.

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: a-naphthyl butyrate, a-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 n

MPX + + *

SBB + + +*

NASDA + + *

ANBE -/+ + diffuse + diffuse -

ANAE -/+ + diffuse + diffuse -

PAS Varied Varied Varied Varied +; blotchy in pronormoblast -/+ localize d

F VII -

ALL AML AMML AMoL Erythro leukemi a

Megaka ryocytci Leukem * = (+) in myeloblast (-) in normoblast ia

+ localize d

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 0 1 2 3 4 Total No. Of Cells 20 45 25 5 5 100 Score x No. Of cells 0 45 50 15 20 130=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 Normal Chronic Myelogenous Leukemia Leukemoid reaction Polycythemia Vera Secondary Polycythemia

SCORE 20-100 <13 >100 100-200 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 Lymphocyte Hairy Cells

Without tartrate + +

With Tartrate +