Hematology stains

1. SPECIAL AND ROUTINE STAINS
IN HAEMATOLOGY
PRESENTOR-Dr SHAHID

2. OUTLINE
• Introduction
• Routine Stain
• Special stains
• Summary

3. INTRODUCTION
 Differentiation of the peripheral blood is an
important procedure in the diagnosis of
hematologic disorders.
 The slide must be absolutely clean to avoid
introducing artifacts.
 Slides are cleaned most effectively by degreasing
in alcohol
for 24 h, drying with a lint-free cloth,

4. • Blood films are usually examined to
investigate hematological problems (disorders
of the blood) and
 Occasionally, to look for parasites within the
blood such as malaria and filaria.
 Examination of thin blood films is important in
the investigation and management of
anaemia, infections, and other conditions
which produce changes in the appearance of
blood cells and differential white cell count.

5. Three basic steps to make blood
film:
1. Preparation of blood smear.
2. Fixation of blood smear.
3. Staining of blood smear.

6. The peripheral blood film (PBF) is of
two types:
1.Thin blood film
2.Thick blood film

7. THIN BLOOD FILM
 Obtained by venepuncutre or from free
flowing finger prick blood by any of the
following three techniques :
1.Slide method
2.Cover glass method
3.Spin method

8. Preparation of blood smears
• Blood collection for thick or thin blood smears
• Capillary blood obtained by fingerstick:

9. • 1. Label pre-cleaned slides (preferably frosted-
end) with patient’s name (or other identifier),
date and time of collection.
• 2. Wear gloves.
• 3. Clean slides with 70 to 90% alcohol and
allow to dry. Do not touch the surface of the
slide where the blood smear will be made.

10. • 4. Select the finger to puncture, usually the
middle or ring finger. In infants, puncture the
heel.
• 5. Clean the area to be punctured with 70%
alcohol; allow to dry
• 6. Puncture the ball of the finger, or in infants
puncture the heel.

11. • 7. Wipe away the first drop of blood with
clean gauze.
• 8. Touch the next drop of blood with a clean
slide. Repeat with several slides (at least two
thick and two thin smears should be made). If
blood does not well up, gently squeeze the
finger.

13. For venous blood obtained by
venipuncture:
• 1. Label collection tubes and pre-cleaned slides
(preferably frosted-end) with the patient’s name
(or other identifier), date and time of collection.
• 2. Clean the site for blood collection well using
70% alcohol; allow to dry.

14. • 3. Collect the venous blood in a vacuum tube
containing anticoagulant (preferably EDTA);
alternatively, collect the blood in a syringe and
transfer it to a tube with anticoagulant; mix
well.
• 4. Prepare at least two thick smears and two
thin smears as soon as possible after
collection.

15. Making thick and thin blood smears
1. Whenever possible, use separate slides for
thick and thin smears.
2. Thin film (a): Bring a clean spreader slide, held
at a 45° angle, toward the drop of blood on
the specimen slide.
3. Thin film (b): Wait until the blood spreads
along the entire width of the spreader slide.

16. • 4. Thin film (c): While holding the spreader
slide at the same angle, push it forward
rapidly and smoothly.
• 5. Thick film: Using the corner of a clean slide,
spread the drop of blood in a circle the size of
a dime (diameter 1-2 cm). Do not make the
smear too thick or it will fall off the slide. (You
should be able to read newsprint through it.)

17. 6. Wait until the thin and thick films are
completely dry before staining. Fix the thin
film with methanol (100% or absolute) and let
it dry completely before staining. The thick
film should not be fixed.
7. If both thin and thick films need to be made
on the same slide, fix only the thin film with
methanol. The thick film should not be fixed.

19. Cover GlassMethod
• Procedure
 Take a clean cover glass.
 Touch it on to the drop of a blood.
 Place it on another similar cover glass in crosswise direction
• with side containing drop of blood facing down.
 Pull the cover glass quickly.
 Dry it and stain it.
 Mount it with a mountant, film side down on a clean glass slide.

21. SpinMethod
This is an automated method
Procedure
 Place a drop of blood in the centre of a glass slide.
 Spin at a high speed in a special centrifuge, cytospin.
 Blood spreads uniformly.
 Dry it and stain it.

23. THICK BLOODFILM
Procedure
Place a large drop of blood in the centre of a
clean glass slide.
Spread it in a circular area of 1.5 cm with the
help of a
• stick or end of another glass slide.
 Dry it
 Stain

25. Parts of aThin Blood Film

27. The shape of blood film

28. Examples of unacceptablesmears

29. Fixation of bloodsmear

30. • Topreserve the morphology of the cells, films must be fixed as soon
as possible after they have dried.
• It is important to prevent contact with water before
fixation is complete.
• Methyl alcohol (methanol)is the choice, although ethyl
• alcohol ("absolute alcohol") can be used.
• Methylated spirit (95% ethanol) must not be used as it contains
water.

31.  Tofix the films, place them in a covered staining jar
or tray containing the alcohol for 2-3 minutes.
 In humid climates it might be necessary to replace
the methanol 2- 3 times per day

33. Various stains for peripheral
blood film:
Romanowsky stains are universally employed
for staining of blood films.
 All Romanowsky combinations have two
essential ingredients i.e. methylene blue and
eosin or azure.

34.  Methylene blue is the basic dye
 Has affinity for acidic component of the cell (i.e. nucleus)
 Eosin/azure is the acidic dye and has affinity for basic
component of cell (i.e. cytoplasm).
 Most Romanowsky stains are prepared in methyl alcohol
so that they combine fixation and staining.

35. Various stains included under
Romanowsky stain are as under
1. Leishman stain
2. Giemsa stain
3. Wright stain
4. Field stain
5. Jenner stain
6. JSB stain

36. Principle And Interpretation
• Leishman stain, is used in microscopy for staining blood
smears. It provides excellent stain quality. It is generally used
to differentiate and identify leucocytes, malaria parasites, and
trypanosomas.
• It is based on a methanolic mixture of "polychromed"
methylene blue. (i.e. demethylated into various
azures) and eosin.The methanolic stock solution is stable and
also serves the purpose of directly fixing the smear eliminating
a prefixing step.

37. Quality Control
Appearance
Blue coloured solution
Clarity
Clear without any particles
Microscopic Examination
Blood staining is carried out and staining
characteristic is observed under microscope
by using oil immersion lens

38. LeishmanStain
Preparation
• Mix and dissolve 0.15 g of Eosin-Methylene blue in 100 ml
Methanol dried p.A. at 56°C. When the stain is dissolved
completely remove the solution from the heater. (Alternatively,
dissolve at RT over night. Cover container with Parafilm in
order to prevent contamination by moisture). After the solution
reached RT, clear the solution by using a dry Whatman paper
filter. Collect filtered solution in a clean and dry brown glass
bottle. Age the solution at least 2-3 days before using it the first
time.

39. Procedure for staining
Pour Leishman’s stain dropwise (counting the drops) on the
slide and wait for 2 minutes.
This allows fixation of the PBF in methyl alcohol.
Add double the quantity of buffered water drop wise
over the slide (i.e. double the number of drops).
Mix by rocking for 8 minutes.
Wash in water for 1 to 2 minutes.
Dry in air and examine under oil immersion lens of the
microscope.

41. GiemsaStain
PROCEDURE
Giemsa May-Grünwald
1. Dilute Giemsa Stain 1:20 with deionized water.
For bluer coloration, water buffered at pH 7.2
may be used in place of deionized water.
2. Place slides in May-Grünwald Stain for 5 minutes.
3. Place slides in Working Phosphate Buffer or
Trizma® Buffer (20-70 mmol/L). pH 7.2, for 1.5
minutes.

42. 4. Place slides in dilute Giemsa solution from
step 1 for 15-20 minutes.
5. Rinse slides BRIEFLY in DEIONIZED water. 6.
Air dry and evaluate.

43. Preparation
• Stock solution of Giemsa stain is prepared
by mixing 0.15 g of Giemsa powder in 12.5
ml of glycerine and 12.5 ml of methyl
alcohol.
• Before use dissolve one volume of stock
solution in nine volumes of buffered water
(dilution 1:9).

44. • .

45. Caution
Thick films need careful rinsing!
(since they are not fixed before staining)
Mistake:
Thick films are not Rinsed
properly! Blood is lost

46. Wright Staining
FIXATION
• Streak thin smears across a sterile slide by
means of a second slide or cover glass placed
at an angle. Air-dry completely. Make sure all
slides are clean prior to making the blood or
bone marrow smear. If slides are not
completely clean the stain will not absorb into
the smear.

47. Procedure
1. Place 1.0 ml of the Wright Stain Solution upon
the smear approximately 1-3 minutes.
2. Add 2.0 ml of distilled water or Cat.
Wright Stain Phosphate Buffer pH 6.8 and let the
slide stand twice as long as in step 1.

48. 3. Rinse off the slide with water or Cat.
• Wright Stain Phosphate Buffer pH 6.8 until a faint
pinkish red appears on the edges.
• If a Giemsa appearance is desired, stain 10
minutes in one volume of Wright Stain Solution
and 4 volumes of Wright Stain Phosphate Buffer
pH 6.8.
4. Dry thoroughly using bibulous paper to blot dry.
Allow the slide to dry at room temperature
before examination to determine if adjustments
are needed in the dilution or timing.

49. Jennerstain
• The Jenner stain Solution is a mixture of
several thiazin dyes in a methanol solvent.
• Ionic and non-ionic forces are involved in the
binding of these dyes
• The staining solution has anionic and cationic
properties

50. • The negatively charged phosphoric acid
groups of DNA attract the purple
polychromatic cationic dyes to the nuclei.
• The blue basophilic granules are
stained by the
• polychromatic cationic dyes.

51. Immersion StainingProtocol
• Thoroughly dry blood or bone marrow smears
• Fix smears in absolute methanol for 15 seconds to 5
minutes
• Stain smears in Jenners Stain Solution for 2 minutes
• Stain in mixture of 50ml of Jenners Stain Solution,
75ml of PH 6.6 Phosphate Buffer Solution and 175ml
deionized water for 5 minute

52. • Rinse in standing deionized water for 1.5
minutes or rinse briefly in running deionized
water
• Air dry smears
• Examine smears under a microscope

53. StainingProtocol
• Place slide with thoroughly dried film in a
horizontal staining rack
• Flood smear with absolute methanol for 15-
30 seconds and then drain
• Flood smear with 1ml Jenners Stain Solution
and let stand for 3 minute

54. • Add 1mL of pH 6.6 Phosphate Buffer
solution and 1 ml deionized water to
smear and let stand for 45 seconds.
• Rinse briefly with running deionized water
• Air dry and examine under a microscope
• Perform immunochemical staining procedure
according to manufacturer.

55. J.S.B.Stain
( Jaswant–Singh–Bhattacharji stain)
Materials and reagents required:
1. Eosin yellow (water soluble)
2. Methylene blue
3. Potassium dichromate
4. Di-sodium hydrogen phosphate (dihydrate)
5. 1% sulphuric Acid.
6. Round bottom flask (2 lit.)
7. Healing mantle
8. Distilled water
9. Staining jars.

56. Preparation of the smear
• Use universal precautions while
preparing the smears for malarial
parasites – use gloves; use only
disposable needles/lancets; wash hands;
handle and dispose the sharp
instruments and other materials
contaminated with blood carefully to
avoid injury.

57. • Hold the third finger of the left hand and wipe
its tip with spirit/Savlon swab; allow to dry
• Prick the finger with disposable needle/lancet;
allow the blood to ooze out
• Take a clean glass slide. Take 3 drops of blood
1 cm from the edge of the slide, take another
drop of blood one cm from the first drop of
blood

58. • Take another clean slide with smooth edges
and use it as a spreader and make thick and
thin smears. Allow it to dry
• Slide number can be marked on the thin
smear with a lead pencil

60. Staining technique
• Prepare thin and thick smears from malaria
cases on micro slides
• De-haemoglobinise the thick smear
• Fix the thin smear in methanol for few
minutes

61. • Take 3 staining jars for J.S.B. I, J.S.B.II and tap
water
• Dip the smears in J.S.B. II for few seconds and
immediately wash in water
• Drain the slides free of excess water

62. • Dip the smears in J.S.B.I for 30-40 seconds
• Wash well in water and dry
• Examine the smears under oil immersion

63. Staining of Thick Smear
•It can be stained with any of the Romanowsky
stains
• listed above except that before staining, the
smear is
• dehaemoglobinised by putting it in distilled
water for 10 minute

64. Jaswant Singh Battacharya (JSB) Stain
for thick and thin films:
• This is the standard method used by the
laboratories under the National Malaria
Eradication Programme in India

68. Special Stains In
Hematology

69. 1. Periodic assay Schiff (PAS)
2. Perl’s Prussian Blue Reaction
3. Leucocyte alkaline phosphatase (LAP)
4. Myeloperoxidase
5. Sudan Black B
6. Toluidine Blue
7. Specific esterases
8. Non- Specific esterases
9. Tartarate resistant acid phosphatase
10. Pappenheim’s Stain (Panoptic Stain)
11. Mayer’s Acid Hemalum Nuclear Stain
12. Heilmeyer’s Reticulocyte Stain
13. Heinz Body Test of Beutler1
14. Nile Blue Sulfate Stain

70. 15. Kleihauer-Betke Stain for Demonstrating Fetal
Hemoglobin in Red Blood Cells
16. Kleihauer-Betke Stain for Demonstrating
Methemoglobin- Containing Cells in Blood Smears
17. Berlin Blue Iron Stain
18. Cytochemical Determination
of Peroxidase

71. Periodic Acid Schiff
Reaction

72. Principle
• Periodic Assay oxidizes 1-2 glycol groups to
produce stable dialdehydes which give a red
reaction product when exposed to Schiff’s
reagent (leucobasic fuchsin)
• Positive reaction occurs with carbohydrates,
principally glycogen

73. Reagents
• Fixative : Methanol
• 1% Periodic acid
• Schiff’s Reagent: 5g basic fushcin in 500ml of hot
distilled water and then saturated with SO2 for 1-12 hr.
• Shake vigorously with 2g activated charcoal for 1 min.
• Counterstain : Aqueous Haematoxylin.

74. Method
• Fix films for 15 min in methanol
• Rinse with tap water
• Flood slides with 1%periodic acid for 10 min
• Immerse in Schiff reagent for 30 min
• Rinse in running tap water for 10 min
• Counterstain

75. Results and Interpretation
 PAS-positive material in the cytoplasm may produce a
diffuse red stain
 Or may appear as pink to burgundy-red granules, flakes,
or clumps of varying size that may occupy large areas of
the cytoplasm.
 Some plasma cells, macrophages, and osteoblasts
may also show a positive PAS reaction
 Megakaryocytes are strongly positive.

76.  Monocytes and their precursors shows diffuse PAS positivity
in AML M5
 Normal erythroid precursors and RBCs are negative but
dysplastic erythroblasts are positive-
AML M6-Coarse granules are seen in cytoplasm
 Megakaryocytes and Platelets are strongly positive
 -AML M7-fine cytoplasmic granularity and blebs are positive
 Lymphocytes shows PAS blocks or granules ie block positivity
 ALL-L1,ALL-L2.

79. Perl’s PrussianBlue
Reaction
Principle
• The reaction occurs with the treatment of
tissue sections with acid ferrocyanide solution.
Any ferric ion (Fe3+) in the tissue combines
with ferrocyanide and results in the formaion
of a bright blue pigment called “prussian
blue” or ferric ferrocyanide.

80. Fixation
• Avoid the use of acid fixatives. Chromates will
also interfare with the preservation of iron

81. Staining solution
• 1% aqueous potassium ferrocyanide = 20 ml
• 2% aqueous hydrochloric acid = 20 ml
• Mix both

82. Procedure
• Deparaffinize and bring the sections to water.
• Treat the sections with freshly prepared acid
ferrocyanide solution for 10-30 minutes
• Wash well in distilled water.

83. • Lightly stain the nuclei with 0.5% aqueous
neutral red or 0.1% nuclear fast red.
• Wash rapidly in distilled water.
• Dehydrate, clear and mount

84. Result and Interpretation
Ferric ion = blue
Nuclei = red
Background = pink

86. Differential diagnosis by iron stain
in the bone marrow

87. Sideroblasts Iron storing reticulum
cells,
sideromacrophages
Special features
Normal bone marrow 20–60 %
finely granular,
1– 4 granules
Isolated,
mostly finely
granular deposits
Siderocytes in
peripheral blood 0-
0.3%
Iron deficiency < 15 % finely granular None None Serum Fe
decreases
– Lead poisoning > 90 % coarsely
granular;
ringed sideroblasts
Greatly increased,
many diffuse or
coarsely granular
deposits
Serum Fe increase
siderocytes
may be increased
Thalassemia > 90 % coarsely
granular;
ringed sideroblasts
Greatly increased,
many diffuse or
coarsely granular
deposits
Serum Fe increses
siderocytes
may be increased
Hemolytic anemias <80 % finely granular Increased finely
granular or (rarely)
coarsely granular
deposits

88. Hemochromatosis 80 % finely granular Increased
Plasma cells
contain
iron
Bone marrow is
not useful for
diagnosis except
positive plasma
cells

89. LeucocyteAlkaline
Phosphatase

90. PRINCIPLE
• Naphthol AS phosphate in the presence of
LAP gets hydrolysed then in the presence of
Coupling Azo-dye: Fast blue RR salt gives
insoluble ppt at the site of enzyme activity

91. REAGENTS
• Fixative: 4% formalin methanol
• Substrate : Naphthol AS phosphate
• Buffer: Tris Buffer (pH 9.0)
• Stock Substrate solution
30mg naphthol AS phosphate in 0.5ml N,N dimethyl
• formamide. Add 100ml Tris buffer
• Coupling Azo-dye: Fast blueRR salt
• Counter stain: Neutral red, 0.02% aqueous solution

92. Method
• Fix air dried smears for 30 s in 4% formalin methanol.
• Rinse with tap water.
• Prepare working solution by adding 24mg Fast blue BB
salt in 40ml stock substrate solution.
• Incubate slides in working solution for 15 min
• Rinse with running tap water
• Counterstain for 3 min

93. Results and interpretation
•Reaction product is blue and granular.
•LAP score is determined by evaluation of the staining
intensity (ranging from 0 to 4+) of 100 counted
neutrophils or bands.
•Normal LAP scores range from 15 to 130

94. USE
• Useful as a screening test to differentiate
chronic myelogenous leukemia from
leukemoid reactions and other
myeloproliferative disorders

95. POSITIVE LAP REACTION NEGATIV LAP REACTION

97. Score Interpretation
0 Negative, no granules
1 Occasional granules scattered in cytoplasm
2 Moderate number of granules
3 Numerous granules
4 Heavy positivity with numerous coarse granules,
frequently overlying the nucleus

98. Low LAP score (<15) High LAP score (>130)
CML Infections
Paroxysmal nocturnal
hemoglobinuria
Growth factor therapy
Myelodysplastic
syndromes
Inflammatory disorders
Pregnancy, oral
contraceptives
Stress

99. Myeloperoxidase

100. Principle of Procedure
• Antigen detection in tissues and cells is a multi-
step immunohistochemical process.
• The initial step binds the primary antibody to its
specific epitope. A secondary antibody may be
applied to bind the primary antibody, followed by
an enzyme labeled polymer; or an enzyme
labeled polymer may be applied directly to bind
the primary antibody.
• The detection of the bound primary antibody is
evidenced by an enzyme-mediated colorimetric
reaction

101. Reagents
• Fixative : buffered formal acetone.
• Substrate : 3,3’diaminobenzidine (DAB).
• Buffer : Sorenson’s phosphate buffer pH7.3.
• Hydrogen peroxide.
• Counterstain: hematoxylin.
• Working substrate solution: 30mg DAB in 60ml buffer,
add 120l H2O2 and mix well

102. Method
• Fix air dried smears in buffered formal
acetone for 30s.
• Rinse thoroughly in running water.
• Incubate for 10 min in working substrate
solution.
• Counterstain

103. Reactions and interpretations
• Reaction product is brown and granular.
• Red cells and erythroid precursors show
diffuse brown cytoplasmic staining.
• Most primitive myeloblasts are negative with
granular positivity appearing progressively as
they mature toward promyelocyte stage.
• Promyelocytes and myelocytes are strongly
staining cells in granulocyte series

104. • Metamyelocytes and neutrophils have
fewer positive granules.
• Eosinophil granules stain strongly and the
large specific eosinophil granules are easily
distinguished from neutrophil granules.
• Monoblasts and monocytes have variable
positive reaction

105. Use
• Todifferentiate a myelogenous or monocytic
leukaemia from acute lymphocytic leukaemia.
• Auer rods are better visualized with MPO than
• Romanowsky stains.

106. Pathological variants
• Some individuals have congenital
deficiency of neutrophil MPO.
• All stages of neutrophil lineage from
myelocyte onwards are negative, although
the eosinophils stain normally.
• Some individuals may have eosinophil MPO
or Monocyte MPO deficiency

107. • Red brown
precipitate

109. References
1. Toth B, et al. Immunophenotyping of acute lymphoblastic leukaemia
in routinely processed bone marrow biopsy specimens. J Clin
Pathol. 1999 Sep;52(9):688-92.
2. Pinkus GS, Pinkus JL. Myeloperoxidase: a specific marker for
myeloid cells in paraffin sections. Mod Pathol. 1991 Nov;4(6)733-
41.
3. Center for Disease Control Manual. Guide: Safety Management, NO.
CDC-22, Atlanta, GA. April 30, 1976 "Decontamination of Laboratory
Sink Drains to Remove Azide Salts.“
4. Clinical and Laboratory Standards Institute (CLSI). Protection of
Laboratory Workers from Occupationally Acquired Infections;
Approved Guideline-Fourth Edition CLSI document M29-A4 Wayne,
PA 2014.

110. Sudan Black B

111. PRINCIPLE
• PRINCIPLE: Sudan Black is slightly basic dye
and will combine with acidic groups in
compound lipids, thus staining phospholipids
also. An alternative stainto the Sudan Black B
stain. CONTROL: Use a positive control of a fat
smeared slide, and a negative control slide of
a paraffin processed tissue, such as lung.

112. PRINCIPLE
• Sudan Black B is a lipophilic dye .
• That binds irreversibly to an undefined
granule component in granulocytes,
eosinophils and some monocytes.

113. Method
•Fix air dried smears in formalin vapors.
•Immerse the slides in working stain solution for
1 hr. in a coplin jar with lid on.
•Transfer the slides to a staining rack and flood
wth 70% alcohol. After 30s, tip off and repeat
three times in total
•Rinse in gently running tap water
•Counterstain with May-Grunwald-Giemsa or
Lieshman stain

114. REAGENTS
• 1. Dye 60 ml
• 2. Buffer 40 ml
ADDITIONAL REAGENTS:
Fixative : Available on request .
- Formaldehyde Fixative . Or
- Formaline – ethanol fixative
Counterstain : Methyl green or Giemsa

115. STABILITY
• Reagents are stable up to the expiration date
given when stored at 4 °C

116. PROCEDURE
Fixation:
1. Fix air – dried films of blood or bone marrow
for 10 min in formalin vapour, formaldehyde
fixative or formaline – ethanol fixative .
2. 2. Wash gently in water for 5 – 10 min ;

117. Staining
Working Reagent:
Mix Reagent R1 and Reagent R2 in ratio of
1. 5 : 1, Filter before use.
1. Incubate the fixed film in 20 drops working
reagent for 10 min. in closed area to avoid
evaporation.

118. • 2. Wash in 70 % ethanol by waving the slides
in the alcohol for 3 - 5 min .
• 3. Wash with water for 2 min, dry .
• 4. Counterstain for 5 min and mount .

119. SudanBlackBstaining
Positive stain in a patient of AML Black stained
cytoplasm in myeloblasts
.

120. RESULT
• The reaction product in the cytoplasm is black;
the nuclei stain blue or red depending on the
counterstain used. (Giemsa, safranin or methyl
green.)

121. REFERENCE
Sheehan , H .L . and Story , G . W . ( 1977 ) J .
Path . and Bacter . 59 , 336

122. Toluidine BlueStain

123. Principle
• Toluidine blue staining is useful for the
enumeration of basophils and mast cells.
• It binds strongly to the granules in these
cells

124. Method
• Place air dried smears on a staining rack
and flood with toluidine blue solution
• Incubate for 5-10min.
• Rinse briefly in gently running tap water

125. RESULTS
• The granules of basophils and mast cells stain
a bright red/purple and are discrete and
distinct.
• Nuclei stain blue and cells with abundant
RNA may show a blue tint to the cytoplasm

126. Toluidinebluestainingshowingstronglypositivebasophils

127. Specific Esterases

128. Use
• The specific (naphthol AS-D chloroacetate)
esterase stain, also called the Leder stain, is
used to identify cells of the granulocytic
series

129. Method
• Fix smears in cold buffered formal acetone.
• Rinse in gently running tap water.
• Immerse slides in working solution for 10min.
• Rinse in tap water.
•Counterstain for 1 min with aqueous
hematoxylin

130. RESULT
• Reaction product is bright red.
• It is confined to cells of neutrophil series and
mast cells.
• Myeloblasts stain rarely.
• Promyelocytes and myelocytes show strong
positvity

132. Nonspecific
esterases

133. Use
• To identify monocytic cells but do not stain
granulocytes or eosinophils
• Include α-naphthyl butyrate and α-naphthyl
acetate

134. α-Naphthylbutyrate
• Reagents
• Fixative: Buffered formal acetone.
• Buffer: phosphate buffer (pH8.0).
• Substrate stock solution
• α-Naphthyl butyrate 100l in 5ml
• acetone stored at -20oC
• Coupling reagent : Fast Garnet GBC 15mg.
• Counterstain : aqueous hematoxylin

135. Method
•Fix air dried smears in buffered formal acetone for
30 sec and then rinse in tap water.
•Add Fast Garnet GBC to 50ml buffer and mix.
•Add 0.5 ml of substrate solution.
•Pour incubation medium in coplin jar containing
fixed slides and incubate for 20-40min.
• Air dry and counterstain for 1 min aqueous
hematoxylin

136. • Results and interpretation
•Reaction product is brown and granular.
•Majority of monocytes stain strongly.
•More specific for identifying monocytic
component in AML

137. α-naphthylacetate

138. • Results and interpretation
•Reaction product is brown and granular.
•Majority of monocytes stain strongly.
•More specific for identifying monocytic
component in AML

139. Method
•Fix air dried smears in buffered formal acetone for 30s.
•Rinse in running tap water.
•Immerse slides for 30-60min in incubation medium.
•Rinse in running tap water.
•Counterstain for 2-5min

140. • Results and interpretation
•Reaction product is red/brown in color.
•Normal and leukemic monocytes stain
strongly.
•Normal granulocytes are negative but in
MDS or AML may give varying positive
reaction.
•Megakaryocytes stain strongly

143. TartrateResistant
Acid Phosphatase

144. Principle
•The tartrate-resistant acid phosphatase (TRAP) is an isoenzyme of acid
phosphatase that is found in high levels in the cells of hairy cell
leukemia and osteoclasts.

145. Method
•Air dry films for at least 24hr.
•Fix for 10 min in methanol.
•Incubate for 1hr at 37oC in working solution A.
•Rinse in tap water and air dry.
•Counter stain with 1% aqueous methyl green or
hematoxylin for 5 min.
•Rinse in tap water and mount

146. • Results and interpretation
•Reaction product is red with a mixture of
granular and diffuse positivity.
•Almost all T-lineage leukemias show string
activity
•In Hairy cell leukaemia, majority of
leukemic cells react equally positively.

147. Tartrate-resistantAcidPhosphatase(TRAP)ActivityOfLymphocytes

148. Pappenheim’s Stain (Panoptic Stain)
 It is based on a combination of the Jenner-
May-Gru¨nwald stain and Giemsa stain.
 Used to differentiate basophilic granules of
erythrocytes and nuclear fragments
Uses-

149. Heinz Body Test of Beutler1
 This test is used to detect defects of red cell metabolism
that do not allow glutathione , maintained in a reduced
state.
 The defect may result from a glucose-6-phosphate
dehydrogenase deficiency, a glutathione reductase
deficiency,diseases
 In which there is“unstable hemoglobin,” or an “idiopathic”
Heinz body anemia.
 The test involves the oxidative denaturation of hemoglobin
to intraerythrocytic“Heinz bodies” following incubation of
the red cells with acetylphenylhydrazine.

150. INTERPRETATION-
 The percentage of erythrocytes that contain
more than four Heinz bodies is determined.
 Normal values range from 0 % to 30 %.

151. Nile Blue Sulfate Stain
 This stain is used for the visualization of
Heinz inclusion bodies.
 The Heinz bodies appear as small,dark blue
bodies situated at the margin of the yellow
to bluish erythrocytes.

152. Kleihauer-Betke Stain for Demonstrating Fetal
Hemoglobin in Red Blood Cells
• Principle
 Normal adult hemoglobin (HbA) is dissolved
out of the red cells by incubating air-dried and
fixed blood smears in citric acid phosphate
buffer (of McIlvain), pH 3.3, at 37 8C.
 Fetal haemoglobin(HbF) is left undissolved in
the red cells and can be made visible by
staining

153. • Method
 Prepare thin blood smears, air dry, and fix in 80 % ethyl
alcohol for 5 min.
 Wash in water and dry.
 If further processing is delayed, the slides may be stored in a
refrigerator for 4–5 days.
 For elution, place the slides upright in a beaker containing
the buffer in a 37 C water bath for 3 min,moving the slides
up and down after 1 and 2 min to keep the buffer mixed.
 Then wash in running water.

154. • Staining
 Stain in Ehrlich hematoxylin for 3 min,then
post stain in 0.1 % aqueous erythrosin
solution for 3 min.
 Examine at 40 using dry or oil immersion
magnification

155. • Interpretation.
• Erythrocytes that contain HbA appear as
unstained “shadows,” while cells that
contain HbF will stain a bright red color.
• The method can be used for the diagnosis of
thalassemia major and for the detection of
fetal erythrocytes that have entered the
maternal circulation.

157. Kleihauer-Betke Stain for Demonstrating
Methemoglobin-Containing Cells in Blood Smears
• Principle.
• Methemoglobin combines with KCN to for
cyanmethemoglobin, while oxyhemoglobin does not
react with cyanides.
• Oxyhemoglobin functions as a peroxidase, whereas
cyanmethemoglobin has very low peroxidase activity.

158. • Interpretation
• Oxy-Hb-containing cells stain a bright red.
• Cells that contain met-Hb (converted to
cyanmet-Hb) are eluted and appear as
shadows

160. Cytochemical Determination of Acid Phosphatase
 Used in the cases of plasmacytomas, the abnormal
plasma cells tend to show stronger activity than
normal plasma cells or plasma cells affected by
reactive changes.
 A dot like staining pattern is seen in T-lymphocytes,
while the blasts of T-ALL usually show a circumscribed
(focal) paranuclear acid phosphatase reaction.

161. SUMMARY

162. Stain Structure stained Use
Periodic assay
Schiff (PAS)
Carbohydrates,
principally
glycogen
In AML and MDS
to identify
abnormal
erythroblasts and
dysplastic
megakaryocytes
Toconfirm the diagnosis
of acute promyelocytic
leukaemia
Perl’s Prussian
Blue Reaction
Iron For demonstration of
ring sideroblasts and
Pappenheimer
bodies
Leucocyte alkaline
phosphatase (LAP)
Neutrophil alkaline
phosphatase
Differentiate chronic
myelogenous
leukemia from
leukemoid reactions
and other
myeloproliferative
disorders

163. Stain Structure stained Use
Myeloperoxidase Myeloperoxidase in
neutrophils, monocytes
and eosinophils
Todifferentiate a
myelogenous or monocytic
leukemia from acute
lymphocytic leukemia.
Tovisualize auer rods
Sudan Black B Intracellular lipid Similar to MPO
Toluidine Blue Basophils and mast cells Toidentify dysplastic
basophils in
myeloproliferative
diseases
Specific esterases Neutrophil series and
mast cells
Marker of cytoplasmic
maturation in myeloid
leukemias
Non- Specific esterases Monocytes Monocytic component in
AML
Tartarate resistant acid
phosphatase
T-cells and granulocytes Diagnosis of T-cell ALL
and hairy cell leukemia

164. References-
Bacus, JW. Erythrocyte morphology and “spinner” blood film preparations. J Histochem Cytochem.
1974;22:506–516.
• Crossref | PubMed |
• Bentley, SA. Quality control and the differential leukocyte count. Clin Lab Haematol. 1990;12:101–109.
• PubMed
• Beutler, E, Lichtman, MA, Coller, BS et al, Williams Hematology. ed 5. McGraw
• Chanarin, I. Laboratory Haematology. Churchill Livingston, Edinburgh; 1989.
• Dacie, JV, Lewis, SM. Practical Haematology. ed 5. Churchill Livingston, Edinburgh; 1975.
• Devices for collection of skin puncture blood specimens. NCCLS Approved Guideline H14-A2. 1990;.
• Gleeson, RM. An improved method for thick film preparation using saponin as a lysing agent. Clin Lab
Haematol. 1997;19:249–251.