment’s maintenance folder.

5.1.3 Ensure that current thermometers are used.

 .

5. References:
Reference Number or Authors Document Title
6.1 Clinical Laboratory Technical Manuals, GP2-A2, 3rd
edition Approved guidelines 1996.
6.2. Reference book Equipment manuals.
6.3

7. Forms and Appendices:

Form or Appendix Number Title
Appendix 7.1 Refrigerator daily maintenance Log (2 to 8ºC)
Appendix7. 2 SOP Copy Control and Updating Log
Appendix 7.3 Training documentation for Sop files
6 EXPLANATION OF ABBREVIATIONS AND TERMS:

6.1 CA-Corrective Action

6.2 CLSI - Clinical Laboratory Standards institute.

6.3 EQP-Equipment

6.4 GEN – General

6.5 NKR- Nkr County Medical Laboratory Department

6.6 Lab- Laboratory

6.7 NIST – National Institute of standards and technology

6.8 N/A - Not Applicable

6.9 PPE- Proper Protective Equipment’s

6.10 QA– Quality assurance.

6.11 QC – Quality Control.

6.12 SOP – Standard Operating Procedure

6.13 TEMP – Temperature.

6.14 ºC-Degrees Centigrade

6.15 %-Percentage
Appendix 7.1

Refrigerator Daily Temperature chart Range: 2 ºC to 8 ºC

Month/Year: _______/_______ EQP Name: ____________________ID: _______________ Location: ________

Daily Maintenance Morning temperature

Temp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
10
9
8
7
6
Thermometer
5
4
3
2
1
Display thermometer
Check cleanliness
Check door seals
Tech Initials
Afternoon temperature
10
9
8
7
Thermometer
6
5
4
3
2
1
Display thermometer
Record CA
Record Scheduled
Maintenance/ repair
Tech Initials
 8. Version Table:

Original: (Current) Dated: SOP No.: No.

Title: Title: MONITORING AND May 2015 Pages:
MAINTENANCE OF LABORATORY
REFRIGERATORS SOP
 SOP Copy Control and Updating Log

DOCUMENT COPY CONTROL

DATE PRINTED: May’ 2012 NUMBER OF COPIES: 5
SOP DISTRIBUTION
COPY 1 OF 1
Main Lab
By Initialing and dating below I understand and approve of the changes to the
attached SOP.
SOP CHANGES Changes Approval
Initials/Date

Date/Initials Nature of Change QA Approving

Authority
 Appendix 7.2
Training Documentation Log for SOP Files
Nakuru County SOP No: COUNTY EQP
Laboratory Department COPY 1 OF 5 001
Standard Operating Procedure Supersedes: None
Effective Date: May 2015
Title: MONITORING AND MAINTENANCE OF LABORATORY REFRIGERATORS SOP
This SOP has been read and understood by:
Printed Name Printed Name
Date: OR Date: OR
DD-MM-YY Initials Signature DD-MM-YY Initials Signature
 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME……………………………………………………………………………………………………….

COUNTY………………………………………………………………………………………………………………….

SUB COUNTY………………………………………………….………………………………………………………..

SOP Title: MALARIA THIN FILM READING SOP No: 027

Version: Original
Effective Date: January 2016 Page 1 of 6

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

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1.1. PURPOSE

1. PURPOSE/scope: To provide guidelines for the proper detection, identification and

quantification of malaria parasites in Giemsa stained MBFs (malaria blood films) at the
Tabaka Mission Hospital laboratories.

1.2 scope:
Tabaka Mission Hospital Lab technologists/technicians .

1.3 PRINCIPLE:
In thick malaria blood film (MBF), the red blood cells (RBCS) are lysed and dehaemoglobinized
while the malaria parasites are left intact and concentrated. This eases detection and
identification of parasites. The thin film MBF, when fixed with absolute methanol, enables the
RBCs to retain their original morphology with malaria parasites, if present, visible inside the
cells

2. Abbreviations and Terms

2.1 TMHL - Tabaka mission Hospital laboratory

2.2. LAB - Laboratory
2.3 TECH - Technologist
2.4 Q.A. - Quality Assurance
2.5 Q.C - Quality Control
2.6 IAW - In accordance with
2.7 SOP - Standard operation procedure
2.8 MBFs - Malaria blood films

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3. Equipment and Materials
• Binocular microscope

3.2 Reagents
• Immersion oil
• Lens cleaning solution
3.3 materials
• MBFs register
• Pen
• Lens paper
• Slide boxes

4. Responsibility
4.1 The laboratory in charge is responsible for ensuring the implementation of this SOP with
the assistance of Quality Officer to ensure strict adherence to this procedure.
4.2 All personnel involved in the execution of this SOP must ensure that they are properly
trained.
4.3 Quality Assurance: SOPs are to be reviewed/approved by the Lab Manager, office of
Quality or designee prior to the responsible approving authority.

5.0 PROCEDURE
a. After staining, the slides are arranged with corresponding request forms
b. Sequential reading order will follow a consistent pattern: i.e. slides with an earlier
number will be read first
c. Place the MBF on the microscope stage, switch on the light and adjust the light source
optimally by looking through ocular and the x40 objective

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 d. Examine the thick film grossing under x40 objective to check the quality of the slide as
follows
§ Is the thick film>90% intact? If no, do not read
§ Except for around the edges, the red cells should be completely lysed. If not, do not
read.
§ Are white blood cells (WBCs) in thick film properly stained (purple granules) visible
within the cytoplasm of the neutrophils? If not do not read.
§ Does the thick film have significant debris? If yes do not read.

e. Place a drop of immersion on the dry stained slide. To avoid cross contamination,
ensure the immersion applicator to not touch the slide
f. Slowly change to the immersion objective, and a thin film of oil will form between the
slide and the lens. Adjust the light source optimally by locking the x10 ocular ( eye
piece) and the x100 objective and use the film adjustment knob to focus the field, the
lens should not be allowed to touch the slide
g. Examine the slide in a systematic fashion: start at the left end of the film and begin
reading at the periphery of the field and finish at the centre. When the field is read,
move the slide right to examine adjacent fields. The slide can move vertically so that
the second sequence can be read from the right to the left. There are about 100
immersion fields in the long axis of 1.5 cm long smear

h. Quantification of parasites in thick film

v The following method will be used for quantifying a sexual malaria parasites forms (in either
single or mixed species infections) as well as sexual (gametocytes) forms. (if different
species are observed, this fact will also be recorded).
§ 1- 10 parasites per 100 HPF……………….. +

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 § 11- 100 Parasites per 100 HPF……………..++
§ 1- 10 Parasites per HPF………………..+++
§ Over 10 parasites per HPF…………………. ++++

v After examination of the blood smears results should be recorded in the lab register
v All results will be verified by the second reader before dispatch.

6.0 REFERENCES
AUTHOR TITLE

AMREF Standard operating procedure(laboratory

utilization for clinicians)
Cheesbrough, Monica(2005) District laboratory practice in tropical
countries, part 1 tropical health
technology
Carter j, Lema O (1994) Practical laboratory manual for health
centres in eastern Africa
John warren L(2000) Selection of basic laboratories with limited
resources. WHO Regional publications
WHO (2008) Maintenance manual for laboratory
equipment. Department of Essential
Health Technologies, WHO
Division of malaria control National guidelines for laboratory
diagnosis of malaria in kenya

7.0 Forms and Appendices

Appendix 1 training Log

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TRAINING LOG:
By signing this log is an acknowledgement that I have read and understood the attached
document

DATE NAME SIGNATURE

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 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME……………………………………………………………………………………………………….

COUNTY………………………………………………………………………………………………………………….

SUB COUNTY………………………………………………….………………………………………………………..

SOP Title: MALARIA THIN FILM READING SOP No: 027

Version: Original
Effective Date: January 2016 Page 1 of 6

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

Supported by Centre for Health Solutions – Kenya (CHS)

1.1. PURPOSE

1. PURPOSE/scope: To provide guidelines for the proper detection, identification and

quantification of malaria parasites in Giemsa stained MBFs (malaria blood films) at the
Tabaka Mission Hospital laboratories.

1.2 scope:
Tabaka Mission Hospital Lab technologists/technicians .

1.3 PRINCIPLE:
In thick malaria blood film (MBF), the red blood cells (RBCS) are lysed and dehaemoglobinized
while the malaria parasites are left intact and concentrated. This eases detection and
identification of parasites. The thin film MBF, when fixed with absolute methanol, enables the
RBCs to retain their original morphology with malaria parasites, if present, visible inside the
cells

2. Abbreviations and Terms

2.1 TMHL - Tabaka mission Hospital laboratory

2.2. LAB - Laboratory
2.3 TECH - Technologist
2.4 Q.A. - Quality Assurance
2.5 Q.C - Quality Control
2.6 IAW - In accordance with
2.7 SOP - Standard operation procedure
2.8 MBFs - Malaria blood films

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3. Equipment and Materials
• Binocular microscope

3.2 Reagents
• Immersion oil
• Lens cleaning solution
3.3 materials
• MBFs register
• Pen
• Lens paper
• Slide boxes

4. Responsibility
4.1 The laboratory in charge is responsible for ensuring the implementation of this SOP with
the assistance of Quality Officer to ensure strict adherence to this procedure.
4.2 All personnel involved in the execution of this SOP must ensure that they are properly
trained.
4.3 Quality Assurance: SOPs are to be reviewed/approved by the Lab Manager, office of
Quality or designee prior to the responsible approving authority.

5.0 PROCEDURE
a. After staining, the slides are arranged with corresponding request forms
b. Sequential reading order will follow a consistent pattern: i.e. slides with an earlier
number will be read first
c. Place the MBF on the microscope stage, switch on the light and adjust the light source
optimally by looking through ocular and the x40 objective

Supported by Centre for Health Solutions – Kenya (CHS)

 d. Examine the thick film grossing under x40 objective to check the quality of the slide as
follows
§ Is the thick film>90% intact? If no, do not read
§ Except for around the edges, the red cells should be completely lysed. If not, do not
read.
§ Are white blood cells (WBCs) in thick film properly stained (purple granules) visible
within the cytoplasm of the neutrophils? If not do not read.
§ Does the thick film have significant debris? If yes do not read.

e. Place a drop of immersion on the dry stained slide. To avoid cross contamination,
ensure the immersion applicator to not touch the slide
f. Slowly change to the immersion objective, and a thin film of oil will form between the
slide and the lens. Adjust the light source optimally by locking the x10 ocular ( eye
piece) and the x100 objective and use the film adjustment knob to focus the field, the
lens should not be allowed to touch the slide
g. Examine the slide in a systematic fashion: start at the left end of the film and begin
reading at the periphery of the field and finish at the centre. When the field is read,
move the slide right to examine adjacent fields. The slide can move vertically so that
the second sequence can be read from the right to the left. There are about 100
immersion fields in the long axis of 1.5 cm long smear

h. Quantification of parasites in thick film

v The following method will be used for quantifying a sexual malaria parasites forms (in either
single or mixed species infections) as well as sexual (gametocytes) forms. (if different
species are observed, this fact will also be recorded).
§ 1- 10 parasites per 100 HPF……………….. +

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 § 11- 100 Parasites per 100 HPF……………..++
§ 1- 10 Parasites per HPF………………..+++
§ Over 10 parasites per HPF…………………. ++++

v After examination of the blood smears results should be recorded in the lab register
v All results will be verified by the second reader before dispatch.

6.0 REFERENCES
AUTHOR TITLE

AMREF Standard operating procedure(laboratory

utilization for clinicians)
Cheesbrough, Monica(2005) District laboratory practice in tropical
countries, part 1 tropical health
technology
Carter j, Lema O (1994) Practical laboratory manual for health
centres in eastern Africa
John warren L(2000) Selection of basic laboratories with limited
resources. WHO Regional publications
WHO (2008) Maintenance manual for laboratory
equipment. Department of Essential
Health Technologies, WHO
Division of malaria control National guidelines for laboratory
diagnosis of malaria in kenya

7.0 Forms and Appendices

Appendix 1 training Log

Supported by Centre for Health Solutions – Kenya (CHS)

TRAINING LOG:
By signing this log is an acknowledgement that I have read and understood the attached
document

DATE NAME SIGNATURE

Supported by Centre for Health Solutions – Kenya (CHS)

 LABORATORY STANDARD OPERATING PROCEDURES
FACILITY NAME……………………………………………………………………………………………………………….

COUNTY………………………………………………………………………………………………………………………….

SUB COUNTY………………………………………………….……………………………………………………………….

SOP Title: FIELD STAINING SOP No: 013

Version: Original
Effective Date: January 2016 Page 1 of 5

Signatures and Dates:

Author: _ __________________________________________________________________
Laboratory Technologist Date

QA Review: ________________________________________________________________
QA Officer Date

Approving Authority: ________________________________________________________

County Medical Laboratory Coordinator Date

Review/Approval for unchanged documents

DATE Author QA Review Approving Authority

Supported by Centre for Health Solutions – Kenya (CHS)

1.1 Purpose

To accurately define a method of staining of thick blood films using field stain

1.2 Scope

This procedure applies to staining of thick blood films using field stain at Tabaka Mission
Hospital Laboratory

1.3 Definitions, Acronyms, symbols

AMREF: African medical and research foundation

TMHL: Tabaka Mission Hospital Laboratory

1.4 Principle

Field stain is an aqueous (water based) Romanowsky stain used for staining blood films.
Romanowsky stains contain Eosin Y, an anionic acidic dye, and Azure B, an ionic basic thiazine
dye obtained by oxidation of methylene blue. The anionic dye stains the basic components
(cytoplasm) of cells red; the ionic dye stains the acid components (nucleus) of cells blue.
When these dyes are combined in an aqueous solution, the dye complex precipitates.
Therefore Field stain is prepared as two separate aqueous solutions. Field stain is the stain of
choice for malaria diagnosis in small laboratories with low to moderate workload

2.0 Safety precautions

Gloves must be worn at all times during the procedure

3.0 Procedures

3.1 Equipment, reagents and materials

1. Field Stain A and B powders – commercially available

2. Sodium azide powder – commercially available
3. Distilled water
4. Filter paper (Whatman No 1)

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 5. Measuring cylinder
6. Conical flasks 250 ml
7. Reagent bottles
8. Staining jars
9. Slide drying rack
10. Cotton wool or gauze
11. Weighing scales

3.2 Preparation of reagent

1. Field stain A solution: Dissolve 12.5 g of Field stain A powder in 500 ml of warm
distilled water. Add 0.5 g sodium azide.
2. Field stain B solution: Dissolve 12.5 g of Field stain B powder in 500 ml of warm
distilled water. Add 0.5 g sodium azide.
3. Filter the stains into separate reagent bottles and label with the name of the reagent
and the date of preparation. For daily use, transfer 100ml into separate staining jars
and label.

3.3 Sample required

Use capillary or venous blood anti-coagulated with EDTA. For parasite examination,
preferably collect blood at times of fever. Prepare and stain thick blood films within 1
hour of blood collection. Stored blood is not suitable for parasite examination.

3.4 Procedure

1. Prepare a thick blood film and leave to dry in air.

2. Stain the thick blood film using the Field stain technique by dipping the slide as
follows:
Field stain A – 4 seconds
Tap water – 5 seconds
Field stain B – 4 seconds
Tap water – 5 seconds.

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 3. Clean the back of the slide and place the slide upright on the drying rack. Allow the
film to dry at room temperature away from direct sunlight and hot objects.

4.0 Procedural notes

• Timing is critical because the staining process is so rapid.
• Keep the Field stain jars covered to avoid dust and evaporation. Filter the stains
regularly to remove dirt and scum.
• Sodium azide is used as preservative.

5.0 Quality control

Prepare two thick blood films:
1. Stain with old stain.
2. Stain with the newly prepared stain.
3. Compare the staining reaction.

References

• F. Baker and S. Silverton (1980). Introduction to medical laboratory technology. 5th

Edition.

• AMREF (2008). Standard operating procedures, Essential Laboratory Tests

Associated records and forms

Not applicable

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Training log

All members of staff who use this document must date and sign in the table below to indicate
that they have read, understood and are going to implement the document in their work.

Print Name Date Signature

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