Practical Guide to Specimen Handling in Surgical Pathology

Authors: Robert Lott, Janet Tunnicliffe, Elizabeth Sheppard, Jerry Santiago, Christa Hladik, Mansoor Nasim, Konnie Zeitner, Thomas Haas,
Shane Kohl, Saeid Movahedi-Lankarani
Practical Guide to Specimen Handling in Surgical Pathology
Authors:
Robert Lott, Janet Tunnicliffe, Elizabeth Sheppard, Jerry Santiago, Christa Hladik, Mansoor Nasim, Konnie Zeitner, Thomas Haas, Shane Kohl, Saeid Movahedi-Lankarani

INTRODUCTION
In spite of the abundant guidelines and recommendations published for specimen handling and testing in a clinical pathology laboratory, relatively little literature is available for guidance of
specimen handling in a surgical pathology laboratory. This document does not relate to cytologic or clinical pathology samples.

The following comprehensive table is intended to serve as a general guideline for proper specimen handling from the time it is taken from the patient to the time a completed slide of the
specimen is given to a pathologist for interpretation.
DISCLAIMER:
This document was created by members of the CAP/NSH Histotechnology Committee and is intended to serve as a guideline ONLY and NOT AN absolute recommendation for specimen
handling. Each laboratory is advised to use these guidelines as a starting point and modify certain parameters to fit state and local institutional requirements, as appropriate. Regulatory
references, standards, and CAP checklist items cited in the guideline are current at the time of publication of this version of the guideline. It is recommended that the user confirm all
references used are the latest version available. The use of the information contained in this guideline does not guarantee compliance with the CAP accreditation requirements or regulations
from other accrediting organizations. Some information may be different or more stringent than the published CAP Checklists.

It is the intent of the CAP/NSH Histotechnology Committee to update this document every 2 years or when required and have the updated version of the document available to members on
the College of American Pathologists (CAP) and National Society for Histotechnology (NSH) websites.

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Table of Contents:
Part I – Specimen Collection and Handling
Collection and Handling
A. Patient Identification pg. 3
B. Proper Labeling pg. 3
C. Transport Media pg. 5
D. Completion of Requisition pg. 6
E. Recommendations for Tissue Collection and Handling pg. 11
F. Accessioning pg. 18
G. Handling prior to Gross Examination pg. 18
H. Intra-operative Consultation pg. 20

Part II – Laboratory Processes

A. Guidelines pg. 24
B. Tissue Cassette Identification pg. 28
C. Fixation Parameters pg. 29
D. Processing pg. 33
E. Embedding pg. 37
F. Microtomy pg. 38
G. Staining pg. 42
H. Coverslipping pg. 52

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VERSION REVISION DATE REVISION
1. Addition of disclaimer on cover page
2.0 November , 2013 2. Addition of version control

3.0 November , 2014 1. Revised per comments received from CAP Chair review

1. Updated references – CAP Checklists: ANP, COM, GEN , 4-21-2014

2. All references reviewed
4.0 January, 2015
3. Table of contents added

1. Updated to reflect LAP Committee 2015 Checklist changes

5.0 September, 2015

6.0 November, 2015 1. Updated to reflect corrected formalin solution to tissue ratio with references

1. Updated to reflect August 21, 2017 CAP Checklist edition changes

7.0 September, 2017
1. Updated to reflect August 22, 2018 CAP Checklist edition changes
8.0 September, 2018 2. Updated to reflect review of all references

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 PART I I. SPECIMEN COLLECTION and HANDLING
Related CAP Checklist Requirements
Guideline Section Statement Additional References
2018 Edition
Collection and Handling
A. Patient Identification • Patient is to be identified in a manner that respects patient privacy with respect to Laboratory General Checklist, GEN.41303 -
their medical records and medical data. Patient Confidentiality

Laboratory General Checklist, GEN.40490 -

Patient Identification
• Patient's identity must be verified at the time of specimen collection.
Laboratory General Checklist, GEN.40491
Primary Specimen Container Labeling
Health Insurance and Portability and
• At least two acceptable unique identifiers are required for patient identification: Accountability Act (HIPAA).
o Full name
o Assigned identification number e.g. health record / master index number Clinical Laboratory Standards Institute
o Date of birth CLSI - GP33A, Accuracy in Patient and
o Photo on government issued or other photo ID card, such as driver's license Sample Identification; 2011: Vol. 30
o Other specific personal identifiers No7.

International Standard ISO 15189:2012

- Medical Laboratories; section 5.4 -
Pre-examination Processes
Collection and Handling
B. Proper Labelling • Specimen is labeled in the presence of the patient Laboratory General Checklist, GEN.40490 –
Patient Identification
• Specimen label must contain at least two unique identifiers:
o Full patient name Laboratory General Checklist, GEN. 40100 -
o Assigned identification number e.g. health record / master index number Specimen Collection Manual Elements
o Date of Birth

• Customizable label elements – additional identifiers that are acceptable: Laboratory General Checklist, GEN. 40491 -
o Patient gender Primary Specimen Container Labeling
o Accession or requisition number
o Ordering physician
o Source of specimen (e.g. skin)
o Site of specimen (e.g. left side of chest)

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 Clinical Laboratory Standards Institute
CLSI – Auto12-A Specimen Labels:
All Common Checklist, COM.06100 – Content and Location, Fonts and Label
• Standardized format for label information should be implemented.
Primary Specimen Container Labeling Orientation: 2011: Vol. 31 No7.
o Last name, first name
o Date of Birth – DD –MMM- YYYY i.e. 12 MAR 1968
o Gender M, F, U ( unknown), T ( Transgender), I (Intersex) All Common Checklist, COM.06200 - Clinical Laboratory Standards Institute
Secondary Specimen Container Labeling CLSI– Auto12-A Specimen Labels:
• Written documentation developed for the correct positioning of the label on the Content and Location, Fonts and Label
collection container. Orientation: 2011: Vol. 31 No7.
o Do not attach label to the container lid (in whole or part) Laboratory General Checklist, GEN.40492 –
o Do not overlap label resulting in patient data being covered Specimen Label Correction

• Written documentation for the correction of labelling errors – to be followed when Laboratory General Checklist, GEN.40825 -
specimens cannot be replaced Specimen ID

Laboratory General Checklist, GEN.40491 -

• All subsequent labelling of patient samples (blocks and slides) must follow same
Primary Specimen Container Labeling
unique identifying process.

• Submitted slides may be labeled with a single identifier but two are preferred.

Collection and Handling

B. Proper Labelling • All parameters used for standard specimen labelling are to be followed. Laboratory General Checklist, GEN.40825 - Zarbo RJ, Tuthill JM, D’Angelo R, et al.
i. Barcoding and/or Specimen ID The Henry Ford Production System:
Radio Frequency • The unique specimen bar code or RFID label must be consistent across all reduction of surgical pathology in-
Identification applications: specimen container, requisition label, cassette and slide labels. process misidentification defects by bar
(RFID) code-specified work process
• Barcode and RIFD specifications within a failure rate established by your facility standardization. Am J Clin Pathol. 2009;
for patient care. 131:469-477.

• Barcode label stock or RFID chip validated to withstand chemicals and Clinical Laboratory Standards Institute
processing used for anatomic pathology specimens. CSLI – Auto02-A2 Laboratory
Automation: Bar Codes for Specimen
• Bar coding and/or RFID documentation must be validated and maintained. Container Identification: 2006: Vol. 25
No 29.
• Automatic identification scanning equipment is validated for accuracy and
resistant to chemicals used for anatomic pathology handing.

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 • If used for specimen chain of custody tracking, the barcode or RFID tracking
system must have intelligent location capabilities.

Collection and Handling

C. Transport Media • Collection, handling and submission procedures must be made available to all Laboratory General Checklist, GEN.40100 - Clinical Laboratory Standards Institute
i. No media / saline health care workers involved in the collection, labeling, submission and transport Specimen Collection Manual Elements CLSI – GP33A, Accuracy in Patient and
of specimens to the pathology laboratory. Sample Identification; 2011: Vol 30
No7.
Laboratory General Checklist, GEN.40125 –
Handling of Referred Specimens Clinical Laboratory Standards Institute
• All specimens must be placed in leak proof container.
CLSI - LIS09A, Standard guideline for
Laboratory General Checklist, GEN.40511 - coordination of clinical laboratory
• Specimens should be transported to the laboratory immediately after collection.
Specimen Tracking/Labeling services within electronic health record
environment and networked
Laboratory General Checklist, GEN.40535 - architectures; 2003: Vol. 23 No 15.
• Specimens that cannot be immediately transferred must be refrigerated until
Specimen Transport QM
transferred to the Pathology laboratory.
International Standard ISO 15189:2007
• For specimens submitted to the laboratory from remote sites, there is a - Medical Laboratories; section 16 Pre-
documented tracking system to ensure that all specimens are actually examination.
received.
Laboratory General Checklist, GEN.40530 -
Specimen Tracking

• Specimens transferred from distant referral site to pathology lab should be

shipped under temperature-controlled conditions to avoid over heating or freezing Laboratory General Checklist, GEN.40535 -
Policies regarding courier service should be established Specimen Transport QM
Bancroft J, Gamble M. Theory and
All specimens must be properly packaged and labelled, indicating materials to be Practice of Histological Techniques, 6th
transported prior to shipping to a centralized or referral laboratory. ed. New York, NY: Churchill Livingston;
2008

• To avoid drying of tissues that are not immediately placed into formalin at time of
procurement: Carson F, Hladik C. Histotechnology A
o wrap solid tissue masses (i.e. lymph node or breast lump) in saline Self- Instructional Text, 3rd ed. Chicago,
dampened gauze prior to placement in labelled container (certain biopsies IL: ASCP Press; 2009
may need special handling)
o add a small volume of saline to tissue with insufficient naturally occurring
fluids (i.e. conceptus for embryopathology/genetic studies)

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Collection and Handling
C. Transport Media • Collection, handling and submission procedures must be made available to all Laboratory General Checklist, GEN.40100 - Clinical Laboratory Standards Institute
ii. Different fixatives health care workers involved in the collection, labelling, submission and transport Specimen Collection Manual Elements CLSI - LIS09A, Standard guideline for
of Specimens to the pathology laboratory. coordination of clinical laboratory
services within electronic health record
environment and networked
architectures; 2003: Vol. 23 No 15.

• All specimens must be placed in leak proof container. International Standard ISO 15189:2012
- Medical Laboratories; section 5.4 -
Pre-examination Processes.

• Specimens must be placed in appropriate fixative as specified in Bancroft J, Gamble M. Theory and
collection/handling and submission procedure. Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
2008
Laboratory General Checklist, GEN.40125 –
• Volume of fixative to tissue ratio must be included in the collection/handling and Handling of Referred Specimens Carson F, Hladik-Cappellano C.
submission procedures. i.e. 10% neutral buffered formalin volume should be 15- Histotechnology A Self- Instructional
20 times the volume of the specimen. Laboratory General Checklist, GEN.40511 - Text, 4th ed. Chicago, IL: ASCP Press;
Specimen Tracking/Labeling 2014
• MSDS must be made available to all staff handling fixatives.
Brown RW. et. al., Histologic
Preparations Common Problems and
Their Solutions. College of American
Pathologists, 2009
• All specimen containers containing fixatives must have appropriate OSHA
Chemical labels attached. Material Safety Data Sheets
Laboratory General Checklist, GEN.40535 -
Specimen Transport QM Clinical Laboratory Standards Institute
• Specimens transferred from distant referral site to Pathology lab should be CLSI – GP 17-A3, Clinical Laboratory
shipped under temperature-controlled conditions to avoid over heating or Safety, 3rd edition; 2012: Vol 32 No 9.
freezing.
Occupational Health and Safety
Administration. Occupational Safety &
Specimens containers should be shipped following appropriate regulations for the Health Standards 1910.1200 toxic and
shipping and handling of formalin i.e. hard sided container with absorbent packing Hazardous Substances.
material.
http://www.osha.gov/dsg/hazcom/index.
html
Collection and Handling
D. Completion of
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 requisition • Written procedures on how to properly complete a pathology requisition must be Laboratory General Checklist, GEN.40700 -
i. Patient identifiers made available to all health care workers involved in the collection, labelling, Requisitions
submission and transport of specimens to the pathology laboratory.
Laboratory General Checklist, GEN.40930 -
• Written or electronic request for patient testing from authorized person. Authorized Requestor

Laboratory General Checklist, GEN.40750 - Clinical Laboratory Standards Institute

Requisition Elements CLSI - GP33A, Accuracy in Patient and
• Required patient identifiers to be included on the requisition / test order: Sample Identification; 2011: Vol 30
o Patient’s name No7.
o Unique identifier i.e. health record or master index number
o Date of Birth International Standard ISO 15189:2012
o Sex - Medical Laboratories; section 5.4- Pre-
examination Processes.
Collection and Handling
D. Completion of • Written or electronic request for patient testing to include: Clinical Laboratory Standards Institute
requisition CLSI - GP33A, Accuracy in Patient and
ii. Specimen o Patient identifiers as listed above Sample Identification; 2011: Vol 30
name/type/site o Name and address or other suitable identifiers of the authorized person No7.
requesting the test Laboratory General Checklist, GEN.40930 -
Authorized Requestor International Standard ISO 15189:2012
o Name and address or other suitable identifier for the individual responsible
- Medical Laboratories; section 5.4 -
for receiving the test results Laboratory General Checklist, GEN.40750 - Pre-examination Processes
o Name and address of the laboratory submitting the specimen Requisition Elements
o Test and or tests to be performed
o Procedure performed
o Specimen site – if more than one specimen is collected during a single
procedure; each specimen should be individually identified by anatomic site
and or specimen type
o Date and time of procedure or specimen collection

o Date specimen received Laboratory General Checklist, GEN.40900 -

Specimen Date Received

Collection and Handling

D. Completion of • Written or electronic request for patient testing to include: Health Insurance and Portability and
requisition Accountability Act (HIPAA).
iii. Pertinent clinical o Clinical history – any additional information relevant or necessary for a Laboratory General Checklist, GEN.40750 -
history specific test to ensure accurate and timely testing and reporting of results, Requisition Elements Clinical Laboratory Standards Institute
including interpretation if required. CLSI - GP33A, Accuracy in Patient and
Sample Identification; 2011: Vol 30
No7.
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 International Standard ISO 15189:2012
- Medical Laboratories; section 5.4- Pre-
examination Processes

Collection and Handling

D. Completion of • The procedure date should be indicated on the requisition following standardized Laboratory General Checklist, GEN.40750 - Hammond EH, Hayes DF, Dowsett M,
requisition format DD - MM - YYYY (i.e. 04 JAN 2012). Requisition Elements Allred DC, et al: American Society of
iv. Procedure Clinical Oncology/College of American
time/date Pathologists Guideline
a. Time removed • The requisition must have a space for the documentation of the warm ischemic Recommendations for
from patient time by the physician obtaining the specimen or designate. Immunohistochemical Testing of
(Warm ischemic time) Estrogen and Progesterone Receptors
in Breast Cancer Archives of Pathology
& Laboratory Medicine 134, (6), June
• Warm ischemic time: 2010: 907-922.
The time measured from the interruption of the blood supply to the tissue/tumor
by the surgeon to the excision time of the tissue specimen.

• Information should be available in the laboratory for review and/or appear on the
patient accession.

Collection and Handling

D. Completion of • The requisition should have a space for the documentation of the cold ischemic Anatomic Pathology Checklist, ANP.22983 – Hammond EH, Hayes DF, Dowsett M,
requisition time by the physician obtaining the specimen or designate. HER;ER/PgR Fixation Allred DC, et al: American Society of
iv. Procedure Clinical Oncology/College of American
time/date Pathologists Guideline
b. Time fixative • Cold ischemic time: Recommendations for
added The time from excision of the specimen from the surgical field to the time the Immunohistochemical Testing of
(if required) tissue is placed in fixative. Estrogen and Progesterone Receptors
(cold ischemic time) in Breast Cancer Archives of
• Information should be available in the laboratory for review and/or appear on the Pathology & Laboratory Medicine
patient accession. 134, (6), June 2010: 907-922.

• The requisition should have a space for the documentation of the date and time
the specimen is placed in fixative by the physician obtaining the specimen or
designate.

Collection and Handling

D. Completion of
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 requisition • The requisition must have a space for documentation of the date and time of Hammond EH, Hayes DF, Dowsett M,
iv. Procedure arrival of the specimen in the AP laboratory to allow for calculation of the Allred DC, et al: American Society of
time/date transport time. Clinical Oncology/College of American
c. Time received in Pathologists Guideline
lab Laboratory General Checklist, GEN.40535 - Recommendations for
(Transport time) • Transport time: Specimen Transport QM Immunohistochemical Testing of
The time tissue specimen was collected in the operating room/doctor’s Estrogen and Progesterone Receptors
office/clinic until it is received in the pathology laboratory for processing (this is Laboratory General Checklist, GEN.40530 - in Breast Cancer Archives of
the time point when the specimen is going to be grossly assessed). Specimen Tracking Pathology & Laboratory Medicine
134, (6), June 2010: 907-922.
• Information must be available in the laboratory for review and/or appear on the
patient accession.

Collection and Handling

D. Completion of • The laboratory has the responsibility to calculate and document total time the Hammond EH, Hayes DF, Dowsett M,
requisition specimen was kept in fixative for required specimens (i.e. breast). Anatomic Pathology Checklist, ANP.22983 – Allred DC, et al: American Society of
iv. Procedure To include: HER;ER/PgR Fixation Clinical Oncology/College of American
time/date o Time specimen held in the operating room Pathologists Guideline
d. Calculation of o Transport time from remote site to AP lab Recommendations for
total fixation time o Time the specimen was kept in fixative while in the lab ( i.e. large Immunohistochemical Testing of
specimens like colon, breast mastectomy were opened/cut to allow for Estrogen and Progesterone Receptors
penetration of fixative) in Breast Cancer Archives of
o Time the specimen(s) are kept in cassettes after grossing Pathology & Laboratory Medicine
o Time in fixative onboard the tissue processor 134, (6), June 2010: 907-922.

Wolff AC, Hammond EH, Hicks,DG,

Dowsett,M, et al: American Society of
Clinical Oncology/College of American
Pathologists Guideline
UpdateRecommendations for Human
Epidermal Growth Factor Receptor 2
Testing in Breast Cancer,Journal of
Clinical Oncology, Vol 31, No. 31, Nov1
2013: pp. 3997-4013.
Collection and Handling
D. Completion of • Tissue handling requirements should be standardized and reported on every Laboratory General Checklist, GEN.40100 - Wolff AC, Hammond EH, Hicks,DG,
requisition specimen. Specimen Collection Manual Elements Dowsett,M, et al: American Society of
iv. Procedure Clinical Oncology/College of American
time/date Pathologists Guideline
e. Fixation time for UpdateRecommendations for Human
• 10 % neutral buffered formalin is the recommended fixative.
breast tissue Epidermal Growth Factor Receptor 2
specimens Testing in Breast Cancer,Journal of

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 • All samples must receive a minimum of six(6) hours of 10% neutral buffered Anatomic Pathology Checklist, ANP.22983 - Clinical Oncology, Vol 31, No. 31, Nov1
formalin fixation HER2; ER/PgR – Fixation 2013: pp. 3997-4013.

• Recommended fixation time is 6-72 hrs. for estrogen and progesterone Werner M, Chott A, Fabiano A, Battifora
receptors. H. Effect of Formalin Tissue Fixation
and Processing on
• Recommended fixation time is 6 to 72 hours for Her2neu receptors. Anatomic Pathology Checklist, ANP.23004 - Immunohistochemistry
Digital Imaging – Preanalytic Testing Phase American Journal of Surgical Pathology.
Validation 24. July 2000:1016-1019.
• Fixation time must be documented, and the following is an example of how the
Spruessel A, Steimann G, Jung M, Lee
data could be recorded on the requisition: SA, Carr T, Fentz AK, Spangenberg J,
Zornig C, Juhl HH, David KA. Tissue
ischemia time affects gene and protein
Time frame Minutes Hours expression patterns within minutes
Warm ischemic time following surgical tumor excision
Cold ischemic time BioTechniques, Vol. 36, No. 6, June
2004:1030–1037.
Transport time from OR /physician office /clinic to
laboratory to time of primary examination Petersen BL, Sorensen MC, Pedersen
Time whole specimen held for additional fixation S, Rasmussen M. Fluorescence In-situ
prior to placing in cassettes Hybridization on Formalin-fixed and
Paraffin-Embedded Tissue: Optimizing
Time cassettes are held prior to loading onto tissue the Method. Applied
processor Immunohistochemistry & Molecular
Fixation time on tissue processor (delay time plus Morphology. 12(3) September
processing time) 2004:259-265.
Total Fixation time
Tanney A, Kennedy RD. Developing
mRNA-based biomarkers from formalin-
fixed paraffin-embedded tissue.
Personalized Medicine (2010) 7(2),
205–211.

Hammond EH, Hayes DF, Dowsett M,

Allred DC, et al: American Society of
Clinical Oncology/College of American
Pathologists Guideline
Recommendations for
Immunohistochemical Testing of
Estrogen and Progesterone Receptors
in Breast Cancer Archives of
Pathology & Laboratory Medicine
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 134, (6), June 2010: 907-922.

Collection and Handling Department of Health and Human

D. Completion of • Establish standardized fixation times for all routine and specialized biopsies. Services, Centers for Medicare and
requisition All Common Checklist, COM.06300 – Medicaid Services. Clinical laboratory
iv. Procedure Specimen Rejection Criteria improvement amendments of 1988;
time/date • Document the recommended fixative for routine and specialized biopsies. final rule. Fed Register. 2003(Jan 24):
f. Fixation time for [42CFR493.1283(a)(3)]
NON-breast • Establish specimen acceptance and rejection policies related to specimen
specimens fixation.

Collection and Handling

D. Completion of • When alternate identifier is used for authorized person requesting test or Laboratory General Checklist, GEN.40750 - Health Insurance and Portability and
requisition receiving test results (medical billing number, hospital ID number), the number Requisition Elements Accountability Act (HIPAA).
v. Requesting must be unique and traceable in the LIS.
physician Clinical Laboratory Standards Institute
a. contact information CLSI - GP33A, Accuracy in Patient and
available in LIS Sample Identification; 2011: Vol 30
No7.

International Standard ISO 15189:2012

- Medical Laboratories; section 5.4 -
Pre-examination Processes.

Collection and Handling • The use of surgical instruments driven by heat should be avoided or limited when . Association of Surgical Technologists
E. Recommendations for possible. (AST) Recommended Standards of
Tissue Collection and Practice for Handling and Care of
Handling Surgical Specimens. www.ast.org
i. Limiting Artifacts • Thermal injury has been known to interfere with diagnosis.
a. Thermal injury

Collection and Handling

E. Recommendations for • The use of surgical instruments should be avoided or limited as much as Association of Surgical Technologists
Tissue Collection and possible when handing the specimen to prevent crushing or damaging the tissue. (AST) Recommended Standards of
Handling Practice for Handling and Care of
i. Limiting Artifacts Surgical Specimens. www.ast.org
b. Crush injury

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Collection and Handling
E. Recommendations for • All tissue should be placed in fixative as soon as possible after removal from the Anatomic Pathology Checklist, ANP.11250 - Association of Surgical Technologists
Tissue Collection and body, unless special studies are ordered that might be affected by the available Adequate storage (AST) Recommended Standards of
Handling fixative. Practice for Handling and Care of
i. Limiting Artifacts Surgical Specimens. www.ast.org
c. Drying artifact
• If fixative cannot be added in a timely manner, the specimen should be placed in
Makary MA, Epstein J, Pronovost PJ,
a sterile basin and kept moist with sterile saline or wrapped in saline-dampened Millman EA, Hartmann EC, Freischlag
sponges until the specimen can be properly placed in fixative. JA. Surgical specimen identification
errors: A new measure of quality in
Laboratory General Checklist, GEN.40535 - surgical care. Surgery. 2007.141:450-
• All unfixed specimens should be transported to the pathology laboratory as soon Specimen Transport QM 455.
as possible and refrigerated until placed into appropriate fixative.

Collection and Handling

E. Recommendations for • Health care facility policy and procedure should be followed for the proper Clinical Laboratory Standards Institute
Tissue Collection and collection, labeling, and transportation of the specimen to the pathology CLSI MM13-A: Collection, Transport,
Handling department. Preparation, and Storage of Specimens
ii. Tissue Transport for Molecular Methods; Approved
a. All fresh Guideline; 2005:Vol 25 No31
specimens • All fresh specimens are to be submitted to the pathology department as soon
as possible with instructions for special testing or processes. Makary MA, Epstein J, Pronovost PJ,
Millman EA, Hartmann EC, Freischlag
• All unfixed specimens should be transported to the pathology laboratory as JA. Surgical specimen identification
soon as possible and refrigerated until placed into appropriate fixative. errors: A new measure of quality in
surgical care. Surgery. 2007.141:450-
455.

Slavin L, Best MA, Aron DC. Gone but

• Specimens not in fixative should be placed in a sterile basin and kept moist
not forgotten: The search for the lost
with sterile saline or wrapped in saline-soaked sponges until the specimen surgical specimens: Application of
can be properly placed in fixative. quality improvement techniques for
Anatomic Pathology Checklist, ANP 10016 - reducing medical error. Quality
Surgical Pathology Exclusion Management in Health Care. 2001.
• Confirmation with surgeon on other types of diagnostic studies to be 10(1): 45-53.
performed, including Gram stain, acid fast and mycological studies.
The Joint Commission. (2014). 2014
National Patient Safety Goals Hospital
Program.

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 • Exceptions to immediate delivery of tissue specimen must be clearly US Dept of Health and Human
described in the policies and procedures. (Example: Placentas must be Services. Summary of the HIPAA
refrigerated until delivery). privacy rule. 2003.

World Health Organization. Guidelines

for the safe transport of infectious
substances and diagnostic specimens.
1997.

Carson F, Hladik C. Histotechnology A

Self-Instructional Text, 3rd ed. Chicago,
IL: ASCP Press 2009

Bancroft J, Gamble M. Theory and

Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
2008

Collection and Handling

E. Recommendations for • Specimen in fixative must be delivered to the pathology laboratory according to Laboratory General Checklist, GEN.40535 - Association of Surgical Technologists
Tissue Collection and the Health care facility policies and procedures. Specimen Transport QM (AST) Recommended Standards of
Handling Practice for Handling and Care of
ii. Tissue Transport Surgical Specimens. www.ast.org
b. Specimens in • Special guidelines are required for the handling of breast tissues to ensure
fixative World Health Organization. Guidelines
fixation guidelines are met. (please see section D, iv, e for specific fixation times)
for the safe transport of infectious
substances and diagnostic specimens.
1997.
• Containers should be rigid, impermeable, unbreakable and non-reactive to
fixative solutions.

Collection and Handling

E. Recommendations for • Documentation of fixation time for Breast specimens is required as outlined in Anatomic Pathology Checklist, ANP.22983 – Hammond EH, Hayes DF, Dowsett M,
Tissue Collection and section C. HER;ER/PgR Fixation Allred DC, et al. American Society of
Handling Clinical Oncology/College of American
ii. Tissue Transport Pathologists Guideline
c. Monitoring of time Recommendations for
and environmental Immunohistochemical Testing of
parameters during • All specimens are received in the pathology laboratory according to the policies Laboratory General Checklist, GEN.40100 - Estrogen and Progesterone Receptors
transport Specimen Collection Manual Elements in Breast Cancer Archives of
and procedures approved, to include the acceptance of specimen protocol as
Pathology & Laboratory Medicine
time received, accessioned and grossed. 134, (6), June 2010: 907-922.

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 Laboratory General Checklist, GEN.40125 – Wolff AC, Hammond EH, Hicks,DG,
• Specimen placed in different environment, i.e. dry ice, must be recorded and Handling of Referred Specimens Dowsett,M, et al: American Society of
delivered with specimen. Clinical Oncology/College of American
Laboratory General Checklist, GEN.40535 - Pathologists Guideline Update
Specimen Transport QM Recommendations for Human
Epidermal Growth Factor Receptor 2
Testing in Breast Cancer. Journal of
Clinical Oncology, Vol 31, No. 31, Nov1
2013: pp. 3997-4013

AST Recommended Standards of

Practice for Handling and Care of
Surgical Specimens.

The Joint Commission. (2014). 2014

National Patient Safety Goals Hospital
Program.

Collection and Handling

E. Recommendations for • Chain of custody ensures continuity of quality care for the patient and provides a The Joint Commission. (2014). 2014
Tissue Collection and method to retrieve needed information. National Patient Safety Goals Hospital
Handling Program.
ii. Tissue Transport • All specimens must be recorded on a chain of custody form or log that includes
d. Chain of custody dates and times, patient identification, specimen number, specimen description, US Dept of Health and Human
1. Specimen and purpose for specimen delivery to the pathology department. Services. Summary of the HIPAA
removal from privacy rule. 2003.
origin of
Collection World Health Organization. Guidelines
(time/date) for the safe transport of infectious
substances and diagnostic specimens.
1997.

Collection and Handling

E. Recommendation for • It is advisable that chain of custody include the personnel involved in the All Common Checklist, COM.06100 – The Joint Commission. (2014). 2014
tissue collection and handling and transportation of the specimen to the pathology lab and within the Primary Specimen Container Labeling National Patient Safety Goals Hospital
handling pathology lab during testing procedures. Program.
ii. Tissue Transport o Name of transporter All Common Checklist COM.06200 -
d. Chain of custody o Title (i.e. RN, Surgical Tech, MD) Secondary Specimen Container Labeling US Dept of Health and Human
2. Personnel o Dates: Collection, transported and received Services. Summary of the HIPAA
transporting privacy rule. 2003.
specimen
(name/title/date) World Health Organization. Guidelines
for the safe transport of infectious
15
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 substances and diagnostic specimens.
1997.

Collection and Handling

E. Recommendation for • Specimen receipt procedure must be available to all personnel in the pathology The Joint Commission. (2014). 2014
tissue collection and department. Laboratory General Checklist, GEN.40100 - National Patient Safety Goals Hospital
handling Specimen Collection Manual Elements Program.
ii. Tissue Transport • All specimens must be signed off on the chain of custody form carried by the
d. Chain of custody transporter and logged into the LIS system of the pathology department for US Dept of Health and Human
3. Specimen accessioning. Services. Summary of the HIPAA
receipt by privacy rule. 2003.
laboratory • The pathology lab must have a logging system that identifies the person
(date/time/name) receiving the specimen, the date and time received. World Health Organization. Guidelines
for the safe transport of infectious
substances and diagnostic specimens.
1997.

• The pathology lab must have a process for documenting who handles the Association of Surgical Technologists
original specimen and all sub-specimens throughout the entire examination, (AST) Recommended Standards of
testing and reporting process. Practice for Handling and Care of
Surgical Specimens. www.ast.org
Collection and Handling
E. Recommendation for • A policy and procedure must be made available that identify the process to follow All Common Checklist, COM.06100 – Association of Surgical Technologists
tissue collection and for labeling discrepancies. Primary Specimen Container Labeling (AST) Recommended Standards of
handling Practice for Handling and Care of
ii. Tissue Transport All Common Checklist, COM.06200 - Surgical Specimens. www.ast.org
e. Quality Assurance Secondary Specimen Container Labeling
Monitors
1. Labeling
discrepancies • In some instances, the specimen can be considered to be a rejection specimen
and only the originator should be making the appropriate labeling changes.

• Label and requisition must be a match. Common mistakes are gender or site. Laboratory General Checklist, GEN.40492 -
Specimen Labeling Correction
• Records of all errors should be maintained.
All Common Checklist, COM.06300 -
Specimen Rejection Criteria

Collection and Handling

E. Recommendation for • The pathology department must have a policy and procedure that handles All Common Checklist, COM.06300 – The Joint Commission. (2014). 2014
tissue collection and specimen acceptance and rejection Specimen Rejection Criteria National Patient Safety Goals Hospital
handling Program.
16
Version:8.0
Revised: September,2018
 ii. Tissue Transport • The information on the specimen container must match the information submitted
e. Quality Assurance on the requisition form. US Dept of Health and Human
Monitors Services. Summary of the HIPAA
2. Specimen • Grounds for rejection may include: privacy rule. 2003.
rejection criteria o Wrong name
o Wrong site Department of Health and Human
o Wrong identifiers Services, Centers for Medicare and
o State of specimen Medicaid Services. Clinical laboratory
improvement amendments of 1988;
final rule. Fed Register. 2003(Jan 24):
[42CFR493.1283(a)(3)]

World Health Organization. Guidelines

for the safe transport of infectious
substances and diagnostic specimens.
1997.

Association of Surgical Technologists

(AST) Recommended Standards of
Practice for Handling and Care of
Surgical Specimens. www.ast.org

Collection and Handling

E. Recommendation for • The specimen collection and handling procedures should include the parameters All Common Checklist, COM.06300 – The Joint Commission. (2014). 2014
tissue collection and for specimens deemed acceptable. Specimen Rejection Criteria National Patient Safety Goals Hospital
handling o Identification of the patient sample (labeling) Program.
ii. Tissue Transport o Completion of the requisition to include all required demographic and clinical
e. Quality Assurance data Department of Health and Human
Monitors o Specimen container to be used Services, Centers for Medicare and
3. Tissue o Type and volume of fixation Medicaid Services. Clinical laboratory
Acceptance o Transport packing, temperature and method improvement amendments of 1988;
o Additional specialized instructions final rule. Fed Register. 2003(Jan 24):
[42CFR493.1283(a)(3)]

Association of Surgical Technologists

(AST) Recommended Standards of
Practice for Handling and Care of
Surgical Specimens. www.ast.org

Carson F, Hladik C. Histotechnology A

Self-Instructional Text, 3rd ed. Chicago,
IL: ASCP Press 2009.

17
Version:8.0
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Collection and Handling • A policy and procedure should be made available that identify the process to Anatomic Pathology Checklist, ANP.11670 - Clinical Laboratory Standards Institute
E. Recommendation for follow for different types of specimens/biopsies: Specimen- Gross Examination CLSI MM13-A: Collection, Transport,
tissue collection and o Muscle - enzyme studies Preparation, and Storage of Specimens
handling o Renal/Skin - Immunofluorescence Anatomic Pathology Checklist, ANP.11275 - for Molecular Methods; Approved
iii. Specimen specific o Nerve/CNS Radioactive Material Handling Guideline; 2005:Vol 25 No31
recommendations o Cardiac .
1. Specialized o Lymphatic tissue - mercuric fixative; thinner sections, etc.
biopsies o Specimens that contain radioactive implants Carson F, Hladik C. Histotechnology A
Self-Instructional Text, 3rd ed. Chicago,
IL: ASCP Press 2009

AFIP, Laboratory Methods in

Histotechnology.
Collection and Handling
E. Recommendation for • Health care facility policy and procedure should be followed for the proper Carson F, Hladik C. Histotechnology A
tissue collection and collection and handling of general biopsies. Procedures to include: Self-Instructional Text, 3rd ed. Chicago,
handling o Type of collection container IL: ASCP Press 2009.
iii. Specimen specific o Type and volume of fixative
recommendations o Transport and holding instructions Bancroft J, Gamble M. Theory and
2. General biopsies Practice of Histological Techniques, 6th
• All fresh biopsies not needing special handling are to be submitted to the ed. New York, NY: Churchill Livingston;
pathology department immediately for processing. Laboratory General Checklist, GEN.40100 - 2008.
Specimen Collection Manual Elements
• If this cannot be completed in a timely manner, the biopsy should be placed in a The Joint Commission. (2011). 2011
sterile container and kept moist with sterile saline or wrapped in saline- National Patient Safety Goals Hospital
dampened sponges until the biopsy can be properly placed in fixative Program.
•
• Specimens must be placed in appropriate fixative as specified in Makary MA, Epstein J, Pronovost PJ,
collection/handling and submission procedure. Millman EA, Hartmann EC, Freischlag
JA. Surgical specimen identification
errors: A new measure of quality in
surgical care. Surgery. 2007.141:450-
455.

Collection and Handling

E. Recommendation for • Health care facility policy and procedure should be followed for the proper Laboratory General Checklist, GEN.40100 - Carson F, Hladik C. Histotechnology A
tissue collection and collection and handling of bone marrow cores and aspirates. Specimen Collection Manual Elements Self-Instructional Text, 3rd ed. Chicago,
handling IL: ASCP Press 2009.
iii. Specimen specific • Bone marrow cores/aspirates should be placed in fixative immediately after the
recommendations procedure. Foucar, KM, Bone Marrow Pathology.
3. Bone marrows 2nd ed. Chicago, IL, ASCP Press: 2001.
• Bone marrow cores/aspirates should be stored at room temperature.

18
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 • Cores/aspirates must be received in the laboratory, as soon as possible, for
immediate handling according to written protocols.

Collection and Handling

E. Recommendation for • Health care facility policy and procedure should be followed for the proper American Society of Clinical Oncology.
tissue collection and collection and handling of specimens. Procedures to include: Laboratory General Checklist, GEN.40100 - (2013). ASCO Guidelines. Retrieved
handling o Type of collection container Specimen Collection Manual Elements.
December 18, 2013, from American
iii. Specimen specific o Type and volume of fixative or no fixative
recommendations o Transport and holding instructions Society of Clinical Oncology (ASCO):
4. Large specimen(s) http://www.asco.org/Guidelines/
• All fresh specimens are to be submitted to the pathology department immediately
with instructions for special testing or processes. Lester, S. C. (2010). Manual of Surgical
Pathology (3rd ed.). Saunders.
• Large specimens require a longer amount of time for tissue to be properly fixed
(Ex. Uterus, spleen, lung, liver, etc.)
Anatomic Pathology Checklist, ANP.22983 –
• Breast tissue must follow the ASCO guidelines for strict fixation timing and HER;ER/PgR Fixation Wolff AC, Hammond EH, Hicks,DG,
processing. (please see section D, iv, e for specific fixation times) Dowsett,M, et al: American Society of
Clinical Oncology/College of American
Anatomic Pathology Checklist, ANP.11250 Pathologists Guideline Update
• Placentas should be refrigerated until delivery to the pathology department.
Adequate Storage Recommendations for Human
Epidermal Growth Factor Receptor 2
Testing in Breast Cancer. Journal of
Clinical Oncology, Vol 31, No. 31, Nov1
2013: pp. 3997-4013
HANDLING PRIOR TO
GROSS HANDLING PRIOR TO GROSS
CAP Checklist Reference
Guideline Section Statement

Collection and Handling • Specimen must be identified/labeled following parameters identified in section B.
F. Accessioning • Each specimen container received must be compared to the requisition to All Common Checklist, COM.06100 –
i. Specimen ensure correct match of at least 2 unique identifiers: Primary Specimen Container Labeling
Identifiers and Clinical Laboratory Standards Institute
Labelling o Full patient name All Common Checklist, COM.06200 - CLSI - GP33A, Accuracy in Patient and
o Assigned identification number e.g. health record / master index Secondary Specimen Container Labeling Sample Identification; 2011:Vol 30 No7.
number
o Date of Birth International Standard ISO 15189:2012
• Additional requisition information to be checked: - Medical Laboratories; section 5.4 -
o Number of specimen containers Pre-examination Processes
Laboratory General Checklist, GEN.40490 -
o Type of specimens submitted
Patient Identification Zarbo RJ, Tuthill JM, D’Angelo R, et al.
o Complete clinical history The Henry Ford Production System:

19
Version:8.0
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 o Name of requesting physician to return report to reduction of surgical pathology in-
o Collection data related to fixation (section D) process misidentification defects by bar
code-specified work process
standardization. Am J Clin Pathol. 2009;
131:469-477

Collection and Handling

F. Accessioning • It is good laboratory practice to avoid accessioning like-specimens back to back All Common Checklist, COM.06100 –
ii. Accessioning order Primary Specimen Container Labeling
a. Avoiding Error • If like specimens must be accessioned in sequence it is suggested to separate
All Common Checklist, COM.06200 -
by size (e.g. skin punch biopsy followed by skin excision followed by skin punch Secondary Specimen Container Labeling
biopsy) or to be identified by use of multi colored inks ( punch one black ink,
punch two is green ink, punch three blue ink etc.)
Laboratory General Checklist, GEN.40100 -
Specimen Collection Manual Elements

Collection and Handling

G. Handling prior to • There should be sufficient space available in the surgical pathology suite to store
Gross Examination surgical specimens in an orderly fashion after accessioning, and prior to gross Laboratory General Checklist , GEN.60000 -
examination: Adequate Space
o Space for the containers and accompanying paperwork/request slips.
o Storage area should be clean, free of clutter, and well ventilated. Laboratory General Checklist, GEN.60100 -
. Adequate Space

Anatomic Pathology Checklist, ANP.11250

Adequate Storage
Collection and Handling
G. Handling prior to • Site specific documentation on how to handle specimens requiring immediate Anatomic Pathology Checklist, ANP.11670 -
Gross Examination gross examination (i.e., microbiological cultures, electron microscopy, Specimen Gross Examination
i. Immediate Gross cytogenetics, flow cytometry or other special studies) must be available to all
Examination and Anatomic Pathology Checklist, ANP.11600 -
staff handling the specimens and should include:
Handling Gross Examination – Pathologist
o Specialized grossing techniques i.e. sterile procedures
Anatomic Pathology Checklist, ANP.11605 -
o Sample collection for submission into specialized media i.e. cytogenetic or
Gross Examination – Non-Pathologist
EM
Department of Health and Human
o Requisition completion for further testing i.e. microbiology or pathology
Anatomic Pathology - ANP.11680 – Cross Services, Centers for Medicare and
referral lab
Contamination Medicaid Services. Clinical laboratory
o Labeling procedure for sub - specimens
improvement amendments of 1988;
o Holding and transport instructions for specialized testing (i.e. refrigerate)
Anatomic Pathology Checklist, ANP.11810 - final rule. Fed Register. 1992(Feb
o Specimen cross contamination
Frozen Section Preparation Quality 28):7183 [42CFR493.1489(b)(6)]

20
Version:8.0
Revised: September,2018
 • Specimens submitted fresh for immediate gross examination (i.e., frozen Anatomic Pathology Checklist, ANP.11670 -
sections, margin determination, etc.) should be kept in their labeled Specimen Gross Examination
containers at room temperature
All Common Checklist, COM.06100 –
• If there is a delay, the fresh specimen should be kept in its labeled container Primary Specimen Container Labeling
and refrigerated until it can be examined.
All Common Checklist, COM.06200 -
• Written procedure to prevent cross contamination Secondary Specimen Container Labeling

Anatomic Pathology Checklist, ANP.11250 -

Adequate Storage

Anatomic Pathology - ANP.21397 – Cross

Contamination - Histology

Collection and Handling

G. Handling prior to • Specimens in fixative requiring gross examination should be assembled/stored in Anatomic Pathology Checklist, ANP.11600 -
Gross Examination an orderly fashion after accessioning, with appropriate paperwork/request slips Gross Examination - Pathologist
ii. Delayed time to and labeled cassettes available.
Gross Examination Anatomic Pathology Checklist, ANP.11605 -
Gross Examination – Non-Pathologist
• The containers should be sealed to avoid spillage, loss of fixative, loss of
Laboratory General Checklist, GEN.40125 –
specimen, and to prevent drying of the specimen prior to gross examination. Handling of Referred Specimens

Collection and Handling

G. Handling prior to • An appropriate room temperature should be maintained, so that specimens are Laboratory General Checklist, GEN.61300 -
Gross Examination neither frozen nor damaged by excessive heat. Climate Control
ii. Delayed time to
Gross Examination
a. Monitoring of • Appropriate ventilation should be maintained so that there is adequate air Laboratory General Checklist, GEN.76720 -
Environmental movement around the specimen containers, without buildup of fixative or other Formaldehyde and Xylene Safety
Parameters noxious vapors.

Collection and Handling

G. Handling prior to • Adequate fixative should be added to the specimen container as soon as Laboratory General Checklist, GEN.40125 – Carson F, Hladik-Cappellano C.
Gross Examination possible. If insufficient fixative is present when the specimen is received in the Handling of Referred Specimens Histotechnology A Self-Instructional
ii. Delayed time to laboratory additional fixative should be added.
21
Version:8.0
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 Gross Examination Text, 4th ed. Chicago, IL: ASCP Press
b. Addition of • Generally, this should be a volume such that there is a 15-20:1 ratio of fixative to 2014.
fixative to tissue specimen. If a large specimen (i.e., uterus, colon, breast, etc.) is
specimen(s) submitted, the specimen should be opened or regularly sliced and covered or Brown RW. et. al., Histologic
wrapped in an absorptive material (i.e., paper towels, etc.) to maximize surface Preparations Common Problems and
exposure to fixative reagents. Their Solutions. College of American
Pathologists, 2009
• The specimen container should remain sealed so that drying or other specimen
damage cannot occur. Bancroft J, Gamble M. Theory and
Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
2008

Collection and Handling

H. Intra-Operative • Health care facility policy and procedure should be followed for the proper
Consultation collection and handling of specimens for intra-operative consultation.
(i.e., Frozen Sections) Procedures to include:
o Gross examination only. Anatomic Pathology Checklist, ANP.11670 -
o Frozen sections Specimen – Gross Examination
o Touch preps, scrap preps
Anatomic Pathology Checklist, ANP.11850 -
• All intra-operative consultation results and tissue diagnoses are made and signed Intra-Operative Results Cibull ML. Q&A. Northfield, IL: College
by a pathologist. of American Pathologists CAP Today.
Anatomic Pathology Checklist, ANP.11660 - 1997;11(7):112
Pathologist Diagnosis

Anatomic Pathology Checklist, ANP.11756 -

Reagents
• Reagents and slides used for intra-operative consultation are properly labeled.
All Common Checklist, COM.06100 –
Primary Specimen Container Labeling

All Common Checklist, COM.06200 -

• Intra-operative consultation preparations are adequate for diagnosis. Secondary Specimen Container Labeling

• Intra-operative slides are retained and made part of the permanent case. Anatomic Pathology Checklist, ANP.11810 -
Frozen Section Preparation Quality

Anatomic Pathology Checklist, ANP.12050 - Nakhleh RE, Fitzgibbons PL, editors.

Frozen Section Slides College of American Pathologists.
22
Version:8.0
Revised: September,2018
 Quality improvement manual in
anatomic pathology, 2nd edition.
• Residual tissue(s) used for intra-operative examination are processed into Northfield, IL: CAP, 2002
paraffin for comparison with the frozen section interpretation. Anatomic Pathology Checklist, ANP.12075 -
Residual Frozen Tissue Rickert RR. Quality assurance goals in
surgical pathology. Arch Pathol Lab
Anatomic Pathology Checklist, ANP.12500 - Med. 1990;114:1157-1162
Record Retention
Association of Directors of Anatomic
and Surgical Pathology.
Recommendations on quality control
and quality assurance in anatomic
pathology. Am J Surg Pathol.
1991;15:1007-1009

Gephardt GN, et al. Interinstitutional

comparison of frozen section
consultations. A College of American
Pathologists Q-probes study
of 90 538 cases in 461 institutions. Arch
Pathol Lab Med. 1996;120:804-809

Novis DA, et al. Interinstitutional

comparison of frozen section
consultation in small hospitals. Arch
Pathol Lab Med. 1996;120:10871093

Nakhleh RE, Fitzgibbons PL, editors.

College of American Pathologists.
Quality improvement manual in
anatomic pathology, 2nd edition.
Northfield, IL: CAP, 2002
Collection and Handling
H. Intra-Operative • When giving a verbal report, the pathologist must be able to speak directly with Anatomic Pathology Checklist, ANP.11900 -
Consultation intra-operative medical/surgical personnel. Verbal Reports
i. Reporting
Anatomic Pathology Checklist, ANP.11950 -
• The patient's identification is checked and confirmed before delivery of any verbal Verbal Report/Patient ID
report.
Anatomic Pathology Checklist, ANP.12000 -
Final Report

23
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 • All intra-operative consultation reports are made a part of the final surgical
pathology report.

Collection and Handling

H. Intra-Operative • There is a documented procedure for the routine decontamination of the cryostat Anatomic Pathology Checklist, ANP.23410 - Clinical Laboratory Standards Institute
Consultation at defined intervals. Cryostat Decontamination CLSI. Protection of Laboratory Workers
ii. Cryostat from Occupational Acquired Infections,
decontamination • Decontamination of the cryostat is documented and records are available for Approved Guideline M29-A4;
examination. 2014;Vol34 No8
http://www.epa.gov/oppad001/list_b_tub
erculocide.pdf

Collection and Handling • Establish operation procedures for H&E staining: Anatomic Pathology Checklist, ANP.24200 – Lott RL. HQIP: H&E Staining. HQIP - A
H. Intra-Operative o Reagents to be used – concentration and volumes Biohazard Waste Disposal Final Critique. Chicago, IL: College of
Consultation o Staining schedule for each staining program American Pathologists; 2010.
iii. Hematoxylin and o Rotation or change schedule for the reagents Anatomic Pathology Checklist, Quality
Eosin stain (H&E) o Disposal and or recycle process for reagents Control, ANP.11756 - Reagents Brown RW. et. al., Histologic
Stain Preparations Common Problems and
• Establish quality assurance criteria for the staining and evaluation of H&E Anatomic Pathology Checklist, ANP.21382 – Their Solutions. College of American
staining. Reagent Expiration Date Pathologists, 2009
Anatomic Pathology Checklist, ANP.11734 –
Slide Quality Carson F, Hladik C., Histotechnology A
Self- Instructional Text, 3rd ed. Chicago,
IL: ASCP Press; 2009

Bancroft J, Gamble M. Theory and

Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
2008

Sheehan DC, Hrapchak BB., Theory

and Practice of Histotechnology, 2nd ed.
Columbus, OH: Battelle Press; 1980

Horobin RW. Troubleshooting Histology

Stains, 1998, Churchill Livingstone

PART II II. LABORATORY PROCESSES - Guidelines

24
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 Guideline Section Statement CAP Checklist Reference

Laboratory Processes
A. Guidelines • The laboratory has sufficient space and utilities are adequate for gross Clinical Laboratory
i. Facility examination and specimen storage. Anatomic Pathology Checklist, ANP.11250 - Standards Institute CLSI:
Requirements Adequate Storage. QMS01-A4: Quality
Management System: A
• Gross examination area has adequate lighting. Laboratory General Checklist, GEN.76720 - Model for Laboratory
Formaldehyde and Xylene Safety Services; Approved
Guideline, 4th Edition
2011,Vol31 No15

• Gross examination area has adequate ventilation system, with policy for
monitoring exposure levels to formalin.

• Formalin exposure level of grossing personnel should be examined annually to

assure proper ventilation.

• Grossing area should have readily available:

o Photographic equipment

o Dictation system (unless grossing personnel enters gross dictation directly

into electronic laboratory information system)

o Access to anatomic pathology laboratory information system

o Access to diagnostic imaging PACS system if located in a clinical hospital

setting

Laboratory Processes
A. Guidelines • All macroscopic tissue examinations are performed by a pathologist or pathology Anatomic Pathology Checklist, ANP.11600 - Department of Health and Human
ii. Personnel resident, or under the supervision of a qualified pathologist. Gross Examination - Pathologist. Services, Centers for Medicare and
• Activities and the nature of supervision is defined in a written protocol Medicaid Services. Clinical Laboratory
Anatomic Pathology Checklist, ANP.11605 - Improvement Amendments of 1988;
Gross Examination - Non-Pathologist. final rule. Fed Register. 2003(Oct
1):1070-1071 [42CFR493.1489], 1071-
1072.

25
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 Anatomic Pathology Checklist, ANP.11610 -
Gross Examination Qualifications. http://www.naacls.org/news/naacls-
• Qualification requirements for non-pathologist or pathology resident personnel who news/archives.asp?article_id=599.
assist in gross examination of specimens:
• An earned associate degree in laboratory science or medical Department of Health and Human
laboratory technology, obtained from an accredited institution, OR Services, Centers for Medicare and
Medicaid Services. Clinical Laboratory
• Education/training equivalent to the above that includes at least 60 semester Improvement Amendments of 1988;
hours or equivalent from an accredited institution. final rule. Fed Register. 2003(Oct
1):1070-1071 [42CFR493.1489], 1071-
• This education must include 24 semester hours of medical laboratory 1072 [42CFR493.1491]
technology courses, OR 24 semester hours of science courses that includes
6 semester hours of chemistry, 6 semester hours of biology, and 12 semester Department of Health and Human
hours of chemistry, biology or medical laboratory technology in any Services, Centers for Medicare and
combination. Medicaid Services. Clinical Laboratory
Improvement Amendments of 1988;
final rule. Fed Register. 1992(Feb
• In addition, the individual must have laboratory training including either
28):7183 [42CFR493.1489(b)(6)]
completion of a clinical laboratory training program approved or accredited by
the NAACLS, ABHES, or other organization approved by HHS (note that this Cibull ML. Q&A. Northfield, IL: College
training may be included in the 60 semester hours listed above), OR at least 3 of American Pathologists CAP Today.
months documented laboratory training in each specialty in which the 1997;11(7):112
individual performs high complexity testing.
Grzybicki DM, et al. The usefulness of
• CLIA regulations include exceptions for grandfathered individuals; Refer to pathologists' assistants. Am J Clin
CLIA regulations 42CFR493.1489 and 1491 for details. Pathol. 1999;112:619-626

• The laboratory director is responsible in determining whether an individual's Galvis CO, et al. Pathologists'
education, training, and experience satisfy the requirements. assistants practice. A measurement of
performance. Am J Clin Pathol.
Anatomic Pathology Checklist, ANP.11670 - 2001;116:816-822
Specimen – Gross Examination.
• Protocols should be in place to specify nature of pathologist supervision of non- The Joint Commission. Laboratory
pathologist for differing types of specimens. Services (CAMLAB) 2012
Anatomic Pathology Checklist, ANP.11605 -
o Protocol for small simple specimens that do not require knowledge of Specimen – Gross Examination non-
anatomy can specify indirect supervision. pathologist

o Protocol for more complex specimens can require direct or indirect

supervision based on laboratory director's determination of each grossing
personnel’s ability to properly examine specimen.

26
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 • Pathologist must define in writing the gross activities and the specimen types the The Joint Commission. Laboratory
individual is permitted to perform. Services (CAMLAB) 2012

• Performance of non-pathologist who performs gross examination should be

evaluated by a pathologist on a regular basis. Anatomic Pathology Checklist, ANP.11640 -
Competency Assessment of Non-
o Annual review with documentation of errors in grossing, to include specimen Pathologists
mix-ups, improperly grossed specimens, and other parameters that are felt
to be important by the laboratory director.

Laboratory Processes
A. Guidelines • Identity of every specimen is maintained at all times during the gross All Common Checklist, COM.06100 –
iii. Specimen Gross examination steps. Primary Specimen Container Labeling
Sectioning
All Common Checklist, COM.06200 -
• There are documented instructions or guidelines available for the proper Secondary Specimen Container Labeling CAP Cancer Protocols and Checklists.
dissection, description, and histologic sampling of various specimen types (e.g., http://www.cap.org/apps/cap.portal
gastrointestinal biopsy, mastectomy, colectomy, hysterectomy, renal biopsy,
nerve biopsy, muscle biopsy, etc). Anatomic Pathology Checklist, ANP.11670 - Barnes CA. False-negative frozen
Specimen – Gross Examination. section results. Am J Clin Pathol.
o Complex specimens should be dissected, described, and histologically 2000;113:900; 6)
sampled in a way that:
▪ Ensures proper microscopic evaluation and diagnosis can be
performed by the pathologist by following established guidelines for
specimen dissection and histologic sectioning.
▪ All required parameters of CAP Cancer Checklists can be assessed
by pathologist.

• There are specific policies and procedures for the safe handling, storage, and Glass EC, et al. Editorial: radiation
disposal of tissues that may contain radioactive material. safety considerations for sentinel node
▪ Procedures should be developed in conjunction with institutional Anatomic Pathology Checklist, ANP.11275 - techniques. Ann Surg Oncol. 1999:6:10
radiation safety guidelines and must comply with state regulations for Radioactive Material Handling.
safe handling of radioactive materials. Miner TJ, et al. Guideline for the safe
▪ Procedures should distinguish policy regarding specimens with low use of radioactive materials during
radioactivity levels (such as sentinel lymph nodes) and high localization and resection of sentinel
radioactivity level specimens such as implant devices. lymph nodes. Ann Surg Oncol.
▪ Procedure should specify specific handling details and laboratory 1999;6:75-82
should include specific storage area of higher radioactive material.
▪ Procedure should include institute specific directions for the disposal Cibull ML. Handling sentinel lymph
of potentially radioactive tissues. node biopsy specimens. A work in
progress. Arch Pathol Lab Med.
1999;123:620-621
27
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 Pfeifer JD. Sentinel lymph node biopsy.
Am J Clin Pathol. 1999; 112:599-602.

Fitzgibbons PL, et al.

Recommendations for handling
radioactive specimens obtained by
sentinel lymphadenectomy. Am J Surg
Pathol. 2000; 24:1549-1551.

Zarbo RJ, Nakleh RE. Surgical

• There is a policy regarding what type of surgical specimens (if any) may be Anatomic Pathology Checklist, ANP.10016 - pathology specimens for gross
exempt from submission to the pathology department. Surgical Pathology Exclusion. examination only and exempt from
▪ Such a policy should be approved by the medical staff or appropriate submission. A College of American
health care committee. Anatomic Pathology Checklist, ANP.10032 - Pathologists Q-Probes study of current
▪ Examples of typical exempt specimens include: prosthetic devices, Surgical Pathology Microscopic Exemptions. policies in 413 institutions. Arch Pathol
tonsils and adenoids in children below a certain age, foreskin in Lab Med. 1999;123:133-139
children, varicose veins, cataracts, and pannus.
Nakhleh RE, Fitzgibbons PL, editors.
College of American Pathologists.
Quality improvement manual in
anatomic pathology, 2nd ed.. Northfield,
IL: CAP, 2002,113-114

Medical devices; device tracking. Fed

• There is a complete list of devices required for tracking under the Safe Medical Reg. May 29,119;57:22966-22981
Devices Act of 1990.
All Common Checklist, COM.06300 –
• There is a policy for handling sup-optimal specimens (unlabeled specimens, Specimen Rejection Criteria College of American Pathologists.
specimens unaccompanied by adequate requisition information, left unfixed or Policies and guidelines manual.
unrefrigerated for extended period of time, received in a container/bag with a Surgical specimens to be submitted to
contaminated outside surface. pathology for examination. Northfield,
Anatomic Pathology Checklist, ANP.11550 - IL: CAP, 1999:Appendix M
Specimen Retention.
• There is written procedure for the storage and disposal of all specimens Nakhleh RE, Fitzgibbons PL, editors.
submitted for examination. The guideline should include: College of American Pathologists.
o Time of retention – minimum of two weeks after report issued and results Quality improvement manual in
reported to the referring physician anatomic pathology, second edition.
o Approved disposal method of fixative as per local and state guidelines Northfield, IL: CAP, 2002
o Approved disposal method of solid waste ( tissue)
Laboratory Processes

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A. Guidelines Document physical parameters of sections submitted for histologic examination: College of American Pathologists.
iv. Tissue Submission • General information Anatomic Pathology Checklist, ANP.12200 – Policies and guidelines manual.
o Sample size must be thin (3-4 mm) enough to ensure adequate fixation and Gross Description Reporting Surgical specimens to be submitted to
processing of the tissue. pathology for examination. Northfield,
o Sample must small enough to fit in the cassette and allow space for IL: CAP, 1999:Appendix M
processing fluids to enter the cassette on all sides.
o Bloody or friable tissues should be wrapped so that the tissue sample is Nakhleh RE, Fitzgibbons PL, editors.
contained within the cassette to avoid cross contamination with other College of American Pathologists.
samples. Quality improvement manual in
o The number of biopsies or cores should be limited to enable proper anatomic pathology, 2nd ed.. Northfield,
embedding; all samples flat and within the same plane. IL: CAP, 2002
o Number of cassettes per sample should be recorded.
o Number of pieces per cassettes should be recorded
o Specialized embedding directions should be documented.

• Small biopsies
o Multiple small pieces for most small biopsies (e.g.: stomach, colon,
endometrium) can be submitted in one cassette. For needle core biopsies,
one or at most a few (less than 5) pieces per cassette.
o
• Larger tissue fragments or samples from whole organs
o If more than one section is submitted in a block, the combined sections meet
the above mentioned parameters and that there is sufficient space between
each piece to allow adequate fixation and embedding.

Laboratory Processes
B. Tissue cassette • All tissue cassettes must be identified with a unique identifier. All Common Checklist, COM.06100 – International Standard ISO 15189:2012
identification Primary Specimen Container Labeling - Medical Laboratories; section 5.4- Pre-
• The unique identifier must be indelible throughout all subsequent procedures. examination Processes
All Common Checklist, COM.06200 -
• The unique identifier can be applied manually or electronically through the use of Secondary Specimen Container Labeling Clinical Laboratory Standards Institute
automated printers. CLSI – LIS02A2 – Specifications for
Transferring Information Between
• Minimum requirements for an unique identifier include: Clinical laboratory Instruments and
Laboratory General Checklist, GEN.40825 - Information Systems; 2004: Vol 24 No
o Accession case identifier – to include year, subsection type (surgical,
Specimen ID 33.
cytology etc.)
o Specimen identifier – alpha or numeric Clinical Laboratory Standards Institute
(see above) CLSI – Auto07A – Laboratory
o Block identifier – alpha or numeric
Automation; Data Content for Specimen
Identification; 2004: Vol 24 No 20.

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 • Additional identifiers: to be used but not required:
o Laboratory name or identifier
o Color coded cassette: tissue type, fixative used, pathologist etc.

• Barcodes must not be the only identifying mark; a human readable identifier is
also required.

• If a barcode is applied to the cassette it should be readable by all tracking

modalities used in the laboratory; LIS, Hospital Information system, associated
testing equipment (slide writers) and third party tracking software

FIXATION LABORATORY PROCESSES – FIXATION

Guideline Section Statement CAP Checklist Reference

Laboratory Processes
C. Fixation Parameters • Guidelines for the correct fixative to use for each specimen type should be Laboratory General Checklist, GEN.40100 - Carson F, Hladik C. Histotechnology A
i. Type of fixative documented and include: Specimen Collection Manual Elements Self- Instructional Text, 3rd ed. Chicago,
a. Formalin, types IL: ASCP Press; 2009
o Fixative to be used
o Recommended duration of fixation Lott RL. HQIP: H&E Staining. HQIP - A
o Required documentation of cold and warm ischemia times Final Critique. Chicago, IL: College of
o References to mandatory fixation guidelines for breast tissues American Pathologists; 2010.
o Safety precautions and spill clean up
Bancroft J, Gamble M. Theory and
Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
2008

Laboratory Processes
C. Fixation Parameters • A written policy and procedure for the use of recycled formalin should include: Section 19 of Occupational Safety and
i. Type of fixative Health Act (OSHA) 1970 - Public Law
b. Recycling formalin o Documentation of the initial verification of quality of recycled formalin. 91-596.
fixatives o Documentation of changes and reverification of quality of recycled formalin 29 CFR 1910.1000 (OSHA) Toxic and
after any procedural changes or repairs to equipment used. Hazardous Substances
o What formalin can be recycled: from tissue samples or tissue processor 29 CFR 1910.1048 (OSHA)
o Recycled formalin be used with new tissue samples, samples to be stored, Formaldehyde
and on tissue processors 29 CFR 1910.1200 (OSHA) Hazard
o Procedure for recycling formalin Communication
o Procedure for testing quality of recycled formalin 29 CFR 1910.1048 (OSHA)
o Procedure for disposal of non-reusable waste Formaldehyde, Irritant and Potential
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 o Procedure for cleaning and maintenance of recycling equipment Cancer Hazard
o Validation studies comparing the filtered/tested solution to new solution are
required. 29 CFR 1910.1450 (OSHA)
o Documentation to show licensing agencies is required. Occupational Exposure to Hazardous
Chemicals in Laboratories
40 CFR 262 (EPA) Standards
Applicable to Generators of
Hazardous Wastes
49 CFR 172.101 (DOT) Table of
Hazardous Materials and Special
Provisions

http://www.osha.gov/dsg/hazcom/index.
html
Laboratory Processes
C. Fixation Parameters • Guidelines for the use of specialized fixatives for each specimen type must be Laboratory General Checklist, GEN.40100 - Carson F. Hladik C., Histotechnology A
i. Type of fixative documented and include: Specimen Collection Manual Elements Self- Instructional Text, 3rd ed. Chicago,
c. Non-Formalin, IL: ASCP Press; 2009
types o Fixative to be used
o Recommended duration of fixation Dapson RW: Glyoxal fixation: How it
o Specialized handling requirements i.e. refrigeration or flammable storage works and why it only occasionally
o Specialized preparation or usage i.e. mix before use needs antigen retrieval. Biotech
o Safety precautions and spill clean up Histochem 82:161; 2007

Bancroft J, Gamble M. Theory and

Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
2008

Michel B, et al., Preservation of tissue

fixed immunoglobulins in skin biopsies
of patients with lupus erythematous and
bullous diseases: preliminary report. J
Invest Dermato 59:449, 1972.

Elias JM, et al, New method for

shipment of renal biopsies. J
Histotechnol 1:15. 1977

Laboratory Processes
C. Fixation Parameters • Using 10% neutral buffered formalin (10%NBF), complete fixation of a 4 mm thick Carson F, Hladik C. Histotechnology A
ii. Fixation section of tissue is achieved in approximately 24 hours. Self- Instructional Text, 3rd ed. Chicago,
Times/Factors IL: ASCP Press; 2009
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 a. Fixative type • As a general recommendation, when using 10% NBF, ALL clinical tissue
specimens should be fixed for a minimum of 6 hours and a maximum of 48 hours. Wolff AC, Hammond EH, Hicks,DG,
Dowsett,M, et al: American Society of
• The general recommendations above are fixative dependent and relate Clinical Oncology/College of American
specifically to the use of 10% NBF. Other fixatives, such as alcoholic formalin or Anatomic Pathology Checklist , Pathologists Guideline Update
Bouin, may have different guidelines. Immunohistochemistry, ANP.22300 - Recommendations for Human
Specimen Modification Epidermal Growth Factor Receptor 2
Testing in Breast Cancer. Journal of
Clinical Oncology, Vol 31, No. 31, Nov1
2013: pp. 3997-4013

Goldstein NS, Ferkowicz M, Odish E, et

al: Minimum formalin fixation time for
consistent estrogen receptor
immunohistochemical staining of
invasive breast carcinoma. Am J Clin
Pathol 120:86–92, 2003

Laboratory Processes
C. Fixation Parameters • Guidelines for the fixation and handling of specific tissue types must be Laboratory General Checklist, GEN.40100 - Wolff AC, Hammond EH, Hicks,DG,
ii. Fixation documented based on: Specimen Collection Manual Elements Dowsett,M, et al: American Society of
Times/Factors Clinical Oncology/College of American
b. Tissue type o Accepted standards – CAP/ASCO guidelines for breast tissues Pathologists Guideline Update
o Tissue anatomy: Recommendations for Human
▪ Brain Epidermal Growth Factor Receptor 2
▪ Fatty tissue – requires extended fixation Testing in Breast Cancer. Journal of
▪ Dense tissue such as uterus or cervix- requires extended fixation Clinical Oncology, Vol 31, No. 31, Nov1
▪ Lung - requires inflation 2013: pp. 3997-4013
▪ Whole organs

• Dense tissues, such as uterus or cervix, and those that are especially fatty or
Carson F, Hladik C. Histotechnology A
bloody , like breast, colon and spleen, usually require extended times in most
Self- Instructional Text, 3rd ed. Chicago,
routine fixatives. IL: ASCP Press; 2009

Bancroft J, Gamble M. Theory and

Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
2008

Laboratory Processes
C. Fixation Parameters • Gross dissection manual should include information about the size and thickness
ii. Fixation of the tissue sample – see section A iv
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 Times/Factors Carson F, Hladik C. Histotechnology A
c. Tissue Size • A gross dissection manual should include specific instructions related to the Self- Instructional Text, 3rd ed. Chicago,
fixation of the specimen to include: IL: ASCP Press; 2009
o Total fixation time required prior to processing
o Preparation of large specimen to improve fixation: Bancroft J, Gamble M. Theory and
▪ Opening / slicing of whole organs Practice of Histological Techniques, 6th
▪ Exchange fixative ed. New York, NY: Churchill Livingston;
2008
• Thickness of tissue specimens is especially important because of its effect on
reagent penetration. Large specimens should be opened or regularly sliced to
maximize surface exposure to fixative reagents. Gross tissue sections should be
no thicker than 3-4 mm. and easily fit between the top and bottom of the
processing cassette.

Laboratory Processes
C. Fixation Parameters • Guidelines for the total fixation of the specimens should be documented. Carson F, Hladik C. Histotechnology A
ii. Fixation • Total fixation time required prior to processing to include: Self- Instructional Text, 3rd ed. Chicago,
Times/Factors IL: ASCP Press; 2009.
d. Total Fixation time o Time from placement in fixative to lab
o Time large specimen is held prior to final dissection Wolff AC, Hammond EH, Hicks,DG,
o Time in cassettes prior to processing – hold time and time on processor Dowsett,M, et al: American Society of
Clinical Oncology/College of American
• Tissues for clinical assessment should be placed into an appropriate fixative Pathologists Guideline Update
immediately after surgical removal. Duration of fixation is an important variable in Recommendations for Human
achieving excellent processing, microtomy, staining, and special staining. Epidermal Growth Factor Receptor 2
Testing in Breast Cancer. Journal of
• Total fixation time should be recorded for each specimen and may be dictated Clinical Oncology, Vol 31, No. 31, Nov1
into the body of the surgical report. 2013: pp. 3997-4013

Laboratory Processes
C. Fixation Parameters • Guidelines for the temperature at which the fixative must be used should be Carson F, Hladik C. Histotechnology A
ii. Fixation documented. Self- Instructional Text, 3rd ed. Chicago,
Times/Factors IL: ASCP Press; 2009.
o Storage temperature of fixative prior to use
e. Environmental
Parameters o Temperature the specimen in fixative to be stored at after collection Bancroft J, Gamble M. Theory and
1. Temperature o Temperature the specimen in fixative to be stored at during Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
transport to testing laboratory.
2008.

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 • Almost all fixatives are effectively used at room temperature (22-25°C).

• Some fixatives such as acetone are more effective when used cold (4°).

Laboratory Processes
C. Fixation Parameters • Guidelines for use and operation of specialized microwave equipment used to Anatomic Pathology Checklist, ANP.27170 - Clinical Laboratory Standards Institute
ii. Fixation assist with fixation should include: Microwave usage CLSI – GP28-A, Microwave Device Use
Times/Factors in the Histology Laboratory; Approved
e. Environmental Anatomic Pathology Checklist, ANP.28290 - Guideline; 2005;Vol25 No10
Parameters o Safety instructions to include radiation testing process Microwave Monitoring
2. Use of o What solutions can be used in microwave Carson F, Hladik C. Histotechnology A
Microwaves o Type of tissues that can be microwave fixed Anatomic Pathology Checklist, ANP.28860 - Self- Instructional Text, 3rd ed. Chicago,
o Size of tissue that can be microwave fixed Microwave Container Venting IL: ASCP Press; 2009.
o Protocols to be applied
Anatomic Pathology Checklist, ANP.29430 - Login GR, Giammara B. Rapid
Microwave Venting microwave fixation, staining and
embedding for light and electron
microscopy. Microscopy Society of
America Workshop; Cincinnati, OH.
1993

PROCESSING LABORATORY PROCESSES – PROCESSING

Guideline Section Statement CAP Checklist Reference

Laboratory Processes
D. Processing • Procedures must be written and validated for each processing schedule used. Bancroft JD, Gamble M. Theory and
i. Time Practice of Histological Techniques.
• Documented processing schedules must include: Anatomic Pathology Checklist, ANP.23120 – New York, NY: Churchill Livingstone, 6th
o Unique title that can be related to program on the tissue processor Tissue Processing Programs. ed. 2008: 53-92.
o Identify what tissue types the schedule can be used for
▪ Rush/urgent, biopsies, breast tissue Anatomic Pathology Checklist, ANP.23130- Brown RW, et. al., Histologic
o Indicate any pretreatment of the tissues Tissue Processing Programs. Preparations Common Problems and
▪ i.e. Tissue must be fully fixed prior to processing as program starts in Their Solutions. College of American
alcohol Pathologists, 2009: 4-8.
o Total processing time Anatomic Pathology Checklist, ANP.21382 –
o Schedule: Reagent Expiration Date Carson F. Hladik C. Histotechnology A
▪ Name of reagent Self- Instructional Text, 3rd ed. Chicago,
▪ Expiration date IL: ASCP Press; 2009: 31-42.
▪ Concentration
▪ Location on processor
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 ▪ Order of application of reagents Sheehan D, Hrapchak B. Theory and
▪ Ensure reagents are compatible with each other- i.e. alcohol following Practice of Histotechnology. Columbus,
neutral buffered formalin must be 70% or less to stop precipitation of OH: Battelle Press, 2nd ed., 1980:59-78.
phosphate salts.
▪ Duration of application Llewellyn, B.D., StainsFile,
▪ Specialized functions: http://stainsfile.info/StainsFile/prepare/p
▪ Heat – actual temperature rocess/auto.htm
▪ Pressure /vacuum – actual levels
▪ Mixing/stirring/agitation – Yes / No Clinical Laboratory Standards Institute
CLSI –GP28-A, Microwave Device Use
in the Histology Laboratory; Approved
Guideline; 2005.

Willis, D., Minshew, J., Microwave

Technology in the Histology Laboratory.
Histologic. 2002; 35:1-4.

Login GR, Dvorak AM. The Microwave

Toolbook. A Practical Guide for
Microscopists. Boston, MA: Beth Israel
Hospital; 1994.

Kok, L.P., Boon, M.E., Microwave

Cookbook of Microscopists. 3rd
All Common Checklist, COM.30675 - Edition, Coulomb Press, Leyden, 1992.
Instrument /Equipment Records
Kok LP, Boon ME. Ultrarapid vacuum-
microwave histoprocessing. Histochem
J. 1995;27(5):411-419
• Maintenance programs for the processor must be established:
o Preventative maintenance and service contracts Clinical Laboratory Standards Institute
▪ Completed by lab staff CLSI GP31-A Laboratory
▪ Completed by vendor service Instrumentation, Implementation,
o Operational maintenance: Validation and Maintenance; Vol.29 No.
▪ Reagent top up / exchange / rotation schedule based on: 11.
• Number of cassettes processed
• Number of time program run
• Monitored and established by processor software
▪ Establish if re-cycled reagents can be used on processor
▪ Cleaning of reagent reservoir containers

35
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Laboratory Processes
D. Processing • Establish and document for fixative to be used on the tissue processor: Anatomic Pathology Checklist, ANP.21382 - Bancroft JD, Gamble M. Theory and
ii. Tissue Processor o Type of fixative to be used Reagent Expiration Date Practice of Histological Techniques.
Reagents ▪ 10% neural buffered formalin (NBF) New York, NY: Churchill Livingstone, 6th
a. Fixative ▪ Zinc formalin Anatomic Pathology Checklist, ANP.23120 – ed. 2008: 53-92.
▪ Alcoholic formalin Tissue Processing Programs.
▪ Formalin substitute or proprietary fixative Brown RW, et. al., Histologic
o Number of reservoirs of fixative to be used Anatomic Pathology Checklist, ANP.23130- Preparations Common Problems and
o Duration of time in fixative Tissue Processing Programs. Their Solutions. College of American
o Temperature / vacuum/ agitation Pathologists, 2009: 4-8.
o Rotation or change schedule
Carson F, Hladik C. Histotechnology A
Self- Instructional Text, 3rd ed. Chicago,
• Verify and document that the fixative used is compatible with the tissues to be IL: ASCP Press; 2009: 31-42.
processed.
Sheehan D, Hrapchak B. Theory and
• Establish if recycled fixative can be used on processor. Practice of Histotechnology. Columbus,
OH: Battelle Press, 2nd ed., 1980:59-78.
• Establish and document procedures for fixative handling that include:
o Storage
o Safety to include:
▪ Use of personal protective equipment
▪ Spill control and clean up
▪ Monitoring of exposure levels
o Disposal methods that follow regulatory guidelines

Laboratory Processes
D. Processing • Develop documentation that establishes the parameters of the dehydrant used on Anatomic Pathology Checklist, ANP.21382 - Bancroft JD, Gamble M. Theory and
ii. Tissue Processor the tissue processor: Reagent Expiration Date Practice of Histological Techniques.
b. Reagents for o Type – alcohol or proprietary product New York, NY: Churchill Livingstone, 6th
dehydration o Type of alcohol – ethanol or isopropanol ed. 2008:53-92.
o Concentration – grades alcohols i.e. 70%, 80%, 95%, 100%
o Number of reservoirs of each alcohol concentration Brown RW, et. al., Histologic
o Duration of time for each alcohol reservoir and total time Preparations Common Problems and
o Temperature / vacuum/ agitation Their Solutions. College of American
o Rotation or change schedule Pathologists, 2009:4-8.

Carson F, Hladik C., Histotechnology A

Anatomic Pathology Checklist, ANP.23100 – Self- Instructional Text, 3rd ed. Chicago,
Tissue Processor Solutions IL: ASCP Press; 2009:31-42.
36
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 • Verify and document that the dehydrant is compatible with the tissues to be Anatomic Pathology Checklist, ANP.23120 – Sheehan D, Hrapchak B. Theory and
processed and changed at intervals appropriate for workload. Tissue Processing Programs. Practice of Histotechnology. Columbus,
OH: Battelle Press, 2nd ed., 1980: 59-
• Ensure that dehydrant following fixative is compatible with fixative: Anatomic Pathology Checklist, ANP.23130- 78.
o 10% NBF- the first alcohol in the dehydrating series should be 70% or less Tissue Processing Programs.
to prevent the precipitation of phosphates from the 10% NBF
o Alcoholic formalin – the first alcohol in the dehydrating series can be 95% as
the tissue has already been in 70% alcohol.
o Formalin substitute or proprietary fixatives – must follow guidelines provided
by the manufacturer

• Validate that the dehydrant is compatible with the reagent that follows in the
processing cycle; this could be xylene or xylene substitute or paraffin.

• Develop a documentation process for recording the purchase, use and disposal
of ethanol. Ethanol is strictly controlled by the federal government.
Anatomic Pathology Checklist, ANP.24200 –
• Develop procedures for alcohol: Biohazard Waste disposal
o Storage
o Safety to include:
▪ Use of personal protective equipment
▪ Spill control and clean up
▪ Monitoring of exposure levels
o Disposal methods that follow regulatory guidelines
o Recycling procedures:
▪ Testing method to prove quality
▪ What alcohol can be recycled
▪ When recycled alcohol can be used
Laboratory Processes
D. Processing • Develop documentation that establishes the parameters of the clearant used on Anatomic Pathology Checklist, ANP.23100 – Bancroft JD, Gamble M. Theory and
ii. Tissue Processor the tissue processor: Tissue Processor Solutions Practice of Histological Techniques.
c. Reagents for o Type – xylene, xylene substitute or proprietary product New York, NY: Churchill Livingstone, 6th
clearing ▪ Verification that clearant is compatible with dehydrants and paraffin Anatomic Pathology Checklist, ANP.23350 – ed. 2008: 53-92.
o Number of reservoirs of clearant Paraffin and Flotation Baths and Embedding
o Duration of time for each reservoir of clearant and total time Stations Brown RW, et. al., Histologic
o Temperature / vacuum/ agitation Preparations Common Problems and
o Rotation or change schedule Anatomic Pathology Checklist, ANP.21382 – Their Solutions. College of American
Reagent Expiration Date Pathologists, 2009 4-8.
• Verification that the clearant to be used is compatible with the tissues to be
processed and changed at intervals appropriate for workload.

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 • Develop procedures for clearant: Carson F, Hladik C. Histotechnology A
o Storage Self- Instructional Text, 3rd ed. Chicago,
o Safety to include: IL: ASCP Press; 2009:31-42.
▪ Use of personal protective equipment
▪ Spill control and clean up Sheehan D, Hrapchak B. Theory and
▪ Monitoring of exposure levels Anatomic Pathology Checklist, ANP.24200 – Practice of Histotechnology. Columbus,
Biohazard Waste Disposal OH: Battelle Press, 2nd ed., 1980: 59-
78.

o Disposal methods that follow regulatory guidelines

o Recycling procedures:
▪ Testing method to prove quality
▪ When recycled clearant can be used

Laboratory Processes
D. Processing • Develop documentation that establishes the parameters of the paraffin to be Bancroft JD, Gamble M. Theory and
ii. Tissue Processor used on the tissue processor: Practice of Histological Techniques.
d. Reagents for o Type – with or without additives New York, NY: Churchill Livingstone, 6th
infiltration ▪ Verification that paraffin is compatible with the dehydrant or clearant ed. 2008: 53-92.
1. Paraffin(s) used
o Melting point of paraffin Brown RW. et. al., Histologic
o Number of reservoirs of paraffin Preparations Common Problems and
o Duration of time for each reservoir of paraffin and total time Their Solutions. College of American
o Temperature / vacuum/ agitation Pathologists, 2009: 4-8.
o Rotation or change schedule
▪ Format of wax to be used; melted wax , pellets, solid block Carson F, Hladik C. Histotechnology A
Self- Instructional Text, 3rd ed. Chicago,
IL: ASCP Press; 2009: 31-42.

Sheehan D, Hrapchak B. Theory and

Practice of Histotechnology. Columbus,
OH: Battelle Press, 2nd ed., 1980:59-78.
EMBEDDING LABORATORY PROCESSES - EMBEDDING
Guideline Section Statement CAP Checklist Reference
Laboratory Processes
E. Embedding • Develop standardized guidelines for routine embedding and handling of special Carson F, Hladik C., Histotechnology A
i. General biopsies: Self- Instructional Text, 3rd ed. Chicago,
Recommendations o Opening of cassettes – one cassette at time Anatomic Pathology Checklist, ANP.23350 – IL: ASCP Press; 2009
o Mold size Paraffin and Flotation Baths and Embedding
o Storage and temperature of molds Stations Bancroft J, Gamble M. Theory and
o Placement of tissue in mold Practice of Histological Techniques, 6th

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 ▪ Similar surfaces in same direction ed. New York, NY: Churchill Livingston;
▪ Direction of surface in orientation to block placement on the microtome 2008
o Orientation of the tissue types
o Method for cooling embedded blocks Luna L. Histopathologic Methods and
o Method for release of blocks from molds and removal of excess paraffin Color Atlas of Special Stains and tissue
o Method for cleaning and reuse of molds Artifacts; American Histolabs Inc;1992
(embedding table)
• Develop quality assurance procedures:
o Manual or electronic workload log used to compare recorded number of
cassettes with the actual number of cassettes.
o Documentation and follow up of discrepancies Anatomic Pathology Checklist, ANP.21350 –
Specimen Preparation Records
• Establish guidelines for the order of embedding cassettes:
o Urgency
o Tissue type; biopsy, routine tissues

• Establish guidelines for the use and operation of the embedding center:
o Temperature of embedding paraffin – monitored daily
o Set temperature of other heated elements: holding paraffin, work surface
and forceps
o Cleaning of forceps and work surfaces
o Addition of paraffin to reservoir: liquid, pellets solid block
o Cleaning of the paraffin reservoir and filter

Laboratory Processes
E. Embedding • Establish type of paraffin wax to be used for embedding: Carson F, Hladik C., Histotechnology A
ii. Paraffin Wax o Specialized paraffin or the same as processing paraffin Self- Instructional Text, 3rd ed. Chicago,
o Additives - beeswax, plastic polymers, diethylene glycol distearate, ceresin IL: ASCP Press; 2009Bancroft J,
o Melting point Gamble M. Theory and Practice of
Histological Techniques, 6th ed. New
York, NY: Churchill Livingston; 2008
MICROTOMY LABORATORY PROCESSES - MICROTOMY

Guideline Section Statement CAP Checklist Reference

Laboratory Processes
F. Microtomy • Written instructions for the operation of all makes/models of microtomes: Anatomic Pathology Checklist, ANP.23400 - Clinical Laboratory Standards Institute
i. Microtome o Manual vs. automated Microtome Maintenance CLSI GP31-A Laboratory
Maintenance o Cleaning and maintenance Instrumentation, Implementation,
o Acceptable cleaning products Validation and Maintenance 2009:Vol.
o Lubrication schedule and reagent 29, No. 11..
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 All Common Checklist, COM.30675 -
• Schedule and document annual preventative maintenance, service, or repair Instrument /Equipment Records Carson F, Hladik C., Histotechnology A
Self- Instructional Text, 3rd ed. Chicago,
IL: ASCP Press; 2009

Bancroft J, Gamble M. Theory and

Practice of Histological Techniques, 6th
ed. New York, NY: Churchill Livingston;
2008
Laboratory Processes
F. Microtomy • Develop technique to standardized position of microtome chuck (block holder) on Clinical Laboratory Standards Institute
ii. Section preparation all microtomes to ensure blocks can be recut on any microtome. CLSI - GP33A, Accuracy in Patient and
a. Block trimming Sample Identification; 2010:Vol 30 No7.
All Common Checklist, COM.06100 –
• Establish guidelines for the orientation of block placement in microtome chuck: Primary Specimen Container Labeling Carson F, Hladik C., Histotechnology A
o Block identifier to face to the right, left, up or down. Self- Instructional Text, 3rd ed. Chicago,
All Common Checklist, COM.06200 - IL: ASCP Press; 2009
Secondary Specimen Container Labeling
• Establish cutting guidelines: Bancroft J, Gamble M. Theory and
o Placement of the slide label Practice of Histological Techniques, 6th
o Limiting one patient tissue to a slide ed. New York, NY: Churchill Livingston;
o Thickness of section 2008
▪ Routine tissues see above
▪ Specialized tissues i.e. brain, lymph nodes
▪ Specialized techniques i.e. amyloid, immunohistochemistry

Tissue Thickness Anatomic Pathology Checklist, ANP.11716 –

Paraffin Microtomy
Routine Paraffin 4 to 5 microns
Renal Sections 1 to 3 microns
Bone Marrow 2 to 3 microns
Nerve histochemical staining 6 to 15 microns
Amyloid demonstration 6 to 12 microns

o Number of sections / ribbons per slide

▪ Sections/ ribbons are same depth
▪ Each section / ribbon is a different depth
▪ Amount of trim between each section/ribbon
o Placement of sections on the slide
o Number of slides per tissue type i.e. 2 slides for biopsy blocks
o Use of specialized slides:
▪ Adhesive or no adhesive
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 ▪ Control slides – specialized markings
o Addition of additives to water bath
▪ Adhesives – i.e. gelatin, agar, Elmer’s glue or proprietary products
▪ Surfactants – i.e. tween

Laboratory Processes All Common Checklist, COM.30675 -

F. Microtomy • Establish guidelines for the use and maintenance of flotation/water bath: Instrument /Equipment Records Carson F, Hladik C., Histotechnology A
iii. Flotation Bath o Temperature of flotation/water bath – documentation of temperature Self- Instructional Text, 3rd ed. Chicago,
a. Temperature o Type of water to be used – tap versus distilled Anatomic Pathology Checklist, ANP.23350 – IL: ASCP Press; 2009
o Use of additives – gelatin, agar, Elmer’s glue, proprietary product(s) Paraffin and Flotation baths
o Cleaning method Bancroft J, Gamble M. Theory and
▪ Frequency Practice of Histological Techniques, 6th
Cleaning products to be used ed. New York, NY: Churchill Livingston;
2008
Laboratory Processes
F. Microtomy • All slides must be clearly labeled to identify the following: All Common Checklist, COM.06100 – Clinical Laboratory Standards Institute
iv. Slides o Specimen accession number Primary Specimen Container Labeling CLSI - GP33A, Accuracy in Patient and
a. Labelling o Block identifier Sample Identification; 2010: Vol 30
All Common Checklist, COM.06200 - No7.
o Slide level number
Secondary Specimen Container Labeling
o Patient name Carson F, Hladik C., Histotechnology A
o Stain identifier Self- Instructional Text, 3rd ed. Chicago,
IL: ASCP Press; 2009

• Establish a labeling procedure to be used; It is good laboratory practice to label

slides only as required and to avoid the practice of pre-labeling large numbers of
slides in advance.
see above
• Establish a quality assurance process of matching slides against the block before
delivery out of the laboratory.

Laboratory Processes
F. Microtomy • Drying times for slides with paraffin sections should be established and made Clinical Laboratory Standards Institute
iv. Slides available to all technical staff. The following recommendations should be CLSI - GP33A, Accuracy in Patient and
b. Slide Drying considered: Sample Identification; 2010: Vol 30
o Air drying of cut sections before placing into the drying oven No7.
o Use of a forced air dryer maintained at a temperature just above the melting
point of the paraffin. Carson F, Hladik C., Histotechnology A
o Drying time and temperature, commonly slides are dried at 58-60°C for 15- Self- Instructional Text, 3rd ed. Chicago,
30 minutes. IL: ASCP Press; 2009

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 • Special techniques, such as immunohistochemistry or in-situ hybridization may
require longer drying times. The required drying time should be included in the
written procedure.
Clinical Laboratory Standards Institute
• Dry slides in an oven for a minimum of 60 minutes at a temperature between 50- CLSI – I/L28-A2, Quality Assurance for
60°C. Optimal results are achieved at room temperature for 24 hours; however Design Control and Implementation of
this is impractical in a clinical laboratory setting. ( Note: Some molecular testing Immunohistochemistry Assays,2011:
protocols require that slides not be oven dried.) Vol. 31 No.4.

Laboratory Processes
F. Microtomy • Guidelines to be established for the retention and disposal of all glass paraffin Anatomic Pathology Checklist , ANP.27150 Clinical Laboratory Standards Institute
iv. Slides blocks and slides. – Glass Slide/Block Disposal CLSI – GP05-A3 Clinical Laboratory
c. Disposal of Waste Management; 2011: Vol. 31, No.
Blocks/Slides 3.

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 STAINING LABORATORY PROCESSES – STAINING
Guideline Section Statement CAP Checklist Reference
Laboratory Processes
G. Staining • Establish operation procedure for manual or automated staining: Anatomic Pathology, ANP.24200 – Clinical Laboratory Standards Institute
i. Hematoxylin & Eosin o Reagents to be used – concentration and volumes Biohazard Waste Disposal CLSI GP31-A Laboratory
(H&E) o Staining schedule for each specific staining program Instrumentation, Implementation,
o Rotation or change schedule for the reagents Validation and Maintenance: 2009: Vol.
o Disposal and or recycle process for reagents Anatomic Pathology, ANP.21382 – Reagent 29, No. 11.
Expiration Date
• Establish quality assurance criteria for the staining and evaluation of hematoxylin
and Eosin stain. Laboratory General Checklist, GEN.30000 –
Monitoring Analytic Performance
• HEMATOXYLIN: When applied correctly, in well-fixed, well processed tissues, Lott RL. HQIP: H&E Staining. HQIP - A
epithelial cells will demonstrate: Anatomic Pathology Checklist, ANP.11734 Final Critique. Chicago, IL: College of
o A well-defined nuclear membrane – Slide Quality American Pathologists; 2010
o Clear, open (vesicular) karyoplasm (cytoplasm of the nucleus)
o Crisp, fine-spiculed chromatin patterns Anatomic Pathology Checklist, , ANP.23021 - Brown RW. et. al., Histologic
▪ Also, in most tissue sections, there are some dense closed Positive Threshold Level Preparations Common Problems and
(hyperchromatic) nuclear patterns present in lymphoid tissue. Their Solutions. College of American
Anatomic Pathology Checklist, ANP.23018 – Pathologists, 2009
o Prominent “eosinophilic” nucleoli. (if present) Daily QC
o Cartilage and calcium deposits stain dark blue Carson F, Hladik C., Histotechnology A
o The hematoxylin should appear blue to blue-black Anatomic Pathology Checklist, ANP.23020 - Self- Instructional Text, 3rd ed. Chicago,
QC Handling IL: ASCP Press; 2009

• EOSIN: When applied correctly, in well-fixed, well processed tissue, eosin Anatomic Pathology Checklist, ANP.23022 – Bancroft J, Gamble M. Theory and
produces, at least, a “tri-tonal” (three-color) effect. QC Confirmation of Acceptability Practice of Histological Techniques, 6th
o Muscle cells (smooth, skeletal, cardiac) and epithelial cell cytoplasm will ed. New York, NY: Churchill Livingston;
stain deep red-pink. All Common Checklist , COM.30675 - 2008
o Collagen will stain a distinct lighter pink. Instrument /Equipment Records
o Red blood cells (RBC) will stain a bright orange-red. Prophet EB, Mills B, Arrington JB, Sobin
o Nucleoli (if present) should exhibit a reddish-purple color due to their high Anatomic Pathology Checklist, ANP.21360 LH. AFIP Laboratory Methods in
protein and RNA content. Automated Stainer. Histotechnology, AFIP;1992

▪ It is essential, when applying eosin, that the smooth muscle/cell Sheehan DC, Hrapchak BB., Theory
cytoplasm and collagen be differentially stained. (different shades of and Practice of Histotechnology, 2nd ed.
red/pink). Columbus, OH: Battelle Press; 1980

Horobin RW. Troubleshooting Histology

Stains, Churchill Livingstone; 1998

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 • Complete and document results of a H&E control prior to staining routine
workload.
o Documentation to include changes or actions taken to correct substandard
staining of the control.

• Establish a preventative maintenance program that includes annual service and

emergency service.
Laboratory Processes
G. Staining • Establish written procedures for manual or automated staining procedures to Anatomic Pathology Checklist, ANP.21395 -
ii. Histochemical and include: Special Stains/Studies
enzymatic stains o Special cutting or preparation of tissue section
(special stains) o Reagents used
▪ Access to material data sheets
▪ Concentration
▪ Storage
▪ Disposal Anatomic Pathology Checklist, ANP.21382 –
o Specific steps of staining procedure Reagent Expiration Date
o Quality assurance process
▪ Define positive control tissue Anatomic Pathology Checklist, ANP.24200 –
▪ Define expected stain results Biohazard Waste Disposal
▪ Records of acceptabilty

• Establish operation procedures for automated staining equipment:

o Validation process All Common Checklist, COM.30675 -
o Cleaning and maintenance procedures Instrument /Equipment Records

• Establish a preventative maintenance program that includes annual service and

emergency service.

• Histochemical stains, or special stains, refer to a group of secondary stains used Bancroft J, Gamble M. Theory and
in conjunction with H&E staining. They were developed to provide differential Practice of Histological Techniques, 6th
coloration and contrast to cell and tissue constituents with the goal of ed. New York, NY: Churchill Livingston;
understanding cell structure and function. 2008

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 • Many are used to identify morphological entities such as bacteria, fungi, nerve Carson F, Hladik C., Histotechnology A
fibers, and for connective tissues including collagen and reticular fibers. Self- Instructional Text, 3rd ed. Chicago,
IL: ASCP Press; 2009
• Other special histochemical stains are used for specific tissue components and
include stains for iron, mucins, glycogen, amyloid, and nucleic acids. Sheehan DC, Hrapchak BB., Theory
and Practice of Histotechnology, 2nd ed.
• Enzyme histochemical staining refers to a subclass of histochemistry that Columbus, OH: Battelle Press; 1980
identifies enzymes by employing substrates containing one of a number of
various naphthol compounds. Kiernan J. Histological and
Histochemical Methods: Theory and
Practice 4th ed. Oxfordshire, England;
2008

Pearse AGE, Stoward PJ.

Histochemistry, Theoretical and
Applied, 4th ed. Vol. 2. Analytical
Technique. Edinburgh: Churchill-
Livingstone, 1985

Lillie RD, Fullmer HM. Histopathologic

Technic and Practical Histochemistry.
4th ed. New York: McGraw-Hill;1976

Laboratory Processes
G. Staining • Establish a procedure for selection and development of antibodies and clones to Anatomic Pathology Checklist, ANP.22983 – Clinical Laboratory Standards Institute
iii. be added to menu: HER2/ER/PgR - Fixation CLSI: ILA28-A2: Quality Assurance for
Immunohistochemical o Fixation of tissue Anatomic Pathology ChecklistANP.22300 – Design Control and Implementation of
stains o cutting of tissue section Specimen Modification Immunohistochemistry Assays;
▪ Paraffin Approved Guideline –2011;Vol31 No4
▪ Frozen Anatomic Pathology Checklist, ANP.22500 -
o Selection and validation of antibody and clone Buffer pH Bancroft J, Gamble M. Theory and
o Selection, validation and monitoring of reagents Practice of Histological Techniques, 6th
o Validation of application method Anatomic Pathology Checklist, ANP.22750 - ed. New York, NY: Churchill Livingston;
▪ Pretreatment Antibody Validation 2008
▪ Antibody dilution
▪ Retrieval method – if required Anatomic Pathology Checklist, ANP.22999 Dabbs D. Diagnostic
▪ Detection method HER2 by IHC - Scoring Immunohistochemistry: Theranostic and
• DAB Genomic Applications, Expert Consult:
• Alkaline phosphatase Anatomic Pathology Checklist, ANP.23003 – Online and Print , 3rd Edition
• Fluorescent Receptor Reporting
o Documentation of scoring methodology
▪ Manual or automated
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 o Documentation of validation; record test tissue, expected results actual Anatomic Pathology Checklist, ANP.22615 – Taylor, Cote; Immunomicroscopy
results and changes to method Endogenous Biotin Volume 19 in Major Problems in
o Storage of antibody and reagents Pathology Series, 3rd ed.
Anatomic Pathology Checklist, ANP.22900 –
Slide Quality Hayat MA.Microscopy,
Immunohistochemistry and Antigen
Anatomic Pathology Checklist, ANP.22760 - Retrieval Methods: For Light and
New Reagent Lot Confirmation of Electron Microscopy, Springer Press;
Acceptability 2002.

Laboratory General Checklist, GEN.30000 – Elias JM. Immunohistopathology: A

Monitoring Analytic Performance Practical Approach to Diagnosis; 2nd ed.
Chicago, IL: ASCP Press, 2003
Laboratory General Checklist, GEN.30070 –
• Establish re- validation procedures after change of: Validation of Accuracy Hayat MA. Immunogold-Silver Staining:
o Methodology Principles, Methods, and Applications,
o Reagent CRC;1995
o Antibody
▪ Clone Javois LC. Immunocytochemical
▪ Lot number Methods and Protocols, 3rd ed.:BIOS
▪ Dilution Anatomic Pathology Checklist, ANP.23085 - Scientific; 2003
o Equipment Pipette Accuracy – Non Class A
▪ New model Polack JM. Introduction to
▪ major service repair Immunocytochemistry, 3rd ed.,BIOS
▪ move or relocation Scientific; 2003

All Common Checklist, COM.30675 - Hayat MA. Microscopy,

Instrument /Equipment Records Immunohistochemistry and Antigen
• Establish procedures for cleaning and maintenance of equipment Retrieval Methods: For Light and
o Calibration of pipettes Electron Microscopy, Springer Press;
o Monitoring of refrigerator and freezer temperature 2002
▪ NIST calibration procedure
o Ancillary equipment Javois LC. Immunocytochemical
▪ Microwave oven Methods and Protocols, 3rd ed:BIOS
▪ Steamer Scientific; 2003
o Stainer
Shi S, Taylor CR. Antigen Retrieval
• Establish a preventative maintenance program that includes annual service and Techniques: Immunohistochemistry and
emergency service. Molecular Morphology, Eaton
Publications;2000

46
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 • Establish procedure for the disposal of reagents as per local , state and national Immunochemical Staining Methods
requirements Handbook, 3rd ed., Dako Corp,
Carpinteria, CA

Clinical Laboratory Standards Institute

• Immunohistochemistry (IHC) staining refers to the method of localizing specific CLSI – I/L28-A2, Quality Assurance for
antigens (e.g., proteins) in cells of a tissue by the principle of an antibody / Design Control and Implementation of
antigen recognition. This reaction is labelled by a detection technique and Immunohistochemistry Assays,2011.
visualized by a chromagen.
Laboratory Processes
G. Staining • Establish Quality Control and Quality Assurance procedures to include: Anatomic Pathology Checklist, ANP.21395 – Bancroft J, Gamble M. Theory and
iv. Special Stains/Studies Practice of Histological Techniques, 6th
Immunohistochemical o Selection of appropriate control material ed. New York, NY: Churchill Livingston;
Stains o Validation of control material Anatomic Pathology, ANP.21850 - QC - 2008
a. Quality Control ▪ Documentation of test of control at accredited lab Immunofluorescence
o Use and application of controls Dabbs D. Diagnostic
▪ Patient and antibody reagent control Anatomic Pathology ChecklistANP.22550 – Immunohistochemistry: Theranostic
▪ Positive and negative QC - Antibodies and Genomic Applications, Expert
Consult: Online and Print , 3rd Edition
Anatomic Pathology Checklist, ANP.22570 –
QC - Antibodies Taylor C, Cote RJ; Immunomicroscopy
• Establish procedures for the review of controls and release of patient slides for Volume 19 in Major Problems in
interpretation Anatomic Pathology Checklist, ANP.22660 - Pathology Series, 3rd ed.
• Records of review need to be retained. Control Slide Review
Hayat MA.Microscopy,
▪ IHC quality control measures are essential to provide and ensure consistency of Laboratory General Checklist, GEN.30000 – Immunohistochemistry and Antigen
performance and reproducibility of the intended target. Monitoring Analytic Performance Retrieval Methods: For Light and
Electron Microscopy, Springer Press;
2002

Elias JM. Immunohistopathology: A

Practical Approach to Diagnosis; 2nd ed.
Chicago, IL: ASCP Press; 2003

Taylor C, Cote RJ. Immunomicroscopy:

A Diagnostic Tool for the Surgical
Pathologist, 3rd ed., WB Saunders; 2005

Immunochemical Staining Methods

Handbook, 3rd ed., Dako Corp,
Carpinteria, CA
Laboratory Processes
G. Staining • Establish procedure for clinical validation of each antibody:
47
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 iv. o Number of tissue sections to be tested per antibody Anatomic Pathology Checklist, ANP.22750 - Clinical Laboratory Standards Institute
Immunohistochemical o Comparison of results to previous stained slides or duplicate slides stained by Antibody Validation CLSI – I/L28-A2, Quality Assurance for
stains accredited lab Design Control and Implementation of
b. Intended Use of Laboratory General Checklist, GEN.30070 – Immunohistochemistry Assays,2011:
the Antibody Validation of Accuracy Vol. 31 No.4

Anatomic Pathology Checklist, ANP.22760 -

• Each antibody MUST be clinically validated to be relevant to its intended target New Reagent Lot Confirmation of
antigen. Acceptability Fitzgibbons PT, Bradley LA, et.al.
. Principles of Analytic Validation of
Anatomic Pathology Checklist, ANP.22550 - Immunohistochemical Assays:
QC- Antibodies
Guideline from the College of American
Anatomic Pathology Checklist, ANP.22570 - Pathologists, Arch Path Lab Med. (In
QC – Antibodies Press)

Anatomic Pathology Checklist, ANP.22976 -

ER/PgR Validation

Laboratory Processes
G. Staining • Establish a procedure for selection and development of probes to be added to Anatomic Pathology Checklist, ANP.22956 - Clinical Laboratory Standards Institute
v. In Situ Hybridization menu: ISH Probe Validation CLSI: MM13-A: Collection, Transport,
o Preparation and cutting of tissue section Preparation, and Storage of Specimens
o Selection of probe Anatomic Pathology Checklist, ANP.22978 – for Molecular Methods; Approved
o Validation of application method HER2 Assay Validation Guideline.2005;Vol25 No9
▪ Pretreatment
▪ Antibody dilution Clinical Laboratory Standards Institute
▪ Retrieval method – if required Anatomic Pathology Checklist, ANP.22964 – CLSI.- MM7-A2 Fluorescence In Situ
▪ Detection method ISH Controls Hybridization (FISH) Methods for
• DAB Clinical Labs,Approved Guideline,2nd
• Alkaline phosphatase Ed. 2013:Vol.33,No.10
• Fluorescent
o Selection and validation of control material Bancroft J, Gamble M. Theory and
o Instructions on how to score slide and expected results Anatomic Pathology Checklist, ANP.23002 - Practice of Histological Techniques, 6th
o Documentation of validation; record test tissue, expected results, actual HER2 (ERBB2) by ISH – scoring ed. New York, NY: Churchill Livingston;
results, and changes to method 2008.
o Storage of probe and reagents

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 o Retention and storage of slides and or images Anatomic Pathology Checklist, ANP.22963 – David J. Dabbs.Diagnostic
ISH scoring Immunohistochemistry: Theranostic and
Genomic Applications, 3rd ed.
Anatomic Pathology Checklist, ANP.22965 - Philadelphia, PA: Saunders Elsevier;
• Establish procedures for change of: Retention - Images and Slides 2010.
o Methodology
o Reagent Awatif I. AL-Nafussi, 2nd ed. Tumor
o Antibody Diagnosis, Practical Approach and
▪ Clone Pattern Analysis. London, Hodde
▪ Lot number Arnold; 2005
▪ Dilution Anatomic Pathology Checklist, ANP.22956 -
o Equipment ISH Probe Validation American College of Medical Genetics
▪ New model Laboratory. Standards and guidelines
▪ major service repair Anatomic Pathology Checklist, ANP.22963 – for clinical genetics laboratories, 2nd
▪ move or relocation ISH Scoring ed. Bethesda, MD: ACMG; 1999.

Anatomic Pathology Checklist, ANP.22964 – Clinical Laboratory Standards Institute

• Establish procedure for clinical validation of each probe: ISH Controls CLSI.- MM7-A2 Fluorescence In Situ
o Number of tissue sections to be tested per probe Hybridization (FISH) Methods for
o Comparison of results to previous stained slides or duplicate slides stained Anatomic Pathology Checklist, ANP.22966 - Clinical Labs,Approved Guideline,2nd
by accredited lab ISH Interpretation Ed. 2013:Vol.33,No.10

Jennings L, Van Deerlin VM, Gulley ML

Anatomic Pathology Checklist, ANP.23002 - (2009) Recommended Principles and
• In Situ Hybridization (ISH) staining refers to a method using probes made up of HER2 (ERBB2) by ISH – Scoring Practices for Validating Clinical
complementary strands used to target sequences of mRNA, viral DNA or Molecular Pathology Tests. Archives of
chromosomal DNA located in tissue cells. Pathology & Laboratory Medicine: Vol.
133, No. 5: 743-755.
• Retention of Images and permanent slides
Wolff AC, Hammond EH, Hicks,DG,
Dowsett,M, et al: American Society of
Clinical Oncology/College of American
Pathologists Guideline Update
Recommendations for Human
Epidermal Growth Factor Receptor 2
Testing in Breast Cancer. Journal of
Clinical Oncology, Vol 31, No. 31, Nov1
2013: pp. 3997-4013

Tanner M, Gancberg D, Di Leo A,

Larsimont D, Rouas G, Piccart MJ, et
al. Chromogenic in situ hybridization: a
practical alternative for fluorescence in
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situ hybridization to detect HER-2/neu
oncogene amplification in archival
breast cancer samples. Am J Pathol
2000;157(5):1467-72.

Di Palma S, Collins N, Faulkes C, Ping

B, Ferns G, Haagsma B, et al.
Chromogenic in situ hybridisation
(CISH) should be an accepted method
in the routine diagnostic evaluation of
HER2 status in breast cancer. J Clin
Pathol 2007;60(9):1067-8.

Gong Y, Gilcrease M, Sneige N.

Reliability of chromogenic in situ
hybridization for detecting HER-2 gene
status in breast cancer: comparison
with fluorescence in situ hybridization
and assessment of interobserver
reproducibility. Mod Pathol
2005;18(8):1015-21.

Hauser-Kronberger C, Dandachi N.
Comparison of chromogenic in situ
hybridization with other methodologies
for HER2 status assessment in breast
cancer. J Mol Histol 2004;35(6):647-53.

Saez A, Andreu FJ, Segui MA, Bare

ML, Fernandez S, Dinares C, et al.
HER-2 gene amplification by
chromogenic in situ hybridisation
(CISH) compared with fluorescence in
situ hybridisation (FISH) in breast
cancer-A study of two hundred cases.
Breast 2006;15(4):519-27.

Bhargava R, Lal P, Chen B.

Chromogenic in situ hybridization for
the detection of HER-2/neu gene
amplification in breast cancer with an
emphasis on tumors with borderline and
low-level amplification: does it measure
50
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 up to fluorescence in situ hybridization?
Am J Clin Pathol 2005;123(2):237-43.

Dietel M, Ellis IO, Hofler H, Kreipe H,

Moch H, Dankof A, et al. Comparison of
automated silver enhanced in situ
hybridisation (SISH) and fluorescence
ISH (FISH) for the validation of HER2
gene status in breast carcinoma
according to the guidelines of the
American Society of Clinical Oncology
and the College of American
Pathologists. Virchows Arch
2007;451(1):19-25.

van de Vijver M, Bilous M, Hanna W,

Hofmann M, Kristel P, Penault- Llorca
F, et al. Chromogenic in situ
hybridisation for the assessment of
HER2 status in breast cancer: an
international validation ring study.
Breast Cancer Res 2007;9(5):R68.

Bilous M, Morey A, Armes J, Cummings

M, Francis G. Chromogenic in situ
hybridisation testing for HER2 gene
amplification in breast cancer produces
highly reproducible results concordant
with fluorescence in situ hybridisation
and immunohistochemistry. Pathology
2006;38(2):120-4.

Di Palma S, Collins N, Bilous M, Sapino

A, Mottolese M, Kapranos N, et al. A
quality assurance exercise to evaluate
the accuracy and reproducibility of
chromogenic in situ hybridisation for
HER2 analysis in breast cancer. J Clin
Pathol 2008;61(6):757-60
Laboratory Processes
G. Staining • Establish Quality Assurance procedures for IHC and ISH procedures to include: Anatomic Pathology Checklist, ANP.22970 -
v.Immunohistochemistry o Compilation of predictive marker results Annual Result Comparison
and In Situ Hybridization ▪ Total cases
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 a. Quality assurance ▪ % positive, % negative
▪ Comparison to benchmarks
▪ Corrective action taken

Anatomic Pathology Checklist, ANP.22973 -

• Documented participation in external proficiency testing for HER2, ER and PR PT for HER2, ER, and PgR

COVERSLIPPING LABORATORY PROCESSES - COVERSLIPPING

Guideline Section Statement CAP Checklist Reference
Laboratory Processes
H. Coverslipping • Establish manual coverslipping procedures that: Bancroft J, Gamble M. Theory and
i. Manual/Automated o Include ergonomic techniques Practice of Histological Techniques, 6th
o Reduce chemical exposure ed. New York, NY: Churchill Livingston;
2008.
• Use mounting media with an appropriate refractive index for proper resolution:
o Aqueous vs. non aqueous Carson F, Hladik C. Histotechnology A
o Non fluorescent Self- Instructional Text, 3rd ed. Chicago,
IL: ASCP Press; 2009
• Identify size and weight of coverslip to be used

• Identify drying method of coverslip and slide

All Common Checklist, COM.30675 -

• Establish validation and operation procedures for an automated coverslipper:
Instrument /Equipment Records
o Speed of operation
o Type of mounting media
o Size and type of coverslip
o Type and volume of transfer fluid ( xylene or xylene substitute)
o Cleaning and maintenance
o Reagent filling or change
o Filter change
o Drying time

• Establish a preventative maintenance program that includes annual service and

emergency service.

END

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