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special stains in hematology and cytology
Special stains are used in hematology and cytology to identify specific molecules that are not visible with routine staining. They can determine if certain molecules are present or absent, where they are located, and how much is present. Examples of special stains discussed in the document include fetal hemoglobin stain, Prussian blue for sideroblasts, methyl violet for Heinz bodies, Papanicolaou stain in cytology, and Masson Fontana silver stain to identify melanin and argentaffin granules. These stains help characterize and diagnose blood and cellular abnormalities.
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special stains in hematology and cytology
- 1. Special stains in hematologyand cytology
- 2. Special stains Special stainsare “special” because they are not routine. They are applied to tissue sections in addition to hematoxylin and eosin. Special stains can answer these questions: Is a certain class of molecules present or absent? Where are the molecules located in the preparation? How many of the molecules are present?
- 3. Contents RBC • Fetal Hb •Sideroblasts • Heinz bodies • Hb H inclusions • G6PD deff cells • Reticulocytes WBC • Neutrophil Alkaline Phosphatase • MPO • Sudan black • Acid phosphatase reaction • Periodic Acid Schiff Cytology • PAP stain • Alcian blue • Masson Fontana stain
- 4. Fetal Hemoglobin Theidentification of cells containing Hb F depends on the fact that they resist acid elusion to a greater extent than do normal cells , thus they appear as isolated darkly stained cells amidst a background of palely staining cells - ghost cells. Reagents Fixative-80% ethanol Elution solution –Solution A-7.5g/l hematoxylin in 90% ethanol. Solution B:FeCl3 ,205mol/l HCL; distilled water. Counter stain-1g/l aqueous erythrosine or 2.5 g/l aqueous eosin.
- 5. Method Fetal cellsstain red and adult ghost cells stain pale pink.
- 6. Sideroblasts Siderocytes arered cells containing granules of non haem iron. The granules formed of a water insoluble complex of ferric iron, lipid , protein and carbohydrates. Increased in haemolytic anemias , megaloblastic anemias ,haemochromatosis and haemosiderosis. In addition to erythroblasts , hemosiderin can normally seen marrow films as accumulations of small granules ,lying free or in macrophages.
- 7. Hemosiderin increasedin patients with increased iron stores , anemia of chronic diseases. Absent in iron deficiency anemias. Stained by Prussian blue Principle-The siderotic material reacts with potassium ferrocyanide to form a blue colored compound ,ferriferrocyanide. Ferric iron stains bright blue, nuclei- red and cytoplasm stains pink
- 8. Heinz bodies inred cells Heinz bodies can be produced by the action of a wide range of aromatic nitro and amino compounds, as well as by in organic oxidizing agents such as potassium chlorate. They also occur when one or other of the globin chains of Hb is unstable , or if the patient has been splenectomised. Methyl violet stains Heinz bodies excellently.
- 10. Hb H inclusions Patients with alpha thalassemia who form Hb H (beta4) have red cells in which multiple blue green spherical inclusions develop on exposure to brilliant cresyl or New Methylene blue as in reticulocytre preparations.
- 12. G6PD deficient cells Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) also known as favism (after the fava bean) is an X-linked recessive genetic condition that predisposes to hemolysis and resultant jaundice in response to a number of triggers, such as certain foods, illness, or medication.
- 14. Staining of reticulocytes Reticulocytes are immature RBCs contain remnant cytoplasmic ribonucleic acid (RNA) and organelles such as mitochondria and ribosomes. are visualized by staining with supravital stains (methylene blue or brilliant cresyl blue).
- 15. Methylene blue orbrilliant cresyl blue These stains cause the ribosomal and residual RNA to precipitate with the few remaining mitochondria and ferritin masses in living young erythrocytes to form microscopically visible dark-blue clusters and filaments (reticulum). The reticulocyte count is a means of assessing the erythropoietic activity of the bone marrow.
- 16. Procedure Add 5drops of new methylene blue solution to the labeled test tube. Add 5 drops of well mixed EDTA anticoagulated blood to the labeled test tube. Mix the contents by gently shaking. Incubate at room temperature (20-24°C) for 10 minutes. After the 10 minute incubation, gently mix the stain/blood solution. Using the wedge smear technique, make two additional smears (not to thick or thin. Allow to AIR dry. (Do not blow to hasten drying)
- 17. The reticulocyte countis elevated in patients with hemolytic anemia, hemorrhage, and following the treatment of iron deficiency anemia. The reticulocyte count is decreased in cases of aplastic anemia and disorders resulting in ineffective erythropoiesis
- 18. Neutrophil Alkaline Phosphotase Alkaline phosphatase activity is found predominantly in mature neutrophils, with some activity in metamyelocytes. New born babies , children and pregnants have high scores. The most significant diagnostic use of the NAP score is in chronic myeloid leukemia-in chronic phase of the disease , the score is almost invariably low , usually zero.
- 19. NAP Low scoresare seen in paroxysmal nocturnal haemoglobinuria. High scores are seen in neutrophilia of infection , polycythaemia rubra vera ,aplastic anemia leukemoid reactions and Hodgkins disease.
- 20. Results and interpretation Scale of1 to 4 0-Negative , no granules 1-occasional granules scattered in the cytoplasm 2-Moderate numbers of granules 3- Numerous granules 4-Heavy positivity with numerous coarse granules crowding the cytoplasm , frequently overlying the nucleus.
- 21. MPO Myeloperoxidase-Located inthe primary and secondary granules of granulocytes and their precussors , and in the azurophil granules of monocytes. MPO splits H2o2 , and in the presence of chromogenic electron donor forms an insoluble reaction product.
- 22. MPO The reaction productis brown and granular. Red cells and erythroid precussors show diffuse brown cytoplasmic staining. Promyelocytes and myelocytes are the most strongly staining cells in the granulocyte series.
- 23. Sudan Black Lipophophilicdye that binds irreversibly to an undefined granule component in granulocytes , eosinophils and some monocytes. In differentiating haematological disorders Sudan black will stain myeloblasts but not lymphoblasts.
- 24. Sudan Black The reactionproduct is black and granular. The only difference with MPO is in eosinophil granules, which have a clear core when stained with sudan black.
- 25. Acid Phosphatase reaction Its main diagnostic use is in the diagnosis of T cell acute leukemias and hairy cell leukemias. The reaction product is red with a mixture of granular and diffuse positivity. In T cell leukemias , the activity is usually highly focalized. Granulocytes are strongly positive. Monocytes , eosinophils and platelets show variable positivity. In bone marrow , macrophages , plasma cells and megakaryocytes are strongly positive.
- 26.
- 27. Periodic Acid Schiff The intensity of the reaction product depends on the quality of the Schiff’s reagent . Granulocytes precursors show diffuse weak positivity, with neutrophils showing intense confluent granular positivity. Eosinophil's granules are negative, with diffuse cytoplasmic positivity. Basophils may be negative but often show large irregular blocks of positive material not related to the granules.
- 29. Special stains incytology Papanicolaou stain is a multichromatic staining cytological technique developed by George Papanikolaou, the father of cytopathology. Pap staining is a very reliable technique. As such, it is used for cervical cancer screening in gynecology. The entire procedure is known as Pap smear.
- 30. PAP The classicform of Pap stain involves three solutions: A nuclear stain, haematoxylin, is used to stain cell nuclei. OG- (Orange G) is used. It stains keratin. EA (Eosin Azure) .
- 31. On a wellprepared specimen, the cell nuclei are crisp blue to black. Superficial cells are orange to pink, intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink at once.
- 32. Alcian blue Itis used to stain acidic polysaccharides such as glycosaminoglycans in cartilages and other body structures, some types of mucopolysaccharides. The tissue parts that specifically stain by this dye become blue to bluish- green after staining and are called "Alcianophilic"
- 33. Cryptoccocus neoformans. Thefungus presents as yeast-like organisms with a centrally placed body and mucoid polysaccharide capsule.
- 34. Masson Fontana silverstain To identify argentaffin granules and melanin. Melanin is a nonlipid, nonhematogenous pigment. It is A brown-black pigment present normally in the hair, skin, retina, iris and certain parts of the CNS. Argentaffin granules are found in carcinoid tumors. PRINCIPLE: A positive argentaffin reaction means the cells take-up silver and then reduce it to a visible metallic state. CONTROL: A nevi for melanin, or small intestine for argentaffin. FIXATIVE: 10% formalin
- 35. Melanin pigment cells inmalignant melanoma.
- 36. RBC • Fetal Hb •Sideroblasts • Heinz bodies • Hb H inclusions • G6PD defn cells • Reticulocytes WBC • Neutrophil Alkaline Phosphatase • MPO • Sudan black • Acid phosphatase reaction • Periodic Acid Schiff Cytology • PAP stain • Alcian blue • Masson Fontana stain
- 37.
Editor's Notes
- #2 SHAHID
- #14 Demonstrated by adding reagents sodium nitrite , di methl tetrazolium , Hypotonic saline
- #21 In normal individuals its rare to find score 3. Normal ALP levels-20 to 140iu/l=biliary obstrn , lymphoma , leukemia
- #27 Focalised , intense localized stainig-Tcell
- #31 2 counter stains first OD then EA. ORANGE GREEN-Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.
- #37 Fetal Hb- Elusion method , Sideroblasts-methylene blue , Heinz bodies-methyl violet , ret-supra vital stain LAP , MPO and sudan black positive myeloid precussors , acid phosphatase- T cell leukemia and hairy cell leukemia , PAS- neutrophils show intense positivity. Other stains in cytology-Shorrs stain-diff b/w kertn and non krtnzd , Wrights stain-fr identfn of red cells.