Author: Wilsie M. Salas M.D.

 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
• Acceptable specimens
– Smears and imprints made from:
1.Bone marrow
2.Lymph nodes
3.Spleen
4.Peripheral blood
– Enzymatic smear specimen: fresh, newly obtained
specimen are preferred
– Non Enzymatic specimen: Periodic Acid Schiff (PAS)
and Sudan Black remains stable even after a month
 Acceptable fixatives should contain:
1. Alcohol (methanol, ethanol)
2. Acetone
3. Formaldehyde
• MYELOPEROXIDASE (MPX)
– Enzyme found in the primary granules of PMNs,
Eosinophils and to certain extent Monocytes
– (-) Lymphocytes
– Differentiates Blasts from Acute Myeloid
Leukemia (AML) from Acute Lymphoblastic
Leukemia (ALL)
– Principle:
Myeloperosidase oxidizes the substrate in the
presence of hydrogen peroxide –black to red
brown
• 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% are
peroxidase positive
 MYELOPEROXIDASE (MPX)
◦ Important: Blast cells be only used as a
differentiation among the acute leukemias
 SUDAN BLACK B
 Differentiation of acute myeloid leukemia

from 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
 SUDAN BLACK B
 Interpretation
 Granulocytes are positive from the
myeloblast throughout the maturation
series
 The staining capacity is directly
proportional to cell growth, maturation and
the asquisition of primary and secondary
granules
 SUDAN BLACK B
 ESTERASES
◦ Used to differentiate the myeloblasts from the
neutrophilic series from the cells of the
monocytic origin
◦ Nine isoenzymes of esterases are present in
leukocytes
◦ Substrates esters commonly used:
1.Non-specific: -naphthyl butyrate, -naphthyl
butyrate
2.Specific: Naphthol AS-D chloracetate esters
◦ Specificity: staining of specifically myelocytic cells
only
 ESTERASES
◦ Principle:
◦ Esterases hydrolyzes an ester.
◦ At the site of any enzyme activity, if it
reacts with a naphthol compund, and
combines with a diazonium salt, it will
form a brightly colored compund
◦ Diazonium salts: pararosaniline,
hexazotized new fuschin or fast blue
 ESTERASES
◦ Interpretation
 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 into
ALDEHYDE
◦ Aldehyde + colorless schiff reagent ---bright red
pink
◦ The intensity of stain is directly proportional to the
number of aldehyde compounds produced
 PERIODIC ACID-SCHIFF
◦ PAS Stain can either be:
1.Fine and diffuse
2.Coarse and granular
 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
Conditio MPX SBB NASDA ANBE ANAE PAS F VII
n

ALL - - - -/+ -/+ Varied -

AML + + + - - Varied -
AMML + + + + + Varied -
diffuse diffuse
AMoL - +- - + + Varied -
diffuse diffuse
Erythro * * * - - +; -
- blotchy
leukemi in
a pronor-
moblast
Megaka - - - - + -/+ +
ryocytci localize localize
Leukem
* = (+) in myeloblast (-) in normoblast d d
ia
 FACTOR VIII ANTIBODIES
 Megakaryoblastic leukemia
 (+) result is from the reaction with

monoclonal or polyclonal antibidies against

Factor VIII-related antigen
 LEUKOCYTE ALKALINE PHOSPHATASE (LAP)
 Differentiates Chronic Myelogenus leukemia

and leukemoid reaction

 Leukemoid reaction is seen in severe

infections
 Principle:

◦ LAP is seen in the membrane of secondary

granules of neutrophil
◦ Substrate naphthol AS-BI phosphate is hydrolyzed
+ dye (fast red violet, fast blue BB) ---produces a
colored precipitate at the site of LAP enzyme
 LEUKOCYTE ALKALINE PHOSPHATASE (LAP)
SCORE No. Of Cells Score x No. Of cells

0 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

 LEUKOCYTE ALKALINE PHOSPHATASE (LAP)
 Reminders in scoring LAP

◦ Subjective method
◦ Two slides be read by 2 different clinical laboratory
technologist
◦ The scores done by the the 2 technologist should
agree by 10%
◦ If nota 3rd should be obtained
◦ Eosinophils should be identified from the
neutrophils
◦ Laboratory values should be established by each
laboratory
 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
 FINDING SCORE
Normal 20-100
Chronic Myelogenous Leukemia <13
Leukemoid reaction >100
Polycythemia Vera 100-200
Secondary Polycythemia 20-100
 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 isoenzymes
are inhibited EXCEPT FOR ISOENZYME 5
◦ TRAP PHENOMENA: ISOENZYME 5 IS RESISTANT TO
TARTRATE
 Cell Type Without tartrate With Tartrate

Lymphocyte + -

Hairy Cells + +