Special Stains In Hematology

1. Periodic assay Schiff (PAS)2. Perl’s Prussian Blue Reaction3. Leucocyte alkaline phosphatase (LAP)4. Myeloperoxidase5. Sudan Black B6. Toluidine Blue7. Specific esterases8. Non- Specific esterases9. Tartarate resistant acid phosphatase

Periodic Acid Schiff Reaction

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

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.

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

Results and Interpretation• Reaction product is red with intensity ranging from pink to

bright red• Granulocyte precursors show diffuse weak positivity, with

neutrophils showing intense confluent granular positivity. • Eosinophil granules are negative, with diffuse cytoplasmic

positivity• Basophils maybe negative but often show irregular blocks

or positive material• Monocytes and their precursors show diffuse variable

positivity.• Normal erythroid precursors and red cells are negative• Megakaryocytes and platelets show variable usually

intense, diffuse positivity.• Lymphocytes show variable PAS positive granules

Granular PAS positivity in proerythroblasts

Homogenous positivity in normoblasts

Lymphoblast showing block and coarse granular PAS staining

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Perl’s Prussian Blue Reaction

Principle

• Treatment with acid ferrocyanide solution results in unmasking of ferric iron as ferric hydroxide Fe(OH)3 which then reacts with a dilute potassium ferrocyanide solution to produce an insoluble blue compound, ferric ferrocyanide (Prussian Blue)

• The stain is used to identify iron in nucleated red blood cells (sideroblastic iron) or to identify Pappenheimer bodies in erythrocytes.

Reagents• Fixative : Methanol• Ferrocyanide solution :

• 20ml 1% aqueous potassium ferrocyanide • 20ml 2% aqueous HCL

Procedure• Methanol fixed smears are placed in freshly prepared

acid ferrocyanide solution for 10-30min• Then counterstained with 1g/l aqueous neutral red or

eosin for 10-15s

Results and interpretation• The ferric ions stain blue and the nuclei stain

red

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Siderotic granules found in some RBCs

Leucocyte Alkaline Phosphatase

Use• Useful as a screening test to differentiate chronic

myelogenous leukemia from leukemoid reactions and other myeloproliferative disorders

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 blue BB salt• Counter stain: Neutral red, 0.02% aqueous solution

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 in 40ml stock substrate solution • Incubate slides in working solution for 15 min• Rinse with running tap water• Counterstain for 3 min

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

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

Low LAP score (<15) High LAP score (>130)

CML Infections

Paroxysmal nocturnal hemoglobinuria Growth factor therapy

Myelodysplastic syndromes Myeloproliferative disorders other than CMLInflammatory disorders

Pregnancy, oral contraceptives

Stress

Myeloperoxidase

Use

•To differentiate a myelogenous or monocytic leukaemia

from acute lymphocytic leukaemia.

•Auer rods are better visualized with MPO than

Romanowsky stains.

Principle

• Myeloperoxidase is located in the primary and secondary

granules of granulocytes and their precursors, in

eosinophilic granules and azurophilic granules of

monocytes.

• MPO in eosinophil granules is cyanide resistant, whereas

that in neutrophils and monocytes is cyanide sensitive

• MPO splits H2O2 and in the presence of a chromogenic

electron donor forms an insoluble reaction product which

is brown and granular.

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

120l H2O2 and mix well

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

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

• 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

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

• Red brown precipitate

• Red granular staining peroxidase activity

Sudan Black B

Principle • Sudan Black B is a lipophilic dye that binds irreversibly to an

undefined granule component in granulocytes, eosinophils and some monocytes

Reagents• Fixative: vapors from 40% formaldehyde solution• Stain: SBB 0.3gm in 100ml absolute ethanol• Phenol buffer: dissolve g crystalline phenol in 30ml absolute

ethanol. Add to 100ml distilled water in which 0.3gm Sodium hypophosphate has been added

• Working solution: add 40 ml buffer to 60ml SBB solution• Counterstain : May-Grunwald-Giemsa or Lieshman stain

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

Results and interpretation• Reaction product is black and granular• Results are essentially similar to MPO staining• MPO negative neutrophils are also SBB negative• The only notable difference is in eosinophil granules which have a clear core

when stained with SBB• Rare cases of ALL show non-granular smudge positivity not seen with MPO• Basophils are generally not positive but may show bright red/purple

staining

Sudan Black B stainingPositive stain in a patient of AML

Black stained cytoplasm in myeloblasts

Toluidine Blue Stain

Principle • Toluidine blue staining is useful for the

enumeration of basophils and mast cells • It binds strongly to the granules in these

cells

Reagents• Toluidine Blue 1%w/v in methanol . Add

1g toluidine blue to 100ml methanol and mix for 24hr.

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

Results and interpretation• 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

Toluidine blue staining showing strongly positive basophils

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Specific Esterases

Use• The specific (naphthol AS-D

chloroacetate) esterase stain, also called the Leder stain, is used to identify cells of the granulocytic series

Reagents

• Fixative: Buffered formal acetone• Buffer: phosphate buffer (pH 7.4)• Naphthol AS-D chloroacetate substrate solution: Dissolve 0.1g naphthol

AS-D chloroacetate in 40ml N,N-dimethyl-formamide• Working solution:• 2ml naphthol AS-D chloroacetate solution• 38ml buffer• 0.4ml hexazotized New Fuschin

• Coupling reagent• 0.2ml Hexazotized new fuschin : 4gm of new fuschin in 100ml 2N HCl• 0.4ml Sodium Nitrate solution : 2.1g sodium nitrate in 100ml water

• Counterstain: aqueous hematoxylin

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

Results and interpretation• 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

Non specific esterases

Use •To identify monocytic cells

but do not stain granulocytes or eosinophils

• Include α-naphthyl butyrate and α-naphthyl acetate

-α Naphthyl butyrate

Reagents• Fixative: Buffered formal acetone• Buffer: phosphate buffer (pH8.0)• Substrate stock solution• α-Naphthyl butyrate 100l in 5ml

acetone stored at -20oC• Coupling reagent : Fast Garnet GBC 15mg • Counterstain : aqueous hematoxylin

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

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

-α naphthyl acetate

Reagents• Fixative: Buffered formal acetone• Buffer: phosphate buffer (pH6.3)• Substrate stock solution

• α-Naphthyl acetate 100mg in 5ml ethylene monomethyl ether• Coupling reagent :

• Stock pararosaniline : 1gm pararosaniline in 25ml 2mol/l HCl• 4% sodium nitrite solution: 200mg sodium nitrite in 5ml distilled

water• Hexazotized pararosaniline: equal volume of pararosaniline and

4% sodium nitrite• Incubation medium: 2ml substrate solution in 38ml buffer. Add

0.4ml hexazotized pararosaniline• Counterstain : aqueous hematoxylin

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

Results and interpretation• Reaction product is red/brown in color• Normal and leukaemic monocytes stain strongly• Normal granulocytes are negative but in MDS or AML may give varying

positive reaction• Megakaryocytes stain strongly

Tartrate Resistant Acid Phosphatase

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

Reagents • Fixative : Methanol• Buffer pH 5.0• Substrate Solution : 25mg naphthol dissolved in 2.5 ml N,N- dimethyl

formamide • Sodium nitrate 4% NaNO2 aqueous solution• Hexazotized pararosanonine (equal volume of pararosanonine and

4%sodium nitrite)• Counterstain: 1% aqueous methyl green or aqueous hematoxylin• Tartaric acid• Pararosanonine Solution :

• 92.5 ml of buffer• 2.5 ml of substrate solution. • 32.5ml distilled water• 4ml Hexazotized pararosanonine

• Working Solution A: (pH 5.0)• 92.5 ml of buffer• 2.5 ml of substrate solution. • 32.5ml distilled water• 4ml Hexazotized pararosanonine

• Working Solution B: • 375mg Tartaric acid • 50ml working solution A

Method• Air dry films for atleast 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

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

positively.

 Tartrate-resistant Acid Phosphatase (TRAP) Activity Of Lymphocytes

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conclusion

Stain Structure stained Use

Periodic assay Schiff (PAS)

Carbohydrates, principally glycogen

In AML and MDS to identify abnormal erythroblasts and dysplastic megakaryocytesTo confirm 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

Stain Structure stained Use

Myeloperoxidase Myeloperoxidase in neutrophils, monocytes and eosinophils

To differentiate a myelogenous or monocytic leukaemia from acute lymphocytic leukaemia.To visualize auer rods

Sudan Black B Intracellular lipid Similar to MPO

Toluidine Blue Basophils and mast cells To identify dysplastic basophils in myeloproliferative diseases

Specific esterases Neutrophil series and mast cells

Marker of cytoplasmic maturation in myeloid leukaemias

Non- Specific esterases Monocytes Monocytic component in AML

Tartarate resistant acid phosphatase

T-cells and granulocytes Diagnosis of T-cell ALL and hairy cell leukaemia

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