SHAHID

1. Special stains in
hematology and
cytology

2. Special stains
Special stains are “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
 The identification 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 cells stain red and adult
ghost cells stain pale pink.

6. Sideroblasts
 Siderocytes are red 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 increased in 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 in red 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 or brilliant 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 5 drops 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 count is 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 scores are 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 of 1 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 in the 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 product is
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
 Lipophophilic dye 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 reaction product 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. Acid phosphatase

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 in cytology
 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 classic form 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 well prepared 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
 It is 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.
 The fungus presents as
yeast-like organisms with a
centrally placed body and
mucoid polysaccharide
capsule.

34. Masson Fontana silver stain
 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
in malignant
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. Thank You