Lecture #14: Blood Cell Cytochemistry

  

The cytochemical stains used most frequently in the identification and morphological differentiation of cells are those detecting DNA, iron, peroxidase, alkaline and acid phosphatases, RNA, lipids, glycogen, esterases, etc.

1. Used in the study of cell differentiation and in the classification of acute leukemias.

2. Used in the differentiation of leukocytosis and leukemoid reactions from genuine myeloproliferative disorders.

3. Used in the characterization of lymphoproliferative disorders.

4. Used in the demonstration of free iron, Hb derivatives, DNA, RNA and red cell enzymes in erythrocytes and leukocytes.

A. Peroxidase Reaction

The peroxidase reaction is based on the principle that in the presence of hydrogen peroxide, myeloperoxidase in leukocyte granules oxidizes benzidine dihydrochloride from a colorless form to a blue or brown derivative which is localized at the site of enzyme.

Methods:

1.     Goodpasteur method

2.     Osgood and Asthworth method

3.     Sato and Sekiya method

4.     Kaplow’s myeperoxidase method

5.     Mattori’s method

This is useful in distinguishing acute myeloid from acute lymphoid leukemia

Acute myeloid leukemia      =          moderate to strongly positive

Acute lymphoid leukemia    =          negative

B. Sudan Black B stain

Sudan Black B stains phospholipid and sterols. Cells of the granulocytic series are generally sudanophilic while cells of the lymphocytic series are sudanophilic.

Methods:

1.     Sheehan and Storey method

2.     Bailiff and Kimborough method

C. Neutrophil Alkaline Phosphatase (NAP)

This enzyme is located in neutrophils from the metamyelocyte to the segmented stage. It can be detected by exposure to the substrate (a napthol phosphate) in the presence of diazonium salt at an alkaline pH 9.5. The substrate is hydrolyzed by the enzyme releasing a phosphate and aryl–naptholamide. The latter is immediately coupled to the diazonium salt, forming an azo dye. After counterstaining, 100 mature neutrophil are scored (0 – 4) according to the intensity of the staining reaction, from negative to the most intense. Adding to scores for 100 neutrophils will give a total score with a possible range of 0 to 400. Reference values must be determined for each laboratory and usually are about 20 to 100.

High NAP score     =          infections, polycythemia vera, Hodgkin’s disease, leukemoid

                                          reaction

Low NAP score      =          chronic myeloid leukemia, acute myeloid leukemia, PMN

D. Esterases

The leukocyte esterase hydrolyzes an ester which is a derivative of naphthalene. A napthol compound is liberated and rapidly couples with a diazonium salt present in the mixture resulting in a brightly colored precipitate at or near the site of the enzyme activity. The cytochemical reactions for esterases are positive in many cell types.

The reaction is useful in distinguishing leukemias.

E. Periodic Acid Schiff (PAS) Reaction

The PAS reaction is based on the principle that periodic acid is an oxidizing agent that converts hydroxyl groups on adjacent carbon atoms to aldehyde. The resulting aldehydes are combined with Schiff’s reagent to give a red colored product. A positive reaction is therefore seen with polysaccharides, mucopolysaccharides and glycoproteins. In blood cells, a positive PAS reaction usually indicates the presence of glycogen.

In erythroleukemia and in thalassemia, some of the erythroid precursors are PAS positive.

This is true to a lesser extent in iron deficiency anemia and sideroblastic anemia.

F.  Acid Phosphatase

Acid phosphatase in the cells hydrolyzes the substrate, napthol AS – BI phosphoric acid. The napthol released is insoluble and couples with “hexazotized” pararosanilin. The colored precipitate in the cytoplasm of the cell indicates acid phosphatase activity.

The most important application of acid phosphatase reaction is in classifying lymphoproliferative disorders.

Chronic lymphocytic leukemia                =          lymphocytes are negative

Hairy cell leukemia                                  =          lymphocytes are moderately to strongly

                                                                              Negative

G. Feulgen Reaction

This is a cytochemical test for DNA, important component of nuclear chromatin. The reaction depends on the liberation of free pentose aldehyde groups from DNA after hydrolysis with HCl and the subsequent combination of these groups with leukobasic fuchsin to give a magenta color.

H. Unna–Pappenheim stain

This is a combination of pyronin with methyl green. The pyronin stains the cell components containing RNA bright red. The methyl green part of the stain demonstrates the nuclear chromatin which stains greenish–black.

I. Lysozyme activity

A simple cyto–bacterial method used to demonstrate lysozyme activity in monocytes and neutrophils. Lysozymes are not positive in lymphocytes.

In acute leukemia, lymphoblasts and myeloblasts are lysozyme negative.

In myelomonocytic and monocytic leukemias, a high proportion of leukemia cells are lysozyme positive.

J. Manson’s stain (Methylene blue)

This is used to demonstrate basophilic stippling of RBC in lead poisoning.

K. Carbol–Thionine Blue

This is also used to demonstrate basophilic granular degeneration or basophilic stippling of RBC

L. Nitro–Blue Tetrazolium Test (NBT)

This is used to differentiate certain non–bacterial disease from true bacterial infections. The test is based on the enzymatic activity of the neutrophils that are capable of destroying certain strains of bacteria once they have been phagocytized. The activity reduces also the nitroblue tetrazolium to formazan (black precipitate). In patients with bacterial infections, 12 to 70% of the PMN are positive and those with granulomatous disease are negative.