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Cytochemistry Lecture

Cytochemistry studies the chemical elements found in cells, including enzymes and non-enzymatic substances. It can be used to detect enzymes like myeloperoxidases, esterases, and phosphatases, as well as non-enzymatic lipids and glycogen. Specific stains are used to differentiate between types of leukemia and stages of myeloid maturation. For example, myeloperoxidase staining identifies neutrophils and distinguishes acute myeloid from lymphoblastic leukemia. Leukocyte alkaline phosphatase scoring also separates chronic myelogenous leukemia from non-cancerous reactions. Esterases and acid phosphatase identify myeloid versus monocytic cells and detect hairy cell leukemia.

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Cytochemistry Lecture

Cytochemistry studies the chemical elements found in cells, including enzymes and non-enzymatic substances. It can be used to detect enzymes like myeloperoxidases, esterases, and phosphatases, as well as non-enzymatic lipids and glycogen. Specific stains are used to differentiate between types of leukemia and stages of myeloid maturation. For example, myeloperoxidase staining identifies neutrophils and distinguishes acute myeloid from lymphoblastic leukemia. Leukocyte alkaline phosphatase scoring also separates chronic myelogenous leukemia from non-cancerous reactions. Esterases and acid phosphatase identify myeloid versus monocytic cells and detect hairy cell leukemia.

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mma1976
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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

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