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.
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Study of chemical elements found in cells This elements can either be:
1. Enzymatic e.g. peroxidases
2. Non-enzymatic
e.g. lipids and glycogen
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Acceptable specimens Smears and imprints made from:
1. Bone marrow
2. Lymph nodes
3. Spleen4. Peripheral blood
Enzymatic smear specimen: fresh, newly obtainedspecimen are preferred
Non Enzymatic specimen: Periodic Acid Schiff(PAS) and Sudan Black remains stable even after amonth
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Acceptable fixatives should contain:1. Alcohol (methanol, ethanol)
2. Acetone
3. Formaldehyde
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MYELOPEROXIDASE (MPX) Enzyme found in the primary granules of PMNs,
Eosinophils and to certain extent Monocytes
(-) Lymphocytes
Differentiates Blasts from Acute MyeloidLeukemia (AML) from Acute LymphoblasticLeukemia (ALL)
Principle:
Myeloperosidase oxidizes the substrate in thepresence of hydrogen peroxide black to redbrown
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MYELOPEROXIDASE (MPX)Interpretation:
AML (w/o maturation, with maturation,promyelocytic leukemia) is 80% positive with MPX
Auer rods strongly positive to MPX Auer rods are found in leukemic blasts and
promyelocyte
Monocytes are MPX negative to weakly positive
Lymphoblasts are negative; ALL 3% areperoxidase positive
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MYELOPEROXIDASE (MPX) Important: Blast cells be only used as a
differentiation among the acute leukemias
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SUDAN BLACK B
Differentiation of acute myeloid leukemiafrom acute lymphoblastic leukemia
More sensitive for early myeloid cells
Principle: Sudan Black stains lipids such as sterols, neutral
fats and phosphilipids
SB is soluble to lipids
Lipids are found on:
1. the primary and secondary granules of PMNS
2. lysosomal granules of monocytes
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SUDAN BLACK B
Interpretation Granulocytes are positive from the
myeloblast throughout the maturationseries
The staining capacity is directlyproportional to cell growth, maturationand the asquisition of primary andsecondary granules
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SUDAN BLACK B
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ESTERASES Used to differentiate the myeloblasts from the
neutrophilic series from the cells of themonocytic origin
Nine isoenzymes of esterases are present inleukocytes
Substrates esters commonly used:1. Non-specific: a-naphthyl butyrate, a-naphthyl
butyrate2. Specific: Naphthol AS-D chloracetate esters
Specificity: staining of specifically myelocyticcells only
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ESTERASES
Principle:
Esterases hydrolyzes an ester.
At the site of any enzyme activity, if it
reacts with a naphthol compund, andcombines with a diazonium salt, it willform a brightly colored compund
Diazonium salts: pararosaniline,
hexazotized new fuschin or fast blue
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ESTERASES Interpretation
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PERIODIC ACID-SCHIFF Diagnosing Acute Lymphocytic Leukemia and other
erythroid tyoe of Acute myeloid leukemia
Principle:
Periodic acid oxidizes glycogen, mucoproteins band
other high molecular weight carbohydrate intoALDEHYDE
Aldehyde + colorless schiff reagent ---bright redpink
The intensity of stain is directly proportional to thenumber of aldehyde compounds produced
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PERIODIC ACID-SCHIFF PAS Stain can either be:
1. Fine and diffuse
2. Coarse and granular
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PERIODIC ACID-SCHIFF Interpretation
Granulocytes are PAS positive
Megakaryocytes has a finely diffuse staining
Platelets are intensely red pink
Erythrocyte precursor does not stain
ALL: Lymphoblast may stain coarse or fine or mixed
ERYTHROID: (+) coarse and granular
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Condition
MPX SBB NASDA ANBE ANAE PAS F VIIALL - - - -/+ -/+ Varied -
AML + + + - - Varied -
AMML + + + +
diffuse
+
diffuse
Varied -
AMoL - +- - +diffuse
+diffuse
Varied -
Erythro-
leukemia
* * * - - +;blotchy
inpronor-moblast
-
MegakaryocytciLeukem
ia
- - - - +localize
d
-/+localize
d
+
* = (+) in myeloblast (-) in normoblast
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FACTOR VIII ANTIBODIES
Megakaryoblastic leukemia
(+) result is from the reaction withmonoclonal or polyclonal antibidies against
Factor VIII-related antigen
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LEUKOCYTE ALKALINE PHOSPHATASE (LAP)
Differentiates Chronic Myelogenus leukemiaand leukemoid reaction
Leukemoid reaction is seen in severe
infections Principle:
LAP is seen in the membrane of secondarygranules of neutrophil
Substrate naphthol AS-BI phosphate is hydrolyzed+ dye (fast red violet, fast blue BB) ---produces acolored precipitate at the site of LAP enzyme
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LEUKOCYTE ALKALINE PHOSPHATASE (LAP)
SCORE No. Of Cells Score x No. Of cells0 20 0
1 45 45
2 25 50
3 5 15
4 5 20
Total 100 130=LAP Score
Please refer to page 403 for the example of LAP scoring
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LEUKOCYTE ALKALINE PHOSPHATASE (LAP)
Reminders in scoring LAP Subjective method
Two slides be read by 2 different clinical laboratorytechnologist
The scores done by the the 2 technologist shouldagree by 10%
If nota 3rd should be obtained
Eosinophils should be identified from the
neutrophils Laboratory values should be established by each
laboratory
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LEUKOCYTE ALKALINE PHOSPHATASE (LAP)
Interpretation of LAP Score Normal 20-100
Untreated Chronic Myelogeous Leukemia: decrease
Leukemoid reaction: high normal to increase
Low LAP Scores:
Paroxysmal Nocturnal Hemoglobinuria
Sideroblatic Anemia
Myelodysplastic Disorders
High LAP Scores: Pregnancy in the third trimester
Polycythemia Vera
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FINDING SCORENormal 20-100
Chronic Myelogenous Leukemia 100
Polycythemia Vera 100-200Secondary Polycythemia 20-100
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ACID PHOSPHATASE (Tartrate Resistant)
Detection of Hairy Cell Leukemia
All cells contain 7 non-erythroid isoenzymes:
Isoensymes: 0, 1, 2, 3, 3b, 4 and 5
Hairy cells is isoenzyme 5 positive Principle:
Acid Phosphatase + AS-BI phosphoric acid + dye(fast garnet GBC) ---Red ppt
Red ppt + L-(+) Tartaric acid ---all isoenzymesare inhibited EXCEPT FOR ISOENZYME 5
TRAP PHENOMENA: ISOENZYME 5 IS RESISTANT TOTARTRATE
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Cell Type Without tartrate With TartrateLymphocyte + -
Hairy Cells + +
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