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Cytochemical Stains In Hematology Pdf Download




Cytochemical Stains in Hematology PDF Download


Cytochemical Stains in Hematology PDF Download




Cytochemical stains are special stains used for staining peripheral blood and bone marrow smears that help in classifying and differentiating different types of leukemias. They are based on enzymatic colorimetric reactions that occur in the cells of interest, revealing specific cellular components that characterize different hematopoietic lineages. Cytochemical stains are useful adjunct assays for the proper diagnosis and classification of acute leukemia, especially in resource-poor settings where immunophenotyping and cytogenetic analysis may not be readily available. In this article, we will review the principles, procedures, interpretation, and results of some of the most common cytochemical stains in hematology.


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Myeloperoxidase (MPO) Stain




Myeloperoxidase (MPO) is an enzyme present in the primary granules of neutrophils, eosinophils, and basophils, as well as in the secondary granules of monocytes. It catalyzes the oxidation of hydrogen peroxide and halides to form hypohalous acids, which have bactericidal properties. MPO stain is one of the most important cytochemical stains for identifying myeloid differentiation in acute leukemia. It is positive in most cases of acute myeloid leukemia (AML), except for M0 (AML with minimal differentiation), M6 (erythroleukemia), and M7 (megakaryoblastic leukemia). It is also positive in some cases of mixed-phenotype acute leukemia (MPAL) with myeloid markers. MPO stain is negative in acute lymphoblastic leukemia (ALL), except for some cases of B-ALL with t(9;22) (Philadelphia chromosome).


The procedure for MPO stain involves incubating the blood or bone marrow smear with a solution containing hydrogen peroxide and a chromogen, such as benzidine or 3,3'-diaminobenzidine (DAB). The chromogen reacts with the hypohalous acids produced by MPO and forms a brown precipitate in the cytoplasm of positive cells. The intensity and distribution of the staining can vary depending on the degree of differentiation and maturation of the cells. The interpretation of MPO stain is based on counting at least 200 cells and calculating the percentage of positive cells. A positive result is defined as more than 3% of blasts showing moderate to strong staining.


Sudan Black B (SBB) Stain




Sudan Black B (SBB) is a lipid-soluble dye that stains the phospholipids and neutral fats present in the membranes and granules of myeloid cells. SBB stain is similar to MPO stain in terms of its specificity and sensitivity for myeloid differentiation in acute leukemia. It is positive in most cases of AML, except for M0, M6, and M7, and negative in ALL, except for some cases of B-ALL with t(9;22). SBB stain can also be used to detect monocytic differentiation, as monocytes show a characteristic ring-shaped staining around the nucleus.


The procedure for SBB stain involves fixing the blood or bone marrow smear with ethanol, incubating it with SBB solution, washing it with water, and counterstaining it with nuclear fast red. The positive cells show a black or dark blue staining in the cytoplasm or around the nucleus. The interpretation of SBB stain is based on counting at least 200 cells and calculating the percentage of positive cells. A positive result is defined as more than 3% of blasts showing moderate to strong staining.


Chloroacetate Esterase (CAE) Stain




Chloroacetate esterase (CAE) is an enzyme present in the specific granules of neutrophils and eosinophils, as well as in the azurophilic granules of mast cells. CAE stain is used to identify granulocytic differentiation in acute leukemia. It is positive in most cases of AML with granulocytic maturation (M1-M5), and negative in AML without granulocytic maturation (M0, M6, M7), ALL, and MPAL. CAE stain can also be used to distinguish eosinophils from neutrophils, as eosinophils show a characteristic orange-red staining due to the presence of eosinophil peroxidase.


The procedure for CAE stain involves incubating the blood or bone marrow smear with a solution containing naphthol AS-D chloroacetate and fast garnet GBC as the chromogen. The positive cells show a red staining in the cytoplasm or in the granules. The interpretation of CAE stain is based on counting at least 200 cells and calculating the percentage of positive cells. A positive result is defined as more than 3% of blasts showing moderate to strong staining.


Nonspecific Esterase (NSE) Stain




Nonspecific esterase (NSE) is an enzyme present in the lysosomes of monocytes, macrophages, and megakaryocytes. NSE stain is used to identify monocytic and megakaryocytic differentiation in acute leukemia. It is positive in most cases of AML with monocytic differentiation (M4 and M5), and in some cases of AML with megakaryocytic differentiation (M7). It is negative in AML without monocytic or megakaryocytic differentiation (M0-M3, M6), ALL, and MPAL. NSE stain can be inhibited by sodium fluoride, which can be used to differentiate NSE-positive monocytes from NSE-positive megakaryocytes, as the latter are resistant to sodium fluoride inhibition.


The procedure for NSE stain involves incubating the blood or bone marrow smear with a solution containing alpha-naphthyl acetate and fast blue BB as the chromogen. The positive cells show a blue staining in the cytoplasm or in the vacuoles. The interpretation of NSE stain is based on counting at least 200 cells and calculating the percentage of positive cells. A positive result is defined as more than 3% of blasts showing moderate to strong staining.


Acid Phosphatase (AP) Stain




Acid phosphatase (AP) is an enzyme present in the lysosomes of various cells, including lymphocytes, monocytes, macrophages, osteoclasts, and prostate epithelial cells. AP stain is used to identify T-cell differentiation in acute leukemia. It is positive in most cases of T-ALL, and negative in B-ALL and AML. AP stain can also be used to detect hairy cell leukemia, a rare chronic lymphoproliferative disorder characterized by B cells with cytoplasmic projections, which show a strong positive staining with AP.


The procedure for AP stain involves incubating the blood or bone marrow smear with a solution containing naphthol AS-BI phosphate and fast red TR as the chromogen. The positive cells show a red staining in the cytoplasm or in the granules. The interpretation of AP stain is based on counting at least 200 cells and calculating the percentage of positive cells. A positive result is defined as more than 3% of blasts showing moderate to strong staining.


Periodic Acid Schiff (PAS) Stain




Periodic acid Schiff (PAS) stain is a histochemical technique that stains carbohydrates and carbohydrate-rich molecules, such as glycogen, glycoproteins, and glycolipids. PAS stain is used to identify B-cell differentiation and erythroid differentiation in acute leukemia. It is positive in most cases of B-ALL, especially those with t(12;21) (ETV6-RUNX1 fusion), and in some cases of AML with erythroid differentiation (M6). It is negative in T-ALL and AML without erythroid differentiation (M0-M5, M7). PAS stain can also be used to detect lymphoblastic lymphoma, a rare type of non-Hodgkin lymphoma that originates from immature lymphocytes, which show a positive staining with PAS.


The procedure for PAS stain involves oxidizing the blood or bone marrow smear with periodic acid, which breaks down the carbohydrates into aldehydes, and then reacting it with Schiff reagent, which forms a magenta-colored complex with the aldehydes. The positive cells show a magenta staining in the cytoplasm or in the granules. The interpretation of PAS stain is based on counting at least 200 cells and calculating the percentage of positive cells. A positive result is defined as more than 3% of blasts showing moderate to strong staining.


Conclusion




However, cytochemical stains have some limitations and drawbacks that should be considered. Some of them are:


  • Cytochemical stains are not specific for leukemic cells, and can also stain normal cells or non-hematopoietic cells. Therefore, they should be interpreted in conjunction with other morphological and clinical features.



  • Cytochemical stains are not sensitive enough to detect minimal residual disease (MRD), which is the presence of a small number of leukemic cells after treatment. MRD can be detected by more sensitive techniques, such as flow cytometry, polymerase chain reaction (PCR), or next-generation sequencing (NGS).



  • Cytochemical stains are influenced by various factors, such as the quality of the smear, the fixation method, the staining protocol, the age of the reagents, and the subjective evaluation of the observer. Therefore, they should be performed and interpreted by standardized and validated methods.



  • Cytochemical stains are not sufficient to diagnose and classify acute leukemia, as they do not provide information about the genetic and molecular abnormalities that underlie the pathogenesis and prognosis of leukemias. Therefore, they should be complemented by other techniques, such as immunophenotyping, cytogenetics, and molecular genetics.



However, cytochemical stains have some limitations and drawbacks that should be considered. Some of them are:


  • Cytochemical stains are not specific for leukemic cells, and can also stain normal cells or non-hematopoietic cells. Therefore, they should be interpreted in conjunction with other morphological and clinical features.



Cytochemical stains are not sensitive enough to detect minimal residual disease (MRD), which is the presence of a small


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