Mtap Batch 2 Midterms

PARASITOLOGY
Medical Technology Assessment Program - 1

October 28, 2021 | Ma. Cristina SJ Liwanag RMT, RN, Ph.D, M.A., MSMLSc
SPECIES AND ITS COMMON NAMES Chinese Liver

Clonorchis sinensis
Fluke/Oriental Liver Fluke
NEMATODES Opistorchis felineus Cat Liver Fluke
Eelworm/Giant Intestinal
Ascaris lumbricoides
Roundworm CESTODES
Enterobius vermicularis Pinworm/Seatworm Fish Broad
Trichuris trichiura Whipworm Diphyllobothrium latum Tapeworm/Russian
Toxocara cati Ascaris of Cats Broad Tapeworm
Toxocara canis Ascaris of Dogs Pork Tapeworm/Armed
Ancylostoma braziliense Cat Hookworm Taenia solium
Tapeworm
Ancylostoma caninum Dog Hookworm Beef
Ancylostoma duodenale Old World Hookworm Taenia saginata Tapeworm/Unarmed
Necator americanus New World Hookworm Tapeworm
Strongyloides stercoralis Threadworm Hymenolepis nana Dwarf Tapeworm
Capillaria philippinensis Pudoc Worm Hymenolepis diminuta Rat Tapeworm
Pork Muscle Dog Tapeworm/Double-
Trichinella spiralis Roundworm/Trichina Dipylidium caninum
pored Tapeworm
worm Echinococcus granulosus Hydatid Tapeworm
Wuchereria bancrofti Bancroft’s Filaria
Loa loa Eyeworm
Brugia malayi Malayan Filaria HABITAT – INFECTIVE STAGE – MODE OF TRANSMISSION
Onchocerca volvulus Convoluted Filaria INTESTINAL NEMATODES
Mansonella ozzardi Ozzard’s Filaria Habitat: Small Intestine
Mansonella perstans Persistent Filaria Infective Stage:
Angiostrongylus Embryonated egg Ascaris lumbricoides
Rat Lung Worm
cantonensis Mode of Transmission:
Guinea worm/Serpent Ingestion
Dracunculus medinensis
worm/Dragon worm Habitat: Large Intestine
Dirofilaria immitis Dog Heart Worm Infective Stage: 1. Trichuris trichiura
Embryonated egg 2. Enterobius
TREMATODES Mode of Transmission: vermicularis
Schistosoma japonicum Oriental Blood Fluke Ingestion
Schistosoma mansoni Manson’s Blood Fluke Habitat: Small Intestine
Schistosoma Infective Stage: 1. Necator americanus
Vesical Blood Fluke
haematobium Filariform Larva 2. Ancylostoma
Paragonimus westermani Oriental Lung Fluke Mode of Transmission: duodenale
Giant Intestinal Skin Penetration
Fasciolopsis buski
Fluke/Busk Fluke Habitat: Small Intestine
Echinostoma ilocanum Garrison’s Fluke Infective Stage:
Von Siebold Fluke/Dwarf Filariform Larva Strongyloides stercoralis
Heterophyes heterophyes Mode of Transmission:
Fluke
Metagonimus yokogawai Yokogawa’s Fluke Skin Penetration
Fasciola hepatica Sheep Liver Fluke Habitat: Small Intestine
Capillaria philippinensis
Infective Stage:
1
PARASITOLOGY
Larva Mosquitoes
Mode of Transmission: Brugia malayi Upper lymphatics (Mansonia
Ingestion/Consumption spp.)
of raw fishes Chrysops
Subcutaneous
Habitat: Small Intestine Loa loa fly, Mango
tissues
(Adult); Muscle (Larva) fly, Deer fly
Infective Stage: Encysted Onchocerca Subcutaneous
Black flies
Larva Trichinella spiralis volvulus tissues
Mode of Transmission: Mansonella
Body cavities
Ingestion/Consumption ozzardi
of inadequately cooked Mansonella Midges
pork Body cavities
perstans (Culicoides
Dermis of the skin fly)
Mansonella
ZOONOTIC NEMATODES (<1 mm of the skin
streptocerca
Infective stage to man: surface)
Embryonated egg
NON-HUMAN ASCARIS
Mode of Transmission: 1. Toxocara canis Habitat: Subcutaneous
Ingestion (accidental) tissue
2. Toxocara cati
Causative agent: Visceral Infective Stage: Third
larva migrans Stage Larva
Infective stage to man: Mode of Transmission: Dracunculus medinensis
NON-HUMAN HOOKWORMS
Filariform Larva Ingestion
1. Ancylostoma
Mode of Transmission: Intermediate Host:
caninum
Skin Penetration Copepods/Freshwater
2. Ancylostoma
Causative agent: fleas
braziliense
Cutaneous larva migrans
Infective stage to man: TREMATODES
Third Stage Larva Angiostrongylus BLOOD FLUKES
Mode of Transmission: cantonensis Habitat: Superior
Ingestion mesenteric veins
Mode of Transmission:
Skin Penetration
BLOOD AND TISSUE NEMATODES Infective Stage: Fork- Schistosoma japonicum
(EXTRAINTESTINAL)
tailed Cercaria
Specimen for Diagnosis:
FILARIAL WORMS Feces
PARASITE HABITAT VECTOR Diagnostic Stage: Eggs
Mosquitoes Habitat: Inferior
(Aedes, mesenteric veins
Wuchereria Culex, Mode of Transmission:
Lower lymphatics Schistosoma mansoni
bancrofti Anopheles, Skin Penetration
Mansonia Infective Stage: Fork-
spp.) tailed Cercaria
2
PARASITOLOGY
Specimen for Diagnosis: HEPATIC/LIVER FLUKES

Feces Second Intermediate
Diagnostic Stage: Eggs Host: Fresh water
Habitat: Vesical veins vegetation
Mode of Transmission: Infective Stage: Fasciola hepatica
Skin Penetration Metacercaria
Infective Stage: Fork- Schistosoma Mode of Transmission:
tailed Cercaria haematobium Ingestion
Specimen for Diagnosis: Second Intermediate
Urine Host: Fish
Diagnostic Stage: Eggs Infective Stage:
Clonorchis sinensis
LUNG FLUKE Metacercaria
Second Intermediate Mode of Transmission:
Host: Crabs Ingestion
Infective Stage: Second Intermediate
Paragonimus westermani
Metacercaria Host: Fish
Mode of Transmission: Infective Stage:
Opistorchis felineus
Ingestion Metacercaria
INTESTINAL FLUKES Mode of Transmission:
Second Intermediate Ingestion
Host: Fresh water
vegetation CESTODES
Infective Stage: Fasciolopsis buski PSEUDOPHYLLIDEA
Metacercaria Infective Stage: (1)
Mode of Transmission: Plerocercoid Larva
Ingestion Mode of Transmission:
Second Intermediate Ingestion/consumption of
Host: Snails inadequately cook fishes
Infective Stage: (Diphyllobothriasis)
Echinostoma ilocanum
Metacercaria
Infective stage to man: (2) Diphyllobothrium latum
Mode of Transmission: Procercoid Larva
Ingestion Mode of Transmission:
Second Intermediate Ingestion/Accidental
Host: Fish consumption of procercoid
Infective Stage: larva (Sparganosis)
Heterophyes heterophyes
Metacercaria
Mode of Transmission: 1st IH: Copepods
Ingestion 2nd IH: Fishes
Second Intermediate CYCLOPHYLLIDEA
Host: Fish Infective stage to man: (1)
Cysticercus cellulosae
Infective Stage:
Metagonimus yokogawai (larva) Taenia solium
Metacercaria Mode of Transmission:
Mode of Transmission: Ingestion (Taeniasis -
Ingestion Intestinal)
3
PARASITOLOGY
DISTINCT MORPHOLOGY
Infective stage to man: (2)
Eggs OVA/EGGS OF NEMATODES
Mode of Transmission: SPECIES MORPHOLOGY
Ingestion (Cysticercosis -
• Produces fertilized
Tissues)
and unfertilized eggs
IH: Pigs/Swine/Hogs Ascaris lumbricoides • Shell with outer
Infective stage to man: layer called
Cysticercus bovis (larva) mamilliary coat
Mode of Transmission: Taenia saginata • If Ascaris egg lacks
Ingestion (Taeniasis) its mammillary coat,
we refer to it as
IH: Cattles/Cows decorticated egg
Infective stage to man: Hymenolepis nana
Cysticercoid
• Barrel
Hymenolepis diminuta shaped/lantern
Ingestion
shaped/football
IH of H. nana and H. Trichuris trichiura shaped with
diminuta: Beetles/Fleas Dipylidium caninum protruded polar
plugs
IH of D. caninum: Fleas/Dog • Commonly referred
lice to as Japanese
Infective stage to man: Eggs Lantern Ova
Echinococcus granulosus Enterobius vermicularis Eggs flat on one side, D-
Ingestion (Hydatid
Disease/Echinococcosis) shaped with thin &
transparent shell, with
TISSUE CESTODES larva inside
INFECTIVE DISEASE
SPECIES Capillaria philippinensis
STAGE CAUSED
Taenia solium Eggs Cysticercosis With typical & atypical
Diphyllobothrium Procercoid Sparganosis forms, peanut shaped,
latum striated shell & with flat
Spirometra spp. Procercoid Sparganosis polar plugs
Taenia multiceps Eggs Coenurosis
• All eggs are
• Habitat of Adult Tapeworm: Small Intestine Hookworms IDENTICAL
o Man harbors the adult tapeworm, serves as • Ovoid, with thin
the definitive host. shell containing 2-8
• Habitat of Larva: Tissues germ cells
resembling
MORULA BALLS
• Similar with
Strongyloides stercoralis hookworm but are
embryonated
4
PARASITOLOGY
PERIODICITY AND MORPHOLOGY OF

MICROFILARIA
SPECIES PERIODICITY MICROFILARIA
SHEATHED MICROFILARIA
Nucleus NOT With cuticular alar
extending to tip expansion & well-defined
Wuchereria Nocturnal esophageal bulb
of tail
bancrofti periodic Enterobius vermicularis
Two terminal
Nocturnal nuclei
Brugia malayi With slender head &
subperiodic
fleshy tail resembling a
Nucleus extends whip
Diurnal to tip of tail Trichuris trichiura
Loa loa
periodic
UNSHEATHED MICROFILARIA
Nucleus NOT
• A. braziliense - 1 pair
Onchocerca extending to tip
of buccal teeth
of tail
volvulus
Nucleus NOT
Mansonella extending to tip
ozzardi of tail • A. duodenale - 2
pairs of buccal teeth
Non-periodic Nucleus
Mansonella extending to tip
perstans of tail Hookworms
• A. caninum - 3 pairs
Nucleus of buccal teeth
extending to tip
of tail; curved
Mansonella
(Shepherd’s
streptocerca
crook)
• N. americanus -
semi-lunar cutting
plates
ADULT NEMATODES
SPECIES CHARACTERISTIC
With 3 oval lips/Trilobate
Ascaris lumbricoides
Lip
5
PARASITOLOGY
With distinct terminal

spine
Schistosoma
haematobium
Uterus with Barber’s Pole
appearance Eggs of Intestinal, Lung and Liver flukes are all
OPERCULATED
Angiostrongylus
cantonensis
•
With flat operculum
With • abopercular
Paragonimus westermani shell thickening
(shell thickening)
Longer buccal cavity;
Hookworm - • Eggs may resemble
smaller genital
RHABDITIFORM those of D. latum
premordium
Shorter buccal cavity; • With narrow
Strongyloides stercoralis - Heterophyes Heterophyes
larger genital operculum
(Thick-shelled)
RHABDITIFORM
premordium • No abopercular
Metagonimus yokogawai
Sheathed with pointed knob
(Thin-shelled)
tail
• With wide
Hookworm - FILAFORM operculum
Clonorchis sinensis
• with abopercular
(broadly ovoid)
knob
• with eggs resembling
Unsheathed with bifid or Opistorchis felineus
an OLD-FASHIONED
notched tail end (elongately ovoid)
ELECTRIC BULB is
Strongyloides stercoralis - Clonorchis sinensis
FILARIFORM
OTHER DISTINCT
SPECIES
MORPHOLOGY
With FORK-TAILED
OVA/EGGS OF TREMATODES Schistosoma
CERCARIA
SPECIES MORPHOLOGY Adult assumes a Coffee
Eggs of Schistosoma/Blood flukes are NON-OPERCULATED
Paragonimus westermani
bean appearance
With minute lateral knob • Appears as if with
Schistosoma japonicum shoulders because
of CEPHALIC CONE
With distinct lateral Fasciola hepatica • May resemble F.
spine hepatica but no
cephalic cone:
Schistosoma mansoni
Fasciolopsis buski
Echinostoma ilocanum Oral sucker with spines
6
PARASITOLOGY
Circumoral Disk with THICKENINGS & POLAR

Spines FILAMENTS, egg contains
• Adult equipped with hexacanth embryo
Central 3rd
Sucker/genital • Non-operculated
sucker eggs with polar
Hymenolepis diminuta thickenings but NO
• May resemble
Heterophyes heterophyes polar filaments
heterophyes
heterophyes but no • Eggs with
3rd sucker: INTRALAMINAL
Metagonimus LAYER giving it a
yokogawai FRIED EGG
Largest blood fluke; with appearance
Schistosoma japonicum Dipylidium caninum
smooth integument
• Smallest blood fluke
Produces egg packets
• integument with
Schistosoma mansoni
coarse
tuberculations Eggs NEVER SEEN in
Blood fluke whose Echinococcus granulosus
HUMAN FECES
Schistosoma integument shows fine
haematobium tuberculations OTHER DISTINCT
SPECIES
MORPHOLOGY
OVA/EGGS OF CESTODES
SPECIES MORPHOLOGY
• OPERCULATED, with
abopercular knob
Diphyllobothrium latum opposite the
operculum & may
contain undeveloped
coracidium • Scolex (head) – for attachment
• EGGS may resemble • Neck – region of growth
those of P. • Proglottids – segments
westermani o Immature – sexually undeveloped
• NON-OPERCULATED o Mature – full sets of male and female
Taenia solium eggs, with radially reproductive organ (testes, ovaries,
Taenia saginata striated shell genital pores)
containing o Gravid/ripe – contains the egg-filled
hexacanth embryo uterus (uterus shape varies)
• Taenia eggs may Diphyllobothrium • With ROSETTE SHAPED
resemble aegis latum uterus in gravid segments
shield
Non-operculated eggs
Hymenolepis nana
with POLAR
7
PARASITOLOGY
Gravid segments with

SACCULAR or sac-like uterus
Hymenolepis nana
Hymenolepis
diminuta
• Adult worm is regarded as

the LONGEST as to length • Adult worm has only
• Adult worm with THREE segments
ALMOND (Immature, Mature,
SHAPED/SPOON SHAPED Gravid)
SPATULATE SCOLEX Echinococcus • Regarded as the
• Two false suckers - granulosus SHORTEST as to size
BOTHRIA
• With vase-
• Gravid segments may shaped/pumpkin seed
contain elongated uterus shaped segments/rice
with cylindrical trunk with grains appearance
8-12/15 lateral uterine • With BILATERAL GENITAL
branches Dipylidium caninum PORES
Taenia solium
• Mature segments – with
3rd ovary called Hymenolepis nana Adult with Y-shaped hooks
ACCESSORY OVARIAN
LOBE PATHOLOGY
May cause intestinal
Ascaris
obstruction-intestinal
lumbricoides
perforation
• Rectal prolapse
o Symptom of
Gravid segments may contain Trichuris trichiura heavily infected
uterus with cylindrical trunk individual
with 15-30 lateral uterine (Trichuriasis)
branches
Taenia saginata
Enterobius
• Pruritis ani
vermicularis
o Symptom of
Enterobiasis
Capillaria • Borborygmi/Gurgling
philippinensis stomach
8
PARASITOLOGY
o Symptom of appropriate blood vessel

Capillariasis Paragonimus • May produce symptoms
The only nematode that can westermani similar to TB
Trichinella spiralis cause INTENSE eosinophilia, • May cause Vit. B12
Periorbital edema deficiency anemia
• COOLIE ITCH/DEW (Pernicious anemia)
ITCH/GROUND ITCH Diphyllobothrium o Adult in the
• Due to latum small intestine
filariform larva competes in the
Hookworms (blood
skin absorption of
feeders)
penetration Vit. B12
• Anemia – Iron Deficiency
Anemia/Microcytic
Hypochromic Anemia OTHER IMPORTANT NOTES
• May produce COIN
LESIONS in the lungs NEMATODES
Dirofilaria immitis
o detected Embryonated egg Fully-developed larva
through X-RAY
• First stage larva
• Elephantiasis of lower limbs • Shorter, more robust,
• Tropical Pulmonary Rhabditiform not infective
Wuchereria Eosinophilia • Feeding stage with
bancrofti • Chyluric/milky urine open mouth
o Obstruction in
• Second stage larva
the lymphatics
• Mature
• Elephantiasis of upper
Brugia malayi • Longer, slender,
limbs Filariform
infective
• Calabar/fugitive/temporary • Non-feeding stage with
Loa loa
swellings closed mouth
Onchocerca • River blindness/Blinding #1 - Enterobius
volvulus filariasis vermicularis Common intestinal
Toxocara canis • Visceral larva migrans or #2 - Ascaris nematodes worldwide
Toxocara cati Ocular larva migrans lumbricoides
Ancylostoma #3 - Trichuris trichiura
caninum • Cutaneous larva migrans/ • Trichuris trichiura Nematodes classified as
Ancylostoma “creeping eruption” • Trichinella spiralis APHASMIDS
braziliense
• Capillaria
• May develop because of philippinensis
CERCARIAL PENETRATION • Ascaris
Swimmer’s Itch o Allergic lumbricoides With lung migration in life
reaction; Form
• Strongyloides cycle and cause
of dermatitis PNEUMONITIS
stercoralis
• Hypersensitivity reaction
• Human
Katayama Fever due to migration of
hookworms
Schistosomule to
9
PARASITOLOGY
• Human • ADULT WORMS may be

hookworms free-
• Ascaris living/FACULTATIVE
UNHOLY THREE
lumbricoides (can survive even w/o
• Trichuris trichiura host)
• Dracunculus • Three life cycles:
medinensis VIVIPAROUS/LARVIPAROUS Parasitic (direct), Free-
• Trichinella spiralis FEMALE (not capable of living (indirect), and
producing eggs)
• Filarial worms Autoinfection
• Ascaris OVIPAROUS (capable of • Muscle biopsy as a
lumbricoides producing eggs without fully means of diagnosis
• Trichuris trichiura developed larva in its shell) Trichinella spiralis • Bacchman intradermal
• Enterobius OVOVIPAROUS (capable of & bentonite
vermicularis producing eggs with fully Flocculation test
• Strongyloides developed larva in its shell) Reservoir of Capillaria
stercoralis Fish-eating Birds philippinensis (local
Ascaris lumbricoides Largest intestinal nematode nematode)
• Examination of • Cannot be detected
Toxocara canis and
Perianal/Scotch tape through stool exam
Toxocara cati
swab • Sero-test
• Eggs are Ancylostoma With CATS/feline as
deposited braziliense DEFINITIVE HOST
in the With DOGS/canine as
Ancylostoma caninum
perianal DEFINITIVE HOST
Enterobius vermicularis region/skin • New name:
• may carry Parastrongylus
Dientamoeba fragilis cantonensis
trophozoite in its eggs • Definitive Host: Rats
• may cause RETRO • Intermediate Host:
INFECTION Angiostrongylus Land snails
• may cause AUTO cantonensis • CEREBRAL
INFECTION ANGIOSTRONGYLIASIS
Human hookworms Blood-sucking nematodes • Eosinophilic
• Smallest intestinal meningitis
nematode • Dx: Histologic
• Finding detection of ADULT
RHABDITIFORM LARVA FEMALE
in feces is diagnostic • ABDOMINAL
Strongyloides ANGIOSTRONGYLIASIS
• Female may be
stercoralis • MOT: Consumption of
PARTHENOGENIC Parastrongylus
• can salad contaminated
costaricensis
produce with excretions from
eggs even infected slugs/snails
w/o male • Dx: Larva or eggs in
10
PARASITOLOGY
tissue secretions • Non-hermaphroditic;

Culture Methods for separated sexes
• Harada Mori
Hookworm and S.
• Copro culture
stercoralis Blood flukes
• They need two hosts
• Two developmental • Intermediate host:
stages: Larva and Adult Snails
• Arthropod-borne • Definitive host: Man
Filarial worms • Infective stage to man: • Hermaphroditic;
Third Stage Larva combined sexes
Lung, Intestinal and
• Infective stage to • They need three hosts.
Hepatic flukes
vector: Microfilaria • First intermediate
Diagnostic stage for hosts: snails/mollusks
Microfilaria
filariasis detection • a sero-test for diagnosis
• Periodicity - it is of S. japonicum
the migration of • Serum + Antigen
larva into the LYOPHILIZED EGGS of
blood S.japonicum
Must be considered in the
• Filaria – LPO • Incubate for 24 hours
collection of blood for the
• Malarial blood • (+) result - BLEB
diagnosis of filariasis COPT/CIRCUMOVAL
collection should Formation or
be done before PRECIPITIN TEST Precipitates attached to
the fever spikes the egg
(OIO)
• Concentration
Knott’s Concentration technique for filariasis
Technique detection
• Reagent: formalin
• Can be detected thru Paragonimus Fresh mountain crabs as 2nd
observation of larva on westermani Intermediate Host
open blisters/skin ulcer
• Heterophyes
after placing a drop of
Dracunculus heterophyes
water
medinensis • M. yokogawai
• Habitat: Subcutaneous 2nd intermediate Hosts: FISH
• Clonorchis
tissue
sinensis
• Intermediate host:
• Opistorchis
copepods/cyclops
felineus
• Fasciolopsis buski 2nd intermediate hosts are
TREMATODES • Fasciola hepatica FRESH WATER VEGETATION
Larva with mouth, GIT and
Cercaria 2nd intermediate Host:
tail Echinostoma ilocanum
SNAILS
Cercaria without tail; upon
Heterophyes
penetration of cercaria to Deadliest trematode
Schistosomule heterophyes
the human skin, it loses its
tail
11
PARASITOLOGY
CESTODES
NON-HELMINTHS
All tapeworm scolex are globular except: D. latum
All tapeworm species have one genital pore (laterally PROTOZOA
located) except: D. caninum • Unicellular/single-celled eukaryotic microorganism
• Adult: May resemble o Classified under Kingdom Protista
Spirometra o Performs all the functions:
Diphyllobothrium latum • Egg: May resemble Reproduction, digestion, excretion etc.
eggs of Paragonimus • Cytoplasm: with ectoplasm and endoplasm
westermani • Trophozoite - protozoan stage in liquid feces;
• Deadliest cestode motile stage
• Tapeworm species • Cyst - Protozoan stage likely to be recovered in
that can complete its formed feces; immotile form
Hymenolepis nana cycle in one host
(HOMOXENOUS) CILIATE
• Can cause SPECIES CHARACTERISTICS
autoinfection • Intestinal protozoa
More than 25 years or Life span of Adult D. with directional
greater latum & Taenia spp. tumbling motility
Causes coenurosis which • Common parasite of
may be acquired through pigs; tissue invader
accidental ingestion of • Largest intestinal
dog feces with eggs; this protozoa
Taenia multiceps can lead to a condition Balantidium coli
• Habitat: Large
dog feces with eggs; this Intestine
can lead to a condition
• Trophozoite: With
called GID, STURDY or
Kidney-shaped
STAGGERS
macronucleus and
• Casoni’s is a sero test Spherical
for diagnosis micronucleus
• Man is dead-end
host.
AMOEBA
Echinococcus granulosus • Hydatid – potentially
SPECIES CHARACTERISTICS
dangerous
• Tissue invading-
depending on
intestinal protozoa
location of cyst;
usually fluid-filled • Infective stage:
Entamoeba histolytica Quadrinucleated
Causes alveolar hydatid
cyst
disease (fatal form of
Echinococcus multicularis echinococcosis; most • Likely to ingest RBC
LETHAL of all helminthic in its cytoplasm
diseases) Sluggish, non-directed
Entamoeba coli
motility
• Smallest intestinal
Endolimax nana protozoa
• With cross-eyed cyst
12
PARASITOLOGY
•Species of amoeba West African Sleeping

Trypanosoma gambiense
with large glycogen Sickness
Iodamoeba butschlii
mass Trypanosoma East African Sleeping
• Iodine cyst rhodesiense Sickness
• Not hematophagous • Transmitted by tse
do not ingest RBC to tse flies/Glossina
Trypanosoma gambiense
its cytoplasm spp.
Trypanosama
Entamoeba dispar • Currently • Causative agent of
rhodesiense
morphologically Old-World
closes to E. Trypanosomiasis
histolytica
FACULTATIVE AMOEBA
Acantamoeba Causative agent of SPOROZOA
Granulomatous amoebic SPECIES CHARACTERISTICS
Balamuthia
meningoencephalitis Protozoan parasite with
An amoeboflagellate, Toxoplasma gondii cats/feline as definitive
causative agent of host
Naegleria fowleri
primary amoebic • Intraerythrocytic
meningoencephalitis parasite transmitted
through tick bites
Babesia microti
• Assumes as maltese
FLAGELLATES cross appearance of
SPECIES CHARACTERISTICS trophozoite
Intestinal flagellate Infective stage and
Cryptosporidium parvum
Giardia lamblia exhibiting kite-like or diagnostic stage: Oocyst
falling leaf-like motility Protozoan parasite that
Cyclospora cayetanensis
Protozoan parasite that shows autofluorescence
Chilomastix mesnili assumes a shepherd’s Plasmodium spp.
crook appearance • Infective stage to man: Sporozoites
Leishmania spp. • Infective stage to vector: Gametocytes
• Transmitted by Sandflies/Phlebotomus o Male: Microgametocytes
• Promastigote - Infective Stage of Leishmania o Female: Macrogametocytes
species to man • Examination of thick and thin blood smear
Visceral o Gold standard for Malaria Detection
Leishmania donovani Leishmaniasis/Dumdum Schuffner’s granules,
Plasmodium vivax
Fever/Kala-azar amoeboid trophozoite
Oriental sore/Cutaneous Maurer’s dots, aplique
Leishmania tropica
leishmaniasis accole forms, with
Plasmodium falciparum
Muco-cutaneous banana shaped, crescent
Leishmania braziliensis
leishmaniasis shaped gametocytes
Chaga’s disease/South Rosette merozoites, band
Plasmodium malariae
American form trophozoites
Trypanosoma cruzi
Trypanosomiasis or New-
World Trypanosomiasis
13
PARASITOLOGY
READING ASSIGNMENT: MAHON PP. 626-631 APPROPRIATE COLLECTION OF SAMPLES

➢ The container must be clean, dry, sealed
FECAL SPECIMENS tightly, and waterproof (plastic container
with lid). There is also collection container
COLLECTION, HANDLING AND TRANSPORT that contain preservatives.
➢ Stool samples must be transported to the ➢ Stool specimen should not be collected
laboratory immediately or a preservative is from bedpans or toilet bowls since it can
immediately placed. contaminate the specimen with water or
➢ Trophozoites and some helminth eggs may urine which can lead to the destruction of
disintegrate if not examined or preserved within trophozoites or introduction of free-living
a short time. protozoa.
➢ For optimal detection of intestinal parasites, a ➢ Alternative method
series of three stool specimens spaced a day or o The specimen could be collected on
two apart, collected within a 10-day period is a clean piece of waxed paper or
recommended newspaper and transfer it to the
o Disadvantage: time consuming and container
labor intensive o Use a disposable collection
➢ Pooling of three formalin-preserved specimens container that can be fitted under
gives a parasite recovery rate comparable to the toilet bowl rim
that of the individual examination of formalin- ➢ Information on the container should include
preserved stools. the patient’s name and the date and time
o Advantage: Saves time the stool was collected.
o Disadvantage: Organisms are present in ➢ Stool specimen must be collected before
small numbers. They can be missed in the start of antimicrobial therapy since it
microscopic examination of wet mounts can reduce the number of organisms
because of dilution effect (reduced present in the sample.
sensitivity) ➢ If the patient undergone barium enema,
➢ The algorithm that laboratories take into stool examination must be delayed for 7 to
consideration to determine if a single or 10 days. Barium obscures organisms when
multiple specimens is needed: specimens are examined microscopically
o Parasitology examination routinely even after concentration procedures.
performed (concentration and ➢ If a purged specimen is to be collected,
permanent stained smear) saline or phosphosoda purgative is used
o Patient population and specific criteria because mineral oil droplets can interfere
(Travel symptoms, immune status, with parasite identification especially
inpatient or outpatient classification) protozoan cysts. The second or third
➢ Permanent stained smears should be made and specimen after the purge may contain the
examined for individual specimens. trophozoites that inhabit the cecum.
➢ Immunoassay for Giardia lamblia or
Cryptosporidium may be requested on
specimens from immunocompromised patients
with diarrhea or symptomatic children from
daycare settings.
14
PARASITOLOGY
PRESERVATION ➢ Used when a permanently stained smear will be

➢ The ratio of 3 parts fixative and 1 part stool made.
must be followed for optimal preservation.
Single vial fixatives
➢ Used for wet mount and permanent stained
LABORATORY
PRESERVATIVE slides that do not use formaldehyde or mercury
EXAMINATION METHOD
compounds.
Permanently stained o Ecofix
smear o Parasafe
Polyvinyl Alcohol o Prot-fix
DNA-PCR ➢ Advantages
o A satisfactory substitute for standard
Formalin-ethyl acetate PVA that contains mercuric chloride
concentration o Smears dry faster
➢ Disadvantages
10% formalin Direct wet mount o Background quality was poor
o Less sharp morphology of organisms
Immunoassays was observed
Permanently stained Formalin
Sodium acetate-acetic smears ➢ Used in wet mount or concentration procedure
acid-formalin (SAF) (sedimentation or floatation)
Concentration
Sodium acetate-acetic acid-formalin (SAF)
Concentration ➢ Preservation of fecal specimens when
Merthiolate-iodine- concentration procedures and permanent stains
formalin (MIF) Direct wet mount will be used
Concentration Merthiolate-iodine-formalin
➢ Preservation of trophozoites, cysts, larvae, and
Direct wet mount helminth eggs for wet mount or concentration
procedures
Single-vial systems ➢ Not routinely used for permanently stained
Permanently stained
smears smears
Immunoassays
➢ A two-vial system using polyvinyl alcohol (PVA),
which is a resin polymer, in the first vial and
10% formalin the other vial is commonly used in
the laboratory.
PVA fixative
➢ Consists of mercuric chloride (for fixation) and
PVA (to increase adhesion of the stool to the
slide)
15
PARASITOLOGY
MACROSCOPIC EXAMINATION ➢ Any portion that contains blood or blood-tinged

➢ The initial laboratory procedure done in an mucus must be selected for wet mount and
unpreserved stool placed in a preservative
➢ Intact worms or proglottids (tapeworm
segments) are found on the surface of the stool
MICROSCOPIC EXAMINATION
➢ Formed stool should be examined 2 to 3 hours
of passage if held in a room temperature Wet Mount Preparation
➢ Examinations may be delayed by 24 hours after ➢ Direct wet mount of unpreserved stool is used
passage if it is placed in a refrigerator to detect the presence of motile protozoan
➢ A portion of formed stool should be placed in a trophozoites in a fresh liquid stool or from
formalin for concentration procedure and sigmoidoscopy material
another portion in PVA for permanently stained ➢ Purged specimens should be examined
smears immediately (within 30 minutes) after passage
➢ Specimens should not be placed in a 37° C to ensure motility of the organisms
incubator to avoid the disintegration of any ➢ A portion should also be placed in a fixative
such as PVA, so that permanent stained smears
organisms and avoid the growth of bacteria
for definitive identification can be made
Consistency ➢ Saline preparation in unfixed stool sample is
➢ This can help determine the type of important for the detection of helminth eggs or
preservative to be used larva, motile protozoa, and protozoan cysts.
➢ It can also indicate the forms of parasite ➢ Iodine can help in emphasizing nuclear details
expected to be present and dictate the and glycogen masses but it can kill trophozoites
immediacy of examination ➢ If the specimen is treated with 10% formalin,
➢ Cysts are most likely found in formed stools and saline preparation should be omitted.
sometimes soft stools ➢ For the microscopic examination, start at low
➢ Trophozoite are usually found in liquid stools power objective then move at a high-power
objective to identify suspicious structures
➢ Oil immersion should not be used on a wet
mount unless the sample has been sealed with
clear polish or vaspar
Test Procedure
➢ The wet mount procedure uses a glass slide on
which a drop of physiologic saline (0.85%) has
been placed at one end and a drop of iodine
(Dobell and O’Connor, D’Antoni, or 1: 5 dilution
of Lugol solution) at the other end.
Color ➢ A small amount (2 mg) of feces is added to each
drop and mixed well.
➢ Normal stool usually appears brown ➢ The sample should be covered with a coverslip
➢ Black stool may indicate bleeding in the upper ➢ The preparation should be thin and should not
GIT overflow beyond the edges of the coverslip. The
➢ Presence of fresh blood in the sample may edges can be sealed with a clear nail polish or
indicate bleeding in the lower portion of the
intestinal tract
16
PARASITOLOGY
vaspar (1:1 mixture of petroleum jelly and ➢ Advantage of Zinc sulfate method
paraffin) to prevent rapid drying o Yields less fecal debris
➢ PVA preserved specimens are not acceptable ➢ Disadvantage of Zinc sulfate method
for wet mounts because it turns cloudy when o Causes operculated eggs to open or
exposed to air collapse
o Distorts protozoan cysts
Concentration Techniques o Infertile Ascaris lumbricoides eggs and
➢ Designed to concentrate the parasites into a Schistosoma spp. eggs may be missed
small volume of fluid and remove debris ➢ Due to their high density, eggs will sink to the
➢ Fresh or formalin-preserved stools can be used bottom of the tube.
➢ The sediment may be examined unstained or ➢ Most organisms tend to settle after 30 minutes.
stained with iodine.
That is why examination should be performed
➢ Cysts, helminth larva and helminth eggs can be as soon as possible after the procedure has
detected by this method but trophozoites do been completed.
not survive this procedure. ➢ Special floatation method:
➢ There are two types of Concentration o Sheather sugar floatation method for
techniques: Cryptosporidium spp.
o Sedimentation method o Replaced by Fluorescent antibody
o Floatation method microscopy or modified acid-fast stain
➢ Both of the methods are based on the
difference in specific gravity between the Permanently Stained Smears
parasites and concentrating solution ➢ Used to detect and identify protozoan
trophozoites and cysts
Sedimentation Method ➢ Characteristics needed for the identification of
➢ The organisms are concentrated at the bottom protozoa
of the centrifuge tube o Nuclear detail
➢ It can detect cysts, larva, and eggs. o Size
➢ Formalin-ethyl acetate sedimentation method
o Internal structures
(FES) is the standard sedimentation method.
➢ Permanent stains that are commonly used
➢ Specimens fixed using 10% formalin, SAF or include hematoxylin and trichrome (Wheatley
Ecofix can be concentrated. modification of the Gomori stain)
➢ The sediments are used in preparing wet ➢ The stain of choice in most laboratories is the
mounts and permanent stained slides trichrome stain because:
➢ Commercially available kits market self- o Less dependent on the technique
contained fecal concentration without the use o Less time consuming
of ethyl acetate. ➢ A trichrome stain can be performed in a fresh
o Advantage: easier disposability and stool fixed in Schaudinn fixative or a sample
cleaner preparation due to the filtration preserved in PVA.
method ➢ Specimens preserved in SAF is stained with iron
o Disadvantage: more expensive hematoxylin since it does not stain well using a
Floatation Method trichrome stain.
➢ Organisms are suspended at the top of a high- ➢ Poor fixation of fecal material results in poor
density fluid staining or non-staining organisms.
➢ Zinc sulfate method is the usual floatation ➢ There are modifications in trichrome staining to
procedure allow the detection of “microsporidia”
17
PARASITOLOGY
Procedure for Trichrome Stained Smear ➢ Nuclear peripheral chromatin – DNA and
➢ Use applicator sticks to smear a thin film of proteins on the edge of the nucleus
stool in a glass slide ➢ Karyosome – Mass of chromatin in the nucleus
➢ The smear must not be allowed to dry and it ➢ Chromatoidal bars – dark-staining cytoplasmic
must be placed immediately in Schaudinn inclusion of chromatin
fixative.
➢ For PVA preserved samples, several drops In a smear stained with Iron Hematoxylin
of specimen are placed on a paper towel to STRUCTURES COLOR
drain excess fluid. The material on the Parasites Gray-black
paper towel is collected and smeared on Nuclear Material Black
the slide. Background material Light blue-gray
➢ The sample is allowed to air-dry before
staining ➢ All permanently stained smears should be
examined by scanning for the thick and thin
In a well-stained Trichrome smear
parts of the smear using lower power
STRUCTURES COLOR
magnification (x10 or x40 objective)
➢ Then, thin areas are examined under Oil
Cysts and Trophozoites Blue-green
immersion (x100 objective) for identification of
*except for Entamoeba organisms
coli *Purple ➢ It should take approximately 10 to 15 minutes
to properly examine the selected areas
➢ Organisms that stain lightly and may be difficult
Nuclear peripheral to identify are the following:
chromatin o Entamoeba hartmanni
o Dientamoeba fragilis
Karyosome o Endolimax nana
Dark Red-Purple o Chilomastix mesnili
o Giardia lamblia
Chromatoidal bars
Modified Acid-Fast Stain

Red Blood Cells ➢ The combination of Iron hematoxylin and carbol
fuchsin is used for the simultaneous staining of
Eggs and Larvae Red Isospora, Cryptosporidium, and protozoa
Kinyoun modified acid-fast stain

*often distorted or ➢ Used to detect oocysts of Cryptosporidium,
destroyed during staining Cystoisospora belli (formerly Isospora belli), and
Cyclospora cayetanensis.
Background debris Green ➢ Oocysts will appear as magenta-stained
organisms against a blue background
Yeasts
18
PARASITOLOGY
OTHER SPECIMENS EXAMINED FOR INTESTINAL AND o The remaining specimen from the string
UROGENITAL PARASITES is placed in a fixative for a permanently
stained smear.
CELLOPHANE TAPE PREPARATION FOR PINWORM o Organisms that can be recovered:
➢ Fecal specimen is not optimal due to the life ▪ Eggs of Fasciola hepatica and
cycle of the pinworm Enterobius vermicularis. Clonorchis sinensis
Female pinworm migrates from the anus at ▪ Oocysts of Crypstosporidium
night to lay eggs in the perianal area. and Cystoisospora belli.
➢ The cellophane tape preparation is routinely
used for detection of suspected pinworm
SIGMOIDOSCOPY SPECIMENS
infections.
➢ Sigmoidoscopy scrapings or aspirates are used
Procedure to diagnose amebiasis or cryptosporidiosis.
➢ Swab the person’s perianal area with a tongue ➢ Immediately examine for motile trophozoites,
blade covered with cellophane tape (sticky side and a portion of sample is placed in PVA fixative
out). for permanently stained smears.
➢ The collection should take place first thing in ➢ Smear for staining with modified acid-fast stain
the morning, before the individual uses the or by fluorescent procedure is prepared if
bathroom or has bathed. suspected for cryptosporidiosis.
➢ After collection, the sticky side of the tape is
placed on a microscope slide and scanned at
URINE, VAGINAL, AND URETHRAL SPECIMENS
low- and high-power magnification.
➢ There are commercially available paddles with a ➢ Organisms can be detected in urine sediment:
sticky surface for the test. o Eggs of Schistosoma haematobium and
E. vermicularis
o Trophozoites of Trichomonas vaginalis
DUODENAL ASPIRATES (can be seen also in wet mount of
➢ Material obtained from duodenal aspirates or vaginal or urethral discharge)
from Enterotest may be submitted in cases of ➢ Envelope culture methods for T. vaginalis such
suspected giardiasis or strongyloidiasis when as InPouchTV are used. Specimen is added to
clinical symptoms are suggestive of infection the medium and incubated and observe the
but negative in routine stool examination. growth within 3 days.
➢ Procedure of Enterotest
o The patient swallows a gelatin capsule
SPUTUM
containing a weighted string.
o One end of the string is taped to the ➢ Organisms seen in Direct Wet Mount of
side of the patient’s mouth; the Sputum:
weighted end is carried into the upper o Filariform larva of Strongyloides
small intestine. stercoralis (hyperinfection)
o After 4 hours, the string is brought up, o Eggs of Paragonimus westermani
and collected a portion of the mucus ➢ Organism seen in Permanently Stained smear
adhered on the string and examined on o Entamoeba histolytica (patient is
a wet mount for motile trophozoites. suspected of having pulmonary abscess)
19
PARASITOLOGY
EXAMINATION OF SPECIMENS FOR BLOOD AND o Disadvantage: color intensity for the
TISSUE PARASITES differentiation of parasites is not as
good as with Giemsa Stain.
BLOOD SMEARS
Identification Procedure
➢ Staining with Giemsa and Wright stain is the
➢ Thick and thin film is useful for blood parasite
most common method of detecting malaria,
and can be prepared on the same slide or on
Babesia, Trypanosoma, and some species of
separate slide.
microfilaria.
➢ Using two slides is more efficient due to
➢ Trypanosoma and microfilariae can be detected
different treatment of the films.
on a wet preparation of a fresh blood specimen
➢ Giemsa stain can be used on thick and thin
under low- and high-power magnification, and
films.
identification is based on characteristics on
o Thin smears are fixed with methanol
stained smear.
before staining with Giemsa stain.
➢ Trypanosoma spp. or microfilariae can be seen
o Thick smear is unfixed on staining
using concentration method using membrane
procedure to lyse the RBC.
filters but rarely used in laboratory.
➢ Wright stain cannot be used for thick film
➢ Tissue biopsy is used to detect Trichinella
because it contains methanol, which will fix
spiralis, Leishmania spp., and Toxoplasma
RBCs. Lyse the RBCs first with distilled water.
gondii.
➢ Serologic methods may be useful to detect Thick Film
current infection if the individual is from a ➢ A thick film is best for the detection of parasites
nonendemic area. (high sensitivity) because of the larger volume
of blood and the fact that organisms are
Collection and Preparation
concentrated in a relatively small area.
➢ The ideal specimen for malarial smear is the ➢ Thick film Procedure:
fingerstick blood because it gives the best o Put several drops of blood on the slide
staining characteristics and spread it.
➢ Blood in EDTA gives adequate staining if o Optimal thickness when newsprint is
processed within 1 hour. barely visible through the drop of blood
➢ Longer than 1 hour preparation of the slide, the before it dries.
organism may distort. And exceeding 4 hours, o Too thick = film peels from the slide
the organism may be lost. o Allow to dry for at least 6 hours before
➢ Giemsa Stain: staining
o Cytoplasm : Bluish o Should not be fixed with methanol
o Chromatin : Red to Purple-red because fixing prevents lysis of the
o (+) Malarial stippling : Pink-red dots RBCs.
o Advantage: gives the best morphologic o Giemsa stain releases hemoglobin by
detail lysing unfixed RBCs.
o Disadvantage: time-consuming o Stained smear at x100 detects
procedure microfilariae
➢ Wright Stain: o Thick smear at x1000 detects malarial
o Advantage: shorter staining period organisms.
➢ White cells, platelets, and parasites are only
visible.
20
PARASITOLOGY
➢ It is difficult to identify organisms and also ➢ Trypomastigote is visible because of the motion
compare the size of the infected and of the flagellum and undulating membrane.
noninfected RBCs. ➢ Requires skills that can distinguish the amebic
➢ Species identification should be made from a motility in a field of neutrophils.
thin film because the characteristics of the ➢ CSF is cultured when Amebic
parasite and the RBCs can be seen. meningoencephalitis caused by Naegleria
fowleri is suspected.
o Non-nutrient agar is seeded with an E.
Thin Film coli overlay, and the CSF sediment is
➢ A thin film is prepared same as with differential inoculated.
cell count. o Sealed and incubated at 35oC.
➢ Fixed in methanol for 1 minute and air-dried o It is examined daily for thin tracks of
before staining with Giemsa stain. bacterial growth that indicates that
➢ Scanned at x100 to detect large organism such amebae have been feeding on the
as microfilariae bacteria.
➢ At least 100 oil immersion fields (x1000) to
examine the presence of organisms such as
IMMUNOLOGIC DIAGNOSIS
Trypanosoma or for intracellular organisms such
➢ Parasites that invade tissue (E. histolytica, T.
as Plasmodium or Babesia.
cruzi, or T. gondii) are the primary organisms
➢ For a symptomatic patient, several blood
that stimulate antibody production.
smears from samples collected at approximate
➢ Serologic tests are useful for identification if
6-hour intervals over 36 to 48 hours should be
invasive methods cannot be used.
examined before a final negative diagnosis is
➢ Antibody tests serve only as epidemiologic
made.
markers, especially in endemic areas.
➢ Parasitemia (percentage of erythrocytes
➢ Detection of IgM
parasitized) can be calculated from the thin
o Advantage: Useful in identifying
blood smear.
infection during acute phase
o Disadvantage: Declines to
BIOPSY SPECIMENS nondetectable levels as if the infection
➢ Used to diagnose Leishmania spp. infections begins to resolve.
because they are intracellular. ➢ Detection of IgG
➢ Amastigote can be detected in tissue such as o Does not distinguish between relatively
skin, liver, spleen, and bone marrow. It depends recent or past infection because it can
on the species present. persist for years
➢ Cutaneous lesions should be sampled below the ➢ Antibody detection is useful to some cases:
edges of the ulcer because the surface does not o a patient who lives in a nonendemic
yield infected cells. area for a parasite has recently traveled
to an endemic area and now shows
symptoms, but the organism has not
CEREBROSPINAL FLUID been detected in a clinical specimen.
➢ Organisms that cause amebic meningitis or o A positive test for antibody would help
sleeping sickness can be occasionally seen in confirm a diagnosis.
CSF specimen.
21
PARASITOLOGY
➢ Disadvantage of Antibody Test: large number of ➢ Not all kits can detect E. histolytica but can
cross reactions that limits their diagnostic differentiate E. histolytica and E. dispar.
usefulness. ➢ Antigen detection kits have replaced
➢ Many serologic tests used by reference microscopic examinations in some hospital labs.
laboratories such as CDC are not commercially ➢ The malarial tests approved in US are based on
available. the principle of immunochromatographic
➢ Parameters that should be considered by a antigen capture that uses whole blood to detect
laboratory in selecting a method to be used: malarial proteins.
o Cost ➢ The patient’s blood is reacted with monoclonal
o Diagnostic yield antibody labeled with dye or gold particles.
o Patient population ➢ Some tests are nonspecies-specific and detect a
o Relative incidence of the parasite in the protein such as parasite Lactate Dehydrogenase
area or Aldolase that is common to all human
o Number of specimens to be processed Plasmodium spp.
➢ Hemagglutination and complement fixation are ➢ Other tests detect species-specific protein such
classical methods used to detect antibodies to as histidine-rich protein that is associated with
parasitic organisms. P. falciparum.
➢ Newer tests use fluorescent or enzyme ➢ Some tests detect both types to provide more
immunoassay (EIA) techniques. complete picture of the infective agent.
➢ Immunoassay test for antibodies to T. gondii or
E. histolytica (extraintestinal infections) are
available for clinical labs. FLUORESCENT ANTIBODY TECHNIQUES
Direct fluorescent Antibody
➢ Direct fluorescent antibody (DFA) techniques
ENZYME IMMUNOASSAYS using monoclonal antibodies developed to
➢ Problems with developing EIA methods for the detect Cryptosporidium oocysts in stool.
detection of antibody to intestinal parasites: o Feces is spread in slide
o Difficulty in obtaining parasite antigen o Reagent containing the antibody is
appropriate for detection added
o Cross-reactivity of antibodies o The specimen is examined with
o Poor sensitivity and specificity of tests fluorescent microscope for
➢ The major use of EIA has been to detect characteristic of apple green structure.
parasite antigens. ➢ Advantage: demonstrate greater sensitivity,
➢ They also provide information about current especially when only rare oocysts are present.
infection, in contrast to antibody tests. ➢ Disadvantage: more expensive than modified
➢ Number of EIAs are available to detect the acid-fast procedure
presence of Giardia lamblia, Cryptosporidium ➢ Monoclonal antibodies eliminate false-positive
spp., E. histolytica, and Entamoeba dispar. and false-negative results that is useful in
➢ Some are able to detect more than one parasite screening large numbers of specimens.
using organism-specific antibodies immobilized
on a membrane. Quantitative Buffy Coat
➢ Rapid EIA kits used with fresh or preserved stool ➢ Quantitative Buffy Coat procedure uses acridine
and kits using monoclonal antibody to detect orange (fluorescent dye) to stain nuclear
Giardia and Cryptosporidium organisms. material for detecting malarial organisms in
blood.
22
PARASITOLOGY
o Microhematocrit tube is coated with

the fluorescent dye
o After centrifugation, parasites can be
seen in a small area at the top of the
RBC column.
➢ Advantage: More sensitive that thick smear in
demonstrating parasites
➢ Instrumentation needed may not be available.
MOLECULAR METHODS
➢ Molecular methods for parasite identification
are still limited to reference laboratories.
➢ Methods can include classic and real-time
polymerase chain reaction (PCR) assays.
➢ Molecular methods of detection exist for
speciating a number of organisms, including
malarial parasites.
23
IMMUNOLOGY AND SEROLOGY
DATE | Mark Rodrigo D. Mendros RMT, MT(ASCPi), MSMT
ANTIGEN, ANTIBODY and IMMUNE TWO PROPERTIES OF ANTIGENS

RESPONSE
1. IMMUNOGENECITY: inherent ability of a
ANTIGENS substance to induce immune response
resulting in the formation of immune
Definition: Any molecular structure that when lymphocytes or antibodies
introduced is capable of Antibody production.
 If an antigen is immunogenic, it is
 Can elicit the immune system to produce capable of inducing antibody
antibodies production or immune response
 Can be microorganism, pathogen, foreign FACTORS THAT AFFECT
substances, etc. IMMUNOGENECITY
PARTS OF ANTIGEN A. FOREIGNESS: the immunogen must be
• Carrier Portion: responsible for the recognized as foreign or non-self to induce
molecular weight of the antigen; the immune response
molecular weight of an antigen can be  The immune system is capable of
expressed as daltons discriminating self from non-self and
• Epitope or Determinant: determines the this unique characteristic would
specificity of the antigen; responsible for the enable the immune system to be
specific antibody production and can be activated when foreign agents
based on the nature of the antigen (invaders) are present in the body.
 The immunogen must be recognized
as foreign or non-self or simply an
invader so that immune response is
produced.
ANTIGEN TYPES
• Autoantigen: self-antigen; derived from the
person’s body
 A healthy immune system should not
be stimulated by an autoantigen
because it is considered as self or part
of the body system.
 The entire microbial cell is the carrier  It should not elicit immune response
portion of the antigen. The specific because it could lead to autoimmune
structures on the membrane of the cell disorder
are the epitopes. The epitopes on the • Alloantigen: derived from other individuals
surface would determine the specific of the same species
antibody that would interact with the (Example: Blood cells of donors)
bacillus.
• Heteroantigen: derived from other species;
can derived from animals or nonhuman
sources; most foreign to humans
[AUTHOR NAME] 1
• Heterophile antigen: a type of heteroantigen Example:

that is produced from unrelated plants or
animals that can induce a common or similar  Albumin (30, 000 to 60, 000 daltons);
immune response considered as a good antigen
 Hemocyanin (100,000 to million daltons);
GRAFT TYPES excellent antigen because it is highly
complex type
Graft: is also known as tissue transplant
• Autograft: obtained from the patient’s own C. CHEMICAL COMPLEXITY
body • Proteins: the best and strongest antigen;
Example: Patients who had second- or third- highly stable because of the peptide bonds
degree burn present which makes it circulate longer in the
• Isograft/ Syngraft: obtained from identical body
individuals (identical twins)  In the circulation, if the antigen could
• Allograft: obtained from non-identical last longer in the circulation, that
individuals of the same species; most would make an antigen highly
common graft transplanted immunogenic. The longer it stays in
Example: Kidney the circulation, the longer the
Blood: commonly transfused tissue exposure of the antigen in the immune
• Heterograft/ Xenograft: obtained from system leading to a greater chance of
other species immune response.
Example: Other mammals or laboratory • Polysaccharides: ex. Endotoxin,
animals (pig’s heart) pneumococcal capsule
• Glycoproteins: like ABO, Rh antigens
• Polypeptide: ex. Insulin
B. SIZE • Nucleic Acid, Lipids and Amino Acids:
• The larger the molecule, the more least immunogenic
immunogenic.
• The number of epitopes increases
proportionally with the size of the molecule. 2. ANTIGENICITY/ SPECIFICITY: the
• Minimum molecular weight for an antigen/ ability to react specifically with the antibody
substance to induce immune response: at or cell that caused it to be produced
least 10,000 daltons of molecular weight or  Specificity is based on the epitope
higher present on the antigen surface
• If less than 10, 000 daltons, the antigen is DEFINITION OF TERMS
considered as hapten (incomplete antingen)
thus, incapable to induce an immune • Hapten: an incomplete antigen; not
response due to its small size immunogenic by itself; if coupled with a
carrier protein, hapten can elicit immune
Increased size – Increased epitope – Enhance
response since molecular weight will be
Immune Response
increased and hapten structure will be
stabilized
o Common carrier protein: Albumin
[AUTHOR NAME] 2
• Immunogen: defined as any substance that 2. Heterogenous antigen: cross reaction;

can induce an immune response; antigen antigen reacts with antibody. It did not induce
 All immunogens are antigenic, but not for its production
all antigens are immunogenic
• Allergen: a special class of immunogen that AFFINITY vs. AVIDITY
induces hypersensitivity reactions
specifically Type 1 hypersensitivity reaction; Affinity: the strength of attraction between an
cause reaction to some people but not all epitope and the antigen combining site (Fab portion)
• Adjuvants: substances added to an of the antibody
immunogen to enhance immune response
Avidity: the sum of interaction/the strength of
 Actions: interaction between complex antigens and antibodies
✓ Prolongs the retention time of
the immunogen in the body – Similarities: Both measures the strength of
longer stay in circulation, attraction/interaction
increases the chance to be
recognized by immune cells Differences: Affinity involves simple interaction
(WBCs), activating immune (single epitope + Fab portion) while Avidity involves
system multiple epitopes are interacting with antibodies
✓ Increases the effective size of
immunogen; the bigger
substance, the more ANTIBODIES
immunogenic and becomes Definition: substances, specifically glycoproteins,
easily recognized produced by B lymphocytes or plasma cells (matured
✓ Stimulates the influx of or differentiated B lymphocytes) in response to
macrophage and/or antigenic stimulation. Aka Immunoglobulins.
lymphocytes – increased
production of Previously known as gamma globulins since it was
immunocompetent WBC found to be concentrated on gamma region of serum
 Examples: protein electrophoresis.
✓ CFA (Complete Freunds
Adjuvants): composed of
water in oil emulsion of TERMINOLOGIES:
Mycobacterium butyricum or
Bordetella pertussis culture Isotype: is the same as the antibody classes.
✓ LPS (Lipopolysaccharide) Antibody classes depends on their heavy chain
✓ Aluminum Adjuvants: more present and the heavy chain of the antibodies is
clinically/commonly used basically used to name the antibodies.
CLASSIFICATION OF ANTIGEN 5 HEAVY CHAINS:

ACCORDING TO ANTIBODY REACTION ✓ Gamma (IgG)
1. Homologous antigen: antigen that induces ✓ Mu (IgM)
an antibody and reacts specifically with it ✓ Alpha (IgA)
✓ Delta (IgD)
[AUTHOR NAME] 3
✓ Epsilon (IgE) 3. Heavy chains are interconnected by

DISULFIDE LINKAGES
Allotype: determined by the difference in the
antibody structure in the constant region, specifically 4. Ig has 2 terminal regions:
the amino acid sequence Carboxyterminal: with constant amino acid
Idiotype: determined according to the difference in sequence (constant region)
the variable region of the antibody  AKA. Fc region (Crystallizable
Fragment/ Fragment crystalline)
Aminoterminal: with varying antibody
STRUCTURE OF IMMUNOGLOBULIN specificity (variable region)
 AKA. Fab - Fragment for Antigen
Binding or Antigen Binding
Fragment: where epitope of antigen
interacts
 Hypervariable portion of Fab
region: specific location where
epitope of antigen interacts with the
antibody
With the use of enzymes, these are used to
disintegrate and study the parts of antibody
structure
5. Treatment of monomer antibody with
enzyme papain (cleave covalent bonds) will
1. Four (4) polypeptide chains
produce 3 fragments:
 Composed of 2 identical light chains
One Fc fragment: fragment crystalline;
and 2 identical heavy chains
involve in Ig biologic function
(different chains are not allowed)
 Biologic functions of Fc fragment is
 Light chains:
involved in:
o Kappa: synthesized with
✓ Complement Fixation
regulation of gene located in
✓ Placental Transfer of antibody
chromosome 2
✓ Binding site for cell/s
o Lambda: formation of this is
regulated by the gene located
Two Fab fragments (monovalent): capable
in chromosome 22
of antigen binding even without the Fc but
 Heavy chains: Heavy chains: cannot agglutinate or precipitate; separated 2
synthesis of Mu, alpha, delta, epsilon Fab fragments
regulated by the chromosome 14
6. Treatment with pepsin results in digestion of
2. Both light and heavy chains are held together Fc fragment → destroyed → leaving 2 Fab
by COVALENT DISULFIDE BONDS fragments. The two Fab fragments have
two antigens combining sites (Bivalent),
[AUTHOR NAME] 4
therefore, it is capable of antigen binding, Hinge Region: found between CH1 and CH2
precipitation and agglutination.
 The Hinge region is the flexible region of the
antibody. The flexibility of the hinge region
is attributed to the presence of abundant
amino acid proline.
J Chain (Joining Chain): glycoprotein structure
that is used to link or connect 2 or more antibody
monomers
 Example: IgM, secretory IgA
DOMAINS OF IMMUNOGLOBULIN
DOMAINS OF IMMUNOGLOBULINS
Variable Region: responsible for the specificity of
the antibody
 The variable region is the antigen binding site
 HYPERVARIABLE PORTION/
REGION - specific portion in the variable
region where antigen binding takes place
CH2 (Constant Heavy Chain 2): binds with the
complement proteins specifically the C1q
 The part of the antibody which initiates the
activation of complement pathway
specifically classical pathways
CH3 (Constant Heavy Chain 3): responsible for the
cytotropic reactions involving macrophage,
monocytes, mast cells, Cytotoxic Killer Cells and B
cells.
CL (Constant Light Chain): responsible for the
light chain type of the antibody
 The light chain type can either be a Kappa or
Lambda. Kappa is predominantly present in
Ab, 65% of light chain is Kappa and 35% is
Lambda.
[AUTHOR NAME] 5
CLASSES OF IMMUNOGLOBULINS
IgG IgM
• Predominant Ig in healthy • Pentameric antibody;
human serum biggest
 antibody with the longest • Primary response
half-life or circulation life antibody; predominant Ab during acute stage
 predominant antibody in of disease
the secondary or the anamnestic • First to appear in phylogeny and the last to
response leave in senescence
• Provides immunity to the newborn • First antibody to be express on the surface of
 only antibody that can actively cross B cells; as mu is the first heavy chain to be
the placenta because it is the smallest synthesized
antibody; can provide neonatal • First appear after a primary antigenic
immunity stimulus
 IgG1 & IgG3: IgG subclasses that can • Considered as cold-reacting antibody that
actively cross placental reacts at ref temperature (4°C)
• Fixation of complement (Classical Pathway) • Functions of IgM:
 IgG3, IgG1, IgG2 (most to least  Complement fixation (Classical; most
potent) potent and effective)
• Opsonization  Agglutination reaction (can initiate
• Toxin and virus neutralization Lattice Formation)—best
• Participates in agglutination and precipitation agglutinating antibody because IgM
reaction is the biggest Ab
 IgG is considered as the best  Opsonization
precipitating antibody  Neutralization of toxin
 Warm-reacting antibodies; Optimally
reacts at 37°C. All are clinically
significant because they are optimally IgA
reacting at body temperature.
• Exist as monomer
(blood) and dimer
4 SUBCLASSES OF IgG (secretion)
Percentage from the
• Responsible for mucosal immunity
No. of
total IgG in the  Transferred by the mother thru
disulfide bods
circulation colostrum (along IgG)
IgG1 66% 2 • 2 subclasses: IgA1, IgA2
IgG2 23% 4 • With secretory component
IgG3 7% 15  Secretory component is responsible
for maintaining the IgA structure in
IgG4 4% 2
the secretion by preventing enzymatic
degradation of the antibody structure
[AUTHOR NAME] 6
Activa Yes Alternativ Yes No No

te (specifical e pathway (spec
IgD Comp ly only ifical
• Function is still unknown but lemen Classical (Provided ly
believes to play a role in t Pathway), that IgA Class
immunoregulation except is in ical
IgG4 aggregate Path
• Found on the surface of B-
s for it to way)
lymphocytes
elicit or ,
• Postulated to be an anti-idiotypic antibody induce the most
activation effici
) ent
IgE
• Least abundant Ig in the Other Name Other Notes
serum ✓ Can cross the
• Heat-labile antibody, originally placenta except
known as Reaginic antibody IgG Serum Ig IgG2
• Binds strongly to mast cell ✓ Major Ig in 2°
(tissues) and basophil Immune response
(intravascular circulation) receptor ✓ Predominant Ig in
• Mediates some types of hypersensitivity secretion
reactions (histamine released is due to the ✓ With J chain
binding of IgE with attached allergen) IgA Secretory Ig
✓ Exist as monomer
• Increased during infections (i.e., helminthic in serum and dimer
infection) in secretion
✓ Predominant Ig in
1° Immune
IgM Pentameric Ig
CLASSES OF IMMUNOGLOBULINS response
✓ Has J chain
IgG IgA IgM IgD IgE ✓ Involved in B cell
Molec 150T 160-400T 900T 180 190 activation
ular T T ✓ Susceptible to
weight IgD --- enzyme
(Dalto
degradation
n)
✓ Heat & Acid
Halflif 21-23 5-6 days 5 2-3 2-3
Labile
e days days day days
✓ Associated with
s
Immediate
Subcl 4 2 2 - -
Hypersensitivity
asses
IgE Reagenic Ig ✓ Binds basophils
Domai 4 4 5 4 5
and mast cells
ns
✓ Elevated during
parasitic infections
[AUTHOR NAME] 7
IMMUNE RESPONSE PRIMARY IMMUNE RESPONSE

Definition: Reaction of the immune system, • Occurs in four phases after exposure to an
specifically the immune cells, when the body is antigen
exposed to foreign agent.  Lag Phase: antibody is not yet
detectable; it is the phase wherein the
Affected by the following factors:
antigen is being processed by the
1. Overall health status immune cells. If the antigen is
 If the person is healthy, it follows that encountered for the first time, the
the immune response is also longer the antigenic process is.
immediate/strong. If a person is Therefore, resulting to a Longer
sickly, the immune response is a bit LAG Phase. If the antigen is already
delayed/weak. exposed the second time (Re-
2. Age exposure of the same agent), the
 Newborns’ immune system have not antigenic process in the LAG phase is
yet fully developed and is still in the Shorter because of recognition by the
process of development, thus having memory cells. In Primary Response,
a weaker immune response. The cells there is a longer LAG Phase.
of the immune system of babies are
naïve. Geriatrics also have weak  Log Phase/Exponential Phase:
immune system, thus having antibody titer increases
weak/delayed immune response. logarithmically; peak titer of the
3. Dose/Route of antigen administration antibody is seen;
 The higher the dose of the antigen/the
greater the antigen exposed to the
immune system, the stronger or faster  Plateau/ Stationary: antibody titer
immune response stabilizes; There is an
 When a person is vaccinated with a equilibrium/balance between
particular antigen, the dose of antigen antibody synthesis or production
is considered to ensure the elicitation versus the antibody catabolism.
of immune response. Common way of
administering a vaccine is
Intramuscular/Subcutaneous.  Decline: antibody catabolism is
 Best route of administering antigen is increasing (Catabolism of antibody
Intravenous (IV); uncommon exceeds the production of antibody);
4. Genetic control of the immune response thus, antibody titer is decreasing
 Major Histocompatibility Complex
(MHC) is genetically controlled, a
manner of activating and eliciting • IgM is the first antibody to appear in the
immune response. primary immune response
[AUTHOR NAME] 8
COMPARISON OF PRIMARY AND

SECONDARY IR
IR 1° 2°
Lag Phase Longer Shorter
Plateau Phase Shorter Longer
Decline Phase Less gradual More gradual
AB Present IgM IgG, IgM
AB Titer Lower Higher
SECONDARY IMMUNE RESPONSE

• A.k.a. ANAMNESTIC/ MEMORY
RESPONSE
• Elicited by a second or any subsequent
exposure to the same antigen
• Exhibits the same four phase as the primary
response
 LAG phase is shorter because there is
already a re-exposure or memory,
thus there is more rapid antibody
response
• Antibody titer of the Secondary Immune
Response is higher than the Primary
Response
• IgG is the predominant antibody in
secondary immune response
• Artificial Active - Adaptive immunity of

vaccines with attenuated antigens
• Non-responders – a person that is not
capable of producing adequate antibodies
against particular agent
[AUTHOR NAME] 9
FLOW CYTOMETRY and injects inside a carrier stream of isotonic

saline (sheath fluid) to form laminar flow
• First used for research purposes in 1960s
• Sample stream is controlled by the carrier
• In 1980s, it was used due to a drop ↓ in stream making it hydronamically focused for
circulating helper T cells the cells to be accurately measured
• Used in identification of HIV infection and
in immunophenotyping cells in identifying Laser Light Source
the surface antigen expression
• Consists of one or more small, air-cooled
Cell Flow Cytometry lasers
• The wavelength emitted by the
• Single cells in fluid suspension are analyzed monochromatic light indicates which
through their: fluorochromes can be used, however not all
o Intrinsic light-scattering fluorochromes can be used. The
characteristics fluorochrome to be used in an assay depends
o Extrinsic properties (such as on the light source used for excitation
presence of specific surface proteins) o Fluorochrome: absorbs light across
• It analyzes using fluorescent labeled a spectrum of wavelengths; emits
antibodies or probes light of lower energy across a
• Advantages: spectrum of longer wavelengths;
o Using several different have distinctive spectral pattern of
fluorochromes enables cytometers to absorption and emission
measure multiple cellular properties • Clinical flow cytometers contain at least one
o Thousands of cells can be analyzed, laser (argon) while newer flow cytometers
thus allows accurate detection of rare have a second laser (helium-neon)
cells • As cells pass through the laser, light is
• The major components of a flow cytometer scattered in many directions where the
are the ff: amount and type of light scatter can dictate
o Fluidics the information about the cell’s physical
o Laser light source properties
o Optics and photodetectors • Intrinsic parameters: used to characterize
o Computer for data analysis and different types of cell
management o Forward-angle light scatter/FSC
INSTRUMENTATION (light scatter @ less than 90 degree):
indicates size
Fluidics o Orthogonal right angle light
• For cellular parameters to be accurately scatter/SSC (light scatter @ 90
measured, cells must pass through a laser degree): indicates granularity or
one at a time in single file. To do so, cells intracellular complexity
suspended are drawn up by the cytometer • Extrinsic parameters: requires addition of
fluorescent probe for detection
[AUTHOR NAME] 10
Fluorochromes Commonly Used in Clinical DATA ACQUISITION AND ANALYSIS

Flow Cytometry
Graphic Representations (Levels of
Excitation Fluorochrome Emission
Representation):
Wavelength or Dye Wavelength
488 nm Fluorescein 580 1. Single-Parameter Histogram
(argon laser) isothiocyanate • Plots a chosen parameter (generally
(FITC) fluorescence) on the x-axis versus the number
Phycoerythrin 580 of events on the y-axis, so only a single
(PE) parameter is analyzed using this type of graph
Peridinin iodide 620 • The operator can then set a marker to
(PI) differentiate between cells that have low
Peridinin 670 levels of fluorescence (negative) from cells
chlorophyll that have high levels of fluorescence
(PerCP) (positive) for a particular fluorochrome
633 nm (He- Allophycocyanin 670 labeled antibody.
Ne laser) (APC)
Cy-5 670
OPTICS
• Light scatter and fluorescence generated due
to cell interaction with the laser are detected
by photomultiplier tubes and detectors
o Fluorescent light reaches the
photomultiplier tube creates
electrical current → voltage pulse → 2. Bivariant Histogram (or also known as
digital signal Dual-Parameter Dot Plot)
▪ Digital signal is proportional • Where each dot represents an individual cell
to the intensity of light or event
detected • Two parameters, one on each axis, are plotted
against each other. Using dual-parameter dot
▪ The intensity is measured on
plots, the operator can then draw a “gate”
a relative scale set into 1 to
around a population of interest and analyze
256 channels various parameters (extrinsic and intrinsic)
• The number of photodetectors limits the of the cells contained within the gated region.
amount of fluorochrome to be measured This allows the operator to screen out debris
• Photodetector specificity to a certain band of and isolate subpopulations of cells of interest.
wavelength is achieved by the arrangement
of series of optical filters
[AUTHOR NAME] 11
• The absolute count of a particular cell

type—for instance, CD4 T lymphocytes—is
• When analyzing a population of cells using a
obtained by multiplying the absolute cell
dual-parameter dot plot, the operator chooses
count of the population of interest (e.g.,
which parameters to analyze on both the x-
lymphocytes) derived from a hematology
and y-axes. He or she then divides the dot plot
analyzer by the percentage of the population
into four quadrants, separating the positives
of interest in the sample (CD3 and CD4
from the negatives in each axis
lymphocytes).
QUADRANT 1 Consists of cells that are • Detailed phenotypic analysis can
positive for determine the lineage and clonality, as well
fluorescence on the y- as the degree of differentiation and
axis and negative for activation of a cell population. This is useful
fluorescence on the x- for differential diagnosis or clarification of
axis. closely related lymphoproliferative
disorders.
QUADRANT 2 Consists of cells that are
positive for
fluorescence on both
SAMPLE ANALYSIS
the x- and y-axes
QUADRANT 3 Consists of cells that are Samples commonly used for analysis:
negative for
• Whole Blood
fluorescence on both
 should be collected into
the x- and y-axes ethylenediaminetetraacetic acid
QUADRANT 4 Consists of cells that are (EDTA), the anticoagulant of choice
positive for for samples processed within 30
fluorescence on the x- hours of collection
axis and negative for  Heparin can also be used for whole
fluorescence on the y- blood and bone marrow and can
axis provide improved stability in samples
over 24 hours old.
 Hemolyzed or clotted specimens
should be rejected. Peripheral blood,
bone marrow, and other samples with
large numbers of red cells require
[AUTHOR NAME] 12
erythrocyte removal to allow for One of the most important components of flow
efficient analysis of white cells. cytometric analysis is the stratification of
• Bone Marrow hematopoietic malignancies by their lineage (i.e., B
• Fluid Aspirates cell, T cell, or myeloid) and the degree of
differentiation.
Tissue specimens - are best collected and
transported in tissue culture medium (RPMI 1640) CD45 is a pan-leukocyte marker present on all
at either room temperature or 4ºC, if analysis will be white cells but with varying levels of expression; this
delayed. results in varying levels of fluorescence. This
variance in expression is based on the cell’s maturity.
 The specimen is then disaggregated to Blasts express lower levels of CD45 (low
form a single cell suspension, either by fluorescence) but show an increase of CD45
mechanical dissociation or by expression as the cell matures, so mature white cells
enzymatic digestion. have much brighter fluorescence compared to their
 Mechanical disaggregation, or earlier progenitor stages. This varying level of CD45
“teasing,” is preferred and is expression is useful in differentiating various
accomplished by the use of either a populations of white cells.
scalpel and forceps, a needle and syringe,
or wire mesh screen. Enumeration of peripheral blood CD4 T cells in
 Antibodies are then added to the HIV-infected patients remains the highest volume
resulting cellular preparation and test performed in the flow cytometry laboratory,
processed for analysis. The antibodies because it is used in classifying stages of HIV disease
used are typically monoclonal, each with and determining treatment options.
a different fluorescent tag  HIV type 1 infections cause a rapid,
profound decrease in CD4 T cell numbers
and an expansion of CD8 T-cell levels
CLINICAL APPLICATIONS during the early course (12 to 18 months)
Routine applications of flow cytometry in the lab: of the illness
 Some individuals continue to rapidly lose
• Immunophenotyping of peripheral blood CD4 T cells and progress to AIDS, while
lymphocytes others maintain relatively stable CD4 T-
• Enumeration of CD34 stem cells in cell counts and remain AIDS-free for
peripheral blood and bone marrow for use in years.
stem cell transplantation,  During the chronic phase of HIV-1
• Immunophenotypic characterization of acute disease, the decline in CD4 T cells can be
leukemias, non-Hodgkin’s lymphomas, and slow over many years due to maintenance
other lymphoproliferative disorders. of homeostatic mechanisms. However, as
these homeostatic mechanisms start to
fail, there is a further decline in CD4 T
Immunophenotyping by flow cytometry has and CD8 T cells, which eventually leads
become an important component of initial evaluation to the development of AIDS.
and subsequent post-therapeutic monitoring in
leukemia and lymphoma management.
[AUTHOR NAME] 13
CD4+ T CELLS SURFACE MARKERS ON T & B CELLS

➢ used to stage HIV disease progression ANTIGE
CELL TYPE FUNCTION
➢ prognostic for the development of AIDS N
➢ used to monitor response to antiretroviral CD2 Thymocytes, T Involved in T-cell
therapy cells, NK cells activation
CD3 Thymocytes, T Associated with
CATEGORIES OF HIV-1 ACCDG TO CDC
cells T-cell antigen
Percentage of receptor; role in
CD4+ T cells CD4 cells w/in TCR signal
(CD4 cells/mm3) lymphocytes transduction
(%)
Stage 1 >500 >28% CD4 Helper T cells, Coreceptor for
Stage 2 200-500 14-28% monocytes, MHC class II;
Stage 3 <200 <14% macrophages receptor for HIV
(AIDS) CD5 Mature T cells, Positive or
thymocytes, negative
subset of B modulation of T-
ADDITIONAL EXAMPLES OF FLOW cells (B1) and B-cell
CYTOMETRY USE
receptor signaling
• Determination of DNA content CD8 Thymocyte Coreceptor for
• Ploidy status of tumor cells subsets, MHC class I
• Leukemia/Lymphoma/Minimal Residual cytotoxic T
Disease post treatment patient monitoring cells
• Detection of hemoglobin F positive cells (in CD10 B- and T-cell Protease; marker
cases of fetal-maternal hemorrhage) precursors, for pre-B CALLA
• Human Leukocyte Antigen (HLA) typing and bone marrow
cross-matching for solid organ transplantation stromal cells
CD11b Myeloid an NK α M subunit of
cells integrin CR3,
MONOCLONAL ANTIBODIES binds complement
➢ Important in immunotyping by flow cytometry component iC3b,
o Fluorescent-labelled monoclonal and CD13 Myelomonocyti Zinc
polyclonal antibodies are used. c cells metalloprotease
➢ Specific for various surface antigens are CD14 Monocytic cells Lipopolysacchari
preferable to using polyclonal antibodies de receptor
➢ Produced through hybridoma and recombinant CD15 Neutrophils, Terminal
DNA technique eosinophils, trisaccharide
monocytes expressed on
glycolipids
[AUTHOR NAME] 14
CD16 Macrophages, Low affinity FC CD45R Different forms Essential in T-

NK cells, receptor, mediates on all and B-cell
neutrophils phagocytosis and hematopoietic antigen-
ADCC cells stimulated
CD19 B cells, Part of B-cell activation
follicular coreceptor, signal
dendritic cells transduction CD 56 NK cells, Not known
molecule that subsets of T
regulates B-cell cells
development and CD 94 NK cells, Subunit of
activation subsets of T NKG2-A complex
CD21 B cells, Receptor for cells involved in
follicular complement inhibition of NK
dendritic cells, component C3d; cell cytotoxicity
subset of part of B-cell NK = Natural killer;
immature TCR = CD3-αβ receptor complex;
thymocytes MHC = major histocompatibility class;
coreceptor with HIV = human immunodeficiency virus;
CD 19 CALLA = common acute lymphoblastic
CD23 B cells, Regulation of IgE leukemia antigen;
monocytes, synthesis; triggers FC = Fragment crystallizable;
follicular release of Il-1, Il- ADCC = antibody-dependent cell cytotoxicity;
dendritic cells 6, and GM-CSF IgE = immunoglobulin E;
from monocytes GM-CSF = granulocyte-macrophage colony-
stimulating factor.
CD25 Activated T Receptor for IL-2
cells, B cells,
IMMUNOASSAY AUTOMATION
monocytes
CD44 Most Adhesion • Reliable immunoassay instrumentation was
leukocytes molecule first available in the early 1990s.
mediating homing o Uses solid support for separating free and
to peripheral bound analytes
lymphoid organs o To automate heterogeneous
immunoassays even for low-level
CD45 All Tyrosine peptides (e.g., peptide hormones)
hematopoietic phosphatase, • Reasons for automation of immunoassay
techniques:
cells augments
a. more accurate and precise method
signaling
b. increased sensitivity
[AUTHOR NAME] 15
c. reducing or eliminating the many Instrument • Maintenance requirements

manual tasks required to perform • Automation compatibility
analytical procedures • Space requirements
d. decreases the likelihood of error • Utility requirements
e. reduce erroneous sampling • Reliability
f. reduce turnaround time and the cost per • Clot error detection
test
Manufacturer • Future product plans
g. ability to provide more service with less
• Reputation
staff
h. saving on controls, duplicates, dilutions, • Technical support
and repeats • Menu expansion plans
i. longer shelf life of reagents and less Operational • Test menu
disposal due to outdating • Throughput
j. better sample identification = with the • Reagent capacity
use of bar codes in sample delivery with • Reagent stability
bar codes • STAT capability
• There are wide variety of automated • Reflex testing ability
immunoassay available; choosing best • Reagent kit size
instrument depends on laboratory needs. • Training requirements
o Main priority in instrument selection: • Operating complexity
analytic quality of assays; analyzer that • Waste requirements
has the properties that can meet the • Reagent storage
demands of the laboratory requirements
• Downtime plans
FACTORS FOR CONSIDERATION IN

SELECTING AN AUTOMATED MAIN TYPES OF IMMUNOASSAY
IMMUNOASSAY INSTRUMENT ANALYZERS
CATEGORY FACTOR 1. Batch Analyzers

Analytical • Sensitivity ➢ Can examine multiple samples and
• Precision provide access to the test samples for the
formation of subsequent reaction mixtures
• Accuracy/test
➢ permit only one type of analysis at a time
standardization
(can be considered drawback → do not
• Linearity
allow processing of STAT samples)
• Interferences o STAT SAMPLES is loaded
• Carryover effects randomly; requires multiple analyses.
Economic • Purchase cost o Batch analyzers are not designed for
• Lease options multiple analyses on any one samples.
• Maintenance costs o Modular System: configuration
• Reagent costs available in next generation batch
• Operator time & costs analyzers that was designed to
• Disposable costs
[AUTHOR NAME] 16
measure multiple analytes from • Reagent use in automated immunoassay

multiple samples. analyzers requires consideration of the
2. Random Access Analyzers following factors: handling, preparing, and
➢ Can analyze multiple test samples storing, and dispensing.
➢ Multiple testing on any test samples • After reagents have been added to the
samples, the next concern is proper mixing to
obtain reliable results. Analyzers use
different methods and whichever methods
used; it is imperative that there be no
splashing between sample wells to prevent
erroneous results.
• Timed incubation is then carried out at
ambient temperatures, or analyzers need to
have built-in incubators for temperature-
controlled incubation. Heated metal blocks
are widely used to incubate reagent wells or
cuvets.
• Colorimetric absorption spectroscopy has
been the principle means of measurement,
due to the ability to measure a wide variety of
compounds.
• Automation occurs in all three stages of • Other methods of detection include
laboratory testing: the preanalytical, fluorescence and chemiluminescene, both
analytical, and postanalytical stages. which require fluorescence detectors.
• The tasks to be considered during the
analytical stage include introducing sample, VALIDATION
adding reagent, mixing reagent and sample, • Regardless of the instrumentation
incubating, detecting, calculating, and considered, proper validation of new
reporting readout or results. instrumentation or methodology must always
• Automatic sampling can be accomplished be performed.
by peristaltic pumps (older technology) and • Validation of the new instrument or method
positive-liquid displacement pipettes (newer must be completed before patient results
technology). using that instrument can be reported.
• Issues to consider with probe technology are
the inclusion of clot detectors that will • As designated by CLIA, the required
automatically reject a sample if a clot is verifications to be determined for a new
detected and the ability of the sample probe method are accuracy, precision, analytic
to detect the amount of the sample liquid, sensitivity, analytic specificity to include
using a liquid level sensor. interfering substances, reportable range, and
• Another issue associated with reusable reference intervals.
pipette probes is carryover or contamination
of one sample with material from the • Lists of other governmental websites with
previous samples. information regarding method/instrument
validation.
[AUTHOR NAME] 17
• The reference interval is the range of values

found in healthy individuals who do not have
the condition that is detected by the assay.
This is used to define the expected value of a
negative test.
SUMMARY
• Flow cytometry is a powerful tool to identify
and enumerate various cell populations.
• A flow cytometer measures multiple
properties of cells suspended in a moving
fluid medium. As each cell or particle passes
single file through a laser light source, it
• Batch analyzers may work best if only one produces a characteristic light pattern that is
type of testing is performed on a large scale. measured by multiple detectors for scattered
• Random access analyzers allow for more light (forward and 90 degrees) and
flexibility, include rapid processing of stat fluorescent emissions (if the cell is stained
samples. with a fluorochrome).
• In either case, any new instrument requires • Forward scatter is a measure of cell size,
extensive validation before patient results can while side scatter determines a cell’s internal
be reported with confidence. complexity, or granularity.
• Instrumentation can increase turnaround time • Determining an individual’s lymphocyte
for testing, remove the possibility of manual population is essential in diagnosing such
errors, and allow for greater sensitivity in conditions as lymphomas, immunodeficiency
determining the presence of low-level diseases, unexplained infections, or acquired
analytes. immune diseases such as AIDS.
• Lymphocytes are identified using
DEFINITION OF TERMS monoclonal antibodies directed against
• Accuracy refers to the test’s ability to specific surface antigens.
measure what it claims to measure. • Flow cytometry is the most commonly used
• Precision refers to the ability to consistently method for immunophenotyping of
reproduce the same result on repeated testing lymphocyte populations.
in the analysis. • Automated systems can generate more
• Analytic sensitivity is defined as the lowest accurate, precise, and sensitive analysis of
measurable amount of an analyte. many analytes compared to manual
• Analytic specificity is the assay’s ability to immunoassays.
generate a negative result when the analyte is • Automation is incorporated in all stages of
not present. laboratory testing: preanalytical, analytical,
• The reportable range is defined as the range and postanalytical.
of values that will generate a positive result • The validation should include at least a
for the specimens assayed by the test determination of accuracy, precision,
procedure. reportable range, reference range, analytic
sensitivity, and analytic specificity.
[AUTHOR NAME] 18
• Automated analyzers are costly but can

reduce turnaround time and hands-on time by
the technical staff and can provide sensitive
and precise results.
[AUTHOR NAME] 19
BACTERIOLOGY
Medical Technology Assessment Program-1
November 11, 2021 | Ma.CristinaSJLiwanagRMT,RN, Ph.D,M.A.,MSMLSc
GRAM [+] BACILLI
Bacillus cereus Bacillus anthracis

Considered as an agent of enteric infections (infections • Most significant; a bioterrorism agent
affecting the GIT) is Bacillus cereus (out of three bacilli • Causes anthrax
mentioned, it is the only agent of enteric infection). − Classified under Category A
− Bioterrorism agents may be classified into three
Commonly referred to as “fried rice bacillus” since this categories
organism is implicated in cases of food poisoning that is  Category A – easily transmitted and high
associated with consuming fried rice.
mortality rate
 Category B
B. cereus, B. anthracis, and B. subtilis are spore-formers.
 Category C
VIRULENCE FACTORS: • Not an agent of enteric infections
✓ Exotoxin/enterotoxin cholera like toxin • NON-MOTILE and GAMMA HEMOLYTIC
• Microscopically, it forms the so-called “disjointed
Can cause Food poisoning bamboo fishing rod appearance”
▪ Diarrheal Type • [+] Lecithinase test
▪ Emetic Type − Inoculation in egg yolk agar
Produces two types of toxins: the reason why it causes When inoculated on plated media (BAP):
food poisoning. − The colonies will produce “swirling
projections” giving it a MEDUSA HEAD,
Compared with B. anthracis, B. cereus is considered LION HEAD appearance or “cut glass”
MOTILE and BETA HEMOLYTIC on BAP. appearance”
− The colonies are so TENACIOUS giving it a
Best specimen for testing: suspected food “beaten egg white” consistency
[+] Gelatin hydrolysis On tubed (semi-solid) media (gelatin media):
[+] Growth on PEA (Phenylethyl alcohol agar) − It forms the so-called “inverted Fir Tree” or
[+] Lecithinase “Inverted Pine Tree pattern”
Egg yolk agar Selective media is PLET
- Used to detect lecithinase & lipase detection, (polymyxin lysozyme EDTA Thallous acetate):
innoculation − Shows “string like pattern” because of its
- (+) opaque zone around the colonies susceptibility to Penicillin
- Inoculate → Incubate → Note for Opaque Zones − To obtain a “string of pearl” pattern:
 Inoculate on MHA (Mueller Hinton agar)
 Place a penicillin antibiotic disk then we
place a cover slip on top of the disk.
 Incubate the entire preparation.
 After 24 hours, we remove the cover slip
covering the penicillin disk and we invert it
[+] Result in Lecithinase test in the slide.
 Bacilli arranged in chains will appear (string
Note: PEA (Phenylethyl alcohol agar), can be used as an of pearl pattern)
isolation media for gram-positive cocci (S. aureus or
streptococci) Note: MHA (Mueller Hinton agar) is used for
susceptibility testing
1
BACTERIOLOGY
CASUSES THREE TYPES OF ANTHRAX:
EXTERNAL INTERNAL
Cutaneous A. Pulmonary A. Intestinal A.
Most common Inhalation of Least common
but less severe. spores while but most
handling wool severe
“Disjointed bamboo fishing rod appearance”
Contact with Ingestion of

infect spores. spores
May develop Also known as: Ingestion of

“black eschar” “Ragpicker’s improperly
on the skin. Disease” cooked
infected meat
“Woolsorter’s
Disease”
“Medusa head colonies”
Observed when the B. anthracis is inoculated in BAP
“Hide Porter
Disease”
Bacillus subtilis
• Bacillus subtilis is a common laboratory
contaminant
• Also known as Hay Bacillus
• Bacillus cereus & Bacillus subtilis are both
MOTILE and BETA hemolytic on BAP
“Inverted fir/pine tree pattern”
VIRULENCE FACTORS:
✓ D-Glutamate capsule
 prevents phagocytosis
 not polysaccharide in nature but is D-glutamate
✓ Ability to produce an exotoxin with 3 components
1. Lethal factor
2. Edema factor
3. Protective antigen
2
BACTERIOLOGY
CLOSTRIDIUM
Clostridium perfringens
Clostridium botulinum • Also considered as an agent of enteric infection
• Also known as “canned good bacillus” • Under genus Clostridium, C. perfringens are
• Also known as “Von Ermengen’s Bacillus” considered as histotoxic
• An agent of GIT infection
• In the laboratory, it is not usually cultivated Other names: Clostridium welchii, Frankel’s bacillus,
• It is not cultured on BAP but usually produces BETA Gas gangrene bacillus
hemolysis.
• Spores are sub terminally located Important Characteristics:
• [+] Lipase • Microscopically forms the “Box Car
• [–] Lecithinase motile Morphology”
• When inoculated on BAP, it produces (+) Target
VIRULENCE FACTORS: or Double hemolysis
✓ Botulinum toxin  Target or Double hemolysis is when
 A neurotoxin that is potent in human a colony is surrounded by an inner
 Blocks the release of acetylcholine causing Beta and an outer Alpha
flaccid paralysis or rag doll paralysis (muscles
are not responsive).  Usually, organisms only have 1
pattern of hemolysis. But, in the
CAN CAUSE: case of Clostridium perfringens, it
• Botulism shows double hemolysis
− A fatal type of food poisoning
− Infant Botulism  The beta hemolysis is due to beta
 May develop as a result of spore ingestion via hemolysin and tetatoxin which is
breast feeding produced by Clostridium
 Affected child will manifest hypotonia perfringens. While alpha hemolysis
(reduced muscle strength) is due to alpha toxin and lecithinase
 Symptom: floppy baby syndrome
• SID / Sudden Infant Death Syndrome / Crib Death • Causes stormy fermentation of milk when
inoculated on a litmus milk medium.
Laboratory confirmation is done by demonstrating  Due to excessive gas production
the presence of toxin in: • It is Lecithinase (+) and Nagler’s test (+)
• Serum  Both performed on an egg yolk agar
• Stool • To identify Clostridium perfringens in the
• Food laboratory, you need to perform 2 tests.
It can also be done by culturing C. botulinum from:  Reverse CAMP Test and to
• Stool demonstrate the Target or Double
• Wound Hemolysis
• Food
3
BACTERIOLOGY
• Reverse CAMP Test is carried out by

inoculating on BAP which is the required media.
Aside from BAP, you will need a known
organism which Streptococcus agalactiae.
 S. agalactiae when inoculated on
BAP, produces beta hemolysis.
 We inoculate first the known
organism which is the S. agalactiae
on BAP. Then we inoculate next the
unknown organism which is
suspected to be C. perfringens LITMUS MILK MEDIUM
perpendicular to S. agalactiae. Uses:
There should be a point where both • Used primarily to differentiate members within
organisms will meet. the genus Clostridium
 The positive result (+) of Reverse • To differentiate Enterobacteriaceae from other
CAMP test is the same as the result Gram-negative bacilli based on the ability of
of CAMP test which is the Enterobacteriaceae to produce litmus
“Enhanced hemolysis as shown by • To cultivate and maintain cultures of lactic acid
arrow-head zone of beta hemolysis” bacteria
 The negative result (-) for both
CAMP and Reverse CAMP test is Components of Litmus Milk Medium
“No enhanced hemolysis” • Skim milk and a pH indicator
 The skim milk provides nutrients for
o CAMP test is for S.
agalactiae and to do CAMP growth, contains lactose for
test, the known organism to fermentation and detection of acid
be used is S. aureus. While, production. Skim milk will also provide
Reverse CAMP Test is for protein in the form of casein
the detection of C.  The pH indicator is called Azolitmin. In
perfringens using the an acidic pH (4.5), the indicator will
organism S. agalactiae appear pink but in alkaline pH (8.3) it
• Causes Gas gangrene and Myonecrosis becomes blue. Between these extremes
(Neutral pH) it is color purple.
The picture below shows the so-called Double hemolysis
or Target hemolysis which surrounded by inner beta and 4 basic reactions in Litmus Milk medium
outer alpha. 1. Lactose Fermentation
 The picture on the right is the positive result of 2. Reduction of litmus
Reverse CAMP test. Take note of the 3. Casein coagulation
development of beta hemolysis with arrow-head 4. Casein hydrolysis
shape which is considered as (+).
 The Group B Streptococcus is the S. agalactiae  These reactions yield a variety of results
 Group A beta hemolytic is S. pyogenes and the and combinations, each of which can
Group B hemolytic is S. agalactiae differentiate bacteria
 Take note that combination of results is
possible. For example, in a pink color
result, there are fissures in clot
4
BACTERIOLOGY
RESULTS INTERPRETATION SYMBOL

Pink Acid reaction A
Pink and
solid (white
in the lower Acid clot AC
portion); clot
not movable
Fissures in Gas G
clot
Clot broken Stormy fermentation S *A curd differs from an acid clot in that it will not
apart dissolve in alkaline conditions
White color Reduction of litmus R
(lower • In a litmus milk medium, there are 4 possible
portion of the
reactions, which can produce a variety of
medium)
combination results, which can be used to
Semisolid Curd C
identify the organism.
and not pink;
clear to gray
fluid at top
Clarification Digestion of peptone; P OR D
of medium; peptonization
loss of
“body”
Bluer Alkaline reaction K
medium or
blue band top
No change None of the above NC
reactinos
Possible Results on Litmus Milk Medium
1. Lactose fermentation acidifies the medium and

turns the litmus to pink
 The thing changed is the pH only (acid
production)
2. Accumulating acid may cause the casein to
precipitate and form an acid clot. Acid clots
solidify the medium and can appear pink or
white with a pink band at the top
 This result is an example of combination
because the pH is acidic and at the same
time there is clot formation
3. Fissures or cracks in the clot are evidence gas
production
 At the bottom of the tube, it is not a
solid white color, it is considered as
fissure or crack which is a sign of gas
production
5
BACTERIOLOGY
Clostridium difficile Clostridium tetani

• Normal flora of the colon Not an agent of enteric infection but classified under
• Causes pseudomembranous colitis or antibiotic genus Clostridium
associated diarrhea
Other names: Tack head bacillus, tetanus bacillus,
Important Characteristics: drumstick, lollipop, tennis racquet bacillus
 Drumstick, lollipop, tennis racquet
• Motile because C. tetani has terminal swollen
• Lecithinase and Lipase (-) spores
• Virulence Factors are Toxin A (enterotoxin) and • C. tetani together with C. botulinum are
Toxin B (cytotoxin) considered as neurotoxic Clostridium
• Even though C. difficile is culturable, seldom do  C. botulinum causes flaccid paralysis,
we perform culture in the laboratory. while C. tetani causes Spastic paralysis
 Cultivation of C. difficile is possible but “little movement”
in the laboratory, to detect C. difficile  In flaccid paralysis, the muscles are
diagnosis or detection is through totally unresponsive compared to
cytotoxin detection. spastic paralysis, there is little
 To detect the cytotoxin produced by the movement
C. difficile we do it through Enzyme • C. tetani causes Tetanus because of its ability to
Immunoassay (EIA). produce Tetanospasmin, an exotoxin which
 Specimen usually submitted is stool. will block the release of neurotransmitters
Freshly passed stool, liquid or unformed  Tetanospasmin is the virulence factor
stool is for culture and toxin assay.  Tetanospasmin is an exotoxin
Formed stool or rectal swab are used to responsible for blocking the release of
detect the “carrier” state. neurotransmitters leading to spastic
paralysis which will trigger locked jaw.
 Locked jaw causes the patient to show
the unique symptom of tetanus which is
risus sardonicus also known as
Media Used for C. difficile sardonic smile
• Tetanus has a triad of symptoms:
• CCFA- Cycloserine Cefoxitin Fructose Agar 1. Locked jaw or trismus
 It is a selective media for C. difficile. It 2. Sardonic smile or risus sardonicus
contatin neutral red as its indicator. 3. Spasm of all muscles or opisthotonos
 On CCFA, C. difficile develops colonies • C. tetani is seldomly cultured and is often
with Horse stable or Barn yard odor diagnosed based on clinical symptoms and
observation of terminally located swollen
• BAP- it will fluoresce CHARTREUSE spores.
 Produces yellow fluorescence
6
BACTERIOLOGY
Staphylococcus aureus
bacitracin
Part of normal flora but causes diseases, both toxin-
mediated and non-toxin mediated. Also considered as an Catalase Test
agent of enteric infection. • Biochemical test for differentiating
• Gram-positive (+) cocci staphylococci and micrococci from streptococci
• Arranged in grape-like clusters when observed  staphylococci and micrococci are
under the microscope using gram stain smears. catalase test positive (+)
• Initial detection is done through catalase test  Streptococci are catalase test negative
• Coagulase test is the definitive test (-)
• Can be done in slide or tube
• Reagent used: 3% hydrogen peroxide
• Positive result: Vigorous bubbling or
effervescence
• In catalase test, colonies/organism growing in
BAP are NOT used because it will lead to false-
positive reaction
TESTS FOR DETECTION OF S. AUREUS
Test S. aureus reaction

Catalase Test Positive (+): Vigorous bubbling
or effervescence
Coagulase Test Slide Coagulase, Positive (+):
Clot formation/Clumping
Coagulase Test
Tube Coagulase, Positive (+): • The definitive/priority test for S. aureus
Clot formation/Clumping • Positive (+) result: Clot formation/Clumping
Mannitol Positive (+): Yellow halo • Done if the presence of S. aureus is suspected
Fermentation Test around the colonies
after catalase test
DNAse Test HCl Precipitation, Positive (+):
• Coagulase test is done to differentiate the
Clearing of agar around the
colonies pathogenic S. aureus from other staphylococci
• Reagent used: Rabbit’s plasma
Dye Method, Positive (+):  Collected using EDTA
Toluidine blue: Pink  In preparing the plasma citrate is never
zone around the used because it can lead to a false-
colonies positive reaction
Methyl green: Clear
zone around the
colonies
Vogues -Proskauer Positive (+) Pink to red color
Test
PYR Test Negative (-)
Hemolysis on BAP Beta hemolytic
Bacitracin Test Resistant to 0.4 units of
7
BACTERIOLOGY
OTHER TESTS FOR IDENTIFYING S. AUREUS
Slide Coagulase Test Mannitol Fermentation Test

• Purpose: detects the clumping factor/bound • S. aureus is positive for Mannitol Fermentation
coagulase Test
• Procedure: • Purpose: detects acid production aerobically or
 Place a Loopful of the organism to be anaerobically
tested on the slide  Fermenter: organism that can produce
 Add rabbit’s plasma and NSS acid with or without air
 Mix then observe for clot formation • Media used: Mannitol Salt Agar (MSA)
 Has phenol red as pH indicator
• If positive (+), then it is reported as positive for • Contains 7.5% salt
S. aureus • Procedure:
• If negative (-), tube coagulase should be done  Inoculate the organism in MSA
 Incubate for 18-24 hours
Tube Coagulase Test  After incubation observe for yellow halo
• Purpose: Detect free coagulase around the colonies
• Similar with slide coagulase but done in a tube • Positive (+): yellow halo around the colonies
and requires incubation for 4 hours at 35-37 °C • Negative (-): Remained red
• Procedure: • A positive mannitol fermentation test would
 Place a loopful of the organism to be mean:
tested on the tube, then add rabbit’s 1. The organism was able to ferment
plasma and NSS (produce acid) mannitol
 Incubate for 4 hours at 35-37 °C 2. The organism was able to tolerate 7.5%
 After incubation, check for clot concentration of salt
formation
 If no clot formation, incubate further for
16 hours at room temperature (25C)
• Total incubation time: 20 hours
• If after incubation, there is a clot formation then
it is positive, if no clot formation then it is
negative
8
BACTERIOLOGY
DNAse Test (Thermonuclease test)

• S. aureus is positive for DNAse test Hemolysis on BAP
• Has two methods: • S. aureus is beta-hemolytic
HCl Precipitation Test Bacitracin Test

• Procedure: • S. aureus is resistant to 0.4 units of bacitracin –
 Inoculate the organism in DNAse agar an antibiotic also known as Taxo A
 Incubate
 Then, add 0.1 normal HCl
• Positive result: Clearing of agar around the
colonies
Dye method
• DNAse agar used with toluidine blue or methyl
green
• Positive (+) result:
 Toluidine blue: Pink zone around the
colonies
 Methyl green: Clear zone around the
colonies
Vogues-Proskauer Test
• S. aureus is positive for VP Test
• Vogues-Proskauer is a biochemical test that is
part of the IMVIC test
• IMVIC is used for gram-negative enteric bacilli
• Positive (+) result: Pink to red color
PYR Test
• S. aureus is PYR negative (-)
• For identification of S. pyogenes which is
positive (+) for PYR
• S. pyogenes - Gram-positive, catalase-negative
cocci
9
BACTERIOLOGY
VIRULENCE FACTORS
Coagulase → Major virulence factor,
considered as MARKER of GRAM NEGATIVE BACILLI
VIRULENCE A. Vibrio cholerae
→ Enzyme that causes bacterial ➢ Enteric pathogen
cell to agglutinate in plasma ➢ Facultative anaerobe (can live w/ or w/o air)
→ Converts fibrinogen into fibrin ➢ Fermenter – can produce acid w/ or w/o air
Beta → AKA Penicillinase ➢ Motile, curved or comma shaped rods
Lactamase → Responsible for S. aureus ➢ Non-halophilic along with Vibrio mimicus
resistance to penicillin and to all ➢ Found in Brackish or marine water and
beta lactams transmission to humans is by ingestion of
Hyaluronidase → AKA Duran Raynal Factor contaminated water, fresh produce meat, dairy
→ Spreading factor products, seafood
→ Enhances ability of organism to ➢ Significant species since it causes the disease
invade tissues CHOLERA (asiatic cholera or epidemic cholera)
DNase → AKA Thermonuclease w/c is characterized by the production of rice
→ When released, decreases watery stool
viscosity of exudates allowing ➢ Virulence factor: Choleragen/ cholera toxin
more mobility ➢ Motility: Shooting star motility
Beta → AKA Sphingomyelinase C or ➢ String test (+): use of sodium desoxycholate
Hemolysin Hot -Cold Lysin  On a slide, place a loopful of organism
→ Causes beta hemolysis then add sodium desoxycholate then mix.
Enterotoxins → Causes food poisoning  After mixing, use the loop/needle to lift
A&B the colonies.
TSST-1 → Toxic Shock Syndrome Toxin 1  (+): thread-like structure
→ Causes Toxic Shock Syndrome  Can also identify Klebsiella pneumoniae
Exfoliatin → Causes skin desquamation/ (under family Enterobacteriaceae;
exfoliation in SCALDED SKIN encapsulated)
SYNDROME/ RITTER’S
disease
PVL/ Panton → Destruction of WBCs
Valentine
Leukocidin
Protein A → Prevents phagocytosis
Cephalosporinase test
➢ Test for Beta Lactamase production Biochemical Test
➢ Uses cefinase disk → has impregnated substrate Oxidase +
Nitrocefin Glucose +
➢ (+) result: Pink to red Lactose -
➢ (-) result: Yellow Sucrose +
10
BACTERIOLOGY
Genus Vibrio B. Campylobacter species

• Oxidase (+), except V. metschnikovii ➢ Oxidase (+)
 Screening test for Genus Vibrio ➢ Two significant species: C. jejuni and C. coli
• Halophilic, except V. cholerae and V. mimicus ➢ Appears as S-shaped bacilli resembling wings of
 Requiring increase salt concentration, seagulls under the microscope
usually 8-10% salt ➢ Motile and shows darting motility
• Glucose (+) ➢ Requires low oxygen 5% (microaerophilic) and
• Lactose (-), except V. vulnificus increased CO2 5-10% (capnophilic)
• Basophilic ➢ Optimum temperature for growth: 42-43 deg C
➢ Most species are asaccharolytic (do not ferment
Diagnostics: any sugar)
✓ Specimen for diagnosis: Stool or rectal swab ➢ Guillain Barre + patients test positive for
✓ Transport media: Cary Blair media Campylobacter antibodies
✓ Enrichment media: Alkaline Peptone water ➢ Causes: febrile systemic syndrome,
✓ Selective-differential media: TCBS – Thiosulfate gastroenteritis, and periodontal disease (dse
Citrate Bile Salt Sucrose affecting the oral cavity)
 Selective – no gram positive will grow
because of the presence of the inhibitor
 Differential – differentiate whether
sucrose (+) or sucrose (-)
 This media contains sucrose as
fermentable carbohydrate, bromothymol
blue and thymol blue as indicators
 Non-sucrose fermenter: Green
 Sucrose fermenter: Yellow
8% NaCl TCBS Oxidase

Yellow –
sucrose (+)
Green –
sucrose (-)
V. parahaemolyticus Halophilic Green +
V. vulnificus Halophilic Green +
V. alginolyticus Halophilic Yellow +
V. mimicus Not Green +
V. fluvialis Halophilic Yellow +
V. furnissii Halophilic Yellow +
V. metschnikovii Halophilic Yellow -
11
BACTERIOLOGY
Aeromonas spp.
Oxidase test [+] (1) Aeromonas hydrophila – water
loving organism associated with
GIT disease Vibrio spp. Aeromonas
(2) Aeromonas caviae – usually spp.
Oxidase Test
isolated in the lab + +
Motility
• Oxidase test [+]; Fermentative
(generally
+ +
(can produce acid w/ or w/o
motile)
air) gram-negative bacilli
NaCl
• Can produce colonies on requirement
+ -
media that is used to isolate 8%-10
members of the family (halophylic
Enterobacteriaceae
Ferments
-can grow on Mac Conkey,
Mannitol
+ +
EMB, SSA, and CIN (growth
Growth on
on this media
TCBS
+ +
indistinguishable to those of
(One way to
Yersinia enterocolitica
separate)
- +
species)
Hemolysis,
Aside from causing GIT disease,
DNase, Esculin
although classified as enteric
Hydrolysis
pathogens, may also cause extra
O129
intestinal infections (septicemia,
Susceptibility
S R
meningitis & wound infection)
Lysine-
Ornithine-
++- +-+
Virulence Factors of Campylobacter & Arginine
Aeromonas Gelatin
Enterotoxin • It alters/change the metabolic Liquefaction
+ +
activity of intestinal epithelial
cells causing the out pouring o D-test
electrolytes and fluid to the • Double disk diffusion test
lumen
• Purpose: to detect inducible clindamycin resistance
• When enterotoxin is released, it among strains of S.aureus
can lead to profuse & watery :to confirm the result of clindamycin antibiotic if
diarrhea resistant or susceptible
• Stool of patients with enterotoxic [+] result : Blunting of the Clindamycin zone to
diarrheal disease → watery & produce a D pattern
profuse (severe) [-] result : report Clindamycin as Sensitive while
Cytotoxin • It disrupts the structure of the if [+] report Clindamycin as Resistant
intestinal epithelial cells
• When destroyed/damaged, it can For example, in performing susceptibility test inoculated
also provoke inflammatory on MHA
response 4 antibiotics:
*Enterotoxin & Cytotoxin are not the only virulence C – Clindamycin
factors for any agent of the GIT disease because most E – Erythromycin
agents of enteric infections can produce other factors P – Penicillin
SXT
12
BACTERIOLOGY
After doing the test and measuring the zone of inhibition, [+] result : Blunting of the Clindamycin zone to produce
it results that the Clindamycin (Class Linconamide) is a D pattern
Susceptible while the Erythromycin (Class Macrolide) *if the organism is susceptible but not D-shaped it is
is Resistant negative, the susceptible result should be in LETTR D
• Regardless of class, both Clindamycin and
Erythromycin mode of action in killing bacteria
is by inhibiting of protein synthesis Staphylococcus aureus
• Since, they have the same mode of action, it is • Considered as a significant spp.
expected the result of organism to both • Even though it is part of the normal flora, it can
Clindamycin & Erythromycin is somehow the cause toxin mediated & non-toxin mediated
SAME diseases
(pls see text in the lower part of the photo) • It produces beta lactamase/penicillinase – it is
resistant to penicillin
In doing D-test: -in treating S. aureus, we never use penicillin
• Inoculate again using MHA but instead of using 4
antibiotics, use only the antibiotics Clindamycin and
Erythromycin
Required in doing D-test:
• MHA
• 15 ug erythromycin and 2 ug clindamycin which are
positioned 15mm apart then incubate
• Measure the zone of inhibition
*before placing the antibody in MHA, inoculate the S.
aureus first
13
BACTERIOLOGY
To detect/treat S. aureus MRSA/ORSA Tests to detect MRSA

infections, we use
penicillinase resistant Methicillin Resistant S. aureus & Oxacillin
drugs Resistant S. aureus
-staphylococcus showing resistance to these

drugs are called MRSA/ORSA
Ex: • Strain of S. aureus resistant to 1. Use of CHROM AGAR (expensive)
Oxacillin Methicillin, Nafcillin and Oxacillin MRSA – Rose or Mauve colony color
Cloxacillin • Resistance of Staphylococcus to Non-MRSA – Growth inhibition or colorless
Methicillin penicillinase resistant drugs is due to or Blue colonies
-it can kill S. aureus PBP2a (Penicillin binding protein 2)
(which is located in the cell wall) 2. CEFOXITIN DISK DIFFUSION TEST
encoded by mec A gene -induces expression of PBP2a
Characteristics of MRSA: 3. GOLD STANDARD for MRSA detection

• Penicillin RESISTANT -PCR to detect mec A gene
• Oxacillin RESISTANT
• Cefoxitin Test POSITIVE
14
ANALYSIS OF URINE AND OTHER BODY FLUIDS
Medical Technology Assessment Program
November 18, 2021 | Mark Rodrigo D. Mendros RMT, MT(ASCPi), MSMT
MICROSCOPIC EXAMINATION OF URINE SAMPLE Confirm pathologic or non-

pathologic cause of turbidity
✓ One of the routine procedures that we do when Blood RBCs/RBC casts
we do routine urinalysis. Protein Casts/cells
✓ Before doing the microscopic examination of Nitrite Bacteria/WBCs
urine, we must perform physical and chemical Leukocyte esterase WBCs/ WBC casts/ Bacteria
examination of urine. We centrifuge and decant Glucose Yeast
the urine. The sediments will be examined in
the microscopic examination.
✓ Purpose: to detect and to identify insoluble SPECIMEN PREPARATION
materials present in the urine o Specimens should be examined while
✓ The urine sediments are contributed by the ff: fresh or adequately preserved.
o Blood - urine is the ultrafiltrate of the o Formed elements – primarily RBCs,
plasma. The insoluble materials in the WBCs, and hyaline casts- disintegrate
blood/plasma are seen in the urine rapidly, particularly in dilute alkaline
o Kidney urine.
o Lower genitourinary tract o Refrigeration (1-6 degrees Celsius) may
o External contamination cause precipitation of amorphous
✓ The urine sediments include: urates and phosphates that can affect
o RBCs, WBCs, epithelial cells, casts the clarity of the urine. Other non-
o Bacteria, yeast, parasites pathologic crystals that can obscure
o Mucus, spermatozoa other elements in the urine sediment
o Crystals, and artifacts = unorganized o Warming the specimen to 37degC prior
types to centrifuging may dissolve some
✓ Examination of the urinary sediment must crystals.
include both identification and quantitation of o The midstream clean-catch specimen is
the elements present. preferred because it minimizes external
✓ The least standardized and most time- contamination of the sediment.
consuming part of the routine analysis
MACROSCOPIC SCREENING AND CORRELATION WITH SPECIMEN VOLUME
MICROSCOPIC SEDIMENTS
o A standard amount of urine, usually
✓ Abnormalities in the physical and chemical
between 10 and 15 mL, is centrifuged in
portions of the urinalysis play a primary role in
a conical tube.
the decision to perform a microscopic analysis
o A 12-mL volume is frequently used
thus the use of the term macroscopic screening,
because multiparameter reagent strips
also referred to as chemical sieving.
are easily immersed in this volume, and
MACROSCOPIC SCREENING CORRELATIONS capped centrifuge tubes are often
Screening Test Significance calibrated to this volume.
Color Blood o If obtaining a 12-mL specimen is not
Hematuria (turbid) versus possible, as with pediatric patients, the
Clarity hemoglobinuria/myoglobinuria volume of the specimen used should be
(clear red) noted on the report form.
1
o The urine output is proportional to the REPORTING THE MICROSCOPIC EXAM

fluid intake and renal function of an
individual o Casts are reported as the average
number per low-power field (lpf)
following examination of 10 fields
CENTRIFUGATION o RBCs and WBCs, as the average number
per 10 high-power fields (hpfs).
o Centrifugation for 5 minutes at 400 RCF ▪ In patients with bleeding in the
produces an optimum amount of urine or suspected UTI = If
sediment with the least chance of many cells are seen in the slide,
damaging the elements. report it as “too numerous to
count”.
VOLUME OF SEDIMENT EXAMINED ▪ Patients with Mild UTI = WBCs
are seen in clumps. It should be
o When using the conventional glass-slide reported as “Pus cells are seen
method, the recommended volume is in clumps or aggregates.”
20 uL (0.02 mL) covered by a 22 mm o Epithelial cells, crystals, and other
glass cover slip. elements are frequently reported in
o Allowing the specimen to flow outside semiquantitative terms such as: rare,
of the cover slip may result in the loss of few, moderate, and many, or as 1+, 2+,
heavier elements such as casts. 3+, and 4+, following laboratory format
as to lpf or hpf use.
EXAMINATION OF THE SEDIMENT ROUTINE URINALYSIS CORRELATIONS
o Observation of a minimum of 10 fields Microscopic
Physical Chemical Exceptions
under both low power field (lpf = 10x) Elements
and high-power field (hpf = 40x) Turbidity Number
RBCs +Blood
o The slide is first examined under low Red Color Hemolysis
power to detect casts and to ascertain +Protein
Number
the general composition of the WBCs Turbidity +Nitrite
Lysis
sediment. +Leukocytes
o In conventional slide method, casts Epithelial
Turbidity Number
have a tendency to locate near the cells
edges of the cover slip; therefore, low- Casts +Protein Number
power scanning of the cover-slip pH
Number
perimeter is recommended. Bacteria Turbidity +Nitrite
and type
o When elements such as casts that +Leukocytes
require identification are encountered, Turbidity Number
Crystals pH
the setting is changed to high-power. Color and type
o When the sediment is examined
unstained, many sediment constituents SEDIMENT EXAMINATION TECHNIQUES
have a refractive index similar to urine.
SEDIMENT STAIN
Therefore, it is essential that sediments
✓ Staining increases the overall visibility of
be examined under reduced light when
sediment elements being examined using
using bright-field microscopy.
2
bright-field microscopy by changing their ✓ Clin. Sig:

refractive index. (Ex. is hyaline cast) o Glomerular membrane damage
✓ Staining also imparts identifying characteristics (glomerulonephritis)
to cellular structures, such as the nuclei, o Vascular injury within the genitourinary
cytoplasm, and inclusions. tract
✓ The most frequently used stain in urinalysis is o Renal calculi
the Sternheimer-Malbin stain (consists of
crystal violet and safranin O) 2. White Blood Cells (Leukocyte/ Pus cells)
✓ Appear as: granular spheres, about 12 microns
in diameter, usually neutrophils
✓ Usually <5 WBC/hpf is present in normal urine
✓ Usually distinguished from RBC by the addition
of 10% Glacial Acetic Acid which dissolves RBC
while making the nuclei of WBC appear more
distinct.
✓ Pyuria- increased WBC in urine
✓ Glitter cells – pale blue leukocytes producing a
“sparkling appearance” in their cytoplasm; seen
in dilute or hypotonic urine.
✓ Clin. Sig:
Bacterial infection
o Pyelonephritis
o Cystitis
o Prostitis
o Urethritis
URINE SEDIMENT CONSTITUENTS Non-bacterial infection

o Glomerulonephritis,
CLASSIFICATION OF URINARY SEDIMENTS
o SLE,
✓ Organized o interstitial,
✓ Unorganized o nephritis, tumors
✓ Eosinophils -uncommon; associated with drug
A. ORGANIZED URINARY SEDIMENTS induced interstitial nephritis (UTI and renal
1. Red Blood Cells transplant)
✓ Colorless disks without nucleus, 7 microns in
diameter 3. Epithelial Cells
✓ Frequently confused with yeast cells, oil a. Squamous cells
droplets, CaOx ✓ Most frequently seen and least significant of
✓ NV: 0-2/hpf or 0-3/hpf epithelial cells
✓ Appearance: ✓ Derived from vaginal lining and lower portions
o Hypotonic urine (dilute and alkaline): of male and female urethra
swollen/ghost cells or shadow cells ✓ Largest cells in the body, has abundant irregular
o Hypertonic urine (concentrated): cytoplasm
crenated ✓ Put a drop of NSS to separate the clumped
Squamous cells found in the sample
3
✓ Clue cells- indicative of vaginal infection by G. 4. Casts

vaginalis which covers most of the cell surface ✓ Formed primarily within the “Lumen of DCT and
and extend beyond the edges of the cell Collecting Ducts”
✓ Excreted at a constant rate by the renal tubule
b. Transitional epithelial cells/ urothelial/ ✓ Provides immunologic protection from infection
caudate ✓ Gels (forms) during urine flow stasis, acidity and
✓ Originate from lining of renal pelvis, bladder, presence of sodium and calcium
upper urethra ✓ Reflect the diameter and shape of the lumen
✓ Smaller than squamous cells, spherical, caudate origin
or polyhedral with central nucleus ✓ Composition:
✓ Has ability of reabsorb large quantities of water a. 1/3: Tamm-Horsfall protein – glycoprotein
✓ Seldom pathogenic importance excreted by RTE cell; forms the matrix of all
cast
c. Renal tubular epithelial cells (RTEc) b. 2/3: Albumin and Globulin
✓ Most significant; presence indicate “tubular ✓ Cylindroids – casts w/ tapered ends produced at
necrosis” and “renal graft rejection the junction of ascending loop of Henle & DCT
✓ Usually round slightly larger than WBC, with ✓ Cynlindriduria or Cynlinduria – presence of cast
single round eccentrically located nucleus in the urine
✓ Presence of >2/hpf RTE cells indicates tubular
injury
✓ Clin. Sig: TYPES OF CASTS
o tubular damage, pyelonephritis, toxic a. Hyaline Casts
reactions, viral infections, allograft ✓ Colorless, homogenous and has same refractive
rejection, secondary effects of index as urine
glomerulonephritis ✓ Made up of entirely Tamm-Horsfall Protein
✓ Indicated “mild renal damage”
d. Oval Fat Bodies – lipid containing renal tubular ✓ 0-2/hpf is considered normal
cells ✓ Clin. Sig:
✓ Seen in conjunction with free floating fat Non-pathogenic
droplets o Strenuous exercise
✓ Under polarize light exhibits “maltese cross o Dehydration
appearance” o Heat exposure
✓ Identification is confirmed by Sudan III or Oil o Emotional stress
Red O (polarized microscopy)
✓ Lipiduria – associated with damage to Pathogenic
glomerulus caused by nephrotic syndrome, o Acute glomerulonephritis
sever tubular necrosis, Diabetes Mellitus, o Pyelonephritis
trauma cases o Chronic renal disease
✓ Lipids are also seen in the urine when the o Congestive heart failure
patient has an increased intake of fatty foods.
✓ Bubble cell – RTEc containing large, nonlipid b. RBC casts
filled vacuoles ✓ Indicated hemorrhage in the Renal Tubules,
active Acute Nephritis”
✓ Found in healthy individual following
participation in “contact sports”
4
c. WBC casts 6. Bacteria

✓ Presence signifies “infection and inflammation” ✓ only reported when observed in fresh
within the nephron specimens in conjunction with WBC
✓ Most frequently seen in pyelonephritis ✓ When the urine is allowed to stand, there will
✓ Presence indicated the need to perform be bacterial multiplication which can cause false
bacterial cultures elevation in the bacterial count
✓ Positive for the presence of WBCs, nitrite and
d. Epithelial cell casts leukocyte esterase
✓ Observed in conjunction with RBC and WBC cast ✓ confirmed with positive urine culture
in Glomerulonephritis and Pyelonephritis ✓ presence is indicative of lower or upper UTI;
usually enterics, Staphylococcus.
e. Granular casts ✓ They are unstained under the microscope,
✓ Usually seen accompanying Hyaline casts therefore it is important to reduce the light
following periods of stress and strenuous intensity of the microscope
exercise
✓ Can be in the form of fine granular cast or 7. Yeast cells
coarse granular cast ✓ seen in Diabetes Mellitus, vaginal moniliasis,
✓ Determine the presence of this cast under lpf immunocompromised patients
and check for its appearance under hpf. ✓ easily confused with RBC, observed with
budding forms
f. Waxy casts ✓ Presence of fungal cells in the urine is an
✓ Indicates “extreme stasis of urine flow” indicator of a severely weak immune system
✓ Refractile with rigid texture, which causes them ✓ Easily confused with RBC, observed with
to be fragmented as they pass through the
budding forms
tubules
✓ Typically seen in the urine = Candida spp.
g. Fatty casts
8. Parasites in Urine
✓ seen in conjunction with Oval Fat Bodies in
✓ Trichomonas vaginalis – acquired thru sexual
disorders causing “lipiduria” such as Nephrotic
contact; This organism is highly motile and it
Syndrome
can cause “Ping pong infection” or
Trichomoniasis.
h. Broad casts
✓ Balantidium coli
✓ presence in urine presents Grave Prognosis,
✓ Schistosoma haematobium
referred to as “Renal Failure Casts”
✓ Enterobius vermicularis
9. Spermatozoa
5. Mucus threads
✓ found in urine after sexual intercourse,
✓ protein material produced by glands and
nocturnal emissions or masturbation
epithelial cells in the genitourinary tract and
✓ (+) Protein reagent strip in increase amount of
RTEC
semen
✓ Tamm-Horsfall Protein – major constituent
✓ We do not report presence of spermatozoa
✓ increased amounts occurs in “vaginal
contamination” and “irritation” of any kinds ✓ Geriatrics: indication of seminal fluid leakage;
there can be a problem in the genitourinary
tract
5
✓ If the patient is minor, do not release the result ✓ Only urate found in alkaline urine
unless advised by the superiors especially in ✓ Converts to uric acid crystal when glacial acetic
possible rape cases acid is added
4. Calcium phosphates
B. UNORGANIZED URINARY SEDIMENTS ✓ Colorless, flat rectangular plates on thin prisms
1. Crystals – found by the precipitation of urine salts often in rosette formation
subjected to changes in pH, temperature, or ✓ Constituent of renal calculi
concentration that affects their solubility 5. Triple phosphate
✓ pH – most valuable in the identification of ✓ Aka ammonium magnesium phosphate
crystals ✓ Referred to as coffin lid crystals
✓ Less often: flat fern leaf form, sheets and flakes
✓ Suggest stasis of urine in kidney and bladder
I. NORMAL CRYSTALS IN ACIDIC URINE ✓ Seen in highly alkaline urine associated with
1. Amorphous urates presence of urea-splitting bacteria
✓ Macroscopic pink upon refrigeration
✓ Microscopic brick red or yellow brown in color III. ABNORMAL ACIDIC URINE
2. Uric acid 1. Cystine
✓ Assumes the greatest variety of shapes ✓ Hexagonal plates; colorless, highly refractile and
(rhombic, whetstone, rosette, burred, thick/thin
quadrates, dumbbell, wedges) ✓ Often mistaken with uric acid but dissolves in
✓ Associated with “gout arthritis”, “Lesch-Nyhan dilute HCl (uric acid does not)
Syndrome” and leukemia patient receiving ✓ A noticeable “sulfur odor” may be present in
chemotherapy the urine in disorders of cystine metabolism
3. Calcium oxalate 2. Cholesterol
✓ Mostly appear as envelope or dumbbell shape ✓ Large flat plates with one or more corners cut-
or ovoid (dihydrate) off; notched plates
✓ monohydrate form – dumbbell and ovoid ✓ Seen in nephrotic conditions and lipid necrosis
rectangle seen in cases of ethylene glycol ✓ Soluble in chloroform
poisoning 3. Leucine
✓ derived from various food notably spinach, ✓ Yellow or brown spheres resembling fat globule
rhubarb, berries, and tomatoes. with delicate radiating and concentric striations
✓ Less seen that tyrosine, soluble in hot
II. NORMAL CRYSTALS IN ALKALINE URINE alkali/alcohol
1. Amorphous phosphate ✓ ” Scallop-lily looking crystal”
✓ Granular, similar to amorphous urates, 4. Tyrosine
macroscopic white turbidity ✓ Colorless fine needles grouped in clusters (may
2. Calcium carbonate appears black in the center), rosettes or
✓ Also seen in neutral urine sheaves crossing at various angles
✓ Colorless granules larger than amorphous ✓ Seen in conjunction with leucin crystal, (+)
phosphates bilirubin test
✓ Often appearing as “dumbbell forms or ✓ Soluble in alkali and heat
spherical forms” ✓ Has “sour and rancid” odor
3. Ammonium biurate 5. Bilirubin
✓ Yellow-brown/golden, “thorny apples”/ spicule- ✓ Yellow-rhombic / ruby red crystals / clumped
covered needles or granules with characteristic yellow
6
✓ Soluble in Acetic acid, HCl, NaOH, ether and

chloroform
6. Sulfonamides
✓ Cause of formation: inadequate patient
hydration
✓ Green color, soluble in acetone
✓ Less encountered: needled, rhombic,
whetstones, sheaves of wheat, rosettes with
colors ranging from colorless to yellow brown
✓ Clin. Sig: Tubular damage
7. Radiographic dye
✓ Similar to cholesterol crystal and highly
birefringent
8. Ampicillin
✓ Appear as colorless needles that tend to form
bundles following refrigeration.
7
CHAPTER 9: URINE SCREENING FOR METABOLIC Cystinosis

DISORDERS Homocystinuria
OVERFLOW VERSUS RENAL DISORDERS
✓ The appearance of abnormal metabolic

substances in the urine can be caused by a
variety of disorders that can generally be
grouped into two categories: OVERFLOW TYPE
and RENAL TYPE
✓ Overflow disorders – result from the disruption
of a normal metabolic pathway thar causes
increased plasma concentrations of the
nonmetabolized substances.
o Either override the reabsorption ability
of the renal tubules or are not normally
reabsorbed because they are only
present in minute amounts. NEWBORN SCREENING TESTS
✓ Renal Disorders – caused by malfunctions in the
✓ Traditionally, urine tests are performed to
tubular reabsorption mechanism.
detect and monitor for IEMs.
✓ The most frequently encountered abnormalities
✓ Defects must be detected early in life because
are associated with metabolic disturbances that
many of the disorders cause the buildup of
produce urinary overflow of substances
unmetabolized toxic food ingredients.
involved in protein, fat, and carbohydrate
✓ Blood – specimen collected by infant heel
metabolism due to vast number of enzymes
puncture because the substances are more
used and the fact that their function is essential.
elevated in the blood than urine.
✓ Disruption of enzyme function can be caused
✓ Tandem Mass Spectrophotometry (MS/MS) –
by:
capable of screening the infant blood sample
o Inborn Error of Metabolism (IEM) -
for specific substances associated with
failure to inherit the gene to produce a
particular IEMs.
particular enzyme, or
✓ Method for specific gene testing is also rapidly
o by organ malfunction from disease or
being developed.
toxic reactions.
ABNORMAL METABOLIC CONSTITUENTS OR AMINO ACID DISORDERS
CONDITIONS DETECTED IN THE ROUTINE
URINALYSIS PHENYLALANINE – TYROSINE DISORDERS
Color Odor Crystals
✓ Many of the most frequently requested special
Homogentistic Phenylketonuria Cystine urinalysis procedures are associated with the
acid phenylalanine-tyrosine metabolic pathway.
Maple syrup ✓ Metabolic defects cause production of excessive
Melanin Leucine
disease amounts of melanin.
Isovaleric
Indican Tyrosine
acidemia
Lesch-Nyhan
Porphyrins Cystinuria
Disease
8
o If detected, dietary changes that eliminate

phenylalanine, a major constituent of
milk, from the infant’s diet can prevent
the excessive buildup of serum
phenylalanine and can thereby avoid
damage to the child’s mental capabilities.
o As the child matures, alternate pathways
of phenylalanine metabolism develop,
and dietary restrictions can be eased.
o Products that contain large amounts of
phenylalanine have warnings for people
with PKU, such as aspartame.
✓ Increased blood levels of phenylalanine must
occur prior to the urinary excretion of
phenylpyruvic acid, which may take from 2 to 6
weeks.
✓ Blood sample must be obtained before the
newborn is discharged from the hospital.
✓ Phenylalanine can be detected as early as 4
hours after birth and, if the cutoff level for
normal results is lowered form 4mg/dL to
1. Phenylketonuria 2mg/dL, the presence of PKU should be
✓ Aminoacidurias detected.
✓ Estimated to occur in 1 of every 10,000 to 20,000 ✓ More girls than boys escape detection of PKU
births and, if undetected, results in severe during early tests because of slower rises in
mental retardation. blood phenylalanine levels.
✓ First identified in Norway by Ivan Follling in 1934 ✓ Urine testing is performed as:
o Mother with other mentally retarded o Follow-up procedure in questionable
children reported a peculiar mousy odor diagnostic cases,
to her child’s urine o Screening test to ensure proper dietary
o Urinalysis showed increased amounts of control in previously diagnosed cases,
keto acids, including phenylpyruvate. and
o This occurs when conversion of o Means of monitoring the dietary intake
phenylalanine to tyrosine is disrupted. of pregnant women known to lack
o Interruption of the pathway produces phenylalanine hydroxylase.
children with fair complexions – even ✓ Microbial Inhibition assay by Guthrie – most
dark-skinned families – owing to the well-known blood test for PKU.
decreased production of tyrosine and its
pigmentation metabolite melanin.
✓ PKU is caused by failure to inherit the gene to PROCEDURE FOR MICROBIAL INHIBITION ASSAY BY GUTHRIE
produce the enzyme phenylalanine
✓ Blood from a heel stick absorbed into filter
hydroxylase.
paper circles.
o Inherited as an autosomal recessive trait
✓ The circle must be completely saturated with a
✓ Screening tests are available for early detection
single layer of blood.
of the abnormality.
9
✓ The blood-impregnated disks are then placed in ✓ Observe color.

culture media streaked with the bacterium ✓ (+) Permanent blue-green color
Bacillus subtilis.
✓ Increased level of phenylalanine: growth will be
observed around the paper disks TYROSYLURIA
o Phenylalanine counteracts the action of ✓ Because tyrosine metabolism involves two
beta-2-thienylalanine, an inhibitor of B. pathways, urine may contain excess tyrosine or
subtilis that is present in the media.
its metabolic products, p-hydroxyphenyl pyruvic
acid and p-hydroxyphenyl lactic acid.
✓ In premature infants, transitory tyrosinemia is
frequently seen due to underdevelopment of the
liver function needed to produce enzyme
necessary for complete tyrosine metabolism.
✓ Perform a Ferric chloride tube test to avoid
✓ Modifications of the Guthrie test can also confusion between PKU and tyrosine during
detect: urinary screening tests. The green color fades
o Maple syrup urine disease, quickly when tyrosine is present.
o Homocystinuria, ✓ Tyrosyluria is also observed in patients with
o Tyrosinemia, acquired severe liver disease with similarity to
o Histidinemia, transitory tyrosinemia but more severe. Tyrosine
o Valinemia, and
and leucine crystals can be observed during
o Galactosemia.
microscopic examination of urine sediment.
✓ MS/MS tests for many substances that can be
also detected from dried blood collected by heel ✓ Hereditary disorders, which cause liver and renal
stick: tubular disease and generalized aminoaciduria,
o Thyroid hormones are usually fatal.
o Trypsin ✓ The nitroso-naphthol test is used to screen for
o Biotinidase tyrosine and its metabolites. It is non-specific
✓ Urine tests for phenylpyruvic acid are based on and reacts with substances other than tyrosine
ferric chloride reaction performed by tube test. and its metabolites. A positive reaction is
o The ferric chloride test is a nonspecific orange-red, indicating that more testing is
reaction and will react with many other required.
amino acids and commonly ingested ✓ Nitroso-Naphthol Test for Tyrosine
medications. o Reagents: 1 mL 2.63N nitric acid, 1 drop
o Brands of disposable diapers also
21.5 % sodium nitrite, 0.1 mL 1-nitroso-
produces false-positive reactions for
2-naphthol
PKU when tested with ferric chloride.
o Positive result in urine: Permanent o Urine: 5 drops
blue-green color. o (+) orange-red color
✓ Tandem mass spectrometry (MS/MS) is available
for screening tyrosinemia types 1 and 2.
PROCEDURE FOR FERRIC CHLORIDE TUBE TEST
✓ Place 1 mL of urine in a tube.

✓ Slowly add 5 drops of 10% ferric chloride.
10
HEREDITARY DISORDERS OF TYROSINEMIA precursor (5,6-dihydroxyindole), which

- deficiency of would then be oxidized to melanogen
fumarylacetoaceta and then to melanin, causing urine to
te hydrolase darken.
- It causes
Type I generalized renal REACTIONS OF MELANIN WITH SCREENING TEST
tubular disorder Screening Tests Positive reaction of
and progressive melanin
liver failure after Ferric chloride test Gray/black precipitate
the infant is born. Sodium nitroprusside Red color
- Deficiency of
tyrosine
aminotransferase ✓ To avoid acetone and creatinine interference,
- It is thought to add glacial acetic acid. When glacial acetic acid is
cause tyrosine added, melanin turns green-black, acetone turns
Type II crystallization in purple, and creatinine turns amber.
cells, resulting in
corneal erosion
and lesions on the ALKAPTONURIA
palms, fingers, and
✓ The third major defect in the phenylalanine-
soles of the feet.
tyrosine pathway is caused by the absence of the
- Deficiency of p-
hydroxyphenylpyr gene required to produce homogentisic acid
uvic acid oxidase. As a result, the phenylalanine-tyrosine
dioxygenase. pathway cannot be completed, and
- Can cause mental homogentisic acid accumulates in the blood,
Type III
retardation if tissues, and urine. Although full-blown
dietary restriction manifestation is not observed during early
of phenylalanine childhood, black and red-stained diapers have
and tyrosine are been reported.
not implemented. ✓ In adults, homogentisic acid deposition in
cartilage causes arthritis.
MELANURIA
o Melanin is the pigment that gives dark ROUTINE SCREENING TEST FOR HOMOGENTISIC ACID
hair, skin, and eyes their color. Albinism Positive reaction
would result from a lack of production of Ferric chloride test Transient deep color
melanin. Clinitest Yellow precipitate
o When urine with high levels of melanin Alkalization of fresh urine Darkening color
is exposed to air, the urine darkens.
Elevated melanin levels may indicate
melanocyte over proliferation or ✓ This test is hindered by high levels of ascorbic
malignant melanoma. These tumors acid, as well as the addition of silver nitrate and
would secrete a colorless melanin ammonium hydroxide.
11
Disorders of the o Reagent: 10 drops of 0.02% 2,4-DNPH in

Phenylalanine-Tyrosine Screening Tests 2N HCl
Pathway o Urine: 1mL
Phenylketonuria Ferric chloride tube test o (+): yellow/white precipitate
Ferric chloride tube test o Interference: Large doses of ampicillin
Tyrosyluria
Nitro-naphthol test
Ferric chloride tube test ORGANIC ACIDEMIAS
Alkaptonuria Clinitest
✓ Organic acidemias can cause severe illness,
Alkalization of fresh urine
including vomiting, metabolic acidosis,
Ferric chloride tube test
Melanuria Sodium nitroprusside test hypoglycemia, ketonuria, and increased serum
Ehrlich test ammonia.
✓ The three most encountered disorders are
isovaleric, propionic, and methylmalonic
BRANCHED-CHAIN AMINO ACID DISORDERS acidemia.
✓ Distinctive characteristic is its methyl group ISOVALERIC ACIDEMIA
attached to the main aliphatic carbon chain.
✓ In general, the presence of ketonuria in a ✓ Patients with this condition have deficiency an
newborn is a significant laboratory finding in isovaleryl coenzyme A in their Lucine pathway.
branch-chain amino acid disorders. The absence of enzyme leads to accumulation of
isovalerylglycine. There is no screening test for
MAPLE SYRUP URINE DISEASE this disease.
✓ Is an inherited autosomal recessive trait caused ✓ The specimen has characteristic sweaty feet
by an inborn error of metabolism. Wherein the odor.
urine specimen produces a strong odor PROPIONIC AND METHYLMALONIC ACIDEMIAS
resembling maple syrup.
✓ Leucine, isoleucine, and valine are the amino ✓ Errors in the metabolic pathway converting
acids involved. isoleucine, valine, threonine, and methionine to
✓ The metabolic pathway begins normally, with succinyl coenzyme A.
the transamination of the three amino acids ✓ Propionic acid is the immediate precursor to
(Leucine, isoleucine, and valine) in the liver to methylmalonic acid in this pathway.
keto acids (a-ketoisovaleric, a-Keto isocaproic, a- ✓ Urine test for methylmalonic aciduria
keto-B-methylvaleric). o Reagent: p-nitroaniline
✓ The lack of the gene required for oxidative o (+): emerald green
carboxylation of these keto acids results in their
accumulation in the blood and urine. TRYPTOPHAN DISORDERS
✓ MSUD is manageable if the disease is detected
by the 11th day and with dietary regulation and ✓ Major concern in the metabolism of tryptophan
careful monitoring of urinary keto acid is the increased urinary excretion of metabolites:
Indican and 5-hydroxyindoleacetic acid (5-HIAA)
concentrations. It is included in many newborn
screening profiles using MS/MS.
✓ Urine screening test for MSUD is 2,4-
dinitrophenylhydrazine (DNPH)
12
INDICANURIA o Major constituent of foods such as

bananas, pineapples, and tomatoes.
NORMAL CONDITIONS ✓ Normally, the body uses most of the serotonin,
Most of the tryptophan that enters the intestine is either: and small amounts of its degradation product 5-
✓ Reabsorb for use by the body in production of hydroxyindoleacetic acid (HIAA) are available for
protein excretion in urine.
✓ Converted to indole by the intestinal bacteria ✓ Carcinoid tumors involving argentaffin
and excreted in feces. (enterochromaffin) cells develop, excess
amounts of serotonin are produced, resulting in
CERTAIN INTESTINAL DISORDERS
elevation of urinary 5-HIAA levels.
o Obstruction ✓ Appearance of purple to black color – due to
o Presence of abnormal bacteria
addition of nitrous acid and 1-nitroso-2-naphtol
o Malabsorption syndromes to urine that contains 5-HIAA.
o Hartnup disease ✓ 2 to 8 mg – normal daily excretion of 5-HIAA
✓ Greater than 25mg/24h – indication of
✓ Increased amounts of tryptophan are converted argentaffin cell tumors.
to indole ✓ Test can be performed on a random or first
✓ Excess indole – reabsorbed from the intestine morning specimen.
into the bloodstream and circulated in the liver. ✓ False-negative results can occur based on
✓ Liver – where it is converted to indicant and then specimen concentration and because 5-HIAA
excreted in the urine. may not be produced at constant rate.
✓ Indicant excreted in urine is colorless until ✓ If 24hr sample is used – preserve with
oxidized (by exposure to air) to the dye indigo hydrochloric or boric acid.
blue. ✓ Medications that cause interference:
phenothiazines and acetanilids. (hold
HARTNUP DISEASE medications for 72hrs prior to specimen
✓ Rare inherited disorder collection)
✓ Early diagnosis: “Blue Diaper Syndrome” – blue
staining of the infant’s diapers.
✓ Urinary indican reacts with acidic ferric chloride
to form deep blue or violet color. (extracted into
chloroform)
✓ Affects not only intestinal reabsorption of
tryptophan but also the tubular reabsorption of
other amino acids resulting in – Generalized
aminoaciduria (Fanconi syndrome)
5-HYDROXYINDOLEACETIC ACID
✓ Serotonin
o Produced from tryptophan by the
argentaffin cells in the intestine.
o Carried through the body primarily by
platelets.
13
o Persons with any form of inheritance

may form renal calculi. (less common in
lysine and cystine are affected)
CYSTINE
✓ Much less soluble than the other three amino
acids
✓ Laboratory screening determinations: based on
observation of cystine crystals in the sediment of
concentrated or first morning specimens.
✓ Only amino acid found during the analysis of
calculi.
✓ Chemical screening test – performed using
cyanide-nitroprusside.
✓ Reduction of cystine by sodium cyanide followed
by addition of sodium nitroprusside = red-
Figure 9-3 - Tryptophan metabolism
purple.
✓ False-positive reactions occurs in presence of
CYSTINE DISORDERS ketones and homocystine.
The two distinct disorders are: CYSTINOSIS
o Inherited
o Exhibit renal manifestations ✓ Genuine IEM
o Noticeable odor of sulfur may be present ✓ Can occur in three variations – ranging from
severe fatal disorder developed in infancy to
CYSTINURIA benign form appearing in adulthood.
✓ Marked by elevated amounts of amino acid Two general categories:
cystine in the urine. 1. Nepropathic
✓ Due to the inability of the renal tubules to ✓ Subdivided into:
reabsorb cystine filtered by glomerulus. (not due o Infantile
to defect in metabolism of cystine) o Late-onset cystinosis
✓ Defect in the renal tubular transport of amino ✓ Defect in lysosomal membranes prevents the
acid release of cystine into cellular cytoplasm.
✓ Incomplete metabolism results in deposit of
Two Mode of Inheritance: cystine in:
1. Reabsorption of all four amino acids – cystine, o Cornea
lysine, arginine, and ornithine is affected. o Bone marrow
2. Only cystine and lysine are not reabsorbed. o Lymph nodes
o Internal organs
Genetic studies have grouped cystinuria based on:
o Two inherited genes ✓ Fanconi syndrome – major defect in renal
o Heterozygous inheritance tubular reabsorption mechanism also occurs.
o Homozygous inheritance
14
✓ Proximal convoluted tubules – affected by the oUse of silver nitrate (in place of sodium
deposits of cystine that interfere with cyanide) – reduces homocystine to its
reabsorption. nitroprusside-reactive form but does not
✓ Continued deposition of cystine results in renal reduce cystine.
failure (if untreated) o (+) reaction confirms presence of
✓ In late-onset, there is gradual progression to homocystinuria
total renal failure. ✓ Fresh urine – should be used when testing
homocystine.
2. Infantile Nephrohepathic
✓ There is rapid progression to renal failure
PORPHYRIN DISORDERS
✓ Relatively benign but may cause some ocular
disorders. ✓ Porphyrin – intermediate compounds in the
✓ Routine laboratory findings: production of heme.
o Polyuria ✓ Three primary porphyrins
o Generalized aminoaciduria o Uroporphyrin
o (+) test result for reducing substances o Coproporphyrin
o Lack of urinary concentration o Protoporphyrin
✓ Porphyrin precursors
o Α-aminolevulinic acid (ALA)
HOMOCYSTINURIA
o Porphobilinogen
✓ Defects in metabolism of the amino acid ✓ Blockage of pathway – results in accumulation of
methionine produce an increase in homocystine the product form.
throughout the body. ✓ Detection and Identification of this product in
✓ The increased homocystine can result in: urine, bile, feces, or blood can aid in determining
o Failure to thrive cause of specific disorder.
o Cataracts ✓ Solubility of porphyrin compounds varies with
o Mental retardation their structure
o Thromboembolic problems most soluble and
ALA, porphobilinogen,
o Death readily appear in
and uroporphyrin
✓ Can alleviate the metabolic problems: urine
o Early detection Less soluble but is
Coproporphyrin
o Change in diet (excluding foods high in found in urine
methionine) Protoporphyrin Not see in urine
✓ Screening for homocystine – included in most
newborn screening program.
✓ Newborn screening tests ✓ Fecal analysis – for detection of coproporphyrin
o Originally performed using modification and protoporphyrin.
of the Guthrie microbial inhibition assay ✓ Bile – more acceptable specimen to avoid false-
o Replaced with MS/MS testing. positive interference.
✓ Cyanide-nitroprusside test – gives positive result ✓ Analysis of whole blood – for the presence of
when there is increased urinary homocystine. free erythrocyte protoporphyrin (FEP) as a
✓ Silver-nitroprusside test – additional screening screening test for lead poisoning.
test for homocystinuria (after a positive cyanide- (recommended by CDC)
nitroprusside test)
o Only homocystine will react
15
PORPHYRIAS o Uses their extraction into a mixture of

✓ Disorders of porphyrin metabolism glacial acetic acid and ethyl acetate.
✓ Can be inherited or acquired from: o Solvent layer – the one to be examined
o Erythrocytic and hepatic malfunctions o Faint blue fluorescence – negative
o Exposure to toxic agents reactions
✓ Common causes of acquired porphyrias: o Violet, pink, red – positive reactions
o Lead poisoning o If there is suspected presence of
o Excessive alcohol exposure interfering substance -> organic layer
o Iron deficiency can be removed to a separate tube ->
o Chronic liver disease add 0.5 mL of hydrochloric acid.
o Renal disease o Porphyrins – only extracted into the acid
✓ Cause of inherited porphyrias (much rarer): layer – then produce bright orange-red
o Failure to inherit the gene that produces appearance.
an enzyme that needed in the metabolic
pathway. ✓ Testing for the presence of porphobilinogen –
✓ Inherited porphyrias – frequently classified by most useful when patient has symptoms of acute
their clinical symptoms: attack.
o Either neurologic/psychiatric or ✓ Increased porphobilinogen – associated with
cutaneous photosensitivity or acute intermittent porphyria.
combination of both ✓ Increased protoporphyrin – best measured in
whole blood.
PORPHYRINURIA
✓ Observation of red or port wine color to the
urine after exposure to air.
✓ Port wine urine color – more prevalent in the
erythropoetic porphyrias.
✓ Staining of teeth may also occur.
✓ Discoloration of an infant’s diaper – sometimes
suspected of congenital porphyria.
✓ Two screening tests for porphyrinuria under
ultraviolet light in the 550-to 600nm range.
1. Erhlich reaction
o Only for the detection of ALA and
porphobilinogen
o Acetylacetone must be added to the
specimen to convert the ALA to
porphobilinogen
2. Fluorescent technique – must be used for other
porphyrins.
o Rules out porphobilinogen and ALA
o Does not distinguish among
uroporphyrin, coproporphyrin, and
protoporphyrin.
16
SUMMARY OF MOST COMMON PORPHYRIAS

✓ Urine that contains mucopolysaccharides
Porphyria Elevated Clinical Laboratory
Compound Symptoms Testing produces a blue spot that cannot be washed
Acute ALA Neurologic/ Urine/Ehrlic away by a dilute acidified methanol solution.
inter- Porpho- psychiatric h reaction ✓ Common mucopolysaccharidoses are Hurler
mittent bilinogen syndrome, Hunter syndrome, and Sanfilippo
porphyria syndrome.
Porphyria Uro- Photo- Urine 1. Hurler syndrome
cutanea porphyrin sensitivity fluore- ✓ Mucopolysaccharides accumulate in the cornea
tarda scence of the eye.
Congenital Uro- Photo- Urine/feces ✓ Skeletal structure is abnormal and there is
erythro- porphyrin sensitivity fluore- severe mental retardation
poietic Copro- scence 2. Hunter syndrome
porphyria porphyrin ✓ inherited as sex-linked recessive and is rarely
Variegate Copro- Photosensiti Bile/ feces
seen in females.
porphyria porphyrin vity/ fluore-
✓ Without treatment, both syndromes are usually
neurologic scence
fatal during childhood.
Erythro- Proto- Photo- Blood FEP
poietic porphyrin sensitivity Bile or feces 3. Sanfilippo syndrome
proto- fluore- ✓ Abnormality is mental retardation.
porphyria scence ✓ Treatment for mucopolysaccharide disorders
Lead ALA Neurologic Acetoacetic o Bone marrow transplants
poisoning Proto- acid o Gene replacement therapy
porphyrin urine/Ehrlic
h reaction PURINE DISORDERS
Blood FEP 1. Lesch-Nyhan
✓ disease is a disorder of purine metabolism. It is
MUCOPOLYSACCHARIDE DISORDERS inherited as a sex-linked recessive results in
✓ AKA. glycosaminoglycans, are a group of large massive excretion of urinary uric acid crystals.
compounds located primarily in the connective ✓ Failure to inherit the gene to produce the
tissue. enzyme hypoxanthine guanine
✓ Products most frequently found in the urine are: phosphoribosyltransferase is responsible for the
o Dermatan sulfate accumulation of uric acid throughout the body.
o Keratan sulfate ✓ Complications:
o Heparin sulfate o motor defects
o Particular substance being determined o mental retardation
by the specific metabolic error that was o self-destruction
inherited o gout
✓ Frequently used screening tests: o renal calculi.
o Acid-albumin ✓ Signs and symptoms
o Cetyltrimethylammonium bromide o First symptom: uric acid crystals
(CTAB) turbidity tests resembling orange sand in diapers.
o Metachromatic staining spot tests.
17
CARBOHYDRATE DISORDERS
1. Melituria COMPARISON OF URINARY SCREENING TESTS
✓ Melituria or increased urinary sugar is most
Test Disorder Observation
frequently due to an inherited disorder.
✓ Fortunately, the majority of meliturias cause no Alkaptonuria Black
Melanuria Black
disturbance to body metabolism.
Color Indicanuria Dark blue
✓ Causes
Porphyrinuria Port wine
o Lactose
o Fructose Phenylketonuria Mousy
o Pentose Maple syrup urine ds. Maple syrup
✓ Pediatric urine should be routinely screened for Isovaleric academia Sweaty feet
the presence of reducing substances using Cystinuria Sulphur
Odor
Cystinosis Sulphur
Clinitest.
Homocystinuria Sulphur
2. Galactosuria
✓ Galactosuria, the inability to properly Sheaths of
metabolize galactose to glucose. Tyrosyluria fine needles
Colorless
✓ Caused by a deficiency in:
Cystinuria hexagonal
o galactose-1-phosphate uridyl
plates
transferase (GALT) Crystals Yellow-
o galactokinase Lesch-Nyhan disease brown
o UDP-galactose-4-epimerase crystals
✓ GALT deficiency causes severe possible fatal Phenylketonuria Blue-green
symptoms associated with galactosemia. Tyrosyluria Transient
✓ Galactose kinase deficiency can result in green
cataracts in adulthood. Alkaptonuria Transient
✓ UDP-galactose-4-epimerase deficiency may be blue
Ferric
asymptomatic or produce mild symptoms. Melanuria Gray-black
chloride
3. Lactosuria Maple syrup urine Green-gray
tube test
✓ May be seen during pregnancy and lactation. disease
Indicanuria Violet-blue
4. Fructosuria
with
✓ Associated with parenteral feeding and
chloroform
pentosuria with ingestion of large amounts of 5-HIAA Blue-green
fruit.
Red
Tyrosyluria
Nitroso- Red
CLINICAL TEST IN IDENTIFYING CHO METABOLISM Maple syrup urine ds.
naphthol Violet with
✓ Finding of a positive copper reduction test 5-HIAA
nitric acid
result combined with a negative reagent strip
glucose oxidase test result is strongly suggestive Phenylketonuria Yellow
of a disorder of carbohydrate metabolism. 2,4- Tyrosyluria Yellow
Dinitro- Maple syrup urine ds. Yellow
✓ Additional tests including chromatography can
phenyl- Isovaleric acidemia Yellow
be used to identify other non-glucose reducing
hydrazine Propionic acidemia Yellow
substances. Methylmalonic acidemia Yellow
18
Maple syrup urine ds. Purple

Isovaleric acidemia Purple
Acetest Propionic acidemia Purple
Methylmalonic acidemia Purple
Melanuria Red
p-Nitro- Emerald
Methylmalonic acidemia
aniline green
Cyanide- Cystinuria Red-purple

nitro- Cystinosis Red-purple
prusside Homocystinuria Red-purple
Silver
Homocystinuria Red-purple
nitro-
Alkaptonuria Black
prusside
Ehrlich Porphyrinuria Red

reaction Melanuria Red
Cetytri-
methyl- White
Mucopolysaccharidoses
ammonium turbidity
bromide
Mucopoly-
saccharide Mucopolysaccharidoses Blue spot
paper
Melituria Orange-red
Clinitest Cystinosis Orange-red
Alkaptonuria Orange-red
19

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PARASITOLOGY

Medical Technology Assessment Program - 1

SPECIES AND ITS COMMON NAMES Chinese Liver

Specimen for Diagnosis: HEPATIC/LIVER FLUKES

PERIODICITY AND MORPHOLOGY OF

With distinct terminal

Circumoral Disk with THICKENINGS & POLAR

Gravid segments with

• Adult worm is regarded as

o Symptom of appropriate blood vessel

• Human • ADULT WORMS may be

tissue secretions • Non-hermaphroditic;

•Species of amoeba West African Sleeping

READING ASSIGNMENT: MAHON PP. 626-631 APPROPRIATE COLLECTION OF SAMPLES

PRESERVATION ➢ Used when a permanently stained smear will be

MACROSCOPIC EXAMINATION ➢ Any portion that contains blood or blood-tinged

Modified Acid-Fast Stain

Kinyoun modified acid-fast stain

o Microhematocrit tube is coated with

ANTIGEN, ANTIBODY and IMMUNE TWO PROPERTIES OF ANTIGENS

• Heterophile antigen: a type of heteroantigen Example:

• Immunogen: defined as any substance that 2. Heterogenous antigen: cross reaction;

CLASSIFICATION OF ANTIGEN 5 HEAVY CHAINS:

✓ Epsilon (IgE) 3. Heavy chains are interconnected by

Activa Yes Alternativ Yes No No

IMMUNE RESPONSE PRIMARY IMMUNE RESPONSE

COMPARISON OF PRIMARY AND

SECONDARY IMMUNE RESPONSE

• Artificial Active - Adaptive immunity of

FLOW CYTOMETRY and injects inside a carrier stream of isotonic

Fluorochromes Commonly Used in Clinical DATA ACQUISITION AND ANALYSIS

• The absolute count of a particular cell

CD4+ T CELLS SURFACE MARKERS ON T & B CELLS

CD16 Macrophages, Low affinity FC CD45R Different forms Essential in T-

c. reducing or eliminating the many Instrument • Maintenance requirements

FACTORS FOR CONSIDERATION IN

CATEGORY FACTOR 1. Batch Analyzers

measure multiple analytes from • Reagent use in automated immunoassay

• The reference interval is the range of values

• Automated analyzers are costly but can

GRAM [+] BACILLI

Bacillus cereus Bacillus anthracis

CASUSES THREE TYPES OF ANTHRAX:

Contact with Ingestion of

May develop Also known as: Ingestion of

“Inverted fir/pine tree pattern”

• Reverse CAMP Test is carried out by

RESULTS INTERPRETATION SYMBOL

Possible Results on Litmus Milk Medium

1. Lactose fermentation acidifies the medium and

Clostridium difficile Clostridium tetani

TESTS FOR DETECTION OF S. AUREUS

Test S. aureus reaction

OTHER TESTS FOR IDENTIFYING S. AUREUS

Slide Coagulase Test Mannitol Fermentation Test

DNAse Test (Thermonuclease test)

HCl Precipitation Test Bacitracin Test

Genus Vibrio B. Campylobacter species

8% NaCl TCBS Oxidase

To detect/treat S. aureus MRSA/ORSA Tests to detect MRSA

-staphylococcus showing resistance to these

Characteristics of MRSA: 3. GOLD STANDARD for MRSA detection

MICROSCOPIC EXAMINATION OF URINE SAMPLE Confirm pathologic or non-