Cytochemistry

Cytochemistry is concerned with studying of chemical elements

found in cells, these elements can either be:

1. Enzymatic – e.g. peroxidases which can be detected by:

_ Myeloperoxidases
_ Esterases
_ Phosphatases

2. Non-enzymatic – e.g. lipids and glycogen by:

_ Periodic Acid-Schiff Stain

_ Sudan Black B

1- Myeloperoxidase:
– Myeloperoxidase is an enzyme found in the primary granules of
neutrophils, eosinophils and to certain extent monocytes.
– It is not present (- ve) in lymphocytes.
Interpretation:
 It gives black to red brown granules in cytoplasm of myeloid series.
 It differentiates acute myelogenous leukemia (AML) from acute
lymphocytic leukemia (ALL).
 It is positive in M1,M2,M3,M4

2- Leukocyte Alkaline Phosphatase (LAP):

LAP differentiates Chronic Myelogenus leukemia (carcinogenic) from

leukemoid reaction (non carcinogenic).

* Leukemoid reaction is seen in severe infections.

Principle:

LAP is seen in the membrane of secondary granules (mature) of

neutrophil.

Results :

Intensity of precipitated dye in the cytoplasm of neutrophilic

leukocytes varies. One hundred are scored from 0 to 4+ based on the
intensity of the precipitated dye in the cytoplasm.

LAP score 4+ LAP score 1+

 LAP score:

Amount of Score X No.

Score Intensity No. of cells
dye/cell % Of cells

0 Zero Nil 20 0

1+ <50 Faint to moderate 45 45

2+ 50-80 Moderate to strong 25 50

3+ 80 - <100 Strong 5 15

4+ 100 Brilliant 5 20

Total = 130

Normal values:

 Normal Leukocyte Alkaline Phosphatase Score is 13 To 130.

 In CML LAP score < 13

3- Esterases:

 Used to differentiate the myeloblasts from the cells of monocytic

origin

 There are nine isoenzymes of esterases are present in leukocytes.

 Non-specific: α-naphthyl butyrate, α -naphthyl acetate.

◦ Strong diffuse in M4 & M5

◦ Localized in M6 & M7
 Specific: Naphthol AS-D chloracetate esters

◦ Specificity: staining of specifically myelocytic cells only

◦ +ve in M2 & M3

◦ Gives specific reaction in M4 eso

M4 AML- Bone marrow aspirate smear, non-specific esterase (NSE) stain

4- Sudan Black B:

 Sudan Black B stain differentiates 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

◦ Lipids are found on:

1. the primary and secondary granules of PMNS

 2. Lysosomal granules of monocytes

Interpretation:

 Myeloid series are positive from the myeloblast throughout the

maturation series.

5- Periodic Acid-Schif:

Periodic Acid-Schif is diagnosing for Acute Lymphocytic

Leukemia (ALL) and other erythroid type of Acute myeloid
leukemia

Principle:

◦ Periodic acid oxidizes glycogen, mucoproteins giving bright

red pink

Interpretation:

◦ ALL: Lymphoblast may stain coarse or fine or mixed

◦ ERYTHROID: (+) coarse and granular
  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

Diffuse pattern in M4 & M5

Localized in M6 & M& and T-ALL