MLS Compiled Based on AO 27 s 2007

Lesson: Clinical Laboratory • According to ownership (government;

private)
What is Medical/Clinical Laboratory? • According to function ( clinical
pathology; anatomic pathology)
• Examination of materials derived from • Institutional character (institution-
the human body (such as fluids, tissues, based; freestanding)
or cells) for the purpose of providing
• According to service capability (primary;
information on diagnosis, prognosis,
secondary; tertiary)
prevention, or treatment of disease.
Institution-based
Classification of Laboratories

According to AO 59 s 2001 • A clinical laboratory that operates

within the premises or part of an
• According to function (Clinical institution such as hospital, school,
Pathology; Anatomic Pathology) medical clinic, medical facility for
• According to institutional character overseas workers & seafarers, birthing
(hospital-based; non-hospital-based ) homes, psychiatric facility, drug
rehabilitation center
• According to service capability (primary,
secondary, tertiary)
Free-standing
Clinical pathology
• A clinical laboratory which is NOT part
• Focuses on the areas of clinical of an established institution like a free
chemistry, immunohematology & blood standing outpatient clinical laboratory
banking. Medical microbiology,
immunology & serology, hematology, Primary category
parasitology, clinical microscopy,
toxicology, therapeutic drug • Licensed to perform basic & routine lab
monitoring, & endocrinology among testing like routine hematology, routine
others stool exam, routine urinalysis, etc
• Concerned with the diagnosis & • Floor area requirement: 10 square m
treatment through laboratory testing of
blood & other body fluids Secondary category (hospital & non-hospital
based)
Anatomic Pathology
• Tests done of primary + routine clin
• A clinical laboratory that focuses on the chem.
areas of histopathology, • Floor area requirement: 20 meter
immunohistopathology, cytology,
autopsy, & forensic pathology among Tertiary category
others
• Concerned with the diagnosis of • Licensed to perform all lab tests
diseases through microscopic performed in secondary lab tests +
examination of tissues & organs immunology + microbiology + special CC
+ special hema + immunhematology
 • Floor area requirement: 60 square • NRL for Hematology, Immunohematology
meter and Immunopathology

• Limited service capability (institution- 4. SACCL ( STD/AIDS Cooperative Central

based) (dialysis centers, social hygiene laboratory )- San Larazo Hospital
clinics)
• Special clinical laboratories ( assisted • NRL for HIV/AIDS, hepatitis, & other
reproduction technology laboratory; sexually transmitted infections
molecular & cellular technology;
molecular biology; molecular pathology, 5. LCP ( Lung Center of the Philippines)
forensic pathology, & anatomic
pathology) • NRL for Anatomic Pathology and
Biochemistry
National reference laboratory (NRL)
Satellite testing sites
Designated by DOH to provide special functions
and services as: • Any testing sites that perform
laboratory examinations under the
• Confirmatory testing administrative control of a licensed
• Surveillance laboratory but outside the physical
• Resolution of conflicts confines of the laboratory
• Training and research
• Evaluation of kits and reagents Mobile clinical laboratory
• External quality assessment program
• A laboratory testing unit that moves
Ex. of NRL from one testing site to another testing
site or has a temporary testing location
1. RITM (Research Institute for Tropical and a “base laboratory”
Medicine) • Shall be licensed as part of the main
clinical laboratory and is permitted to
• NRL for dengue, influenza, TB & other collect specimens only
mycobacteria, malaria & other • Shall be allowed to operate only within
parasites, bacterial enteric diseases, a 100-km radius from its main
measles & other exanthems, mycology, laboratory
enteroviruses, antimicrobial resistance,
& emerging disease Clinical lab Laws
• NRL for confirmatory testing of blood
donors & blood units RA 4688 s 1966

2. EAMC (East Avenue Medical Center) • The clinical laboratory law

• Approved: June 18, 1966
• NRL for environment & Occupational • Regulates the operation, maintenance
Health; Toxicology and Micronutrient and registration of clinical laboratories
Assay in the Philippines

3. NKTI (National Kidney and Transplant AO 59 s 2001

Institute)
 • RULES AND REGULATION GOVERNING Leucocyte Type Number
THE ESTABLISHMENT, OPERATION AND Fraction (Differential
MAINTENANCE OF CLINICAL Count), Qualitative Platelet
LABORATORIES IN THE PHILIPPINES Determination
• Approved : November 19, 2001 o 5.3.1.2. Routine Urinalysis
o 5.3.1.3. Routine Fecalysis
Section 5 Classification of Laboratories o 5.3.1.4. Blood typing –
hospital based
• 5.1. Classification by Function o 5.3.1.5. Quantitative
platelet determination –
 5.1.1. Clinical Pathology – includes hospital based
Hematology, Clinical Chemistry,
Microbiology, Parasitology, • 5.3.2. Secondary – provides the
Mycology, Clinical Microscopy, minimum service capabilities of
Immunology and Serology, a primary category and the
Immunohematology, Toxicology following:
and Therapeutic Drug Monitoring o 5.3.2.1 Routine Clinical
and other similar disciplines. Chemistry – includes
 5.1.2. Anatomic pathology – Blood Glucose
includes Surgical Pathology, Substance
Immunohistopathology, Cytology, Concentration, Blood
Autopsy and Forensic Pathology. Urea Nitrogen
Concentration, Blood
• 5. 2. Classification by Institutional Uric Acid Substance
Character Concentration, Blood
Creatinine
 5.2.1. Hospital–based Concentration, Blood
laboratory – a laboratory that Total Cholesterol
operates within a hospital. Concentration
 5.2.2. Non–hospital–based o 5.3.2.1. Cross matching
laboratory – a laboratory that
operates on its own. • 5.3.3. Tertiary – provides the
secondary service capabilities
• 5.3 Classification by Service Capability and the following:

• 5.3.1 Primary – provides the o 5.3.3.1. Special

minimum service capabilities Chemistry
such as: o 5.3.3.2. Special
o 5.3.1.1. Routine Hematology
Hematology (Complete o 5.3.3.3.
Blood Count or CBC) – Immunology/Serology
includes Hemoglobin Mass o 5.3.3.4. Microbiology
Concentration, Erythrocyte
Volume Fraction • The working space for all categories of
(Hematocrit), Leucocyte clinical laboratories (both hospital and
Number Concentration non–hospital based) shall have at least
(WBC count) and the following measurements:
 • It is a branch of medical science that is
involved in the analysis of biological
materials, usually bodily fluids, to
provide diagnostic results on the state
of the human body
• Blood & urine are common specimens
in this section
• Commonly uses analytic techniques like
III. EQUIPMENT/INSTRUMENTS spectrophotometry.

1. There shall be provisions for sufficient • Spectrophotometry is the

number and types of appropriate measurement of the intensity of light at
equipment/instruments in order to undertake selected wavelengths. It measures the
all the activities and laboratory examinations. concentration of a colored solution.
This equipment shall comply with safety
requirements.

Minimum rqts for equipment/instruments

Spectrophotometer basic parts are:

o Light source - provides incident light for

the whole system
o Entrance slit - reduces stray light;
prevents scattered light from entering
the monochromator
o Monochromator - separate and
transmit a certain wavelength of the
Sections of the clinical lab spectrum
o Exit slit - receives specific spectrum of
• A clinical laboratory is made up of light before it passes through a sample
different sections cohesively & cell
comprehensively performing different o Cuvette ( analytical cell) - a kind of test
activities & procedures for each tube designed for optical analysis
specimen collected from patients to o Photodetector: converts transmitted
produce reliable test results. radiant energy into an equivalent
• At the forefront of these activities are amount of electrical energy
the clinical laboratory personnel o Meter or read-out device – digital
headed by the pathologists display of absorbance or transmittance

Lesson: Clinical Chemistry

Clinical Chemistry
 • RBS- random blood sugar
• OGTT- oral glucose tolerance test
• 2hPPBS- 2h post prandial blood suga
• HBA1c- glycosylated hemoglobin

• Diabetes mellitus (DM) is a defect in

the beta-cells of the pancreas. There is
decrease insulin production
• DM is usually manifested by polyuria
(excessive urination); polydipsia (
• Spectrophotometer applies the Beer- excessive thirst); polyphagia (excessive
Lambert Law eating)
• Beer’s Law states “the concentration of • Hypoglycemia (low blood sugar);
a substance is directly proportional to • hyperglycemia (high blood sugar)
the amount of light absorbed or
inversely proportional to the amount of B. Blood lipid profile
transmitted light“
• Absorbance /optical density is • Lipids (fats) can provide 9 kcal of heat.
synonymous to the amount of blocked They act as primary energy source and
light important constituent of cellular
• Percent transmittance is synonymous to membrane
the reflected light
• TAG – triacylglycerol; storage form ;
Formula? makes serum turbid
• HDL- high density lipoprotein; regarded
as “good cholesterol”- transports back
to liver
• LDL- low density lipoprotein; regarded
as “bad cholesterol”- transport
cholesterol to peripheral tissues
Routine clinical chemistry assays • Total cholesterol - source of hormones,
vit D, bile salt
A. Blood glucose – requested with diagnosis of
diabetic conditions C. Kidney (Renal) Function Tests

• Glucose may elevate after eating, in • Kidneys are considered as the body’s
glycogenolysis or gluconeogenesis “waste sweeper”
• Glucose short term storage sites include • Functional unit: nephron
liver and muscles while long term • Nephron forms urine through 3 major
storage sites are in adipose tissues processes: glomerular filtration, tubular
• Normally ranges from 60-100 mg/dL reabsorption, & tubular secretion
(fasting)
• Can occur in urine if renal threshold • Creatinine – waste product of muscle
(160-180 mg/dL) has been exceeded. = metabolism derived from creatinine
glucosuria phosphate which is stored in the
muscles & is used for energy; in renal
• FBS-fasting blood sugar function there is 50% increase in blood
 • Blood Urea Nitrogen (BUN) - waste • Hypoproteinemia: low total protein
product of protein catabolism; 90% of in the blood suggestive of liver
BUN is excreted. Low levels seen during disorder, kidney disorder
starvation, pregnancy, & low-protein • Hyperproteinemia: high total
diet. High levels seen in high protein protein in the blood; seen with
diet, after steroids use, kidney disease chronic inflammation or infections (
viral hepatitis or HIV)
o Azotemia: elevation of BUN o • Albumin: helps keep blood from
o Uremia: azotemia + renal failure leaking out of blood vessels; carrier
protein of medicines & other
• Glomerular filtration rate (GFR) - substances in the body
estimates how much blood passes • Globulins: help regulate the
through the glomeruli each minute function of circulatory system

D. Liver Function Tests • A low albumin/globulin (A/G) ratio

may reflect an overproduction of
• Liver synthesizes organic substances, globulins ( seen in multiple
detoxifies the body against noxious myeloma or autoimmune diseases);
substances an underproduction of albumin (
seen in cirrhosis); or selective loss
1. D.1. Bilirubin - brownish yellow pigment in of albumin from the circulation (
the bile; comes from old RBC; B1 & B2 seen in nephrotic syndrome)
• High A/G ratio: overproduction of
2. D.2. Liver Enzyme tests Ig ( seen in genetic deficiencies and
leukemia)
• Aspartate aminotransferase (AST) - old
name is serum glutamic oxaloacetic 4. D.4. Prothrombin Time
transaminase (SGOT)
• Alanine aminotransferase (ALT) - old • Test for plasma -clotting activity
name is serum glutamic pyruvic and reflects the activity of vitamin
transaminase (SGPT); considered as K-dependent clotting factors
liver specific synthesized by the liver
• De Ritis Ratio ( AST/ALT ratio) - helps
identify the cause of hepatic disorders; E. Cardiac Function Tests
>1: non-viral in origin;
• Alkaline phosphatase (ALP) - diagnosis 1. E.1. Troponin test: most sensitive and
of bone and liver diseases specific test for myocardial damage;
• Gamma-glutamyl transferase (GGT) - peak 12 hours after infarction
diagnosis of chronic alcoholism 2. E.2. Myoglobin: along with troponin, is
resulting in liver damage considered as cardiac biomarker; rise
• Cholinesterase - for assessing within 2-3 hours of a heart attack or
insecticide and pesticide poisoning other muscle injury; peak within 8-12
hours; generally returns to normal level
3. D.3. total serum protein test within one day. Increase is detectable
later than troponin & is not specific for
heart damage
3. E.3. Cardiac enzymes
Electrolytes

• Sodium - major ECF cation

• Chloride - major ECF anion RBC (Erythrocytes)
• Potassium - major intracellular cation
• Produced in the bone marrow by stem
Special Chemistry Tests cells
• Consist of hemoglobin that carries
• Tumor marker: a biomarker indicative oxygen from the lungs to different parts
of an inherent cancerous condition of the body & carbon dioxide back to
• Oncology: science of cancer lungs

Ex. of tumor makers

WBC (Leukocytes)

Lesson: Hematology • Mobile blood cells

• Produced in bone marrow by stem cells
Hematology • Defend the body against infection

• Hematology section deals with the Types of Leukocytes

enumeration of cells in the blood and
other body fluids
• Coagulation studies focus on blood
testing for the determination of various
coagulation factors
• The study of blood and its components
 • Determine the relative distribution of
various types of cells;
• Measure biochemical abnormalities of
the blood; &
• Assess hemostasis and coagulation.

Hemoglobin
Platelets (Thrombocytes)
• Iron-containing oxygen-transport
• Platelets are cell fragments also metalloprotein in the red blood cells
produced in the bone marrow by stem • Forms are oxyhemoglobin,
cells carboxyhemoglobin ( Hb w/carbon
• Functions: monoxide), methemoglobin ( Hb w/iron
in ferric state), sulfhemoglobin (Hb by
1. adhere to the walls of the blood reaction w/ sulfur-containing drugs)
vessels to plug ruptures, • Reported in grms/dL or grms/L
thereby, preventing the release
of blood Hemoglobin tests
2. Release chemicals that cause
clots to form in the blood

Hematology Tests

• Complete blood count (CBC) is the most

common procedure done in
hematology
Normal Value:
• male: 120-170 g/L
• female: 110-150 g/L

• Also includes MCV, MCH, MCHC Hematocrit

The procedure performed in Hematology are to: • Known as packed cell volume (PCV) or
erythrocyte volume fraction (EVF)
• Count the number or concentration of • Volume percentage of red blood cells in
cells; a sample of whole blood
 • Low hematocrit: anemia, increased
WBCs, vitamin deficiency, recent or
long-term blood loss
• High hematocrit: dehydration, severe
burns, diarrhea, polycythemia vera,
lung or heart disease
• Normal Value:
o male: 0.40-0.54
o female: 0.38-0.47
• Can be manual or automated
Materials used for manual blood cell counting

• Diluting pipet
• Cell counter
• Counting chamber

RBC count

• Can be automated or manual

• High count (Erythrocytosis)
• Low count (anemia)
• Use HPO Diluting fluids
• Formula for manual count:

• Normal Value:
o male: 4.6-6 x 1012/L
o female: 4.0-5.5 x 1012/L

WBC count

• High WBC count (leukocytosis)

• Low WBC count (leukopenia) Platelet count
• Use LPO
• Formula for manual count: • High count (thrombocytosis)
• Low platelet count (thrombocytopenia)
• Use HPO
• Normal Value: 150-400 x 109/L
• Normal Value: 5-10 x 109/L • Manual counting
 Immunology & Serology

• Immunology is the study of all the

aspects of the immune system
• Serology is a division of immunology
that specializes in the laboratory
detection & measurement of antigens
& antibodies

Peripheral Blood Smear Principles of Immunologic & Serologic Methods

• To determine percentage of each cell • The binding of antigen to a specific

type antibody plays an important role in the
• Evaluation of cell morphology diagnosis of diseases using the different
• Use OIO to count 100 cells tests.

Erythrocyte Sedimentation Rate (ESR)

Stages of agglutination
• The rate at which the RBCs fall in a
1. Sensitization -represents the physical
column
attachment of antibody molecules to
• Westergren method is regarded as the
antigens
method of choice by Clinical &
2. Lattice formation - establishment of
Laboratory Standards Institute (CLSI)
crosslinks between sensitized particles
• Tube is placed in a vertical rack for 1 hr & antibodies, resulting in aggregation
@ RT F: 0-20 mm/hr, M: 0-10 mm/hr
Types of agglutination reactions

• Direct agglutination
o Ex. Blood typing
• Passive/indirect agglutination
o Ex. Rheumatoid factor latex
test
• Reverse passive agglutination
o Ex. C-reactive protein test

• Agglutination inhibition is based on the

competition between particulate &
soluble antigens for limited
Lesson: Immunology & Serology
antibodycombining sites. Lack of
agglutination means positive test
 Rh blood group system

• Second most important blood group

system after the ABO blood group
system
• Derived from Rhesus macaque monkey
• 5 important Rh antigens: D,C,E, c & e
Immunohematology/Blood Banking • D antigen is the most important &
immunologic antigen
• Branch of immunology which deals with
the uses of immunologic principles to Interpretation of D blood group
study & identify the different blood
groups
ABO blood group system

• Discovered in the in 1900s by Karl

Landsteiner
• Type “AB” is discovered by Alfred von
De Castello & Adriano Sturli Compatibility testing

ABO typing methods • Performed before blood transfusion

• Involves 2 parts:
• Cell typing (direct or forward typing) o Major crossmatch (PS-DR) -
- To determine what antigens are patient serum + donor RBC
present o Minor crossmatch (PR-DS) -
• Serum typing (backward, reverse, Patient RBC + donor serum
indirect typing)
- To determine what antibodies are
present

Lesson: Clinical Microscopy

Clinical Microscopy

Urinalysis

• overall evaluation of renal function

• Basic functional unit of kidneys:
nephron
 • NEPHRON has glomerulus ( tuft of • Urease-producing organisms will
capillaries), renal tubules ( PCT, loop of degrade urea to ammonia
Henle, DCT, collecting duct) • Loss of CO2
• 3 processes of urine formation:
GLOMERULAR FILTRATION, TUBULAR Steps in RU
REABSORPTION, TUBULAR SECRETION
A. Physical examination – gross appearance

A.1. Color

• is attributed to pigments:
urochrome, urobilin,
uroerythrin
• Is a rough indication of the
state of hydration of an
RU
individual
• When color is dark=more
A. Types of specimen
concentrated
• Early morning urine: preferred because
it is more concentrated from overnight
retention in the bladder; good for
protein analysis
• Random urine: collected any time of
the day
• Fasting/post prandial urine: for glucose
determination
• Timed urine: for clearance test

B. Method of Collection A.2 Odor

• Clean midstream catch • Has little diagnostic

• Catheterization significance; not included in
the routine result
C. Specimen handling • Aromatic due to presence of
volatile acids
• Must be analyzed within 1 hour of • Suggestive of freshness of the
collection if stored at room sample
temperature
• Refrigerate @ 2-8C for not more than 8
hours ( if delay of examination)

D. Effects of unpreserved urine

• Bacterial multiplication causes false (+)

nitrite test
• pH alkalinization leads to cast
degeneration and RBC lysis
A.3 Turbidity (transparency/ clarity) • Normal Value: 1.005-1.030
• Weight of urine / weight of water
• Degree of cloudiness standard
• depends on pH and presence of • Indication of the density of a fluid
dissolved solids depending on the concentration of
• Reported as clear, slightly turbid, dissolved total solids
turbid, cloudy • Marker for the amount of
hydration/dehydration
• Darker urine = higher specific gravity

Methods of SG determination

• Refractometer (total solid meter)

PI - refractive index of urine specimen varies

directly with the total amount of dissolved
solids in the sample

• Urinometer or hydrometer
• Dipstick

A.4 volume

• Indicates balance between fluid

ingestion and water lost from lungs,
sweat, & intestines

• Clinical correlation

• High SG: DM, congestive heart failure,

dehydration, adrenal insufficiency, liver
diseases, & nephrosis
• Low SG: DI, pyelonephritis, &
glomerulonephritis

A.6. PH

• Refers to the negative logarithm of the

hydrogen ion concentration
• Normal pH of a random urine is 4.5-8

A.5 Specific Gravity (SG)

 b) WBC: pyuria

• Reported as ave count/HPO

• Seen in cases of pyelonephritis,
UTI, inflammation
• “glitter cells”: WBC becoming
enlarged exhibiting a sparkling
effect in their cytoplasmic
granules and noticeable
B. CHEMICAL EXAMINATION: REAGENT STRIP Brownian motion
METHOD
c) Epithelial cells (EC)
• Reagent strips contain test pads
impregnated with reagents that
specifically react with a test analyte and • Cells sloughed off the lining of
register specific color change the nephrons and urinary tract
• Color change is compared to a • Squamous cells (large and flat),
comparator chart renal epithelial cells (round and
uninucleate), transitional
bladder EC ( urothelial)

C. MICROSCOPIC EXAMINATION

• 1-2 drops of urine sediments from a

centrifuged urine is placed on a glass
slide to examine under LPO & HPO
d) Casts: Cylinduria or cylindroiduria
• Cellular elements: average of 10
microscopic fields
• Formed primarily within distal
a) RBC: Hematuria convoluted tubule & collecting
duct
• Reported as ave count /HPO • Precipitated cylindrical
• Seen in glomerulonephritis, impressions of the nephrons
severe exercise, menstrual
composed of Tamm-Horsfall
blood contamination, vascular
injury, renal/urinary calculi mucoprotein (uromucoid)
obstruction, pyelonephritis
Types of cast
Crystals

• Formed by the precipitation of urine

salts subjected to pH, temperature, or
concentration
• May collect & aggregate together to
form renal stone or “calculus“
 • Atrichous - absence of flagellum; non-
motile
• Monotrichous -one polar flagellum;
usually exhibits darting motility
• Amphitrichous -single flagellum on
both ends
• Lophotrichous - tuft of flagella on either
end or both ends
Lesson: Medical Microbiology • Peritrichous - flagella is all around the
organism
Medical Microbiology

• Study of organisms too small to be seen

by the unaided eyes
• Branches are:

Gram positve & negative

Parts of Bacterial cell

Shapes of Bacteria

Organisms according to flagella

 o Ex: Haemophilus influenzae,
Neisseria gonorrhea,
Streptoccocus penumoniae

Factors needed for bacterial growth

• Oxygen
• CO2
• Nutrients
• Temperature
• Hydrogen & ion concentration

Type of Bacteria according to O2 requirement

• Some bacteria are capnophiles -

require 5- 10% CO2 (candle jar) to live.
Common techniques in Microbiology
• Antimicrobial Susceptibility Test (AST)
• Staining helps in visualizing is a test that provides a clinician with
microorganisms therapeutic guidelines.

Types of antimicrobial assay

1. Disk diffusion susceptibility test

• Also known as Kirby-Bauer method

• Based on growth inhibition
surrounding antibiotic impregnated
disks
• A 4 mm-thick Muller-Hinton agar
(MHA) is the standard medium used
• Turbidity of standard inoculum is
compared to 0.5 McFarland standard

2. Broth dilution susceptibility test

 • Broth dilution susceptibility is used to
determine both minimal inhibitory
concentration (MIC) & minimal
bactericidal concentration (MBC)
• MIC is the lowest concentration of Lesson: Parasitology
antibacterial agent that inhibits
bacterial growth Basic terminology
• MBC is lowest antibiotic concentration
that results in 99.9% death of bacterial • Parasitology: study of parasites , their
population life cycle
• Parasites: organism that depends on
• Antibiotics are drugs that are another organism for shelter or
administered to either kill bacteria or nourishment
inhibit their growth by preventing • Host: could be definitive (harbors adult
reproduction stage) or intermediate (harbors larval or
asexual) form of parasite
Types of Antibiotics • Mode of transmission: manner of how
parasite successfully enters a
• Bacteriostatic antibiotics: only inhibit
susceptible host
microorganism growth in vitro
• Pathogenic parasites: disease causing
• Bactericidal antibiotics: lethal to
• Nonpathogenic parasites: “commensal”
standard inocula
• Ectoparasites (infestation): thrive
externally to host.
o Ex. Flies, fleas
• Endoparasites (infection): found inside
the body
• Eosinophilia: increase in eosinophil
count Eosinophils act as cellular
protectors against parasites.
• Stool examination/fecalysis: stool or
feces examination to include
macroscopic, microscopic, chemical
exam
Specimen collection 1. Gross stool examination

• Thumb-sized (2-4 grm) specimen is • Analysis with naked eye - to

appropriate for routine fecalysis include color, consistency,
 Collect appropriate amount if watery presence of adult worm
 Specimen must be collected in a dry
• Useful in the diagnosis of
container
intestinal disorders
 Specimen must be placed in a dry,
• Normal stool color is attributed
clean, wide mouth container with tight
to stercobilin, stool coloring
fitting cover
pigment
 Label body of container with patient’s
• Bright red: indicates bleeding
name, date, time of collection
hemorrhoids or bleeding in the
 After ingestion of anti-diarrheal
lower intestinal tract
medication, radio-opaque compounds,
• Bloody mucus (loose or liquid
or oil laxatives, wait until 1 week before
specimens): high suggestive of
collecting the specimen.
amocoic ulcerations in the large
intestine
• Stool specimens can be examined fresh
• Occult blood: indicates parasitic
or preserved.
infection but is more likely a
 Stool should not be frozen
sign of other gastrointestinal
 If delays cannot be avoided, the
disorders like colon cancer
specimen should be preserved to
maintain protozoan integrity and Stool consistency
prevent development of certain
helminths: • Serves as predictor for possible stages
 refrigeration for up to one day of parasites present in the sample
 adding 10% formalin • Normal pH is 7 to 7.5. Variation is due
to diet or pathogenic agents.
Indication
2. Microscopic
• Liquid, diarrheic or watery stool is best
to detect contain trophozoites should
• Needs preparation of specimen
be examined within 30 minutes of
passage (not within 30 minutes of
arrival in the laboratory)
• Soft specimens (which may contain
both trophozoites and cysts) should be
examined within one hour of passage.
• Formed stool is best to detect ova and
cysts.

Method of Stool examination

 diagnostic laboratories because they
are easier to perform and less prone to
technical errors.

The sedimentation technique used at

CDC is the formalin-ethyl acetate
technique, a diphasic sedimentation
technique that avoids the problems of
flammability of ether, and which can be
Concentration Methods used with specimens preserved in
formalin, MIF or SAF.
• Concentration procedure separate
Formalin-Ethyl Acetate Sedimentation
parasites from fecal debris and increase
Concentration
the chances of detecting parasitic
organisms when these are in small • Mix the specimen well
numbers.
• Strain 5ml of the fecal suspension
• They are divided into floatation through wetted cheesecloth-type gauze
techniques and sedimentation placed over a disposable paper funnel
techniques. into a 15 ml conical centrifuge tube.
(Conical paper cups with the tips cut off
• Flotation techniques (most frequently are sufficient).
used: zinc sulfate or Sheather's sugar) • Add 0.85% saline or 10% formalin
use solutions which have higher specific through the debris on the gauze to
gravity than the organisms to be floated bring the volume in the centrifuge tube
so that the organisms rise to the top to 15 ml. Distilled water may be used;
and the debris sinks to the bottom. however, Blastocystis hominis may be
deformed or destroyed.
• Centrifuge at 500 × g for 10 minutes.
• Decant supernatant. Add 10 ml of 10%
formalin to the sediment and mix
thoroughly with wooden applicator
sticks.
• Add 4 ml of ethyl acetate, stopper the
tube, and shake vigorously in an
inverted position for 30 seconds.
• Sedimentation techniques use Carefully remove the stopper.
solutions of lower specific gravity than • Centrifuge at 500 × g for 10 minutes.
the parasitic organisms, thus • Free the plug of debris from the top of
concentrating the latter in the the tube by ringing the sides with an
sediment. Sedimentation techniques applicator stick. Decant the top layers
are recommended for general of supernatant.
 • Use a cotton-tipped applicator to • Mask is worn during collection to avoid
remove debris from sides of the inhalation parasite
centrifuge tube. • Negative for 5-7 swabs before declaring
• Add several drops of 10% formalin to absence of infection
resuspend the concentrated specimen.
Proceed with applicable testing. Collection via Vaginal Swabs

• For Trichomonas vaginalis

• Commercial fecal concentration tubes • Use of BD Affirm VPIII Ambient
are available that decrease processing Temperature Transport System- a
time and supplies needed for molecular system for the detection of
concentrating specimens (e.g., Fecal vaginitis
Parasite Concentrator, Evergreen
• A speculum without lubricant is used.
Scientific).
• Specimen is transferred at room
temperature
• Specimens preserved in PVA are mostly
used for permanent staining with Blood Collection
trichrome. Prior to staining, they are
processed as follows: • To detect Trypanosoma and Leishmania
o Insure that the specimen is well • Definite time for collection is
mixed considered
o Prepare a smear using 2 to 3
Major Groups of Medically Important Parasites
drops of the specimen
depending on density. A. Protozoans
o Heat fix on slide warmer set at
60°C for 5 minutes or air dry • Comprised of the simplest
completely at room organisms
temperature • Most are free-living but some
o Slides may be trichrome stained are mutualistic, commensalistic
or kept for several months in a or parasitic
protective slide tray or box for • Stages: trophozoite
future staining (motile/active stage) & cyst (
infective stage)
Scotch Swab method
• Encystation is the
• For enterobiasis ( both for adult and transformation of trophozoites
worms of Enterobius vermicularis- into cyst
pinworm) • Excystation is the reverse
• Collection is done is 3 days, few hours process
after specimen has retired in the • Protozoans have locomotor
evening or immediately after arising structures that help them move
,but before bathing or bowel during their vegetative stage
movement. and basis for classification
 • Protozoans without locomotor
structures belong to phylum
Apicomplexa

Protozoan-amoeba

• Has pseudopods (“false feet”)

which appear as an ectoplasmic
extension of the parasite
• Transmitted through ingestion
of contaminated food or water
• Entamoeba histolytica - only
pathogenic amoeba
Protozoan-flagellates
• Amoeba can resemble other
insignificant artifacts so careful
examination of fecal smear • Has whip-like structures called
flagella ( for locomotion)
• All demonstrate the
trophozoite stage but not all
are capable of encystation
[Genus Trichomonas ( T
vaginalis, T hominis, T Tenax) &
Dientamoeba fragilis]
• Most live in small intestine but
hemoflagellates (Trypanosoma
sp & Leishmania sp) are vector
– transmitted
 Protozoan-Malaria
Protozoan-ciliates
• Caused by P falcifarum, P vivax, P ovale,
P knowlesi, P malariae
• Move through hair-like structure called
• Plasmodium knowleski resembles P.
“cilia”
falciparum or P. malariae
• Balantidium coli is the only species
• Definitive host is Anopheles minimus
pathogenic to humans
flavirostris (sporogony cycle happens)
• People handling stool samples of pigs
• Intermediate host is human
are of high risk for infection
(Schizogony cycle occurs)
• Parasite also invades tissues
• Malarial parasites invade liver
(preerythrocytic or exoerythrocytic
phase) then destroy red blood cells
(erythrocytic phase) of the infected
patients
• Malaria is characterized by high fever
due to pyrogenic metabolites ( when
malaria leave the blood circulation);
anemia ( result of RBC destruction);
jaundice ( result of hemoglobin
breaking); shaking; chills; flu-like illness
• Paroxysm is a typical manifestation and
coincides with the bursting of infected
RBC. It is observed every 48 hrs (tertian
malaria); 36-48 hrs (subertian malaria);
72 hrs ( quartan malaria)
• Mode of transmission is bite of female
anopheles mosquitoes.
 • Sporozoite is the infective stage
released during the blood meal of the
infected vector
• Diagnosis uses
o Giemsa-stained thick and thin
smear ( gold standard)
o rapid diagnostic test
o molecular approaches

Nematodes

• Also known as round worms

• Adult worms have tapered, cylindrical
bodies with an esophagus and
longitudinal muscles
• Dioecious parasites ( separate male &
female)
• Male is smaller and has “curling tail”,
maybe provided with copulatory
spicules and bursa
Trematodes

• Known as flukes
• 2 orders: monogenea and digenea ( of
medical importance)
• Flat leaf-like and hermaphroditic (
except Schistosome sp or blood fluke)
• Have 2 suckers: oral sucker serves as
mouth; ventral sucker ( acetabulum)
serves an attachment organ
• Requires 2 intermediate hosts: first
snail (always) & second may vary
• Transmission is through ingestion of
infective stage (metacercaria)
• Diagnosis is finding adult worms & ova.
• Can inhabit lungs, liver, intestines
Lesson: Anatomic Pathology

Anatomic Pathology Laboratory

• The section in the laboratory which is

focused on the disease diagnosis based
on the gross, microscopic,
immunohistochemical, and molecular
examination of body tissues and solid
tissue specimens

Sources of specimens

• Autopsy: taken from dead (morgue)

• Biopsy: collected from an alive person (
O.R.)

Routine Histopathologic Exam

• Numbering : process of recording the

tissue specimen in a log book and
assigning identification numbers to the
specimen received in the laboratory
• Fixation: preserving the tissue
specimen in as lifelike manner as
possible. Routine fixative is 10%
formalin
• Dehydration: process of removing
water from the specimen by using
increasing grades of alcohol
• Clearing (or de-alcoholism): removing
excess alcohol in the tissues and making
the tissues transparent. Routine
clearing agent is xylene
 • Infiltration: filling up tissue spaces or Papanicolaou smear
cavities using melted paraffin wax
• Pap smear is a method of screening for
• Embedding/Molding: process of placing
cervical cancer and pre-cancerous
the infiltrated tissue inside a mold.
changes in the cervix. It is also used to
Melted paraffin wax is poured over the
detect STDs
tissue and allows it to solidify
• Trimming: removing excess paraffin
wax from the block until it assumes the
shape of a truncated pyramid
• Sectioning ( microtomy): tissue is cut
into thin slices called ribbons/sections
using a microtome. After sectioning,
ribbons are placed on a floatation water
bath and fished out using tissue Cytopathological Technique
adhesive and glass slide. The ribbons
are placed in an oven to melt the • Cell block - this is a paraffin-embedded
paraffin wax that surrounds the tissue specimen prepared from dried mucus,
ribbons sputum,& debris found in pleural fluids,
pericardial fluids, other sites that
• Staining: use of hematoxylin and eosin
cannot be processed in the routine
dyes to differentiate the cells and
procedure
cellular constituents
• Cytospoin - It is a cytological technique
• Mounting: process of putting the cover
specifically designed to concentrate
slip on the stained tissue using a
cells on a slide in a uniform monolayer
mounting medium
using a high speed centrifuge
• Labelling: identifying the specimen
Frozen Section
Fine needle aspiration biopsy (FNAB)
• Called as Cryosection technique is
• A diagnostic procedure to investigate
performed when an immediate or rapid
superficial masses or lumps. It is less
microscopic analysis of a specimen is
invasive compared to excisional (open)
needed.
surgical biopsy.
Breast Panel

• Consists of biomarkers that are

important in the genetic testing for
breast cancer. Includes: estrogen
receptor assay, progesterone receptor
assay, C-erb-2 (Her2- neu) p-53, and
DNA ploidy analysis

Special staining (Histochemistry)

 • Use of special stains to determine the
chemical compounds and their
distribution within and in between the
biological cells of the body. Ex: alcian
blue, Congo red, oil red O, periodic
acidschiff

Immunohistochemical staining

• Detecting antigens in the cells by using

principle of antibodies binding to
specific antigen

Post mortem examination

• Also known as autopsy or necropsy

• Used to determine the cause of death,
to evaluate any disease or injury that
may have been present