PRELIM LABORATORY: CYTOCHEM REACTION AND INTERPRETATION Flashcards
Enzyme that catalyzes the oxidation of substances by
hydrogen peroxide
Peroxidase
What does the peroxidase enzyme catalyze?
the oxidation
of substances by hydrogen peroxide
Peroxidase catalyzes the oxidation
of substances by hydrogen peroxide, to form what precipitate? (describe the precipitate)
an insoluble
reddish-brown precipitate.
Recommended method for the demonstration of Auer rods
myeloperoxidase (mpo)
T/F: Myeloperoxidase helps differentiates acute myelogenous/monocytic leukemia from ALL
true
Fixative reagent of myeloperoxidase
buffered formalin-acetone
Counterstain reagent of myeloperoxidase
giemsa
How many seconds for the buffered formalin-acetone of myeloperoxidase?
30 seconds
How many minutes for the incubation mixture of myeloperoxidase?
15 minutes
How many minutes for the counterstain of myeloperoxidase?
40 minutes
Positive control of myeloperoxidase activity
neutrophil
Negative control of myeloperoxidase activity
lymphocytes
Myeloperoxidase is primarily expressed in and associated with these cells (cell specificity)
granulocytes & monocytes
Color if positive (+) for MPO
reddish brown/dark brown
Stains various lipids such as sterols, phospholipids and neutral fats
sudan black b
T/F: Myeloperoxidase is more sensitive than sudan black b
false (sbb is believed to be more sensitive)
Reagents of sudan black b
- Formalin-acetone
- Sudan Black B solution
- 70% ethanol
- Nuclear Fast red
Reagents of MPO
- buffered formalin-acetone
- incubation mixture
- giemsa
Fixative of sudan black b
Formalin-acetone
Counterstain of sudan black b
nuclear fast red
Sudan black b is found in which granules?
- primary/secondary granules of the neutrophils
- lysosomal
granules of Monocyte
Most specific for identification of AML
myeloperoxidase
Most sensitive cytochemical stain in identifying AML
sudan black b (non-enzymatic test for lipids)
Used as transport by enzymes
substrate
Marker for mature and immature neutrophil/mast cells
chloroacetate esterase
T/F: Chloroacetate esterase is used to differentiate granulocytes from monocytes
true
Chloroacetate esterase is (less or more?) sensitive than peroxidase stain in terms of primitive myeloid
less
Positive control for chloroacetate esterase
neutrophils
Color of granules for chloroacetate esterase positive cells
bright red granules
Useful in identifying monocytes, promonocytes, and monoblasts
alpha naphthyl butyrate esterase
T/F: Alpha naphthyl butyrate esterase can identify acute/chronic myelomonocytic, acute monocytic, from
other nonlymphocytic leukemia
true
Cells that alpha naphthyl butyrate esterase do not stain
- lymphoblast
- plasma cell
- megakaryocytes
Indication for alpha naphthyl butyrate esterase positive
dark red precipitate
LYMPHOCYTE indication for alpha naphthyl acetate esterase positive
show focal dotlike staining
MONOCYTE positive indication for alpha naphthyl acetate esterase
red-brown
Present in all HSCs and is found in lysosomes. This also provides a simple test for identification of T-acute lymphocytic
leukemia.
acid phosphatase
Acid phosphatase provides a simple test for identification of what?
T-acute lymphocytic
leukemia
Acid phosphatase identification of T-acute lymphocytic leukemia should be evaluated with what 2 enzymes?
peroxidase & esterase
Color of granules positive for acid phosphatase
discrete purplish to dark red granules
In this non-enzymatic staining, carbohydrates present in blood cells are oxidized to glycogen by?
periodic acid
Schiff’s reagent then reacts with the aldehydes to
form what precipitate?
an insoluble red colored precipitate
Periodic acid schiff pattern of reaction
- diffuse
- granular
- or a mixture of the
two
Common stain used for carbohydrates
Periodic acid schiff
Color if positive for periodic acid schiff
bright fuchsia pink
Help in diagnosing ALL and erythroid type of AML(M6)
periodic acid schiff
When differentiating ALL and erythroid type of AML(M6), periodic acid schiff positive (+) indicates for?
AML(M6)
When differentiating ALL and erythroid type of AML(M5),
acute leukocytic
Dye that can bind with acid mucopolysaccharide in blood cells to
form metachromatic complexes
toluidine blue
Most useful in recognition of mast cell disease and acute or chronic basophilic leukemia
toluidine blue
Reagents for toluidine blue
- Mota’s fixative
- 0.1% Toluidine Blue (2 mins)
How many minutes for toluidine blue mota’s fixative
1 minute
How many minutes for 0.1% toluidine blue
2 minutes
Positive control for toluidine blue
buffy coat precipitation of normal peripheral blood
Interpreted as positive for dorm
Nuclei will stain light blue metachromatic granules will
appear reddish violet
Leukocyte alkaline phosphatase (LAP) is also known as
neutrophil alkaline phosphatase
Why is it called neutrophil alkaline phosphatase
because only Neutrophils
are the only leukocyte that normally contains various alkaline
phosphatase
LAP helps in differentiating what disorders?
Chronic myelocytic leukemia from leukemoid
reactions or other myeloproliferative disorders
Explain the principle of alkaline phosphatase
Alkaline phosphatase activity is present in varying degrees in the neutrophil and band form.
The amount of dye precipitated is proportional to the
amount of enzyme present.
Generally, a high WBC with increased neutrophils is
associated with increased enzyme.
Reagents of LAP
-formalin and methanol
- napthol
- fast blue BBN
- nuclear fast red
Fixative of LAP reagent
formalin and methanol
Substrate solution of LAP reagent
naphthol
Working solution of LAP reagent
fast blue bbn
Counterstain of LAP
nuclear fast red
When performing LAP test, what must be the temperature when immersing blood films in cold? How long?
4-10 degrees C & 30 seconds
Provide definition of grading: 0+
No granules
Provide definition of grading: 1+
very few granules (faint, diffuse staining)
Provide definition of grading: 2+
moderate granules scattered
Provide definition of grading: 3+
numerous granules
Provide definition of grading: 4+
cytoplasm is packed
Normal LAP values according to steininger
40-100
Identify if increased/decreased in LAP:
- leukemoid reaction
- pregnancy
- polycythemia vera
- aplastic leukemia
increased
Identify if increased/decreased in LAP:
- CML
- Paroxysmal Nocturnal Hemoglobinuria
- Sickle cell anemia
- Hypophosphatasia
decreased
Test to screen CGD (Chronic Granulomatous disease)
nitroblue tetrazolium test (NBT)
A yellow, water-soluble dye, NBT on reduction is converted into?
an insoluble blue formation formazan
In NBT, (+) Blue color indicates?
granulomatous absorb the formazan blue
Primary color of NBT
(-) Red
At least 2 px slide are obtained with 2 observers &
should count how many neutrophils scoring from 0 to 4+ on the
basis of the quantity & intensity of the precipitated red
dye in their cytoplasm?
50
Procedure for LAP test
- Immerse blood films in cold (4-10 degrees Celsius fixative
for 30 seconds) - Wash well in either tap or distilled water
- Immerse fixed slides in working solution for 20 minutes
at room temperature - Wash well in tap water
Control for LAP test is blood drawn from a woman in her ___ trimester, or within ___ post-partum
- third
- 2 days
Principle for NBT
- 0.1 mL of blood is mixed with 0.1 mL of 0.2 % nitro blue
tetrazolium in saline and 0.1 mL phosphate buffered
saline - This mixture is incubated at 37% for 15 min
- Then held at room temperature for 15 min.
