Ultimate Final Coaching

Olibrian Mallari, RMT, MLS(ASCPi),CPT(IMET), MT(AIMS)
Word Bank

Prefix/Suffix Meaning Example
Cyano- Blue Cyanobacteria (Blue-green algae)
Pyro- Fire Pyrococcus (Thermophile)
Pyo- Pus Streptococcus pyogenes (Pus-forming)
Gen- Generator Antigen (Antibody generator)
Rhoda- Red Pyorhodamine (Red pus)
Cytosis- Increase in Lymphocytosis
Penia- Decrease in Cytopenia (Decrease in cell count)
Pan- All Panmyelocytosis (Increase in all myeloid
cells
Erythro- Red Erythrocyte
Leuko- White Leukocyte
Thrombo- Clot Thromobocyte
Blast- Immature Normoblast (immature RBC)
Pro- Comes in between “Blast” and “Cyte” Promegakaryocyte
Cyte- Mature Megakaryocyte
In-vivo Inside
In-vitro In the glass
Meta- After cyte Metamegakaryocyte
Oxia- Oxygen Hypoxia
Capno- Carbon Dioxide Capnophiles (Carbon dioxide loving)
ULTIMATE FINAL COACHING

-Ase Enzyme Hydrogenase
-in Protein Albumin,Thrombin
-ol Alcohol Methanol, Retinol, Cholesterol
Releasing Hypothalamus Thyroid releasing hormone
Stimulating Pituitary Thyroid stimulating hormone
Kinase Phosphate transfer Hexokinase
-Philia Love/Affinity Hemophilia

1

Positive: Electric Blue Fluorescence
Microbiology Negative: Lack of Fluorescence

QC Positive: E.coli
QC Negative: P. aeruginosa
9. Lepromin Test for Leprosy
Must Know Recall Questions Early Reaction: Fernandez Reaction (24-48hours)
1. Chain of infection Late Reaction: Mitsuda Reaction (3-4 weeks)
– Continous link between a source, a method of transmission and 10. Rota Virus leading cause of gastroenteritis in winter months
a host 11. Unholy Three: Hookworm, Ascariasis, Trichuriasis
– Components: 12. Taenia asiatica also known as the “Taiwan Taenia” resembles T.
a. Pathogen saginata
b. Reservoir 13. Tissue aspirate is recommended for recovery of pathogenic
c. Portal of Exit microorganisms
d. Mode of Transmission ü Cotton swab – Neisseria gonorrhea
e. Portal of Entry ü Wooden shaft – Chlamydia trachomatis
f. Host ü Calcium alginate – HSV
ü General Swabs – Anaerobic organisms, fungi and
2. Bipolar bodies can be stained with the use of Wayson Stain Mycobacteria
3. Water Testing – Detection of fecal contamination 14. Snails is the first intermediate host of Paragonimus westermani
a. Presumptive Test – Lactose Broth; + gas formation while freshwater crabs are the second intermediate host

b. Confirmatory Test – EMB + for colonies 15. Haplorchis taichui was discovered in Mindanao, Philippines in
c. Completion Test – Agar Slant for G – nonspore 2004
4. Baumgarten’s Stain – Differentiates MTB (blue) from M. leprae 16. Periplanta Americana is a vector of Ascaris and Trichuris
(red) 17. Parasite LDH (pLDH) – all Plasmodium spp.
5. E. coli IMVIC Reaction (+ + - - ) Histidine rich protein (HRP-2) – Plasmodium falciparum
6. Iodine Tincture refers to an Iodine diluted in alcohol – Tested by Rapid Diagnostic Test
7. Kovak’s Method is a test for Indole, a positive result is indicated
by the formation of a purple color within 10 seconds 18. Horse or Rabbit RBC can be used for testing Hemolysin
8. MUG Test producing Haemophilus
B-D-Glucopyranosid-uronic B-D-glucoronidase> Aglycons + D-glucoronic 19. DNA Hydrolysis Test

2
S. aureus S. epidermidids Virulence Factor

POSITIVE CONTROL NEGATIVE CONTROL

Characteristic Endotoxin Exotoxin

1. Source ü Gram negative ü Gram positive and
ü Gram Stain Gram negative
Primary Stain Crystal Violet 2. Release ü Lysis of the ü Living cell
Mordant Gram’s Iodine cell
Decolorizer Alcohol 3. Composition ü Lipds Proteins/ peptides
ü
4. Heat Stability ü Heat stable Heat labile except
ü
Secondary Stain Safranin
Staphylococcus

5. Toxicity ü Low toxicity ü Highly toxic
ü Acid Fast Staining:
6. Lethal Dose ü Larger ü Smaller
Stain Comments
7. Disease ü UTI, Typhoid ü Tetanus and
Ziehl-Neelsen/ Hot 3 grams of Carbol fuschin + 5%
Associated botulinum
Staining Method phenol
• Limulus Lysate test – uses Horse shoe crub serum (Limulus
Kinyuon’s/ Cold 4 grams of Carbol fuschin + 9%
polyphemus), with the clumping of blood as a positive result
Staining Method phenol

Primary Stain Carbol Fuschin
Mordant Turgitol/Heat
Decolorizer Acid Alcohol

Secondary Stain Methylene BLue

ü Special Stains:

1. Dyar stain – cell wall
2. Hiss, Gin, Anthony and Welch – Capsule
3. LAMB, Neisser, Albert, Ljubinsky – Metachromatic
granules
4. Dorner, Wirtz, and Conklin, Schaeffer-Fulton – endospore
5. Gray, Leifson, Fisher and Conn – Flagella
6. India ink/ Nigrosin – Candida albicans
7. Feulgen Stains – DNA
8. Fontana- Levadite Silver Impregnation - Spirochete

3
Plating Media for Routine Bacteriology New York City agar Neisseria gonorrhoeae,
Ureaplasma urealyticum and some
Medium Organism Mycoplasma
Bile Esculin Agar (BEA) Group D Streptococci and Regan Lowe Bordetella pertussis
Enterococci Salmonella-Shigella (SS) agar Salmonella and some Shigella spp.
Bile esculin azide agar with Vancomycin-resistant enterococci
vancomycin Schlaeder agar Campylobacter and Helicobacter
Blood Agar Cultivation of non-fastidious spp.
microorganisms, determination of Selenite broth Enrichment of isolation of
hemolytic reactions Salmonella spp.
Bordet-Gengou agar Isolation of Bordetella pertussis Skirrow agar Campylobacter
and Bordetella parapertussis Streptococcal selective agar (SSA) Streptococcus pyogenes and
Brain Heart Infusion agar or broth Cultivation of fastidious organisms Streptococcus agalactiae
Buffered Charcoal Yeast Extract Legionella spp., Francisella and Tetrathionate broth Salmonella and Shigella spp.
agar (BCYE) Nocardia except Salmonella typhi
Buffered Charcoal Yeast Extract Legionella spp. Thayer-Martin agar (modified N. gonorrheae and N. meningitidis;
agar with antibiotics Thayer-Martin agar) Francisella and Brucella spp.
Burkholderia cepacia selective B. cepacia from cystic fibrosis
agar patients Thioglycollate broth Supports the growth of anaerobes,
Campy-blood agar Campylobacter spp. aerobes, microaerophiles, and
Campylobacter thioglycollate Campylobacter spp. incubated at fastidious organisms
o
broth 4 C for cold enrichment
Cefsulodin-Irgasan-Novobiosin Yersinia spp. and Aeromonas Thiosulfate citrate-bile salts (TCBS) Vibrio spp.
(CIN) agar agar
Chocolate agar Haemophilus spp., Brucella spp., Todd-Hewitt broth supplemented Streptococcus agalactiae

Neisseria spp. with antibiotics (LIM)
Cysteine-tellurite blood agar C. diptheriae
Eosin Methylene Blue (EMB) agar Green metallic sheen by E. coli Trypticase Soy broth (TSB) Enrichment broth used for
(Levine) subculturin various bacteria from
Hektoen Enteruc (HE) agar Salmonella and Shigella spp. from primary agar plates
other Gram-negative enteric bacilli Xylene lysine desoxycholate (XLD) Salmonella and Shigella spp.
agar
Loeffler’s medium Corynebacterium
MacConkey agar Gram negative organism
MacConkey sorbitiol agar E. coli O157:H7 in stool specimens “O, Lord grant us success.”
Psalm 118:25
Mannitol salt agar Staphylococci
4
Antimicrobial Agents and Antibiotics Sterilization and Disinfection

Sources Antibiotics Biological Indicators:
1. Bacillus subtilis Bacitracin Indicator Comment

Geobacillus stearothermophillus/
Autoclave
2. Bacillus polymyxa Polymyxin Bacillus stearothermophillus
Ionizing Radiation Bacillus pumitis
3. Streptomyces nodosus Amphotericin Dry Heat Oven Bacillus subtilis var Niger
ETO Bacillus subtillis var Globujii
4. Streptomyces venezuelae Chloramphenicol

Level of Specimen Prioritization
5. Streptomyces erytheus Erythromycin

Level Description Specimen
6. Streptomyces noursei Nystatin
1 Critical/Invasive/ Life Amniotic Fluid, blood, brain,
Threatening CSF, Heart valve, pericardial
7. Streptomyces fradiae Neomycin
fluid
2 Unpreserved/ Contaminated Bone, feces, sputum, tissue,
8. Micromonospora purpurea Gentamicin
by Normal Flora other body fluids
3 Quantity is required Catheter tip, urine, tissue
9. Cephalosporium Cephalosporin
4 Preserved Urine, feces, swabs in media
10. Penicillium notatum Penicillin (TCBS)


Susceptibility Testing

1. Disk Diffusion (Kirby Bauer)

Component Comment

Agar MHA

pH of Agar 7-2-7.4

Depth of Agar 4mm
8
Size of Inoculum 1.5 x 10 CFU/L
o
Incubation Time 16-18 hrs. at 35 C Keep calm, and study on.
Size of Filter paper 2.4-2.6 mm

5
Organisms 4. Streptococcus pyogenes ( Group A)- beta- hemolytic

- Streptolysins (toxins that haemolyze red cells)
1. Staphylococcus aureus
- Catalase: decomposes hydrogen peroxide to water Antigenic O2 Stability
and oxygen Streptolysin S Non-antigenic Stable
o Anaerobic Catalase Test – 15% H2O2
Streptolysin O Antigenic Labile
o Aerobic Catalase Test – 3% H2O2

- Coagulase: Clots plasma, interferes with
phagocytosis, facilitates spread in the tissues - Streptokinase, a protease that lyzes fibrin.
o Slide Coagulase/Cell-bound coagulase – - Hyaluronidase: Facilitates spread in the tissues by
bound to cell wall destroying hyaluronic acid. Streptococcal
o Tube Coagulase/Unbound coagulase/Free hyaluronidase is antigenic (antibody formed after
coagulase – free from the cell wall infection).
_____________________________________________________________ - M-proteins (antigens): Anti-phagocytic virulence
factors (different for different strains). Antibodies to
2. Staphylococcus epidermidis M antigens are protective. Selected M serotypes
appear to be associated with rheumatic fever, acute
- Contaminant of medical equipments
glomerulonephritis, and severe S. pyogenes
- Endocarditis and bacteremia following infection by
infections.
implantation of contaminated heart valve
- Erythrogenic toxin: Responsible for the rash seen in
_____________________________________________________________
scarlet fever and is also associated with streptococcal

toxic shock syndrome. It is produced as a result of a
3. Staphylococcus saprophyticus
lysogenic phage present in the streptococci.
- Urinary tract infections in sexually active women - Streptococcal throat

- Scarlet fever (Strawberry tounge)
Test S. aureus S. epidermidis S. saprophyticus
- Erysipelas – erythema and edema

Coagulase + - -
Sucrose + + + _____________________________________________________________
fermentation
Novobiocin S S R

_____________________________________________________________

6
5.
Streptococcus agalactiae (Group A), beta- hemolytic - Schick’s Test, Elek’s test
- CAMP test- arrow- shaped hemolysis _____________________________________________________________
_____________________________________________________________

6. Bacillus Anthracis – “Anthrax Bacillus” 11. Listeria monocytogenes
- large, square ends, spore centrally located, “bamboo - Tumbling end- over- end motility (25C), flagella
rods” _____________________________________________________________
Blood agar Media Medusa Head colony
Gelatin Liquefaction Test Inverted Fir Tree 12. Gardnerella vaginalis
Pearl-String Test Positive - Bacterial vaginosis
- Clue cells
_____________________________________________________________ _____________________________________________________________

7. Clostridium botulinum – “canned good bacillus” 13. Neisseria
-
Animal feces, “snow shoe” subterminal spore, Media Component
swollen Thaye-Martin
- Botulism- food poisoning (incubation pd- 18-24 hrs) Martin-Lewis
- Infant botulism- “floppy infant syndrome” –flaccid; NYC Media
sudden infant death syndrome (3- 20 weeks of age)
_____________________________________________________________
Test N. gonorrheae N. meningitidis M.

catarrhalis
8. Clostridium tetani – “tetanus”
Glu + + -
-Feces of horses and other animals, Mal - + -
“lollipop,drumstick”, terminal spore, swollen
Suc - - -
_____________________________________________________________

lac - - -

9. Clostridium perfringens - “ Welch’s bacillus”
- Surgical incision, soft tissue infection, central spore, _____________________________________________________________
swollen
- BAM – target hemolysis 14. Enterobacteriaceae
_____________________________________________________________
TSI Reaction
10. Corynebacterium diphtheria – “Klebs- Loeffler” bacillus A/Ag - Escherichia, Klebsiella, Enterobacter
- “ Chinese letters or palisades”, metachromatic K/Ag + Salmonella, Proteus
granules K/A - Shigella
- “ Babes- Ernst granules” K/K - Pseudomonas
7

____________________________________________________________
Organism TSI Gas H2S Ind MR VP Cit 16. Pseudomonas aeruginosa
E. coli A/A + - + + - - - Able to grow at 42C, optimum 35C, grape like odor,
S. sonnei K/A - - - + - - tortilla like odor
E. tarda K/A + + + + - - _____________________________________________________________
Salmonella K/A + + - + - +
K. A/A ++ - - - + + 17. Leigonella pneumophilia
pneumonia - Isolate in air conditioning ducts
S. K/A + - - -/+ + + - Legionnaire’s dse (pneumonia, Pontiac fever, wound
marcescens abscesses, encephalitis)
P. vulgaris K/A +/- + + + - -/+ o Indirect Fluorescent Antibody (IFA) –
P. mirabilis K/A + + - + +/- +/- common test for Legionaire’s Disease
P. stuartii K/A - - + + - + _____________________________________________________________
M. morganii K/A + - + + - -
Y. K/A - - +/- + - - 18. Haemophilus
enterocolitica

Distinguishing X factor V factor
LIA
characteristics
K/K + (purple/purple with Salmonella H. influenza Mousey/bleach odor + +
black) H. aegypticus + +
K/A – (purple/yellow) Shigella H. haemolyticus B- hemolytic + +
R/A – (red/yellow) Proteus, Providencia, H. ducreyi School of fish + -
Morganella H. parainfluenzae Mannose fermentation - +

H. paraphrophilus Lactose and mannose - +
_____________________________________________________________ fermentation
_____________________________________________________________
15. Vibrio cholera (Fresh and salty water) “comma bacillus” 19. HACEK group
- Chicken erythrocyte test (CET) • Haemophilus aphrophilus –most commonly encountered
- Shooting star motility • Actinobacillus actinomycetemcomitans- human oral
Classical El Tor cavity, star- like appearing colonies
Vogues Proskauer - + • Cardiobacterium hominis- teardrop appearance
Chicken Erythrocyte - + • Eikenella corodens- “corroding bacilli”, sharp odor of
Polymixin B (50ug) susceptible resistant bleach
• Kingella kingae- capnophilic
8
20. Rickettsiaceae Acid fast bacilli

Organism Disease ü Distinct Acid Fast: Mycobacterium, Cryptosporidium, and Isospora
Typhus group ü Partially Acid Fast: Nocardia, Rhodococcus, Tsukemalia, Legionella
Rickettsia Epidemic typhus micdadei
prowazekii
Rickettsia typhi Endemic typhus Mycology
Orienta Scrub typhus
tsutsugamushi Fungi Disorder Other
Spotted fever group Remarks
Rickettsia rickettsii Rocky mountain Candidia albicans Oral thrush and vaginitis Germ Tube
spotted fever positive
Rickettsia akari Rickettsial pox
Rickettsia australis Queensland tick Cryptococcus Associated with pigeon feces Produces
typhus neoformans melanin-
Rickettsia conorii Fievre boutenneuse like
Rickettsia sibirica Siberian tick typhus pigment in
Q fever caffeic agar
Coxiella burnetii Q-fever Sporothrix schenkii Rose gardeners fever
Ehrlichie
Ehrlichia chaffeensis Human monocyte Coccidioides immitis San Juaquin Valley Fever
ehrlichiosis
Neorickettsia Human monocyte Histoplasma Mississippi Fever; Darling’s Bird and
sennetsu ehrlichiosis capsulatum Disease bat
Anaplasma Human granulocyte droppings

phagocytophilium anaplasmosis
Disease
Ehrlichia ewingii Human granulocyte
mimics
ehrlichiosis
tuberculosis

Blastomyces North American Blastomycosis;
21. Mycoplasma pneumonia (Eaton agent)
dermatitis Gilcrest disease
- Resembles fried eggs, stained by Dienes stain

- Atypical pneumonia (walking pneumonia)

- Tetracycline or erythromycin
_____________________________________________________________

9
Paracoccidoides South American Blastomycosis; Pox virus DNA Largest DNA

braziliensis Mariner’s Wheel Bunya RNA California Enveloped
Virus Encephalitis
Calcivirus RNA Norwalk Virus Naked
Malassezia furfur Tinea versicolor Flavivirus RNA St. Louis Enveloped
Encephalitis;
Phaeoannellomyces Tinea nigra Japanese
werneckii Encephalitis
Trichosporon beigelii White Piedra Reovirus, RNA Double stranded
Rota Virus Winter months
Trichophyton Athlete’s foot Cherry red fever (Rota virus)
Picorna RNA Smallest RNA
colonies
virus
Epidermophyton Ring worm
Summary of Bacteriology
*Reference: Microbiology Notes by Ms. Jessica Joanna Ramos and Mr. Olibrian Mallari
Virology Morphological
Life Cycle BACTERIA Other Name Appearance Disease Condition
1. Attachment Spider-like
2. Penetration Actinomyces colony, Molar-
3. Uncoating israelli tooth colony Lumpy jaw
4. Assembly Actinomycet Madura foot
5. Release Woolsorter's
disease, Black

Virus Nucleic Morphology Other Remarks Bamboo fishing eschars, Emetic
Acid Bacillus anthracis rod appearance syndrome
Hepadna DNA Double and Single Medussa head
Virus Stranded (Dane colony, inverted
Particle) fir tree in
Herpes DNA Icosahedral Enveloped Bacillus cereus gelatin agar Fried rice poisoning
Simplex Bartonella
Virus quintana Trench fever
Epstein DNA Kissing Disease Bordet-
Barr Virus Bordetella Gengou
HPV DNA Genital Warts Naked pertussis bacillus Mercury droplet Whooping cough

10
Borrelia Daisy head

burgdorferi Lyme fever Corynebacterium colonies in
Borrelia diphtheriae var cystein tellurite
recurentis Relapsing fever gravis agar
Bang's Undulant/Malta Corynebacterium
Brucella abortus bacillus fever diphtheriae var Frog eggs
Burkholderia Vietnamese time intermedius colonies
pseudomallei bomb Corynebacterium
Campylobacter diphtheriae var Coolie hat
jejuni Sea-gull shape mitis colonies
Capnocytophaga Gliding motility Coxiella burnetti Q fever
Chlamydia Lymphogranuloma EHEC HUS
trachomatis venereum Pitting of the
Canned Eikenella agar with Human bite
Clostridium good's Snow-shoe corodans bleach like odor infection
botulinum bacillus appearance Fish-eye
Oval and colonies on
Clostridium subterminal Pseudomembranou EMB, yellow
difficile spore s colitis pigment
Box car intensifies at
appearance, Enterobacter 25C
Horse manure Test tube brush
Welch's odor in CCFA, Erysiplothrix appearance of
bacillus, Double zone of rhusiopathiae colony Erysipeloid
Clostridium hemolysis, Green metallic

welchii, Gas stormy clot Escherichia coli sheen on EMB
Clostridium gangrene appearance in Montezuma's
perfringens bacillus Litmus milk Gas gangrene revenge, Traveler's
Round and ETEC diarrhea
Tackhead terminal Francisella
Clostridium tetani bacillus swollen spore Risus sardonicus tularensis Rabbit fever
Metachromatic Gardnerella
granules, vaginalis Bacterial vaginosis
Kleb- Poached egg Haemophilus Koch-Week's
Corynebacterium Loeffler's colonies in Pai's aegypticus bacilli Pink-eye infection
diphtheriae bacillus slant
11
Haemophilus Ducrey's School of fish Soft chancre/ Walking

ducreyi bacilli appearance Chancroid pneumonia
Exhibits Gonococcal
satelitism vaginitis,
Haemophilus Pfeiffer's around S. Neisseria Ophthalmia
influenzae bacillus aureus gonorrhea neonatorum
Helicobacter Waterhouse-
pylori Peptic ulcer Neisseria Friedrichsen
Klebsiella Friedlander's meningitidis Syndrome
pneumoniae bacillus Nocardia Partially acid
Legionella asteroides fast
pneumophilia Pontiac fever Orientia
Leptospira tsutsugamushi Scrub typhus
interogans Weil's disease Mushroom
Tumbling Pasteurella smell Shipping fever
motility, Swarming
Umbrella-like Proteus motility
Listeria appearance in Granulomatis Glander's
monocytogenes Gelatin agar infantiseptica Pseudomonas bacillus
Morganella Morgan's Sweet or grape-
morganii bacillus like odor,
Mycobacterium Tap water Pyocyanin and
gordonae bacillus Pseudomonas pyoveridin,
Mycobacterium Yellow aeruginosa tortilla like odor Swimmer's ear
kansasii bacillus Rickettisa conorii Boutonneuse fever

Mycobacterium Hansen's
leprae bacillus Rickettsia akari Rickettsial pox
Mycobacterium Swimming pool Rickettsia
marinum granuloma prowazekii Brill-Zinsser Disease
Mycobacterium Koch's Rocky mountain
tuberculosis bacillus Rickettsia ricketsii spotted fever
Mycobacterium Rickettsia typhi Murine typhus
ulcerans Inert bacillus Salmonella Gartner's
Eaton's agent, enteritidis bacillus
Mycoplasma Fried egg Primary atypical Enteric fever, pea-
pneumoniae appearance pneumonia, Eberth's soup stool, Typhoid
Salmonella typhi bacillus fever
12
Red coloration Gram-positive

Serratia of medium due Streptococcus Diplococcus lancet
marcescens to Prodigiosin pneumoniae laneolatum diplococci Lobar pneumonia
Serratia odoferi Potato like odor Scarlet fever, Flesh-
Boyd's Streptococcus eating bacteria,
bacillus, pyogenes Erysipelas
Newcastle- Streptococcus Bacterial
Manchester viridans endocarditis
Shigella boydii bacillus T. pallidum ssp.
Shigella Shiga's carateum Pinta
dysenteriae bacillus T. pallidum ssp.
Strong's pertenue Yaws
bacillus, Pfeiffer's
Flexner's phenomenon;
baciilus, Comma shooting star Rice water stool,
Stanley Vibrio cholerae bacillus motility Gastroenteritis
Shigella flexneri bacillus Kanagawa
Sonne-Duval phenomenon;
Shigella sonnei bacillus Vibrio summer
parahemolyticus diarrhea
Spirillum minor Sodoku disease
Yersinia Bull's eye
Jet black colony enterocolitica appearance
( Tellurite
Safety pin
Glycine Agar);
Plague appearance,
Staphylococcus Mauve colored Scalded skin
bacillus, stalactite
aureus colony syndrome

Black plague pattern in broth
Staphylococcus Slime Yersinia pestis bacillus culture
epidermidis production

Staphylococcus
saprophyticus Honeymoon cystitis
Streptobacillus Ratbite fever,
moniliformis Haverhill fever
Streptococcus Neonatal
agalactiae meningitis
Dental carries
Streptococcus (Ruthia,
mutans Fusobacteria)
13
Chilomastix
Nipple-
like
mesnili
cyst
Parasitology Endolimax

Cleft-
like

intestinalis cytoso
me
Other Other Old
Family Type Name DS IS Disease World,
Name Notes
Oriental
Sarcomastigophora sore,
Leishmania
"glss Aleppo
tropica
like button,
Tear- Jericho
cytopla
drop boil,
Entamoeba sm"/
ulcer/Fla Dehli boil
histolytica Swiss
sk Shape American
cheese Dr.
ulcer cutaneou
cytopla Monte
sm Leishmania s, Bubas,
negro
Gay braziliensis Espundia,
Skin
Entamoeba bowel Chiclero
Sarcodin Test
Amoeba hartmanni syndrom ulcer
a Kala-
e
Non- azar,
Entamoeba Dum-
pathologi Leishmania
coli dum
c donovani
Crosse fever,
Endolimax Black-
d-eye
nana fever
cyst
No Trypanoso Chaga's
Entamoeba
Cystic ma cruzi Disease
gingivalis

stage East
Trypanoso
Jerking African
Trichomona ma
motilit Sleeping
s vaginalis rhodesiense
y Sicknes
Smalle West
Trypanoso
Trichomona st African
ma
s tenax Tricho Sleeping
Mastigop gambiense
Flagellates monas Sickness
hora Macro
Pear
shape, nucleu
Old s
Giardia Flatule Balantidiu
Man's Ciliata (kidney
lamblia nce m coli
Glasses shape),
odor
stool Micron
ucleus
14
(dot Embr
Dwarf Embry
shape) Hymenolepi yonat
Tapew onate
s nana ed
orm d Egg
Plasmodium egg
Flour
Schuff with
Plasmodiu Tertian Hymenolepi Cystiic
ner's Egg rat
m vivax Malaria s diminuta ercus
Dots droppi
Plasmodiu Quartan Zeiman ngs

m malariae Malaria n's Dot Giant Une
Game
Sporoz Cresce Fasciolopsis Intesti mbry Metac
tocyt
Plasmodiu oite Malignan nt buski nal onate ercaria
e
m t Tertian shape Fluke d egg
falciparum malaria gamet Une
ocyte Garriso
Echinostom mbry Metac
Plasmodiu James n's
a ilocanum onate ercaria
m ovale Dot Fluke
d egg
Platyhelminths Intestinal Smalle
Flukes Heterophye Embr st
Fish
s yonat Metac Deadli
Tapew Une
Diphyllobot Procer heterophye ed ercaria est
orm/Br mbry
hrium coid Anemia s egg fluke in
oad onate
lattum larva man
Tapew d egg
Embr
orm Metagonim
yonat Metac
Eggs/ us
ed ercaria
Pork Gravi yokogawai
Taenia Cystiic Neurocys egg
Tapew d Tremato
solium ercus ticercosis Old
orm Progl da
Embr Fashio
ottid Chines
Chlonorchis yonat Metac n
Eggs/ e liver
sinensis ed ercaria Electric
Beef Gravi fluke

Taenia Cystiic egg bulb
Cestoide Tapew d
Tapeworms saginata ercus egg
a orm Progl
Embr
ottid Cat
Opistorchis yonat Metac
Eggs/ liver
felineus ed ercaria
Gravi Embry fluke
Multiceps Liver Fluke egg
d onate Counerus
multiceps South
Progl d Egg Embr
East
ottid Opistorchis yonat Metac
Asian
Echinococc Hyda Embry viverini ed ercaria
Hydatid Liver
us tid onate egg
disease fluke
granulosus cyst d Egg
Embr
Gravi Dicrocelium
yonat Cercari
Dipylidium d Cystice Cysticerc dendriticu
ed a
caninum progl rcoid oid m
egg
ottid
15
Une Filarifo Ground

Sheep
Fasciola mbry Metac Hookworm Egg rm itch/ Dew
liver
hepatica onate ercaria larva itch
fluke
d egg New
Filarifo
Scistosoma Necator World
Cercari Egg rm
haematobi Egg americanus Hookw
a larva
um orm
Schistosom Old
Blood Fluke Cercari Ancylostom Filarifo
a Egg World
a a Egg rm
japonicum Hookw
duodenale larva
Schistosom Cercari orm
Egg
a mansoni a Ancuylosto Dog Filarifo
Une ma Hookw Egg rm
Paragonim Orienta Coffee
mbry Cercari caninum orm larva
Lung Fluke us l Lung bean
onate a Ancyloosto Cat Filarifo
westermani Fluke shape
d egg ma Hookw Egg rm
Minda braziliense orm larva
Other Haplorchis
nao, Rhab
Flukes taichui Filarifo Cochin-
2004 Stongyloide Thread ditifo
rm China
Nematode s stercoralis Worm rm
larva Diarrhea
Larva
Une Wuchereria Micro Elephanti
Embry Rectal L3
Trichuris mbry bancrofti filaria asis
onate prolaps
trichiura onate Mic
d Egg e Brugiya Malayan
d egg rofila L3
Capillaria malayi Filariasis
Pudoc ria
philippinen Onchocerca River
Worm Filarial
Adenoph sis volvolus Blindness

orea Egg is Eye
Capillar
Capillaria pitted Worm,
y liver
hepatica with Loa loa African
worm
pores

Eyewor
Trichin Encys Encyst m
Trichinella
a ted ed Guinea
spiralis
worm larva larva Worm,
Giant Servan
Ascaris Unfer
Intesti Fertiliz Ascaris t
lumbricoide tilize Dracunculu
nal ed egg bolus Worm,
s d egg s L1 L3
Worm Fiery
Oxyuris medinensis
Secernen Serpen
vermic t of
tea Embr
Enteroobius ularis, Embry Israelit
yonat Pruritis
vermiculari Pin onate es
ed ani
s worm, d Egg
egg
Seat
worm

16
7. Abnormal Lipoproteins
ü B-VLDL – “Floating B-lipoprotein”
Clinical Chemistry ü Lp(a) – similar with LDL but migrates with VLDL, “sinking B-
Lipoprotein”

ü LpX – LCAT deficiency, obstructive biliary disease
Must Know Recall Questions Type 1 DM Type 2 DM
1. Visible light has a wavelength of 400-700 nm Frequency 5-10% 90-95%
380-420 nm Violet Age of Onset Children and in More common in
young adults advancing age
420-450 nm Indigo
Pathogenesis Destruction of NO autoimmunity
450-490 nm Blue pancreatic beta cells
490-570 nm Green due to autoimmunity
570-590 nm Yellow C-peptide level Very low Very high
590-630 nm Orange Pre-diabetes Autoantibodies (GAD Absent
630-750 nm Red 65) autoantibodies

Medication therapy Insulin is necessary Oral agent

2. Diacetyl Method is an example of a colorimetric method for Urea
Nitrogen and the enzymatic method is based on the formation of
Ketosis Prone Absent
NH3. Isotope dilution mass spectrometry is considered as the 8. Glucose metabolism at room temperature is 7 mg/dL and 2 mg/dL
o
reference method at 4 C
3. Jaffe Reaction 9. Enzymatic Tests
Creatinine + Picric Acid ------------à Janovski Complex/ Red a. Fixed Time Method – reaction will proceed at a designated
tautomer time, reaction is then stopped by means of adding a weak acid
4. Van Den Berg Reaction for Bilirubin b. Kinetic Method/Continous Monitoring – multiple

Bilirubin + Diazotized Sulfanilic Acid ----à Azobilirubin measurements, usually of absorbance change during a reaction
5. Evelyn Malloy Test is a test for Indirect bilirubin, it is performed in 10. Michaelis-Menten Curve refers to a graph showing the relationship
an acid pH and utilizes Methanol as a dissociating reagent, the end between the reaction velocity and substrate concentration which
product is a red or reddish purple solution can be measured as:
6. Hyperlipoproteinemia Pattern V = V max (S)/Km +(S)
11. IV infusion should be stopped 2 min prior to venipuncture, presence
of at least 5% dextrose can increase blood glucose level of up to 500
Type I IIA IIB III IV V mg/dL
Pattern TAG LDL LDL,VDL TAG, B- VLDL VLDL, 12. Drugs of Abuse

VLDL Chylo ü Ethanol – grain alcohol
17
ü Phencyclidine – angel dust 3. Gaussian Curve – Also known as bell shaped curve
ü Cannabis – Hashish 4. Levey-Jennings Chart – Widely used QC chart in the
ü Opiates – source of Heroin and Hydrococcaine laboratories
Analytical Methods
13. Parts of adrenal cortex:
1. Atomic Absorption Spectrophotometry
ü Zona glomerulosa – mineralocosticoids (SALT) 2. Spectrophotometry
ü Zona fasciculate – glucocorticoid (SUGAR) 3. Volumetry/ Titrimetry
ü Zona reticularis – (SEX) 4. Potentiometry
14. Reinsch Test for Heavy Metal poisoning present in biological 5. Polarography
samples. Utilizes 10% copper in aluminum foil 6. Coulometry
ü Mercury – Silver 7. Amperometry
8. Electrophoresis
ü Arsenic – Flat black
ü Antimony – Blue-Black

Laboratory Supplies
1. Pipets
a. TD – water calibrated
b. TC – mercury calibrator
c. Blow-out – continuous ring at the mouth
d. Self-draining – absence of a continuous ring at the mouth
2. Centrifuge – Calibrated using a tachometer
3. Chemicals
a. ACS – American chemical society; highest in purity

b. USP – for drug manufacturing
4. Water
a. Type I – most stringent purity
b. Type II – most commonly used in the laboratory
c. Type III – used for cleaning laboratory materials
Quality Control
1. Twin Plot / Youden Plot – Used when analyzing interlab
data
2. Cumulative Sum Graph – Used to identify the difference
between results and the target value
18
Metabolic Pathway
9. Chromatography
Thin layer – clinically used for urine drug screening Pathway Comment
Glycolysis metabolism of Glu to pyruvate or lactate
i. Gas-liquid – used to separate volatile
solutes Gluconeogenesis Formation of G-6-PD from non carbohydrate
ii. High performance liquid – uses pressure for sources
fast separations; used to separate
hemoglobin, lipids, drugs Glycogenolysis Breakdown of glycogen to glucose for energy use
iii. Ion exchange – used for separation of
amino acids, proteins and nucleic acids Glycogenesis Conversion of glucose to glycogen
iv. Affinity – used to separate lipoproteins, Lipogenesis Conversion of carbohydrates to fatty acids
carbohydrates, and antibodiees
10. Chemiluminiscence Lipolysis Breakdown of fat
11. Flame Emission Photometry
12. Turbidimetry
13. Fluorometry
14. Nephelometry Glycogen storage disorders
ü Liver damage – Type I, III, IV, VI, IX, 0
Carbohydrate ü Muscular damage – Type V, VII
ü Type I – the most common type
Glucose
- Glucose can be directly used or stored into glycogen Type Other name Enzyme deficient
- storage form of glucose
in the liver Ia Von Gierke Glucose-6-phosphatase
- measured almost in all body fluids

• metabolized at RT with a rate of (7 mg/dL/hour) II Pompe 1, 4 glucosidase
o
• at 4 C the loss is approximately 2 mg/dL/hour
IIIa Cori Forbes De brancher (liver and muscle)

IV Andersen Brancher

V Mc Ardle Muscle phosphorylase

VI Hers Liver phosphorylase

19
VII Tarui Phosphofructokinase - Converted in the liver to form cholic acid (bile)

VIII Adenyl kinase 5. Lipoproteins
- Usually separated by centrifugation (Ficolle
IXa Phosphorylase kinase (liver) Medium/Potassium Bromide)
Lipoprotein S.G. Apolipoprotein
IXb Phosphorylase (muscle and liver) Chylomicron <0.95 Apo B-48
VLDL 1.006 Apo B-100 & Apo B-48
X Cyclic AMP dependent kinase
LDL 1.063 Apo B-100
XI Fanconi-Bickel Glucose transporter-2 HDL 1.21-1.23 Apo-A
Lp a Apo-A and Apo B-100
0 Glycogen synthetase
a. Very Low Density Lipoprotein (VLDL)
o Fridewald Method
Lipids Profile VLDL (mg/dL) = Triglyceride/5
VLDL (mmol/L) = Triglyceride/2.175
Members
o De Long Method
1. Fatty Acids VLDL (mg/dL) =
- Linear chains with a carboxyl group Triglyceride/6.5
VLDL (mmol/L) = Triglycerid/
2. Triglycerides or Triacylglycerol 2.825
- Contain three fatty acids attached to one molecule of
glycerol b. Low-Density Lipoproteins

- Saturated form will be solid at room temperature while
unsaturated will be liquid at room temperature LDL = Total Cholesterol – HDL – (Triglyceride/5)
Chemical Composition of Lipoproteins
3. Phospholipid *Table from Dean Teresita Rodriguez
TAG Chole Free Phospholipid Protein

- Forms of Phospholipid:
Ester Chole
o Lecithin (70%)
Chylomicron 80- 2-4% 1-3% 3-6% 1-2%
o Sphingomyelin (20%)
95%
o Cephalin (10%)
VLDL 45- 16-22% 4-8% 15-20% 6-10%
4. Cholesterol 65%
LDL 4-8% 45-50% 6-8% 18-24% 18-22%
- Exists as a mixture of unesterified (30%) and esterified (70%)
form HDL 2-7% 15-20% 3-5% 26-32% 45-55%
20
Proteins Blood Urea Nitrogen

Major Proteins - Major non protein nitrogen compound in the blood

1. Prealbumin Pathophysiology
- Aka Transthyretin
- Transporter of thyroid hormones Decreased Pre renal Increase Renal Increase Post Renal
2. Albumin Urea Increase
- Binds with many analytes to be transported in the blood Ø Decreased Ø congestive Ø acute and Ø urinary tract
- Major protein responsible for maintaining oncotic pressure protein heart failure chronic obstruction
3. Alpha-1 Antitrypsin intake Ø shock, renal
- Acute phase reactant and a protease inhibitor that neutralizes Ø Severe hemorrhage failure
trypsin type enzymes that can damage structural proteins liver Ø dehydration Ø glomerular
4. Alpha-1 fetoprotein disease Ø increased nephritis
- Synthesized in the yolk sac and liver of the fetus Ø Pregnancy protein Ø tubular
- Peaks at 13 weeks and declines at 34 weeks Ø diarrhea catabolism necrosis
5. Alpha 1 acid glycoproteins Ø corticosteroid
- Aka orosomucoid therapy
6. Haptoglobin
- Binds with free hemoglobin Uric Acid
*Reference: Table by Ms. Ana Leah NAvarro
7. Ceruloplasmin
Method Comments
- Copper containing protein
Colorimetric Interferes with drugs
8. Alpha-2 macroglobulin
UV Enzymatic Needs optical cells
- Inhibits thrombin, trypsin and pepsin
Peroxidase Test Reducing substances cause
9. Transferrin
interference

- Beta globulin that transports iron

Creatinine/ Creatine
Methods for Testing

- Waste product of muscle contraction
1. Biuret Method

- Violet end product

2. Kjeldahl Method
“Every accomplishment starts
- Considered as the reference method
with the decision to try
- It is based on the quantitation of nitrogen content of protein

21
1. Creatinine Clearance Type of Non polar polar Polar

- Used to assess the Glomerular Filtration Test compound
- Requires serum sample
ü Delta Bilirubin
o Delta Bilirubin = Total Bilirubin – Direct
Bilirubin + Indirect Bilirubin

Disorders Related to Conjugation and Excretion

Disease Comments
Gilbert’s syndrome (Increase B1) Transport defect; associated with

Uridine diphosphate
Liver Function Test
Criggler-Najar syndrome Abnormal conjugation
Types of Bilirubin (Increase B1) Has 2 types: Type 1 (Severe) and Type
*Table from Dean Teresita Rodriguez
2 (less severe)
Unconjugated Conjugated Delta
Dubin-Johnson syndrome Impaired liver handling of conjugated
Solubility in No Yes Yes (Increase B2) bilirubin

water

Affinity for brain Increased Decreased Decreased Rotor syndrome (Increase B1) Viral infection; impaired excretion of
tissue conjugated bilirubin

Solubility in Yes No No
Enzymes

organic solvents

Half life Less than 24h Short Long Introduction
ü Enzyme Theories:
Site of Reticulo- Liver Serum o Emil Fisher’s Theory/ Lock and Key Theory – substrate
production Endothelial (key) must fit to the enzyme (lock)
o Kochland’s/Induced Fit Theory – substrate binding to the
System of
active site of the enzyme
spleen
- Apoenzyme is the protein portion of the enzyme
Reaction Indirect Direct Direct
- Co-factors are the non-protein portions of the enzyme; can be
organic or inorganic
22
o Co-enzyme – organic portion Acid Phosphatase

o Activator – inorganic portion
- Zymogens are inactive forms of enzyme - Can be found in high concentrations in prostate gland secretions
and erythrocytes
Inhibition of Enzymatic Reaction - Catalyzes at pH 5.0 – 6.0
1. Competitive inhibition – inhibitor binds to the active site
2. Non-competitive inhibition – inhibitor binds to the allosteric Alkaline Phosphatase
site
3. Uncompetitive inhibition – inhibitor binds to the Enzyme- - Similar with ACP but reacts at pH 9.0-10.0
substrate complex
Isoenzyme Separation
Classification of Enzymes
1. Electrophoresis
Frequent Enzymes (Cathode) Intestinal > Placental > bone > Liver
Prostatic Disorder ACP (Anode)
Hepatic Disorder ALT, ALP, AST, GLD, GGT, LDH, 5’- 2. Heat Denaturation/ Serum inactivation
NT, PChE, GST (Heat Stable) Placental > Intestinal > Liver > bones
Skeletal Muscle Disorder ALD, AST, CK, (Heat labile)
Bone Disorder ALP
3. Chemical Inhibition
Acute Pancreatitis AMS, LPS, TRY
Inhibitor Isoenzyme
Blood Pressure Regulation ACE
Leucine Nagao
Myocardial Infarction AST, CK, GP, LDH
Urea Bone
Chronic Pancreatitis CHY, E-1
Levanosol Bone and liver
Drug induced hemolytic anemia G-6-PD
Phenylalanine Placenta, intestine, Regan,
Hemolytic anemia PK, LDH,
Nagao

Acronyms:
Lactate Dehydrogenase
ACE – Angiotensin converting enzyme
- It is a nonspecific enzyme
CHY – Chymotrypsin
- For AMI it rises within 12-24hours and peaks at 48-72 hrs and
E-1 – Elastase – 1
normally returns after 10days
GLD – Glutamate dehydrogenase

GST – Glutathione S-transferase
Creatine Kinase
GP – Glycogen phosphorylase
Isoenzyme Concentration
PChE – Pseudocholinesterase
TRY – Trypsin CK MM 99% skeletal muscle; 80% heart
CK BB 90% brain
CK MB 20% heart
23
Aspartate Aminotransferase ACTH Addison’s

Disease (dec)
- Formerly known as SGOT (Serum glutamic oxaloacetic GH Bone growth
transaminase) Prolactin Milk production
Posterior Vasopressin Blood pressure
Alanine Aminotransferase Oxytocin Constriction of
Neurohypophysis uterus
- Formerly known as SGPT (serum glutamic pyruvic transaminase)
Adrenal Gland
Amylase Adrenal Cortex Aldosterone Salt Conn’s
Disease
- Catalyzes the hydrolysis reaction of Ca and Cl (increase)
Cortisol Sugar Cushing
Lipase Syndrome
(Increase)
- Used as a marker for acute pancreatitis Androgen Sex
Adrenal Medulla Catecholamines Blood pressure
Gamma Glutamyl Transferase regulation
- Elevated levels may indicate alcoholism Reproductive
Gland
Endocrinology Male Testosterone Sperm
development
Gland Hormone Function Disease Female Estrogen Implantation of
Hypothalamus GRH egg
TRH Progesterone Female

Corticotropin characteristic
Somatostatin Inhibitor of TSh Thyroid Gland Thyroxine Cretinism
and GH (decrease T4)
Dopamine Prolactin Grave’s
release Disease
Pituitary Gland (Increase)
Anterior LH Egg maturation Klinefelter’s Calcitonin Calcium and
FSH Syndrome Phosphate
Adenohypophysis (increase) regulation
TSH T3 and T4 Parathyroid PTH Calcium
production Gland regulator

24
Hypothyroidism Hyperthyroidism Toxicology

T4 DECREASE INCREASED
T3 DECREASE INCREASED
TBG NORMAL NORMAL Blood Alcohol Levels
TSH INCREASED DECREASED
LEVEL INTERPRETATION
Electrolytes 0.01-0.05 No obvious impairment, changes observable
A. Sodium only on performance testing
- Major cation in the extracellular fluid 0.03-0.12 Mild euphoria, decreased inhibitions, some
- Principal osmotic particle outside the cell impairment of motor skills
0.09-0.25 Decreased inhibitions, loss of critical
B. Potassium judgement, memory impairment
- Major cation intracellular 0.18-0.30 Mental confusion, dizziness, impaired motor
skills
C. Chloride 0.27-0.40 Impaired consciousness, vomiting, failure to
- Major extracellular anion stand or work
0.35-0.50 Coma and possible death
D. Bicarbonate
-
nd
2 most abundant anion in the extracellular fluid Tests for Clinical Chemistry
- Major component of the buffering system in the blood
Tests for Clinical Chemistry
Analyte Test Reagent Result Comment
E. Calcium
th Alkaline
- 5 most common element in the body
Copper Cupric to

Reduction Cuprous

F. Phosphate
Alkaline
- Fiske Subbarow method – 600-700nm
Ferric

Reduction Colorless Hagedorn
G. Magnesium Glucose
nd
Method Ferrocyanide Method
- 2 most prevalent intracellular cation Phosphomol
o Calgamite Method – reddish violet complex (532nm) ybdenum
o Formazen Dye – colored method (660nm) Folin Wu Blue First Test
o Methylthymol blue Nelson Arsenomolyb
o AAS – reference method Somogyi denum Blue

25
Yellow- Acetic Acid,

Neocuprine Yellow Sulfuric Acid,
Method Orange Leibermann- Acetic
Modificatio Burchard Anhydride Green
Benedict's Brick Red n of Folin Zak and Acetic Acid,
Test ppt. Wu Boyle Sulfuric Acid
o-Toluidene Dubowski Violet;
Method Method Peptide
Glucose Most Biuret Test Copper Bond
Oxidase specific Nitrogen
Method Test Protein Kjeldhal Test Bonde
Most Dye-Binding
Hexokinase accurate Test BCG
Method test Ninhydrin
2-hour Post Test Violet
Prandial Urease, and
Test 75g of Glu Glutamate
Janney- Berthelot's Dehydrogena Classical
Isaacson Urea Test se Test
Method 75g of Glu Caraway Phosphotungs Tungsten Classical
Exton Rose Uric Acid Test tic Acid Blue Test
Method 150g of Glu Red
HbA1c 2-3 months Tautomer/Cr
Fructosamin eatinine Classical
e 2-3 weeks Jaffe Test Picric Acid Picrate Test

Zilversmit- 4-step Red
TAG Van Handel method Tautomer/Cr
Pearson, eatinine
Creatinine
Stern, Mac Folin Wu Picric Acid Picrate
Gavack 1-step Sodium
Alumina
Cholesterol Bloor's 2-step Lloyd's Test Silicate
Abell- Aluminum
Kendall 3-step; CDC Fuller's Magnesium
Parekh and 4-step Earth Test Silicate
Jung method
26
Creatinine Requires
King- Phenylphosph
Clearance protein
Armstrong ate
Test 24-hour urine removal
Van Den Sulfanilic acid, p-
Bessy- Endpoint
Berg Sodium Diazotizati nitrophenylph
Lowry-Brock or kinetic
Reaction Nitrite, HCl on osphate
Caffeine p-
Bowers- Reference
Jendrassik- Sodium nitrophenylph
Bilirubin McComb method
Groff Benzoate osphate
Evelyn Forward
Wacker Test
Malloy Methanol Pyruvate Test
LDH
Potassium Wrobleuski Reverse
Icterus Index Dichromate La Due Lactate Test
The assay Tanzer- Creatine Forward
B-
is long and Gilvar Phosphate Test
Bodansky glycerophosp CK
is not Oliver Reverse
hate
specific Rosalki Creatinine Test
Gutman, Cherry-
Phenylphosph
King- Nonspecific Crandall
ate
Armstrong Lipase Test Olive Oil Triolin
p- The assay
Hudson nitrophenylph is fast but
osphate not specific
ACP
Recommen
a-
Babson and ded for
naphthylphos

Reed continuous
phate
monitoring
Thymolphthal Most
Roy
ein specific
4-
Reitz, methylumbell Fluorescen
Guilbault iferonephosp t assay
hate
Shinowara- B- Long “Success doesn’t come to you,
ALP Jones- glycerophosp incubation you GO for it.”
Reinhart hate time

27
9. Red Cell Grading:

Morphology Grading
Stomatocyte
Target Cell

Hematology Ovalocyte 1+ = 3 to 10/field
Poikilocyte 2+ = 11 to 20/field
Burr Cell 3+ = >20/field
Elliptocyte
Must Know Recall Questions! Howell-Jolly Bodies
1. Apply tourniquet around the arm around 3-4 inches above the site
Pappenheimer Bodies Positive
of choice
Basophilic Stippling
2. 200-cell differential count is made if there are more lymphocytes Sickled RBC
than neutrophils
10. Grading of Hypochromia (Low hemoglobin)

3. Causes of too Blue Stains:
1+ = Area of central pallor is 1/2 of diameter
ü Thick film
ü Prolonged staining time
ü Decrease washing
4+ = Thin rim of Hemoglobin
ü Too alkaline stain
11. The major cause of cell death in sickle cell anemia is bacterial
4. Causes of too Red Stains:
infection
ü Thin smears
12. Heparin is the preferred anticoagulant of choice when performing
ü Decreased staining time
Leukocyte Alkaline Phosphatase (LAP) staining, which is then
ü Prolonged washing
counted using the Kaplow Method

ü Too acidic stain
13. Citrated whole blood is the sample of choice when performing a
5. Eosinophils are increase in the afternoon due to increase in ACTH,
Sugar water test, a positive result can be confirmed using Ham’s
and lowest in the morning
Acidified Test which utilizes whole blood
6. Platelet satellitosis is due to excessive EDTA
14. Grading of Rouleaux:
7. Thrombocytopenia is caused by an excessive blood transfusion
1+ = Aggregates of 3-4 RBC
8. Spun Hematocrit:
st
2+ = Aggregates of 5-10 RBC
ü 1 Layer: Fatty layer/Chylomicrons
nd
3+ = Numerous aggregates with only few free RBC
ü 2 Layer: Plasma
rd
15. Rule of three:
ü 3 Layer:Buffy Coat
3x RBC = Hemoglobin

3 x Hemoglobin = Hematocrit + 3%

28
Hematopoiesis produces Gower 1,

cells, granulocytes, and committed
Gower II, and monocytes and progenitor cells are
- Production and differentiation of blood cells Portland, the megakaryocytes maintained in the
- Occurs in the RES (Reticuloendothelial system) embryonic
make their marrow.
o Medullary Hematopoiesis – Blood cell production hemoglobins.
appearance.
within the red bone marrow
o Extramedullary Hematopoiesis – Blood cell Hemoglobin A and
production outside the bone marrow A2 become present
- Stem Cell Theory – development of Pluripotent stem cells to at this stage.
committed progenitor cells
Erythrocytes
Committed Progenitor Growth Factors Mature Cells
Cells MATURATION AND MORPHOLOGY
CFU-MEG Thrombopoietin, Thrombocyte 1. Pronormoblast/Rubriblast
GM-CSF 2. Basophilic normoblast/ Prorubricyte
CFU-GM CFU-M GM-CSF, M-CSF, Monocyte o Hemoglobin synthesis begins in this stage
IL-3 3. Polychromatophilic normoblast
CFU-GM CFU-G GM-CSF, G-CSF, Neutrophils - Last stage of mitosis occurs in this stage
IL-3 4. Orthochromatic normoblast/metarubriciyte
BFU-E CFU-E Erythropoietin, Erythrocyte - No more cell division
GM-CSF, IL-3 5. Reticulocyte
CFU-EO Gm-CSF, IL-3, IL- Eosinophils 6. Erythrocyte
5
CFU-BA IL-3, IL-4 Basophils Hemoglobin


MESOBLASTIC HEPATIC PHASE MYELOID/MEDULLARY - 1 globin molecule and 4 heme molecules
PHASE PHASE - Each gram contains 1.34 mL of oxygen.

th th
Starts at first 2-8 Starts at 6 week of Begins at 5 month of Hemoglobin structure
weeks of life. gestation. gestation. The bone 1. Heme portion
Formation of a nd
By the 2 month of marrow becomes the - this structure involves four iron atoms in the ferrous state
primitive vascular surrounded by Protoporphyrin IX, or the porphyrin ring, a structure
gestation, liver main organ of
system. formed in the nucleated red cells
First hematopoietic becomes the main hematopoiesis.
cells are generated site of Myeloid: Erythroid ratio 2. Globin portion
in the yolk sac hematopoiesis. Aside becomes 3:1. - consist of amino acids linked together to form a polypeptide chain,
(erythroblasts) and from the red blood Hematopoietic stem cells a bracelet of amino acids.
29
- Alpha chains have 141 amino acids in a unique arrangement, and Methemoglobin ü Causes “cyanosis” which can be 630 nm
beta chains have 146 amino acids in a unique arrangement treated with Methylene blue
ü Chocolate brown in color
Oxygen dissociation curve
Cyanmethemoglobin ü Forms when ferricyanide binds with 540 nm

ferrous iron in the presence of
potassium cyanide
Sulfhemoglobin ü Combination of inorganic sulfides 618 nm
and hemoglobin
ü It is irreversible
ü Mauve-lavander colour

Types of Hemoglobin
ü Chromosome 11 contains the genes for the
production of epsilon, beta, gamma, and delta chains
ü Chromosome 16 is responsible for the alpha and
zeta genes.

SHIFT TO THE RIGHT SHIFT TO THE LEFT HEMOGLOBIN GLOBIN CHAIN TYPE
pH ↓ pH ↑
O2 ↓ O2 ↑ Gower I 2 zeta, 2 epsilon
PCO2 ↑ PCO2 ↓
Gower II 2 alpha, 2 epsilon
Temperature ↑ Temperature ↓
2,3 DPG ↑ 2,3 DPG ↓ Portland 2 zeta, 2 gamma

Hemoglobin F 2 alpha, 2 gamma
Hemoglobin derivatives Hemoglobin A1 2 alpha, 2 beta
Hemoglobin A2 2 alpha, 2 delta
Type of Hemoglobin Comments WL
Oxyhemoglobin ü Scarlet red in colour
Abnormal RBC
Deoxyhemoglobin ü Dark red in colour ACANTHOCYTE Abetalipoproteinemia; severe liver
ü Carbaminohemoglobin disease
BLISTER CELLS Trauma induced during passage
Carboxyhemoglobin ü Carries Carbon Monoxide 576 nm through injured and altered blood
ü Cherry red in color vessels

30
BURR CELL Uremia, neonatal liver disease, Leukocyte

gastric carcinoma, peptic ulcer,
pyruvate kinase deficiency, artifact Leukopoiesis
(alkaline glass effect)
ELLIPTOCYTE Hereditary elliptocytosis, Cell N:C Nucleus Granulation
pernicious anemia, congenital Myeloblast 7:1-4:1 Round nucleus with Absent
anomaly of red cells fine reddish-puple
MACROOVALOCYTE Megaloblastic anemia staining chromatin
KERATOCYTE Microangiopathic hemolytic Promyelocyte 3:1 Round nucleus with Primary Granules
anemias slightly coarsening
Myelocyte 2:1 Secondary
SCHISTOCYTE Microangiopathic hemolytic chromatin granules
anemias, severe burns,
Metamyelocyte 1.5:1 Kidney bean shape,
Disseminated intravascular
clumped chromatin
coagulation.
Band granulocyte 1:2 “C” or “S” Shape, Primary and
SICKLE CELL Hb SS
with lacking secondary
SPHEROCYTE Hereditary spherocytosis, ABO HDN segmentation granules
STOMATOCYTE Hereditary stomatocytosis, acute

alcoholism, alcoholic cirrhosis,
Qualitative Disorders (Toxic Changes)
glutathione deficiency, infectious

mononucleosis, lead poisoning,
CONDITION
thalassemia minor
ALDER-REILLY Hurler-hunter syndrome
TARGET CELL Liver disease, Hemoglobinopathies
TEARDROP CELL Myelofibrosis, microangiopathic AMATO/DOHLE BODIES Bacterial infections, May-
hemolytic anemia Hegglin anomaly
PYKNOCYTES G-6-PD deficiency, CHEDIAK-HIGASHI Abnormal neutrophils

microangiopathic hemolytic GIANT NEUTROPHILS Pernicious anemia
anemia, infantile pyknocytosis TOXIC GRANULATION Bacterial infections, burns,
LEPTOCYTES Flat cells seen in iron deficiency chemotherapy
anemia, thalassemia, HYPOSEGMENTATION Pelger huet anomaly
hemoglobinopathies, obstructive ATYPICAL LYMPH Infectious mononucleosis,
jaundice viral infections

31
Acute Myeloproliferative Leukemia/ Acute Nonlymphocytic Leukemia Acute Lymphoblastic Leukemia

French-American British Classufication of Acute Leukemia Acute Lymphoblastic Leukemia
FAB Cytology L1 L2 L3
Category Designation Morphology Small cells Large, Large
Size of Cell
Acute Myelocytic w/o Myeloblasts are predominate heterogenous homogenous
M1
Maturation dominant Variable Finely
Chromatin Homogenous
Acute Myelocytic w/ Myeloblasts with heterogenous stippled
M2
Maturation other blasts Regular and
Regular, oval
Promyelocytes with Shape of Nucleus occasional Irregular
to round
Acute Promyelocytic, many conspicous clefting
M3 Nucleoli Not visible One or more Prominent
hypergranular granules; 5-% Auer
Rods Variableoften Moderately
Amount of Cytoplasm Scanty
Promyelocytes abundant abundant
Acute Promyelocytic, Slight or Variable deep
M3V without conspicous Basophilia of Cytoplasm Very deep
hypogranular moderate basophilia
granules
Monocytic-type Often
Cytoplasmic Vacuolation Variable Variable
Nucleus and prominent
M4 Myelomonocytic
monocytic type
cytoplasm
Lymphomas
same as M4 but with
Myelomonocytic with
M4-EO eosinophils in Bone
Eosinophilia Classification of Lymphomas
marrow
1. Hodgkins Lymphoma
Monoblasts without - Thomas Hodgkins

Monocytic without
M5A granulocytic type o Reed-Sternberg Cell
Maturation
cytoplasm § Giant cell in the lymph node
nonblastic monocytic § “pop corn cell”, “lacunar cell”
Monocytic with cells without § “owl’s eye appearance” due to mirror
M5B
Maturation granulocytic image of two nucleus
cytoplasm
Megablastoid 2. Non-Hodgkins Lymphoma
M6 Erythroleukemia pronormoblasts and - Predominant B-cell abnormality
myeloblasts 3. Mycosis Fungoides and Sezary Syndrome
Megakaryocytic Immature - Rare lymphomas
M7 - Mature T helper cells are involved
Leukemia Megakaryocytes
32
Platelets ALPHA GRANULES DENSE GRANULES

- Life span: 8-11 days. Maturation: 5 days Platelet Derived Growth Factor Calcium
- Originates from MEGAKARYOCYTES/fragments of megakaryocytes Platelet Factor ADP
- Normally disc-shaped, transforms into pseudopod shape when Fibrinogen Pyrophosphate
activated Von Willebrand’s Factor ATP

Thrombospondin Serotonin
MEGAKARYOPOIESIS
1. Megakaryoblast β-thromboglobulin
2. Promegakaryocyte β-TGF
3. Megakaryocyte Albumin
Alpha 2-macroglobulin
Major Parts of the Platelet Alpha 2-antiplasmin
1. Peripheral Zone
- Contains glycocalyx/ glycoproteins § ALPHA Granule deficiency: Gray platelet syndrome
a. GP Ia – adhesion to collagen § DENSE Granule deficiencies: Hermansky-Pudlak, Chediak-Higashi,
b. GP Ib – binds vWF Wiskott Aldrich Syndrome
c. GP IIb-IIIa – Binds Fibrinogen and vWF § Bernard Soulier disease: deficiency of GLYCOPROTEIN I b, GIANT
d. GP IV – binds Thrombospondin platelet disorder, individual experiences varying degrees of
e. GP V – binds Thrombin bleeding tendency
f. GP IX – associated with Ib complex § Glanzmann’s thrombasthenia: deficiency of GLYCOPROTEIN IIb-
2. Submembrane area – linking membrane IIIa, individual experiences varying degrees of bleeding tendency
3. Sol-Gel Zone – granules, microfilaments, microtubules
4. Organelle Zone – mitochondria, alpha and dense granules, 2. Secondary Hemostasis
lysosomes - Formation of insoluble, cross-linked fibrin by activated

coagulation factors, specifically thrombin. Fibrin stabilizes the
HEMOSTASIS primary platelet plug
ü PRIMARY GOALS: to retain blood within the vascular system once
damaged, localize reactions involved, and to repair and normalize
blood flow

1. Primary Hemostasis
- Vasoconstriction to decrease the blood flow in the damaged
blood vessel
“Don’t disturb because I am
working hard for the exam.”
33
NOT ADSORBED by ADSORBED by BaSO4 NOT ADSORBED by

BaSO4 or Al(OH)3 or Al(OH)3 BaSO4 or Al(OH)3
Require CALCIUM as Require contact
COAGULATION NOMENCLATURE cofactor for binding with (-) charged
to phospholipid surface for
ASSIGNED NAME surfaces activation
NUMBER ü All coagulation factors are SERINE PROTEASES upon activation
I Fibrinogen EXCEPT Factors XIII (transaminidase) and Fibrinogen
II Prothrombin ü Factor XIII is not normally found in circulating blood
III Tissue Factor/Tissue Thromboplastin ü Aged serum (FACTORS VII, IX, X, XI, XII)
IV Calcium ü Absorbed plasma (V, ,VII, XI, XII, XIII)
V Proaccelerin/LABILE factor
VII Proconvertin/STABLE factor Tests for Hematology
VIII Antihemophilic factor/Antihemophilic globulin/
Antihemophilic Factor A 1. Erythrocyte Sedimentation Rate (ESR)
IX Plasma thromboplastin component/CHRISTMAS factor/ - Determines the rate which RBC fall to the bottom of a vertical
Antihemophilic factor B tube within a specific period
X Stuart-prower factor/Autoprothrombin III - It is influenced by the ability of RBC to form a rouleaux
XI Plasma thromboplastin antecedent/Antihemophilic factor - Used in detection of inflammatory process
C - Used as a monitor of diseases
XII Hageman factor/Glass factor/Contact factor - Used for occult inflammation
XIII Fibrin Stabilizing Factor/LAKI-LORAND factor a. Macrotechnique
Prekallikrein/FLETCHER Factor § Wintrobe-Landsberg Method
High Molecular Weight Kininogen/FITZGERALD factor - Double oxalated blood is allowed to stand for 1
hour

PROPERTIES OF COAGULATION FACTORS - Anticoagulant:Blood (1:4)

FIBRINOGEN GROUP PROTHROMBIN CONTACT GROUP § Westergren Method
GROUP - Anticoagulant:Blood (0.5:4.5)
I, V, VIII, XIII II, VII, IX, X XI, XII, PK, HMWK - Standard Method
CONSUMED during NOT CONSUMED STABLE,
coagulation during coagulation NOT CONSUMED b. Microtechnique
Susceptible to STABLE, well during coagulation § Crista or Hollige-Volmer Method
DENATURATION preserved in STORED Do NOT depend on § Micro Landau
Do NOT depend on Plasma vitamin K for § Barrett’s Micro-ESR
vitamin K for DEPEND on Vitamin K synthesis
synthesis
34

Increased Decreased 4. Sickle Cell Examination
Inflammation Polycythemia - characterized by the presence of HbS
RA Spherocytosis
TB Sickle Cell a. Sealed Whole Blood/ Scriver and Waugh Method
Waldenstrom’s Hb CC - Wrapping of rubber band around the middle finger
Macroglubinemia - Read every hour at 2-3 hours interval
Hepatitis
- Diagnostic of sickle cell anemia
Menstruation
Pregnancy b. Sodium Metabisulfate Method/ Daland and Castle

- Hemoglobin is converted into reduced hemoglobin by
2. Osmotic Fragility Test (OFT) sodium metabisulfate
- Evaluates RBC Membrane
- Exposure of RBCs to increasingly dilute saline
5. Eosinophil Count/ Thorn Test
- Determine internal flow of water to which water causes
- Uses Pilot’s Solution as a diluent
lysis of cells
- Uses WBC Thoma pipet
- Associated to spherocytosis
- Incubated in a “wet house”
- Can be performed with defibrinated blood
- Counted at LPO

- There is a diurnal trend (30% increase in night; 20% mid
Increased Decreased
morning)
SPHEROCYTES LEPTOCYTES

MACROCYTES MICROCYTES
6. Reticulocyte Count
AIHA
- Determines efficacy of erythropoiesis

- Stage that leaves the bone marrow
3. Smear Preparation for Malaria
- Supravital stains are used

a. Thin Smear o BCB (Sucrose, Sodium Citrate, BCB)
- Usual way of smearing o NMB Solution (NMB, Potassium Oxalate)
- For morphologic examination and specie identification of
the malarial parasite 7. Basophil Count
- Stain with Wright-Giemsa (pH 7.2)
- Cooper and Cruickshank Method

- Uses Fusch-Rosenthal Hemocytometer or Speirs-Levy
b. Thick Smear Hemocytometer
- Not fixed but air dried
- Used for screening parasites 8. LE Preparation

- It is a neutrophilic or an eosinophilic leukocyte with an
engulfed purplish hyaline mass
35
- Chelation of Mg and Ca with EDTA and Ammonium

Test Method Characteristic Oxalate
- 1:50 dilution
Rotating Glass Beads Most sensitive method - Reservoir contains 1.225 mL of diluent
- Count in 5 RBC squares
Wire Trauma/Davudsohn Method Satisfactory test
iii. Brecker-Cronkite Method
Heparinized Vacuatiner Method Too much heparin inhibits -
Uses Phase-Contrast Microscopy
formation of LE cell -
Involves the 2-syringe method of collection
-
Uses EDTA as anticoagulant and an RBC diluting pipette
Mashed Clot/Maceration Method Destroys cellular morphology -
Spencer-Briteline No. 1475 is used as a hemocytometer

Antinuclear Antibody Fluorescein-conjugated iv. Guy and Leake’s
antihuman immunoglobulin - Diluting fluid is comprised of crystal violet, sodium
citrate, distilled water and 40% formalin
- Compute: Plt. Ct./Cu.mm= Plt. Counted x 10 x 200 x 1
9. Platelet Count
a. Indirect Method 10. Platelet Function Test
- Platelets are counted in relation to 1,000 RBCs in a PBS a. Platelet Adhesion Test
- Not reliable 1. Glass Bead Method/ Salzman Method
i. Fonio’s Method - 2 whole blood samples is needed
- 14% MgSO4 is used 2. Borshgervinct Method
- count at least 1000 RBC - 2 samples are needed (venous and
capillary)
b. Direct Method

- Whole blood is diluted with a platelet diluting fluid in an Increased Decreased
RBC pipet and counted in a hemocytometer
i. Rees and Ecker’s Thrombosis Glanzmann’s
- Diluting fluid is comprised of BCB, Sodium citrate, and Pulmonary embolism Thrombasthenia
distilled water Carcinoma VWD
- Compute: (Plt. Counted x 10 x 200)/4 Pregnancy Chediak-Higashi

Splenectomy Uremia
ii. Unopette System
Oral Contraceptive
- Uses Trisodium EDTA
- Based on the lysis of RBC by hypotonicity

36
b. Coagulation Test b. Negative Pressure Test/ Hess Test// Suction Test

i. Bleeding Time - 200-250 Torr
ü VonWillebrand’s Disease: Increase
ü Factor VIII Deficiency: Normal 12. Prothrombin Time (PT) or Quick’s One-stage Prothrombin Time
1. Duke Method Method
- Earlobe is punctured using a lancet - Test for extrinsic pathway
2. Ivy Method
- Useful in screening deficiency in Factor II, V, VII, and X
- 40 mmHg sphygmomanometer (inhibited by coumarin)

- Used in monitoring the course of anticoagulant therapy
c. Clot Retraction Test in patients with coumarin treatment
- As a normal blood clot retracts, it o INR= (PT of patient/PT of geometric mean of
retracts a serum ISI
normal)
i. Qualitative
- Hirshboeck/ Castor Oil Method 13. Activated Partial Thromboplastin Time (APTT)
o Adding of a drop of fresh blood into - Single most useful procedure available for routine
castor oil screening of coagulation disorders
o Observe dimpling of serum - Uses calcium chloride
o NV. 15-45 min
- Used to monitor heparin therapy

- Used to detect lupus anticoagulant
ii. Quantitative - Test for intrinsic pathway except Factor XIII
- Stefanini Method
- MacFarlane Method 14. Duckert’s Test or 5.0M Urea Test
o Use 5mL of whole blood - The fibrin stabilizing factor is responsible for converting
o Compute: %CR= (Amt. of serum left in the fibrin clot to a more stable form
tube)/Amt. of blood used x 100

o NV. 44-67% 15. Fibrin-Split Product / Thrombo-Wellcotest
- FDP may be demonstrated in the blood of patients with
11. Tourniquet Test/Capillary Fragility Test primary fibrinolysis and during the process of DIC
a. Positive Pressure Test/ Rumple-Leede Test/ Tourniquet - Sensitive test for FDP
Test
- 100mmHg PRIMARY SECONDARY
o 0-10 – 1+ (normal) FIBRINOLYSIS FIBRINOLYSIS (DIC)
o 10-20 – 2+ PROTAMINE SULFATE - +
o 20-50 – 3+ D-DIMER - +
o >50 – 4+ EUGLOBIN CLOT LYSIS + -
TEST

37
Pointer WAIHA CAIHA PCH

o o o
Reaction 37 C 0-4 C 0-4 C-
Temperature Agglutinate
o
37 C –
hemolysis
o o o
Thermal Bond 20-37 C 0-32 C <15 C
Immunoglobulin IgG IgM IgG
Complement Binds complement

Activation
Protein Structure Polyclonal
Blood Group Rh, Kell Ii Pp

Specificity
Mechanism of Extravascular Extravascular Intravascular
Anemia
Severity Severe Mild Severe
Treatment Steroids, Avoid Colds Avoid Colds

Splenectomy

Transfusion Needed Not needed Not needed

“Exam please be nice.”
38
16. Oligohydramnios refers to a decrease in the amniotic fluid of a

person
Clinical Microscopy 17. Fanconi syndrome is a disorder caused by a defective tubular

reabsorption which can be manifested by the presence of cysteine

crystals

Must Know Recall Questions!
1. hCG is a hormone secreted by the trophoblasts of a developing Light Microscopy
embryo - Refers to the use of any kind of microscope with the use of visible
light to observe the specimen
2. Inulin Test is the gold standard for GFR
- Involves the general principle, which states that:
3. The volume needed for a urinometer is 10-15 mL The shorter the wavelength of the instrument, the
Active Transport Passive Transport greater the resolution
Glucose, Amino acids, salts, Water, Urea, Sodium
chloride, sodium ü Resolution
4. - Aka resolving power
- Refers to the ability of the lenses to
5. Tamm-Horsfall Protein is also known as uromodulin
distinguish fine detail and structure
6. Lignin Test is a test for sulfonamide crystals which has a yellow-
- Refers to the ability of the lenses to
orange positive result when dropped in a newspaper distinguish between two points of a s
7. Amniotic fluids for fetal lung maturity tests must be delivered in the specified distance apart
testing laboratory for up to 72 hours prior to testing, while amniotic Eg. 0.4 nm (resolving power), it can
fluids for cytogenic studies are maintained at room temperature distinguish between two points if they
are at least 0.4 nm apart
8. Bronchial asthma is indicated by the presence of Curschman spiral,

Charcot-Layden crystal, and Crayola bodies (columnar cells)

Types:
9. Neutrophils present in a fecal sample can be tested with the use of a. Compound Light or Bright Field Microscopy
a wet preparation, dried preparation and lactoferrin agglutination - It has a series of lenses and utilizes visible light
test
10. A definitive test for a steatorrhea is known as Van de Kamer Test b. Darkfield Microscopy
11. The size of the coverslip is 22 x 22 mm - Used in observing living microorganism that are invisible
in the ordinary light microscope, and can not be stained,
12. The dilution fluid in a sperm concentration test are sodium
or distorted when stained
bicarbonate and formalin - Utilizes a condenser containing opaque discs
13. Trisomy 18: Edward Syndrome - the specimen appears light against a dark background
14. Trisomy 13: Patau Syndrome - commonly used in specimens suspended in a liquid
15. Trisomy 21: Down Syndrome medium
39
- The electrons are produced by an

c. Phase-Contrast Microscopy electron gun which will migrate
- Used in observing the internal structures of living through a specially prepared
microorganism ultrathin section of the specimen
- Fixing or staining the slide is omitted - The final image produced is called as
Transmission electron micrograph
d. Differential Interference Contrast Microscopy
– Utilizes 2-beam of light 2. Scanning Electron Microscope (SEM)
– Will create a 3-Dimensional image on the specimen - It contains 2 electron gun producing
2 waves of electrons
e. Fluorescence Microscopy
- Utilizes the principle of Fluorescence h. Scanned-Probe Microscopy
o This refers to the ability of the organism to - Usually available in two types depending on the probe
absorb short wavelength of light (UV) and give used:
off long wave of light (visible) o Scanning Tunneling Microscopy (Tungsten
- A fluorochromedye is commonly used to stain the Probe)
specimen o Atomic Force Microscopy (Metal Diamond
Probe)
f. Confocal Microscopy
- The specimen is stained using a fluorchrome (in order to Hazards in the Laboratory
emit light)
- Commonly used in evaluating cellular physiology by
monitoring the distribution of ATP (adenosine
triphosphate) and calcium ion


g. Electron Microscopy
- Used in observing objects smaller than 0.2 um such as
viruses or internal structures of the cell
- The beam of light is replaced by a beam of electrons that
travel in waves
- It has the greatest resolving power (short wavelengths)
- The image produced is commonly in black and white
- The glass lenses are replaced by electromagnetic lenses

o 2 Types of Electron Microscope
1. Transmission Electron Microscope (TEM)
40
Hilus Part in which blood vessels and nerves are

attached
Renal Artery Entry of blood to the kidneys
Renal Vein Exit of blood from the kidneys
Renal Cortex Outer most layer of the nephron
Renal Medulla “stringy” in appearance
Renal Lobe Overlying cortex area
Calyces Cup-like divisions of the renal pelvis surrounding
renal papillae
Pelvis Funnel shape
Nephron Functional unit of the kidneys

Urinalysis
Parts of the Nephrons

Urine Formation
Part Comment
– Occurs in the kidneys (nephrons)
Glomerulus Malphigian Body; Renal Corpuscle

Tuft of capillaries found at the blind end of
ü Urine Composition
renal tubules
Organic Constituents Inorganic Constituents
Renal Tubules Concentration and modification of the
glomerular filtrate
Urea Chloride
Bowman’s Capsule Layer that surrounds the glomerulus
Creatinine Sodium
Afferent Arteriole Carries blood from the renal artery to the

Uric Acid Phosphorus kidneys
Ammonia Potassium Efferent Arteriole Carries blood from the nephron to the renal
Nitrogen Sulfur tubular area
Sugar Calcium Collecting Tubule Carries urine to the ureter
Ketone bodies Magnesium
Carbohydrates Iron Method of Urine Collection

Pigments
Method Comment

Parts of the Kidneys
Bottle Method Receptacles are clean

Part Comment
41
st
Gauze-Pad Method Centrifuged in a tube with Golf Tee Three-glass collection 1 Bottle – First urine passed
nd
2 Bottle – Control for bladder and
kidney infection
rd
Catheterization Clean tube inserted in the urethral 3 Bottle – Prostate fluid + Urine
st
orifice Stamey-Mears 4 Glass 1 – Initial void; Urethral infection
nd
Not recommended Method 2 – Midstream urine; UTI
rd
3 – Expressed prostatic secretion;
Midstream Catch Middle Portion Culture
th
4 – Post-prostatic massage; Culture
st
Suprapubic Aspiration Puncturing of pubic region PPMT (Pre-& Postmassage 1 – Clean Catch
nd
Used for quantitative bacteriology Test) 2 – Urine after prostate massage

Types of Urine Specimens Urine Preservation

Urine Specimens Comment Low Temperature Preservative Comment
Occasional Specimen Anytime of the day Refrigeration Prevents the growth of bacteria
Timed Specimen Collected in a specific time Dry Ice Same with refrigeration
24-hour urine Chemical Comment

12-hour urine a. Night Specimen – 8:00pm-8:00am Toluel/Toluene Ideal for steroid analysis
b. Day Specimen – 8:00am-8:00pm
Afternoon Specimen Specimen is collected between 2:00-6:00pm 40% Formalin Used for Addis Count
*Urobilinogen (2:00-4:00pm) Inhibits indicans (Obermayer Test)
Reduces Alkaline Copper (Fehling
Test)
o Specimen for Prostatic Infection Precipitates urea

Thymol Inhibits Biles
Test Comment
42
False Positive in SSA/ any albumin – Day urine (3-4x larger)

tests
Normal Volume (ml/24 hours)
Chloroform Ideal for Aldosterone analyisis
Not an ideal preservative Adult 800-1,600
Reduces Alkaline Copper
Children (1-6 years) 300-1,000
Sodium Fluoride and Benzoic Acid Prevents glycolysis
Children (6-12 years) 500-1,500
Aids in reducing insulin dosage for
diabetic patients

Phenol or Tricresol Transportation of urine samples

Excellent preservative “You can and you will!”
HCl Preserves 24-hours urine
Vanillyl Mandelic Acid preservative Abnormal Urine
Precipitates uric acid
Polyuria Oliguria Anuria Nocturia Residual
Formaldehyde Tablet (Life Increases specific gravity Urine
insurance Companies’
preservative) DM Acute Acute Instability to
Sulfuric Acid Preserves catecholamines DI nephritis nephritis regulate
Chronic Tumor in the Bricholoride water
Nephritis kidneys poisoning excretion

üChanges in Unpreserved Urine Nervous Severe Transfusion AGN
ANALYTE
condition diarrhea reaction
DECREASE GLUCOSE
Edema Dehydration Toxic Agents
KETONE
Uremia Collapse
CELLULAR ELEMENTS
INCREASE BACTERIA Shock
NITRITE Transfusion
pH reaction
ETG
Physical Examination of Urine Sulfonamides
a. Urine Volume
43
b. Urine Color Milky Chyle, fats, pus,

– Due to urochrome, urobilin, and uroerythrin epithelial cells, bacteria
Color Condition Normal/Pathologic Brownish black or Melanin pigment,
brown to black carcinoma of liver
Orange Pyridium
Black Alkaptone, homogentisic
Dark-smoky Phenol poisoning
acid
Blue/green Methylene blue injection

Greenish fluorescence Acriflavin, indican,

riboflavin

Red in alkaline urine Phenol sulfonaphthalein,

beets

Yellow Carotene, santonin, Non-pathologic
pyridium
Changes in Urine due to Drugs
Brown Rhubbarb, senna
cascara, argyrols Ethanol Pale
Greenish-orange Bilirubin
Olive green Phenyl salicylate Amitryptyline Blue-green
Red Antipyrine, trigonal
Laxatives Reddish (alkaline), yellow-brown (acid)
beets
Brown/Black Pyrogallol Chlorzoxazone Red
Reddish amber Barbital poisoning,
Pernicious anemia Defiroxamine mesylate Red

Brownish Bile pigment,
yellow/green with hepatocellular damage Ethoxazene Orange, red
yellow foam
Fluorescein sodium Yellow
Smoky read to brown Blood or blood pigments, Pathologic
infarcts in kidneys Furazolidone Brown
Clear red to reddish PNH, malaria,
brown transfusion reaction Indigo carmine dye Blue
Greenish foam Biliverdin
Iron sorbitol Brown on standing
Greenish blue Pseudomonas
aeruginosa
44
Levodopa Red then brown Fruity, sweetish Ketones
Mepacrine Yellow Mercaptant/pungent Asparagus, garlic and egg

ingestion
Methocarbamol/Robaxin Blue-green; Green-brown
Fecaloid Recto-vesical fistula
Methyldopa Red to brown; black
Bleach Semen contamination
Methylene Blue Blue, blue-green
Maple syrup MSUD
Metronidazole Black; Brown;Reddish-brown
Mousy PKU
Phenazopyridine Orange foam
Rotting fish Trimethylaminuria
Phenoindione Orange (alkaline)
Rancid Tyrosenemia
Phenol poisoning Brown that turns green when oxidized
Cabbage, hops Methionine Malabsorption
Phenophthalein Red purple in alkaline pH
Sweaty feet Isovaleric academia, glutaric
Phenolsulfonphthalein Pink-red, alkaline pH acidemia
Rifampin Bright orange-red d. Specific Gravity

ü Normal: 1.003-1.035
Riboflavin Bright yellow ü Random: 1.015-1.025

Sulfasalazine Orange-yellow in alkaline pH Isosthenuria Hyposthenuria Hypersthenuria
1.010 1.007 >1.010

c. Urine Odor Severe renal Diabetes insipidus Dehydration
Odor Condition
disease Pyelonephritis Adrenal insufficiency
Glomerulonephritis Hepatic disease
Lack of odor Acute tubular necrosis
Congestive Heart Failure
Foul, ammonia-like UTI Excretion of
radiographic dye
45
in proportion to the density of the

v Methods for Determination solution
1. Urinometer
– Based on density 4. Falling Drop Method
o
– Calibrated at a temperature of 20 C – Direct method
– Not recommended by CLSI – Falling time is proportionalto the
specific gravity
Correction
5. Reagent Strip
o
Temperature +/- 0.001/3 C – Uses Bromthymol Blue as indicator
– Change in acid dissociation
Protein -0.003/g of protein constant in an alkaline medium
– the concentration of urine
Glucose -0.004/g of glucose – Measures only ionic solutes
– Sensitivity: 1.000-1.030
Dilution Multiply dilution with the initial reading
e. Transparency
Clarity Comment
2. Refractometer
– Based on refractive index Clear No visible particulates, transparent

Calibration Hazy Few particulates, newsprint is easily seen
through urine
3% NaCl 1.015
Cloudy Many particulates, newsprint can be seen

5% NaCl 1.022 +/- 0.001 but letter are distorted or blurry
9% Sucrose 1.034 +/- 0.001 Turbid Newsprint cannot be seen through urine
Distilled water 1.000 Milky May precipitate or clot

3. Harmonic Oscillation Densitometry Microscopic Examination of Urine
– Linearity of 1.080
– Based on the frequency of sound Reporting
wave entering a solution changes
HPF LPF
46
RBC, WBC Casts, epithelia Round or pear shape, occasionally

(count per cell) Epithelial Cells (few, moderate, (200um) binucleated
abundant) Endo-ecto cytoplasmic rim
Derived from the transitional epithelium
Microscopy (bladder, pelvis of the kidney, and
a. Bright-field ureters
– commonly used
b. Phase-contrast Most frequently seen
– beneficial for more translucent element Large flat cells
– hardens the margins of the cast Large number is seen in the urine of
c. Polarized Squamous women
– Distinguish crystals and fibers from cellular or protein epithelia
Least significant
cast material
– “anisotropy” Margins are often folded
§ Bright against dark background "Clue cells"
§ Principle of polarized microscopy
Smooth; non-nucleated, biconcave disk
Constituents of Urine Sediment
6-8u in diameter
Red Blood Cell
Urinary Sediments Examined under HPO
(RBC)
Sediment Type Characteristic Reported in average of 10 hpfs
Organized Confused with fat droplets and yeast
Most significant Ghost Found in diluted urine; hypoosmotic
Cells/Shadow
Small round cells, mononuclear Cells of cells Hemoglobin had leaked out

Slightly larger than leukocytes Hematogenou Dysmorphic
Originated from deep layer of the s origin RBC Renal glomerular Bleeding/ G1 Cells
Renal tubular urinary tract Pus cells
Desquamated epithelial cells
Stain with Papanicolau Leukocytes Shrink in acidic urine; swollen/lysed in
Epithelial Cells Coarsely granular with eosinophilic alkaline urine
cytoplasm “Glitter cells”
Suggestive of AGN, nephrosis, hemolytic
Neutrophil Use leukocyte esterase strip to confirm
anemia, hemochromatosis
Seen in pyuria, cystitis, prostatitis, UTI
Caudate and Aka transitional epithelia, urothelial cells
cylindrical Eosinophil Not normally seen in urine
2-4x larger than pus
47
Papanicolau and Hansel stain is Bilirubin Cast Pyridium medication

commonly used
Broad Cast Tubular dilation
Lymphocyte Renal transplant rejection Escherichia
Increased in AGN, APN, HTN, CRD, heart Bacteria coli
Hyaline Casts failure, diabetic nephropathy Candida
Aka renal failure cast Yeast albicans Pseudohyphae
“Glass-like with yellow color” Trichomonas,
Can be characterized as: “cut of Schistosoma,
Waxy Cast
paraffin”, “cork-screw winding” Parasite Entamoeba
Seen in tubular inflammation and Spermatozoa
degeneration Platelets HUS
Seen in iga nephropathy, SLE, renal
infarction, AGN Unorganized sediments
o
Erythrocyte Usually colorless or lavender in a pink Soluble in alkali like naoh, and at 60 c
Cast matrix Amorphous Urate "Brick dust” in appearance
Better visualized under phase-contrast (Ca, Mg, Na, K Convert to uric acid upon acidification
microscopy with acetic acid
Leukocyte Crystals Urate
Cast Pyelonephritis (Na, K, NH4)
Casts
Papanicolaou is used as a stain Acid Urine Crystalline Uric Occur at low ph (5-5.5)
Seen in acute tubular necrosis, heavy Acid
metal poisoning, ethylene glycol, Usually in “Whetstone” crystals
RTE Cast
salicylate intoxication Calcium Oxalates Ethylene glycol poisoning
Seen in allograft rejection after 3 days

"Cigarette-butt” in appearance
from the operation
Calcium Sulfate Soluble in Acetic Acid
Granular Cast Remnants of Calcium
Elongated prismatic tablets
Maltese cross Amorphous
Fatty Cast
Nephrotic syndrome Phosphate
Hemoglobin Blood cast (Calcium and
Cast Alkaline Magnesium)
Tubular bleeding Crystalline Triple Phosphate (ammonium
Hemosiderin Urates
Phosphates magnesium phosphate)
Cast RTE Cell-derived
Myoglobin "Coffin lid” in appearance
Cast Muscle damage “rhomboid”-Magnesium phosphate
48
Calcium Distinguished by their production of CO2 Ammoniacal Silver

Carbonate in the presence of acetic acid Nitrate Test 5% Silver Nitrate Gray-black color
o
Ammonium Dissolve with heat 60 c and with acetic Screening Test Alkali Brown color
Biurate acid Yellow
Confused with hexagonal uric acids Bromine Water Test Bromine water precipitate
Will dissolve in hcl but uric acid will not 10% Ferric
Cystine Chloride & 10% Gray precitateof
Most important crystal to be identified Ferric Chloride Test HCl melanin
Confirmed using the cyanide Brown to black
nitroprusside reaction Melanin
color which can
Forms fine silky needles that may be be dissolved
Abnormal Tyrosine arranged in sheaves after refrigeration Blackberg and Wanfer Potassium per with sodium
Crystals
Leucine Radial concentric striations Test sulfate, Methanol hydroxide
Urine
Sodium
Sulfonamide Sulfadiazine crystal
nitroprusside, Dark green or
Crystal Confirm using diazo reaction, and HPLC Thormoblen Test Acetic acid blue color
Ampicillin Speegrav Method Photometry
Meglumine diatrozoate Specific Similar with
Radiographic
Correlates with a high specific gravity Gravity copper sulfate
Media
(1.040) Bromobenzene, method for
Dogramaci Method Kerosene blood
Chemical Examination of Urine Litmus Paper Method
Compare with
Nitrazine Paper Method chart

Chemical Test pH Range Paper Test
Analyte Name of Test Reagent Result pH Hydrion pH Paper Test
Sodium Hydroxide Test 10% NaOH Brown color Anachemia Papers pH range is 1-12
Bluish Potassium
10% Ferric green/Evenesce Folin's Oxalate, Alcoholic
Alkaptones Ferric Chloride Test Chloride nt blue-green Method/Henderson Phenolphthalein,
Benedict's Qualitative Benedict's Brown to black Method NaOH
Test Reagent color Bence Turbidityat 40C
Lemon yellow Jones Heat and Acetic Acid SSA, 33% Hac and 60C
Million's Test Million's Reagent precipitate Proteins Bradshaw Test conc. HCl Precipitation
49
Precipitation at Heat Coagulation

Heat Precipitation Test 60C Method Henry Tets
2M Acetate White ring at
Putnam Test Buffer Precipitation the zone of
Toluene Sulfonic Acid Toluene Sulfonic Sulfosalicylic Acid Test Exton's Test contact
Test Acid Precipitation White opaque
Turbidity: 1+, ring at the zone
2+. 3+, 4+ Heller's Ring Test Nitric Acid of contact
(distinct Sodium Chloride,
turbidity, Purdy's Test Acetic Acid
Moderate Picric Acid Test Flocculence
turbidity, Heavy Magnesium White ring at
tubidity, Heavy sulfate, Nitric the zone of
and flocculent Robert's Ring Test Acid contact
Sulfosalicylic Acid Test SSA turbidity)
Acetic Acid, Salt
Qualitative
50% Acetic Acid, Osgood Haskin Test solution Precipitation
Osgood-Haskin Test NaCl Precipitation
Potassium Ferrocyanide
Acetic acid, salt Test Cloudiness
Simple Presumptive Test solution Precipitation
Mercuric
Precipitation Chloride, Succinic
Jacobson and Milner that dissolves Acid, Sodium White opaque
Test HNO3, on cooling Chloride, Distilled ring at the zone
Hot sodium Spiegler's Test water of contact
Protein Paper Test chloride 10% NaOH/KOH, Pure violet
No. 26 cellophane Biuret Test Cupric sulfate color

Paper Electrophoresis dialysis tubing Best test Green
Dissolution of Albustix coloration
mucin
Mucin Hepler's Test 10% NaOH precipitates Hema-Combistix
Aluminum Deep rose to Tetrabromphenol
Mucicarmine Test Chloride red coloration Uristix blue Protein
1+Diffuse cloud, Labstix
2+ Granular Bili-Labstix
cloud, 3+
Picric acid, citric
Quantita
Distinct flocculi,
tive
acid, Tsuchiya's
Albumin Heat and Acetic Acid Buer Test 4+ large flocculi
Esbach's Test Rgt
50
Shevky and Stafford's Green/Yellow/O

Test Tsuchiya's Rgt Precipitation range
Life Insurance Clinitest Method Distilled Water coloration
Kingsbury's Test Method Galatest Method Black coloration
Kingsbury-Clark Method Acetic Acid Tes-Tape Method
Kwilecki's Test Clinistix Method
Purdy's Test Benedict's
Sugar Benedict's Test Reagent Gray color
Glucose Fehling's Test

Robert's Fermentation
Quantitative
Benedict's
Benedict's Test Reagent Brick Red Test Wallnut size Yeast
Cupric Sulfate, Yellow Einhorn's Fermentation Change in
Fehling's Test Rochelle Salts precipitate Saccharometer Yeast Specific Gravity
Yellow to red Millard Smith Micro Benedict's
Haines's Test precipitate Modification Test Reagent
Fine Bright Sodium
Phenylhydrazine Test Osazone Test Yellow Needles Somogyi Method Carbonate
Glacial acetic
Kowarsky's acid, sodium Seliwanoff's Reaction Resorcin, HCl Red Precipitate
Phenylhydrazine Test chloride Yellow needles
Qualitative
25% HCl,
10% NaOH/KOH, Red Fructose Berchardt's Test Resorcinol Red coloration
Trommer's Test Cupric sulfate Sedimantation Tauber and
Nylander's Test Rochelle Salts Black coloration Kleiner

1% Canary Barfoed's Test Modification Blackish hue
Yellow, 1-2% Green
Wine Yellow, 2- Bial Orcinol Test BialReagent coloration
3% Sheary Pink to red
Yellow, 3-4% Tauber's Test Benzidine, Hac coloration
Rum Color, >4% Cherry Red
Pentose
Black Color or Tollen's Test HCl Color
Moore-Heller's Test 10% KOH Dark Brown Merck Blood Green
Orthoroluidine Cole Test Charcoal coloration
Glucose Oxidase Method blue Bright Red
Aniline Test Hac Coloration
51
Brick Red with Bordeaux Red

Cherry Red Gerhardt's Test Ferric Chloride Color
Rubner's Test Lead Acetate coloration Diacetic 30% Acetic Acid,
Lactose Methylamine Intense Red Acid Lindemann's Test Iodin, Chloroform Reddish-violet
Ormsby Test Hydrochloride coloration Ketostix
White
Mucic Acid Test Nitric Acid precipitate Labstix
B- Hydrogen
Barfoed's Test Copper Acetate
Oxybutyric Hart's Test peroxide Red ring
Maltose Ormsby Test Acid Osterberg's Test
Mucic Acid Test Smith's Test Iodine Solution Emerald Green
Mucic Acid Test Gmelin's Test Nitric Acid Play of Colors
Galactose Tollen's Test Green
Phenylhydrazine Test Ultzman's Test 25% KOH coloration
Ammonium Foam's Test
sulfate, sodium Red to purple Barium Chloride,
Rothera's Test nitroprusside coloration Fouchet's Blue to green
Lavander to Harrison Spot Test Reagent color
Acetest Tablet Acetone purple Methylene Blue Test Methylene Blue Blue coloration
Yellow Band of colored
Lieben's Iodoform Test KOH precipitate Rosenbach Test Nitric Acid rings
Gunning's Test Iodine Solution Bilirubin Paratoluene, Green
Purplish Red Maher Test sulfonic acid coloration
Frommer's Test KOH Ring Calcium Green-colored

Ketone
Bodies Purplish Red Huppert's Test hydroxide compound
Lange Test Hac Ring Barium Chloride,
Lavander to Fouchet's Green
Denco Test purple Ferric Chloride Test Reagent coloration
Rantzman's Lavander to Barium Chloride,
Rantzman Test Reagent purple Fouchet's Green
Sodium Watson Method Reagent coloration
Nitroprusside, Fouchet's
Legal's Test Acetic acid Violet Color Fouchet Test Reagent Greenish Blue
Ammonium Green
Jackson-Taylor's Test sulfate Purple-red color Franklin Test coloration
52
Bili-Labstix Greenish
Peptone, Salicylic Urobilin Schlesinger's Test Lugol's solution fluorescent
Oliver Test Acid Milky Turbidity Scwartz-Watson Test
Bile-Acids Hay Test Flower of Sulfur Sinking of sulfur Oxalic acid, Fine white
Red ring at the Ammonium precipitate/Milk
Calcium Sulkowitch Test oxalate precipitate
Pettenkofer Test Sulfuric Acid zone of contact
HCl, Ferric Barney Test Sulkowitch rgt. Turbidity
Chloride, Silver nitrate,
Obermayer Test Chloroform Blue coloration Potassium 6-12 drops until
Indican Calcium Chloride Fantus Test chromate change incolor
Jaffe Test hypochlorite Blue coloration Schales and Schales
Jolles Test Lead Acetate Violet Color Method 0.5 Nitric Acid
Benzidine Green or Blue Blue-green
Benzidine Test Solution coloration Ferric Chloride Test color
Glacial Acetic Acid Phenylpyru Ferric Chloride Diaper Gray-green
and 3% Hydrogen Green to blue vic Acid Test coloration
Guiac's Test Peroxide coloration Grayish green
Phenistix coloration
Hema-Combistix Glacial acetic
Hematest Blue color Porphyrin Ham Method aacid Fluorescence
Blood Hemastix Paper Stix 5-
Hydroxyin
Labstix dole Acetic Nitrous Acid, Purple color in
Occultest Tablet Test Acid Sjoerdsma Test Ethanol top layer

Bili-Labstix 17-
Ketosteroi
Orthotoluidene Test ds Zimmerman Reaction HCl
Glacial acetic 17-
Teichmann's Test aacid Rhombic Plates Hydroxyco Yellow
Ehrlich's Test HCl Red coloration rticoids Porter-Silber Method coloration
Cherry Red
Urobilinog Wallace Diamond Test conc. HCl Color
en Ehrlich's rgt, Fluorescent
Scwartz-Watson Test Chloroform violet
Urobilistix
53
Renin-Angiotensin-Aldosterone System

C. Concentration and Dilution Test
a. Mosenthol Test
– Comparison of specific gravity during day and
night
Kidney Function Test b. Fishberg Concentration Test
A. Dye Excretion Test – Determines the ability of the kidneys to
a. Indigo Carmine Test (Used by Urologists) maintain excretion solids under conditions of
– Blue dye is excreted by both kidneys reduced water intake plus a high protein diet
– Confirmatory test for unilateral kidney disease
b. Phenolsulfonpthalein Test Reagent Strip
– Excretion test
Test Readin Principle Reagent
B. Clearance Test Analyte g Time

– Considerations: pH 60 sec Double Indicator Methyl red
ü Substance must be neither reabsorbed nor System Bromthymol blue
secreted Protein 60 sec Protein Error of Tetrabromphenol blue
ü Stability of the substance Indicator
st
Urea Clearance Creatinine Clearance Inulin Clearance Glucose 30 sec Glucose Oxidase 1 Reaction: GOD
nd
2 Reaction: POD
Ketones 40 sec Sodium Sodium Nitroprusside
Nitroprusside or
Nitroferricyanide
Blood 60 sec Pseudoperoxidas
e
54
Bilirubin 30 sec Diazo Reaction NH4OH

Urobilinoge 60 sec Ehrlich’s p- NaCN Bett-red color
n aldehyde diethylaminobenzaldehyd Cystine
Na
reaction e
Nitroprusside
Nitrite 60 sec Reduction of

Nitrite (Greiss’

reaction)

Leukocyte 120 sec Hydrolysis of an

Esterase acid ester to

produce
Murexide Test
aromatic

compound
Type of Calculi Reagent Result
Specific 45 sec pKa change
Gravity
Uric Acid Ammonium hydroxide Deep yellow

Renal Calculi
Xanthine Ammonium hydroxide Greenish yellow
Spot Plate Test

Protein Ammonium hydroxide Pale yellow

Type Reagent Positive Result
Sulfonamides Ammonium hydroxide Mahogany brown
Carbonates NaCO3 Foaming effervescence

Deep blue color Fecalysis
Uric acid or urates HCl, MnO2
Ammonium Specimen Collection and Processing

Distinct yellow Precipitate
Phosphates
Molybdate
Tiny bubbles of gas - Should be collected every other day or 3 samples within 10 days
Oxalates o Amebiasis – 6 specimens for 14 days
explosively released from the
HCl o Anti-malarial drug – collect sample 2 weeks after
bottom
medication
Fine, white precipitate - Size should be 2-5g (Wallnut)
Calcium NaOH
o Toilet water kills Schistosome egg and Amebic
Blue precipitate trophozoite (usually present in watery stools)
Magnesium NaOH, Mg
- Should be transported within 30 min for semi-formed and watery
NaOH, Yellow-orange precipitate stools but up to 24 hours for formed stools
Ammonia
Nessler’s Rgt
55
v Stool Fixatives v Stool Processing

- 3 parts fixative + 1 part stool A. Macroscopic Examination
a. Consistency
Fixative Component Usage
Formalin ü 5% for protozoan Protozoa and Helminth Stool Consistency Condition
cyst Direct fecal examination Pea-soup Salmonella typhi
ü 10% for helminth
Rice-water Vibrio cholera
egg and larvae
Polyvinyl Plastic powder Used as an adhesion for Scyhalous stools Sever constipation
Alcohol Schaudinn Solution parasitic slides Scybala stool Goat droppings
(PVA) (zinc sulfate, copper Used for permanent Gaseous Sprue
sulfate, mercuric staining of slides Pipestem stools Spastic colitis
chloride) Small caliber Spasm
Sodium Acetate Alternative for PVA
Large caliber Hirschung’s disease
Formalin (SAF) Used for staining
Coccidian oocyst
b. Color
Modified PVA Copper sulfate or zinc Good for concentration
sulfate technique and Stool Color Condition
permanent stains Yellow Amylorrhea
Single-Vial Fecal immunoassay Green Meconium and porphyrins
System Yellowish-green Absence of stercobilin
Refrigeration
o
40-45 F Bright-red Bleeding in lower GIT, neo-
prontosil
Cumming Glycerin
Dark red or Hemolytic anemia (excess
Method 0.9% Sodium Chloride

chocolate urobilin)
Merthioloate- Preservative and fixative
brown
Iodine- For teaching, mailing,
Clay or putty Barium meal in X-ray
Formaldehyde and survey studies
examination

Black or tarry Upper digestive bleeding

c. Odor

- Due to indole and skatole

56
B. Chemical Test Huppert’s Test Lime Juice Green color

Analyte Chemical Tests Components Result Urobilin Schmidt’s Test 10% mercuric Urobilin – Red
chloride color
Nitrazine Paper Colored pH chart Bile – Green
color
pH Alizarin Test 1% aqueous Alizarin Redish violet –
Solt’n. Alkaline pH Schlesinger’s Lugol’s solution Greenish
Light yellow – Test fluorescence
Acid pH
Trypsin X-ray film Test Clearing of the
Mucous Hecht Test Briliant green Mucous – red film
Neutral Red Nuclei and cell-
membrane –
Reddish violet C. Microscopic Examination

Test Characteristics Remarks Type of
Examination
Blood Benzidine Test Benzidine Blue to green
Direct Wet Unfixed stool Detection of Direct Slide
Hydrogen Peroxide is positive
Preparation mixed with trophozoite Preparation
saline motility
Guiac Test Guiac Reagent Blue to green
*use Iodine for
product cyst detailing
Formalin-Ethyl Ethyl acetate Most widely Concentration
Orthotouluidine Hydrogen peroxide Blue to green Acetate Formalin Fixed used Method

Test Orthotoluidine product Sedimentation sample Best for
Iodine helminth eggs
Occult Test Zinc Sulfate ZnSo4 (sg. 1.18- Cleaner Concentration
Flotation .120) preparation Method
Bilirubin Gmelin’s Test Barium chloride Play of colors Technique Eggs of
Fouchet’s reagent or (green helminthes do
Nitric Acid periphery with not float
blue, violet, Wheatley Most widely Staining
red, and Trochrome used Method
permanent
yellow centers)
stain
57
Long shelf life C. Color

Gray Presence of pus and epithelial cells
Anchovy sauce or Amoebic abscess of the liver
Iron Excellent for Staining
rusty brown
Hematoxylin intestinal Method
protozoa Prune juice Chronic cancer of the lungs
Black Anthracosis
Sputum Examination Grass green Cancer

Macroscopic Examination
A. Volume D. Consistency and Specific Gravity
a. Mucoid – 1.004-1.008
b. Purulent – 1.015 – 1.060
>100 cc. >500 cc. >1,000cc. Small amount
c. Serous – more than 1.037

Pulmonary edema Entamoeba Bronchiectasis Diffused
E. pH (6.5-7.0)
Bronchiectasis histolytica Tuberculosis bronchitis

Tuberculosis Chronic Pulmonary
F. Macroscopic Structures
Lung abscess bronchitis tuberculosis Dittrich’s plugs Pinhead/ millet seed caseous masses
Bronchomoniliasis Lobar
Pulmonary pneumonia Bronchial casts Branching/ tree like casts of the bronchi
hemorrhage
Cheesy masses Fragments of necrotic tissue or

cartilaginous rings
B. Odor
Sweetish odor Pulmonary tuberculosis with cavities, bronchiectasis Lung Stones Broncholiths

Putrid or foul presence of fusobacteria and spirochetes
Microscopic Analysis
Cheesy odor necrosis or malignant tumors and perforating
empyemas Structure Comment
Elastic fiber Destructive lungs
Fecal odor Ruptures subphrenic or liver abscess, enteric G-
Curschmann’s Spiral Coiled into balls
Products
Charcott-Leyden Crystal Disintegration of eosinophils; Asthma
Hematoidin Crystal Breaking down of old blood
Cholesterol Crystal
Fatty Acid Crystal
Heart Failure Cell Hemosiderin, congestion of the lungs
58
Carbon-Laden Crystal Angular black granules - serum glucose should be simultaneously collected 2-4
Myelin Clobules Mistaken as Blastomyces hours prior to puncture
Actinomyces hominis Sulfur granules - specimen is divided into three tubes:

Cerebrospinal Fluid a. Tube 1- Chemistry and immunology (store at freezer)
b. Tube 2- Microbiology (store in room temperature)
- First recognized by Cotugno c. Tube 3- Hematology (store at refrigerated
- collected by means of lumbar puncture temperature)
d. Tube 4- for Cytology, malignancy, and tuberculosis
Major Components infection

1. Protein Gross Examination
- Low with no fibrinogen
2. Glucose Abnormal CSF Color Disease Associated
- Approximately 2/3 that of blood sugar Pink RBC lysis/ Hemoglobin breakdown products
3. Chloride
- 25% higher than the plasma chloride Yellow RBC lysis/ Hemoglobin breakdown products
4. Lactose CSF Proteins >150 mg/dL
- Major waste product of glucose
Orange RBC lysis/ Hemoglobin breakdown products
Specimen Handling and Collection Increase in Vit. A intake (carotenoids)

Yellow-green Hyperbilirubinemia
• Lumbar puncture
Brown Meningeal metastatic melanoma
- Performed by inserting a needle in the lower lumbar
region of the spinal vertebra (L3-L4 or L4/L5)

- The patient is requested to assume a fetal position Traumatic Puncture Intracerebral Hemorrhage
- A Gauge 25 needle is commonly used Presence of blood
- Manometer should be attached before fluid removal to Ø The greatest amount of Ø Homogenous
record the opening pressure (varies among posture) blood is present in the distribution of blood
first tube throughout all the
Pressure Condition Ø Colorless supernatant tubes
10-100 mmHg Normal adults and children is obtained after Ø Supernatant is still
centrifugation cloudy after
90-180 mmHg Normal lateral lying down position
centrifugation because
10 mmHg Sitting patience
cells will lyse after 1-
250 mmHg Obese Clot formation 2hours

59
Ø Presence of clot due to Smear Preparation

the introduction of Ø Does not contain
plasma fibrinogen fibrinogen - Cytocentrifugation is the easiest method, but least preferred
because is may damage the cells
Xanthochromia - Sedimentation is the best method
Ø Absent
Ø present Cell Count
D-Dimer Test Clarity Dilution Amount of Amount of
Ø negative Sample Diluent
Ø positive
Slightly Hazy 1:10 30 uL 270 uL
Microscopic Examination Hazy 1:20 570 uL

Slightly Cloudy 1:100 2970 uL
Normal CSF
3
- small amount of lymphocyte (adult= 0-5 cells/mm ; infants= 30 Slightly bloody/ 1:200 5970 uL
3
cells/mm ) Cloudy 1:10,000 0.1 mL of a 9.9 mL
- presence of RBC suggests contamination with blood during Bloody/Turbid 1:100 dilution
lumbar puncture
- an increase in cell count must be confirmed by staining a smear
Predominant Cells Seen in CSF
th
Types of Meningitis (Strasinger, 4 Ed. P.155, Table 10-3)

Bacterial Tubercular Viral Fungal Type of Cells Major Clinical Microscopic Finding
Significance
ü Positive ü Pellicle ü Normal ü Lactate level Lymphocyte Normal All stages, HIV and AIDS

Neutrophil Bacterial Meningitis
limulus formation glucose >25 mg/dL
Monocyte Multiple Sclerosis Mixed with Lymphocyte
lysate test ü Lacate level and lactate ü Positive India Macrophage RBC in CSF Phagocytized RBC
with Gram >25 mg/dL ink with C. appearing as ghost cells
negative neoformans Blast Form Acute Leukemia Lymphoblast
organism Malignant Cells Carcinoma Seen in cluster
ü Lactate >35
mg/dL

60
Chemical Tests
1. CSF Protein 5. CSF Enzymes
- Most frequently performed test in CSF - CK-BB Isoenzyme, which is seen in post cardiac
- Normal value is 15-45 mg/dL arrest patients indicating poor prognosis
- “Tau” is the only protein unique to the CSF
a. Electrophoresis Microbiology Test
- Determine the presence of 1. India ink
oligoclonal bands - Negative stain for Negative stain for Cryptococcus
o Located in the gamma neoformans
region (Ig) - Starburst appearance

Presence of Oligoclonal Band 2. Limulus lysate test
- Test for endotoxin
Disease CSF Serum - Used the blood of Limulus polyphemus (Horse
Leukemia, Lymphoma Absent Present shoe crab)
o Contain amebocytes
Viral infection, HIV, Present Present
- Blood cells containing a copper
Syphillis complex giving a blue color
Multiple Sclerosis, Present Absent
Guillain-Barre
Syndrome, Neuropathy

2. CSF Glucose
- 60-70% of serum level
- blood glucose level should be collected 2 hours before

puncture

3. CSF Lactate
- End product of glucose (glycolysis) in cases of hypoxia
Seminal Fluid Analysis

4. CSF Glutamine
Specimen Collection

Ammonia + alpha ketoglutarateà Glutamine
- Majority of sperm is concentrated in the first ejaculate
- Essential in the removal of metabolic waste of
ammonia in the CSF - Abstain for 3 days but not longer than 5 days
- For fertility testing 2 or 3 samples are collected at 2 weeks interval
- Elevated results are also associated with liver

diseases
61
Semen Analysis - Evaluating by observing 20 HPO

o Computer-Assisted Semen Analysis (CASA)
a. Appearance § Automated machine to determine
- Gray-white, opaque in color for normal semen the velocity and trajectory of the
- Musty odor sperm cell

b. Volume Sperm Motility Grading
th
- Normally is 2-5 mL (Strasinger 4 Ed., Table 11-2)
- Use a graduated cylinder
- Decreased volume is associated with infertility Grade Criteria
4.0 Rapid, Straight motility
c. Viscosity 3.0 Slow speed, some lateral movement
- Refers to the consistency of the fluid 2.0 Slow forward progression, noticeable lateral
- Pours in droplets 1.0 No forward progression
0 No movement
d. pH
- alkaline ( 7.2-8.0) g. Sperm Morphology
- Evaluated by observing the head and the tail
e. Sperm Concentration o Head= oval shape and 5 um long x 3 um wide
- 20-160 million sperm per milliliter o Flagella = approximately 45 um long
- Performed using the Neubauer Counting Chamber o Acrosomal cap = one half of the head
o 1:20 is the most common dilution - Count the number of abnormal cells and report in
percentage
§ Sperm Count = Sperm Concentration x Volume of
Semen h. Sperm Viability

- Used by mixing the sample with Eosin-Negrosin Stain
- Makler Counting Chamber - Evaluate 100 sperm cells
o Used for counting undiluted samples o Living Cells = bluish-white color
o Sperm is immobilized by heating the o Dead Cells = red against purple background
specimen prior to charging - Normal cells should be 75% living

f. Sperm Motility i. Sperm Fructose (Resorcinol Test)
- Movement of the sperm is critical for fertility - 50mg of Resorcinol in 33 mL conc. HCL + 1 ml of sample
- Perform by using an undiluted specimen - Should have a normal 13 umol/ejaculate (red-orange color)
- Determined by getting the percent of motile sperm and the
quality of motility Gastric Juice Analysis
- Performed using a liquefied semen Introduction
62
Organic Inorganic a. Histamine Phosphate Test

Component Component b. Insulin Test
Pepsin Water c. Caffeine Test
Lipase HCl
Rennin Electrolytes Physical Examination
Soluble mucin A. Amount
Glairy mucin Increased Decreased

Collection of Gastric Juice Gastric hypomotility Gastric hypermotility
Ewald’s/ Hard tube that is inserted through propulsion Ulcer Pernicious anemia
Boas used for gastric washing Zollinger-Ellison Syndrome Chronic alcoholism
Kaslow’s Introduced via propulsion, gastric contents are High Intestinal Obstruction Leather-bottle stomach
collected with a syringe
Syphilis in the stomach Achylia
Rehfuss Soft rubber administered via swallowing with
Chronic Dilation of stomach
the aid of gravity
Levine A soft rubber that is swallowed or passed
through the nostril B. Odor
Sawyer Connected to a syringe for aspiration Odor Comment
Alcoholic
Stimulation of Gastric Secretion Ammoniacal Uremia
A. Test Meal Fecal Intestinal obstruction
Boa’s Meal Rolled oats in water with a pinch of salt (for Rancid Butyric acid and Lactic acid
lactic acid detection) Putrid Odor Necrotizing Cancer
Heckman’s Egg albumin, distilled water and methylene Rare Bacillus infection
Meal blue

Riegel’s Meal mashed potatoes or broiled beef steak with
C. pH Reaction (1.6-1.9)
bouillon soup
D. Specific Gravity (1.001 – 1.010)
Dock’s Meal Shredded whole biscuit

Ewald’s Meal Breakfast; not recommended for lactic acid
Chemical Examination
Fischer’s Meal Ewald’s Meal, with hamburg stock
Alcohol Meal/ Used in the determination of regurgitating Analyte Test Reagent Result
Lavine’s Meal alkali materials in the duodenum Phenolphthalein Phenolphthalein, Deep Pink
Salzer Motility Roast beef, milk, boiled rice Test NaOH Color
Total Acidity
Salmon
Topfer's Method HCl Pink
B. Chemical Stimulation
Free HCl Topfer's Method
63
Resorcinol, Rose-red Hogben’s Test Female frogs

Boa's Test Sugar Cane color
Galli Mainini Male frogs
Phloroglucin, Purplish
Gunzberg's Test Vanilin, Alcohol Red Bufo bufo Male frogs
SodiumAlizarin Violet with Rana Pipiens Male frogs
Free Acidity Sulfonate blue tinge
Phenol, Ferric Canary
Uffelman'sTest Chloride Yellow Transudates and Exudates
Deep
Kelling's Test Ferric Chloride Yellow
Ether, Ferric Green
Lactic Acid Strauss Test Chloride Coloration
Simon
Modification of Deep
Kelling Test Ferric Chloride Yellow
Phenol, Ferric Canary
Reitman Method Chloride Yellow
3% Hydrogen Green blue
Occult Blood Benzidine Test peroxide color
Display of
Bile Gmellin's Test Nitric acid colors
Coagulation
Reitmen Test Milk of milk
Renin
Phenolphthalein,
Riegel Test NaOH

Bauer Test HCl
Pepsin Hammerschlag
Test 1% Egg albumin

Pregnancy Testing

Test Animal
Friedmann’s Test Virgin rabbits
Hoffman’s Test Virgin rabbits
Ascheim Zondek Test Female mice
64
5.
IS-Blood Bank

Must Know Recall Questions!
1. C2 deficiency is the most common type of complement deficiency
2. C3 is the most fatal type of complement deficiency
3. Common Autoimmune Disorders:
Disorder Autoantibody
Type 1 DM Anti-GAD 65
Grave’s Disease Anti-TSH 6. Diapedesis refers to the migration of monocytes from the blood
Hashimoto’s Disease Anti-Thyroid globulin vessels going to the surrounding tissues
Pernicious Anemia Anti-IF 7. Goats and rabbits produces anti-human CRP which is then utilized
Good-Pasteur Syndrome Ati-glomerular in the Latex agglutination Test
SLE Anti-phospholipid
Sjogren Syndrome Anti-Salivary gland and anti- Immunity
lacrimal gland Organs involved in Immunity
Scleroderma 1. Thymus – Maturation of T lymphocyte
RA Abnormal IgM 2. Bone Marrow – Pluripotent cells; B cells and Plasma cells

4. Cells involved in Immunity

1. T cells (60-80%)
ü T Cell Development:
Development Stage Comment
Double negative
Double positive
Mature

Antigen Remarks
CD3 TCR transduction
CD10 Marker for Pre-B CALLA (Common Acute
Lymphoblastic Leukemia); B cell
65
CD25 Receptor for IL-2 T Cell B Cell

CD56 No known function; NK Cells
CD94 Inhibition of NK cell cytotoxicity
CD2 Sheep RBC receptor

CD4 MHC II

CD8 MHC I

2. B cells (20-35%)

Antigen Remarks
CD19 Signal transduction molecule that regulates B cell
development and activation Phagocytosis
CD21 Receptor for complement component C3d; part of Stages of Phagocytosis
B-cell co-receptor with CD19; EBV receptor 1. Initiation
CD25 Receptor for IL-2 2. Chemotaxis
3. Engulfment
Development Stage Comment 4. Digestion/Degranulation
Pro-B Cell CD19 and CD45
Rearrangement of genes in Chromosome 14 Inflammation
(Heavy Chain coding) ü Cardinal Signs of Inflammation:
1. Functio Lasa
Pre-B Cell Has u chain in the cytoplasm 2. Tumor/swelling
Contains heavy chain 3. Dolor/pain
Rearrangement of genes for Kappa (Chrom 2)

4. Calor/heat
and Lambda (Chrom 22)
5. Rubor/redness

ü Chemical Response of Inflammation
Immature Mature B Expression of IgM on the surface of CD21 and
a. Interferons
Cell CD35

Type 1 Type 2
Mature B Cell Release from the bone marrow to the Non-immune Immune
lymphoid organs
Interferon Alpha Interferon Beta Interferon Gamma
IgD expression occurs
Also known as Also known as Also known as Immune
Activated B Cell CD25 starts to appear
Leukocyte Fibroepithelial Interferon

Interferon Interferon
Plasma Cell
66
Passive Natural
b. Acute Phase Reactants
rd
*Reference: ISBB Lecture notes by Ms. A.L. Navarro/ Stevens 3 Ed. Artificial
APC Response Concentration Increase Function
Time (mg/dL)
Antigens and Antibodies
(Hours)

CRP 6-10 0.5 1000x Opsonization, Definition of Terms:
complement Term Meaning
activation Epitope Part of an antigen which binds specifically to
Antibodies & T cell receptors
Serum 3.0 1000x Removal of Adjuvants Enhances the immune response by surrounding the
amyloid A cholesterol antigen
ü Alum precipitate (stimulate phagocytosis)
Alpha- 24 200-400 2-5x Protease inhibitor ü Squalene (obtained from shark oil; for
Antitrypsin phagocytosis)

Fibrinogen 24 110-400 2-5x Clot Formation ü Freund'saduvant (killed B. pertussis and
water in oil emulsion)
Haptoglobi 24 40-200 2-10x Binds hemoglobin
n
Ceruloplas 48-72 2-40 2x Binds to copper, Affinity Tendency of the binding of the antigenic epitope to
min oxidizes iron the binding site of an antibody
Avidity Strength of overall antigen- antibody binding;
C3 48-72 60-140 2x Opsonization and antibody may bind to more than one epitope

lysis (multivalence)

MBP 0.15-1.0 Complement
Activation Antibodies

Basic Immunoglobulin structure
Adaptive Immunity Heavy Chain Light Chain
Number 2 2
Active Natural Domain 1 variable and 3 constant 1 variable and 1 constant
Composition 400 amino acids 200 amino acids
Artificial Location Chromosome 14 Chromosome 2 and 22

67

Properties of Immunoglobulin 2. Alternate Pathway
- Aka Properdin Pathway
*Reference: ISBB Lecture notes by Ms. A.L. Navarro
- It becomes deactivated by Factor I and Factor H
IgG IgM IgA IgD IgE
Structure Monom Pentam Monomer/di Monom Monom
er er mer er er 3. Mannose Binding Lectin Pathway
% of total 70-75 10 10-15 <1 0.002 - Initiated by mannose containing glycoproteins in cell wall or outer
immunoglob membrane of microorganisms.
ulin
MW 150,000 900,00 160,000 180,000 190,000 Clinical Significance of Complement Deficiency
(Daltons) 0 1. C1 INH
Sediment 7 19 7 7 8 - Hereditary Angioneurotic Edema
coefficient 2. C2 and C4
(S)
- Immune Complex Disorders such as SLE
Serum-Half 23 6 5 1-3 2-3
life (days) - Pyogenic infections with encapsulated bacteria
C’ Fixation Yes Yes Yes No No 3. C3
Cross Yes No No No No - glomerulonephritis
Placenta 4. Recurrent Neisseria infection
- C5,C6,C7,C8,C9
Complement System
3 Complement Pathways Hypersensitivity
Classification
1. Classical Pathway Type I Type II Type III Type IV

- Initiation requires antigen-antibody complex Immune IgE IgM and IgG IgM and T cell
a. Recognition Unit (C1q,C1r,C1s) Mediator IgG
o Associated with Lupus like syndrome C’ No Yes Yes No
involvement
Effector Cell Basophil RBC, WBC Host Tissue APC and
b. Activation (C4,C2,C3)
and Mast and Cell Macrophage
o C2 (Most common deficiency) is associated with Lupus Cells Platelets
syndrome and Atherosclerosis Immune Release of Lysis Deposition Cytokine
o C3 (most serious) is associated with glomerulonephritis Mechanism cells of immune release
complexes
c. MAC (Membrane Attack Complex) Examples Food Transfusion Serum Poison Ivy
Allergies reaction Sickness
68
Asthma AIHA Arthus Contact Complement Antibody + Positive:

Hay Fever HDN Reaction Dermatitis Fixation Antigen + No
Anaphylaxis SLE Tuberculin Indicator hemolysis
Test Negative:
Pneumonitis Hemolysis
Radial Sample: a. Fahey (kinetic) Kinetic: 6-
Basic Principles of Serologic Reactions Immunodiffusi Antibody b. Mancini (end- 12 hours
on point) End-point:
Test Principle Example Other 24-48
Remarks hours
Precipitation Soluble Ag + a. Double Diffusion Laurel Sample:
Soluble Ab (Ouchterlony) Immunoelectr Antigen
b. Single Diffusion ophoresis
ELISA a. HIV Testing
(Radial
b. Serum HCG
Immunodiffusion)
c. Hepatitis Antigen
c. Immunoelectropho
and Antibody
resis
d. Immunofixation

o Grading of Agglutination Reactions
Agglutination Particulate Ag a. Direct
+ Particulate Agglutination (ABO
Grade % Description
Ab Typing) Grading Cells Supernate
b. Passive 0 No agglutinates Dark, turbid,
Hemagglutination homogenous

c. Passive Latex W+ Many tiny Dark, turbid
Agglutination agglutinates
Many free cells
Agglutination Sample: a. Hemagglutination Positive: May not be visible
Inhibition Patient Ag Test for Rubella No without a
Reagent: agglutinati microscope
b. Latex Agglutination
Antibody on 1+ 25% Many small Turbid
test for other agglutinates
Negative:
viruses Agglutinati Many free cells
on 2+ 50% Many medium-sized Clear
agglutinates
69
Moderate number VDRL RPR

of free cells Specimen CSF/Serum Serum
3+ 75% Several large Clear
agglutinates Principle Microflocculation Flocculation
Few free cells Ceramic Ring 16/14 mm 18 mm
4+ 100% One large, solid Clear
Rotation of 4 min at 18 RPM 8 min at 180 RPM
agglutinate
No free cells Slide
Gauge of Qlty: 18g 20 g
Syphilis Serology needle Qty: 19g/23g
Drops of Qlty: 60 drops 60 drops
Causative agent antigen Qty: 75 drops/100 drops
Treponema pallidum subspecies pallidum
o Wasserman antigen
b. Treponemal tests
- diphosphatidyl glycerol

- akacardiolipin antigen
Test Other Remarks
o Reiter strain – Non virulent variant of T. pallidum
o Nichol's strain – Virulent
Treponema pallidum Reference Method
Immobilization Test
Stage Indication Treatment
FTA-ABS Primary Stage of Syphillis
Primary Hard Chancre Arsenic
Secondary Condylomata lata Penicillin
Hemagglutination Test Erythrocyte coated with Nichol’s
Latent Vertical transmission
Strain
Tertiary Gummas, Tabes Jerisch-Herxhimer

dorsalis Treponema Pallidum Glutaraldehyde Turkey Erythrocyte

Hemagglutination
Serological tests for syphilis
a. Non Treponemal tests MHA-TP Tanned Sheep Erythrocyte
- non specific

- detects reagin Hepatitis
Antigen Component Function
Lecithin Anti-complement 1. Hepatitis B
Cholesterol Enhances binding
Cardiolipin Main anitgen - Tests for HBsAg
o First Generation Test
70
§ Ouchterlony Double Diffusion Stages of HIV infection

o Second Generation Test (Complement Fixation) 1. Primary Stage
§ Counterelectrophoresis - General non specific symptoms :flu like symptoms, general
§ Rheophoresis malaise, low grade fever,etc
o Third Generation Tests (Most sensitive) - May be asymptomatic
§ RIA
§ Reversed passive Hemagglutination 2. Intermediate
§ ELISA - aka AIDS-related complex
§ Reversed Passive Latex Agglutination - must be a combination of 2 or more of the following
symptoms and 2 or more laboratory abnormalities
Hb S Ag Anti-HBc Anti-HBS o Laboratory manifestations
No HBV - - -
- CD4+ T cells <400
Infection
Early HBV + - - - CD4/CD8 ratio <1:1 (N:2:1)
3. Final stage
Acute HBV + + -
- aka Full blown AIDS
Window - + -
- Presence of >2 secondary symptoms and addition of
Past Infection - + +
aggravating symptoms
Immunization - - +
- Kaposi’s Sarcoma

- 2-10 years after initial infection
2. Hepatitis C
o Laboratory Diagnosis
o Screening: ELISA
- CD4+ count less than 200 (N:400-1000)
o Confirmatory: Western blot
- CD4/CD8 less than 0.5:1 (N. 2:1)


Human Immunodeficiency Virus (HIV) o Antibody tests
o Screening:
o HIV markers ü ELISA- standard test for screening
ü GP41 – Spans throughout the lipid bilayer o Agglutination tests
ü GP120 – Protruding at the surface; appears as a knob ü IFA,RIA
ü P24 – Pore coat; nucleocapsid core protein o Confirmatory:
ü P17 – Protein found in the matrix shell ü Western Blot
Acquired Immunodeficiency syndrome (AIDS) Streptococcal Serology

71
1. Antistreptolysin O (ASTO) test ü Sheep Cell Agglutination Test (Rose et. al)
- ASTO titer is the reciprocal of the highest dilution that ü Latex Fixation Test (Singer and Plotz)
exhibits no hemolysis. (Todd units) ü Sensitized Alligator Erythrocyte Test (Cohen et al)
- Normal values: less than 166 todd units ü Bentonite Flocculation Test (Bloch and Bunim)
- A 30% increase in a previously measured ASTO titer is
Heterophile Antibodies
indicative of a recent streptococcal infection

- False positive: RF Laboratory Tests for Heterophile Antibodies
1. Paul Bunnell Test
EPSTEIN-BARR VIRUS (EBV) Serology
- Presence of Heterophile antibodies agglutinate sheep red blood

- Primary target for EBV are B cells cells
- CS: titer of 1:56 is positive (presumptive) of IM given that clinical
Systemic Lupus Erythematosus (SLE) and cytological manifestations also suggest IM
Laboratory Diagnsosis
2. Davidsohn Differential Test
- Distinguishes between the three: Heterophile antibodies in
human serum against Forssmanantigen, IM and serum sickness.
- Patient serum is first adsorbed by GPKA and Beef erythrocytes
Adsorption Pattern
Heterophil Antibody Guinea pig kidney Beef extract
extract
Forssman Adsorbed Not Adsorbed
IM Not Adsorbed Adsorbed

Serum sickness Adsorbed Adsorbed
Agglutination with SRBC after adsorption
Heterophil Antibody Guinea pig kidney Beef extract
extract
Forssman Not Adsorbed Adsorbed

IM Adsorbed Not Adsorbed
Rheumatoid Arthritis (RA) Serum sickness Not Adsorbed Not Adsorbed
- Associated with HLA-DR4
- Affects synovial membrane Blood Banking

Laboratory Diagnosis Neutralizing Substances
72
- Karl Landsteiner (1901) discovered the first human blood group

Blood Group Neutralizing Substance system
§ 2 Methods of Typing
ABO
Lewis 1. Forward Typing (Forward Typing) –
uses commercial antisera to detect
P1 antigens
2. Reverse Typing (Back Typing) - uses
a
Sd known antigen against unknown
antibody
Chido, Rogers
Types of Chains

- Type 1 Chains
Lectins
o B-1,3

o Commonly found in all secretions except the CSF
Lectin Specificity
o Can found in secretions (glycoprotein)
- Type 2 Chains
Dolichos biflorus
o B-1,4
o Usually bound in RBC
Ulex europaeus o Found in plasma (glycolipid)

Vicea graminea H System
- H gene (FUT 1 gene)
Arachishy hypogea - Se gene (FUT 2 gene)


Glycine max H-Deficient Phenotype
1. Category 1: RBC H-deficient, nonsecretor (Bombay phenotype)
Salvia 2. Category 2: RBC H-partially deficient, nonsecretor
3. Category 3: RBC H-deficient, secretor (hh, Se)

ABO Discrepancy
A. Major Blood Groups - Occurs when the forward and reverse typing do not match

ABO Blood Group System 1. Group I Discrepancy
o Due to unexpected reverse typing caused by weak or
History missing antibodies
o Most common
73
2. Group II Discrepancy 004-003 E rh’’ Rh3

o Associated with unexpected forward typing due to 004-004 c hr’ Rh4
missing or weakly reacting antigens 004-005 e hr’’ Rh5
o Least frequently encountered
§ “Acquired B” phenomenon
3. Group III Discrepancy Immunogenecity of common Rh Antigens
o Associated with unexpected both forward and reverse (most immunogenic) D>c>E>C>e
typing
o Roleaux formation Lewis Blood Group System
4. Group IV Discrepancy
o No roleaux formation ü Lewis Antigen

- Receptor of Helicobacter pylori
x
ü Le antigen
Rh Blood Group System
- Marker fr Reed Sternberg cells of Hodgkin’s Lymphoma
a
ü Sialyl- Le
- Epitope for tumor marker CA-19-9

MNSs Blood Group System
ü MN Antigens
- Found on sialic acid containing glycophorin A
o M contains serine and glycine
o N has leucine and glutamic acid
ü SsU Antigen
- Located on glycophorin B
o S has Methionine

o s has threonine
ü Anti-M
- Reacts best at pH 6.5
International Society of Blood Transfusion (ISBT): Numeric Terminology - Some react only upon the exposure to glucose
- Six-digit number for each blood group specificity ü Anti-N or Autoanti-N
- First three digit represent the blood group system, and - Found in dialysis patients, with equipments sterilized
the remaining three represents the antigenic specificity with formaldehyde
ü Anti-S and Anti-s
ISBT Fisher-Race Weiner Rosenfield - Implicated with severe hemolytic transfusion reaction
004-001 D Rho Rh1 with hemoglobinuria, and HDN
004-002 C rh’ Rh2 ü U-phenotye
- Most common null phenotype, seen in blacks
74
- Lacking of most GYPA ü Kell Antigen

- Expression is very weak on McLeod phenotype cells
ü Anti-K
- Common antibody encountered in blood bank
Duffy Blood Group System
6
ü Fy
- Receptor to Plasmodium vivax
k
P and P Blood Group System
Kidd Blood Group System
a b
ü P1 Antigen ü Anti-Jk and Anti-Jk
- P1- like antigen has ben found in plasma, and droppings - Detected in antiglobulin test
of pigeons and turtledoves - Common cause of delayed type of hemolytic
- It has been identified in the hydatid cyst fluid, the transfusion reaction
earthworm
- Receptor for parvovirus B19 (Fifth disease) Lutheran Blood Group System
a b
- Receptor for Shiga toxin (EHEC and Shigella) ü Lu and Lu Antigens
ü Anti-P1 - Poorly developed at birth until age 15
- Most common antibody
- It is strongly observed in individuals infected with Crossmatching Interpretation
Echinococcus granulosus *Reference: ISBB Lecture Notes by Ms. Anna Leah Navarro, RMT
k
ü Anti-PP1P
a
- Formerly known as anti Tj Blood Antibody Optimal Reaction Enzyme
- Associated with spontaneous abortion in early pregnancy Group Class Reaction Phase Treatment
ü Anti-P System Temperature
o
- IgG found in patients with PCH Kell IgG 37 C AHG No effect

o
- Demonstrated by Donath-Landsteiner antibody Duffy IgG 37 C AHG Destroyed
o
Kidd IgG 37 C AHG Enhanced
a a o a
I Blood Group System Lutheran Lu IgM Lu 4 C Lu Room Variable
b b o
ü Anti-I Lu IgG Lu 37 C Temp
b
- Associated with Mycoplasma pneumonia infection and Lu AHG
cold agglutinin disorders Lewis IgM Most often Room Enhanced
o
ü Autoanti-I 4 C temp,
o
- Mycoplasma pneumonia and Listeria monocytogenes 37 C, and
ü Anti-i AHG
o o
- Associated with EBV I IgM 4 C IS, 37 C Enhanced
o o
P IgM 4 C IS, 37 C, Enhanced
Kell Blood Group System and AHG
75
o o o o
MN IgM 4 C IS, 37 C, Destroyed 4. Temperature: 35.5 C or 99.5 F
and AHG 5. Blood pressure: 180/100 mmHg
o
Ss IgG 37 C AHG Variable 6. Minimum hemoglobin and hematocrit
• Hemoglobin: > 12.5 g/dL
B. Minor Blood Groups • Hematocrit: > 38%
o Hemoglobin by CuSO4 or
Diego Blood Group System Philips method
a
- Di is used as a marker for studying the Mongolian ancestry
- Can be seen in hereditary spherocytosis Blood Preservation: Anticoagulants and additives
ü Citrate: binds to Calcium
Cartwright Blood Group System ü Dextrose: provides energy for RBC; ACD (Acid citrate
- Involved in neurotransmission dextrose) 21 days
ü Citric Acid: decrease pH; prevents caramelization of blood
Chido/Rogers Blood Group System ü Phosphate buffer: increase ATP; CPD – 21 days
- Associated with Human Leukocyte Antigen (HLA) ü Adenine: improve survival of red cells; CPDA1- 35 days

Gerbich Blood Group System Approved Preservatives
- Expressed on glycophorin C and or D Acid Citrate Dextrose 21 days
- Erythrocytes formes an elliptocyte Citrate Phosphate Dextrose 21 days
Citrate Phosphate Dextrose 35 days
Blood Donation Adenine 1
Citrate Phosphate Double Dextrose 21 days
Basic Qualifications of a Potential Blood Donor
Additive Solutions
1. Good health 1. Saline

2. Age: 18-65 years old 2. Adenine
3. Weight: 10.5 mL of blood/kg or 110lbs/50kg 3. Glucose
a. Volume of blood to be collected 4. Mannitol – RBC stabilizer
𝐷𝑜𝑛𝑜𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 14 ü Commercial names
𝑥 o Adsol (AS-1)- 42 days
𝐼𝑑𝑒𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 100
b. Volume of anticoagulant needed o Nutricel (AS-3)- 42 days
450 mL + 30 mL = 63 mL of Anticoagulant o Optisol (AS-5)- 42 days

c. Volume of anticoagulant to be removed from the blood
Rejuvenating Solutions
bag
31.5 mL (AC): 200 mL (blood) - Regeneration of ATP, and 2,3-DPG
o
- 1-4 hours at 37 C
63 mL (AC): 450 mL (blood)
- PIGPA- Phosphate, inosine, glucose, pyruvate, and adenine
76
- PIPA- Phosphate, inosine, pyruvate and adenine 4. Forms of Calcium Salts:

- Rejuvesol – approved by the FDA ü Calcium urates
ü Calcium phosphates
Changes in Storage of RBC ü Calcium fluoride
ü pH: deacrease ü Calcium oxalate
ü ATP: decrease 5. Nitric acid is the most common and the most rapid decalcifying gent
ü 2,3 DPG: decrease 6. Type of Gland Secretion:
ü Hemoglobin: increase ü Merocrine Gland – no loss of cytoplasm
ü Potassium: increase ü Apocrine Gland – With cytoplasmic loss
ü Sodium: decrease ü Holocrine Gland – total lysis of the secretory cell
7. Collagen is considered as the major ingredient in all connective
Preparation of Blood Components tissues
ü Heavy spin 8. Persons:
o 5000 g for 5 minutes (PRBC, plt conc) ü Hippocrates – Father of Medicine
o 5000 g for 7 minutes (cryo, platelet poor plasma) ü Rudolf Virchow – Father of Cellular Pathology
ü Light spin ü Karl Landsteiner – Father of Blood Trasnfusion
o 2000 g for 3 minutes (platelet rich plasma) ü George Papanicolau – Father of Exfoliative Cytology
o
- centrifugation for platelet concentrate is performed at 20-24 C ü Butschleii – First to use Paraffin
o
- for other blood components, centrifugation is performed at 1-6 C ü Persival Paul – Identified that chimney soot can cause cancer
ü Conheim – First to use frozen technology
ü Cornelius Celsus – First to describe the 4 signs of infection
ü Waldier – First to use hematoxylin dye
HTMLE 9. “Sar” refers to connective tissue malignancies while “Car” refers to

epithelial tissue malignancies
10. Body Changes during death:

o
ü Algor mortis – 7 F/hour
Must Know Recall Questions! ü Rigor mortis – hardening of the body (head)
1. Carbon Tetrachloride is similar to chloroform. It may be used in ü Livor mortis (purpling of skin)
clearing tissues for embedding, It also produces considerable ü Postmortem Clot
hardening of tissues and is dangerous to inhale on prolonged ü Dessication –drying of eyes
exposure due to its highly toxic effect. ü Putrefaction
2. Fixation Requirements: ü Autolysis
ü Tissue size: 10 mm X 4 mm thick 11. Egg albumin should NEVER be used in cytology
ü Volume of fixative: 20 X the volume of the specimen; Osmium 12. Strawberry cervix is caused by Trichomonas vaginalis
tetroxide: 5 – 10x of the tissue volume; Museum: Not < 50 – 100x Reports Storage
ü Tissue container: adequate size, wide mouthed and non-rusting Autopsy forensic reports Indefinite
2
3. The tissue size for Electron Microscopy is 1-2 mm Bone Marrow repots 10 yrs
77
Cytogenic reports 20 years 22. Removal of fixative artifact is performed after dehydration and
Clinical pathology reports 2 years prior to the application of primary stain
Surgical reports 10 years 23. Acridine Orange Stain:
13. ü RNA: Red fluorescence
14. Dry gangrene occurs in the arteries ü DNA: Green fluorescence
15. Wet gangrenes occur in the veins 24. Stains for Collagen include:
16. Cold Knife sectioning utilizes Carbon Dioxide: ü Van Gieson – most common and simplest
ü Knife: -40 to -60C ü Masson Trichrome
ü Tissue: 5 to -10 C ü Malloy Aniline Blue
ü Cutting Environment: 0 to -10C ü Azocarmine
17. Common Gases for freezing: ü Krajan’sAniline Blue
ü Carbon Dioxide gas Specimen Type Storage
ü Aerosol spray Serum 48 hours
ü Isopentane Routine blood smears 7 days
ü Liquid Nitrogen Bone marrow slides 10 years
18. Border’s Classification Cytogenic slides 3 years
Grade Differentiated Cells Undifferentiated Cells Pathology Blocks 10 years
I 100-75% 0-25% Microbiology smears 7 days
II 75-50% 25-50% Crossmatching samples 7 days-post transfusion
III 50-25% 50-75% 25.
IV 25-0% 75-100% 26. IgG is commonly used during immunohistochemistry
27. Rabbits are the common source of Polyclonal antibodies, while

19. Epithelial cells that are produced during HPV infections are referred
to as Koilocytes Mice are commonly used as a source of Monoclonal antibodies
20. Labeling of slides is the last step for tissue processing 28. Reference Laboratories in the Philippines (As of 2000)
ü Fixation Institution Specialization

ü Dehydration RITM Dengue, Influenza, TB,
ü Clearing Mycobacteria, Malaria,
ü Impregnation Parasites, Enteric Bacteria,
ü Embedding Measles, Viruses, Fungi, Blood
ü Trimming Units
ü Sectioning San Lazaro Hospital HIV/AIDS, Hepatitis, Syphillis,
ü Staining STI
ü Mounting EAMC Environment and Occupational
ü Labelling Health, Toxicology
21. Heidenhain SuSa is an acronym of Sublimat (Mercuric chloride) and NKTI Hematology, BB, Anatomic
Saure (Acid) Pathology
78
Lung Center of the Phil Biochemistry/Clinical Chemistry RA Newborn Screening April 7, Pres. Gloria M.
9288 Act of 2004 2004 Arroyo

29. Air embolism is frequently seen in scuba divers while fat embolism
is seen in car accidents
30. Teratomas are referred to as monstrous tumors

Medical Technology Laws
Law Title Date of
Approval
President/Signatory
Laboratory Exercises
RA Philippine Medical June 21, Pres. Ferdinand E.
5527 Technology Act of 1969 Marcos
1969 Acid-Base Balance
RA The PRC December
8981 Modernization Act of 5, 2000
2000
RA Clinical Laboratory June 18,
4688 Law 1966
RA Blood Banking Law June 16,
1517 1956
RA National Blood May 5, 1994

7719 Services Act of 1994

AO # 9 Rules and Regulations May 12, Jaime Galvez Tan

S. 1995 Implementing RA 1994

7719

RA Philippine AIDS February

8504 Prevention and 13, 1998

Control Act of 1998

EO 309 Family Code of the
Mixing Studies
Philippines

RA Organ Donation Act January 7,

7170 1991

RA Comprehensive January 13,
9165 Dangerous Act of 2003
2002
RA Dangerous Drug Act June 7, 2002
6425 of 2002
79

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Olibrian Mallari, RMT, MLS(ASCPi),CPT(IMET), MT(AIMS)

ULTIMATE FINAL COACHING

Microbiology Negative: Lack of Fluorescence

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S. aureus S. epidermidids Virulence Factor

Characteristic Endotoxin Exotoxin

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Antimicrobial Agents and Antibiotics Sterilization and Disinfection

1. Bacillus subtilis Bacitracin Indicator Comment

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Organisms 4. Streptococcus pyogenes ( Group A)- beta- hemolytic

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20. Rickettsiaceae Acid fast bacilli

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Paracoccidoides South American Blastomycosis; Pox virus DNA Largest DNA

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Borrelia Daisy head

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Haemophilus Ducrey's School of fish Soft chancre/ Walking

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Red coloration Gram-positive

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Une Filarifo Ground

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Medication therapy Insulin is necessary Oral agent

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TAG Chole Free Phospholipid Protein

Proteins Blood Urea Nitrogen

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1. Creatinine Clearance Type of Non polar polar Polar

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o Co-enzyme – organic portion Acid Phosphatase

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Aspartate Aminotransferase ACTH Addison’s

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Hypothyroidism Hyperthyroidism Toxicology

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Yellow- Acetic Acid,

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9. Red Cell Grading:

10. Grading of Hypochromia (Low hemoglobin)

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Hematopoiesis produces Gower 1,

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Cyanmethemoglobin ü Forms when ferricyanide binds with 540 nm

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BURR CELL Uremia, neonatal liver disease, Leukocyte

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Acute Myeloproliferative Leukemia/ Acute Nonlymphocytic Leukemia Acute Lymphoblastic Leukemia

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Platelets ALPHA GRANULES DENSE GRANULES

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NOT ADSORBED by ADSORBED by BaSO4 NOT ADSORBED by

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