4. 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.
5. 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
6. 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
10. 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.
11. 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
15. 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
16. 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
17. 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
18. 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
19. Low LAP score (<15) High LAP score (>130)
CML Infections
Paroxysmal nocturnal hemoglobinuria Growth factor therapy
Myelodysplastic syndromes Myeloproliferative disorders other
than CML
Inflammatory disorders
Pregnancy, oral contraceptives
Stress
21. Use
•To differentiate a myelogenous or monocytic leukaemia
from acute lymphocytic leukaemia.
•Auer rods are better visualized with MPO than
Romanowsky stains.
22. 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.
23. 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
24. 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
25. 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
28. 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
29. 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
32. 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.
33. 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
36. Use
• The specific (naphthol AS-D
chloroacetate) esterase stain, also
called the Leder stain, is used to
identify cells of the granulocytic series
37. 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
38. 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
42. 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
44. 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
46. 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
47. • 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
48. 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.
51. Stain Structure stained Use
Periodic assay Schiff
(PAS)
Carbohydrates,
principally glycogen
In AML and MDS to
identify abnormal
erythroblasts and
dysplastic
megakaryocytes
To 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
52. 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
