2. TypesTypes
1.1. WedgeWedge
2.2. Spun – most uniform distribution ofSpun – most uniform distribution of
blood cellsblood cells
3.3. ThickThick
4.4. Buffy layerBuffy layer
5.5. WetWet
4. Thick filmThick film
ParasitesParasites
View large volumeView large volume
4 drops4 drops
Join them over an area of 1 cm2Join them over an area of 1 cm2
5. Buffy layer filmBuffy layer film
1.1. Pernicious anemia – demonstratePernicious anemia – demonstrate
megaloblastsmegaloblasts
2.2. AIHA – erythrophagocytosisAIHA – erythrophagocytosis
3.3. Tumor cells in aleukemic leukemiaTumor cells in aleukemic leukemia
4.4. LE cellsLE cells
6. Wet filmWet film
Examination of blood cells suspendeExamination of blood cells suspende
in plasma +- supravital stainin plasma +- supravital stain
1.1. Demonstrate parasitesDemonstrate parasites
2.2. Induce red cell sicklingInduce red cell sickling
3.3. Study the red cells by NomarskiStudy the red cells by Nomarski
interference microscopy afterinterference microscopy after
fixation in Gluteraldehydefixation in Gluteraldehyde
7. Blood smear preparationBlood smear preparation
EDTAEDTA
Ideal smearIdeal smear
1.1. Length of the smear shud be 2/3 rd ofLength of the smear shud be 2/3 rd of
the slidethe slide
2.2. Uniformly thickenedUniformly thickened
3.3. Thin – printed matter can be readThin – printed matter can be read
4.4. Margin shud be free and centrally placedMargin shud be free and centrally placed
5.5. Tongue shapedTongue shaped
6.6. Shud have adequate thin area –free fromShud have adequate thin area –free from
vacoule ,serration etcvacoule ,serration etc
8.
9. Biological causes of poor filmBiological causes of poor film
1.1. Cold agglutininCold agglutinin
2.2. LipemiaLipemia
3.3. RouleauxRouleaux
17. Romanowsky stainRomanowsky stain
Basic dye forBasic dye for
1.1. Nucleic acidNucleic acid
2.2. NucleoproteinNucleoprotein
3.3. Basophilic granulesBasophilic granules
4.4. Weakly to neutrophil granulesWeakly to neutrophil granules
Acidic dyes forAcidic dyes for
1.1. HgHg
2.2. Eosinophil granulesEosinophil granules
18. Leishman’s stainLeishman’s stain
William Boog Leishman –BritishWilliam Boog Leishman –British
PathologistPathologist
In India he studies Enteric Fever andIn India he studies Enteric Fever and
Kala AzarKala Azar
Buffered water PH 6.8Buffered water PH 6.8
ComponentsComponents
1.1. Methylene Blue – Nuclear stainMethylene Blue – Nuclear stain
2.2. Eosin – cytoplasmic stainEosin – cytoplasmic stain
3.3. Acetone free Methyl Alcohol – solventAcetone free Methyl Alcohol – solvent
and fixativeand fixative
19. ProcedureProcedure
1.1. Enough stain to cover entire smear-Enough stain to cover entire smear-
keep for 2 min – fixation bykeep for 2 min – fixation by
methanolmethanol
2.2. Add distilled water - double theAdd distilled water - double the
amount—10 min –actual stainingamount—10 min –actual staining
3.3. Keep for 7 – 10 minKeep for 7 – 10 min
4.4. WashWash
5.5. DryDry
20. Good stainingGood staining
Pinkish in colourPinkish in colour
RBC – coppery redRBC – coppery red
Eosinophil – orange red – withEosinophil – orange red – with
yellowish tinged granulesyellowish tinged granules
No depositNo deposit
No cell shrinking or swellingNo cell shrinking or swelling
If under atained add stain + waterIf under atained add stain + water
for another 5minfor another 5min
If over stained –add stain onlyIf over stained –add stain only
21. Special stainingSpecial staining
1.1. Peroxidase stainPeroxidase stain
2.2. Sudan Black B stainSudan Black B stain
3.3. PAS stainPAS stain
4.4. Chloro Acetate esterase stainChloro Acetate esterase stain
5.5. Non Specific EsterasesNon Specific Esterases
6.6. Combined NSE & CAE stainingCombined NSE & CAE staining
7.7. Iron stainIron stain
8.8. Reticulin stainReticulin stain
22. Peroxidase stainPeroxidase stain
Enzyme found in Primary andEnzyme found in Primary and
Secondary granules in WBCSecondary granules in WBC
MPO – localised in the AzurophilicMPO – localised in the Azurophilic
granules of Neutrophils & Monocytesgranules of Neutrophils & Monocytes
It can be demonstrated inIt can be demonstrated in
Eosinophils and Basophils alsoEosinophils and Basophils also
23. MPO occurs onlu in the Primary GranulesMPO occurs onlu in the Primary Granules
of Myeloid cellsof Myeloid cells
It can be detected from theIt can be detected from the
Promyelocyte upto the granularPromyelocyte upto the granular
stagestage
Lymphoid series are strongly MPOLymphoid series are strongly MPO
negativenegative
Reddish brown appearanceReddish brown appearance
All myelocytic cell line except MyeloblastsAll myelocytic cell line except Myeloblasts
shows Positiveshows Positive
The more mature the cells become , theThe more mature the cells become , the
weaker the reaction becomesweaker the reaction becomes
Auer rods are strongly PositiveAuer rods are strongly Positive
24. Indications for peroxidase stainingIndications for peroxidase staining
Recognition of Acute Leukemias ofRecognition of Acute Leukemias of
myeloid origin – negative makesmyeloid origin – negative makes
lymphocyte origin – but does notlymphocyte origin – but does not
exclude a myeloid originexclude a myeloid origin
PositivePositive sub division of Myeloidsub division of Myeloid
Leukemias by FABLeukemias by FAB
25. ResultResult
Cells of Myeloid series positive withCells of Myeloid series positive with
increasing intensity and no. of granules asincreasing intensity and no. of granules as
the cells maturethe cells mature
Early blasts negativeEarly blasts negative
Lymphoid cells are negativeLymphoid cells are negative
Erythroid cells and RBC may be Positive –Erythroid cells and RBC may be Positive –
due to Non enzymatic Reaction betweendue to Non enzymatic Reaction between
Hb and stainHb and stain
Imp to distinguish AML from ALLImp to distinguish AML from ALL
Neutrophils show decreased staining inNeutrophils show decreased staining in
congenital MPO deficiency andcongenital MPO deficiency and
Myelodysplastic syndromesMyelodysplastic syndromes
26.
27.
28.
29. Sudan Black B stainSudan Black B stain
Fat soluble substanceFat soluble substance
StainsStains Fat cells ,Macrophages andFat cells ,Macrophages and
GranulocytesGranulocytes
Positive is dark brown to black in thePositive is dark brown to black in the
cytoplasm of the cell containing lipidcytoplasm of the cell containing lipid
Granulocytes +ve inc with maturityGranulocytes +ve inc with maturity
Cells of Burkitts’ Lymphoma +veCells of Burkitts’ Lymphoma +ve
Large vacoulated ALL –L 3 cells +veLarge vacoulated ALL –L 3 cells +ve
30. Myeloblasts –veMyeloblasts –ve
Lumphoblasts and Lymphocytes areLumphoblasts and Lymphocytes are
–ve–ve
Some cases of AML and CML –ve orSome cases of AML and CML –ve or
reduced sudanophiliareduced sudanophilia
31.
32.
33. PASPAS
Positive -Diffuse / granular pinkish colourPositive -Diffuse / granular pinkish colour
in cytoplasmin cytoplasm
Normal positiveNormal positive
1.1. GranulocytesGranulocytes
2.2. MonocytesMonocytes
3.3. MegakaryocytesMegakaryocytes
4.4. LymphocytesLymphocytes
Normal negativeNormal negative
1.1. Erythroid seriesErythroid series
34. Abnormal PASAbnormal PAS
1.1. Lymphocytes in IMN +veLymphocytes in IMN +ve
2.2. Late Erythroblasts in Thalssemia +veLate Erythroblasts in Thalssemia +ve
3.3. Lymphoblasts in all ALL –Large PositiveLymphoblasts in all ALL –Large Positive
GranulesGranules
4.4. 10 – 15% Myeloblasts in AML –diffuse10 – 15% Myeloblasts in AML –diffuse
+ve+ve
5.5. Erythroleukemia – AML –M6 –earlyErythroleukemia – AML –M6 –early
normoblasts – shows coarse granularnormoblasts – shows coarse granular
positivitypositivity late diffuselate diffuse
6.6. Hairy cells +veHairy cells +ve
Myeloblasts inAML -veMyeloblasts inAML -ve
35.
36. Non specific EsteraseNon specific Esterase
Positive for Monocytic SeriesPositive for Monocytic Series
Diagnosis of AML M5Diagnosis of AML M5
37.
38.
39. Iron stainIron stain
PEARL’S PRUSSIAN BLUE StainPEARL’S PRUSSIAN BLUE Stain
Absent in Fe def. AnemiaAbsent in Fe def. Anemia
Increased inIncreased in
1.1. Transfusion HaemosiderosisTransfusion Haemosiderosis
2.2. HaemochromatosisHaemochromatosis
3.3. Refractory Sideroblastic AnemiaRefractory Sideroblastic Anemia
4.4. Aplastic AnemiaAplastic Anemia
5.5. Pernicious anemiaPernicious anemia
40.
41. Nucleus and Cytoplasm pinkNucleus and Cytoplasm pink
Hemosiderin – bright blue – greenHemosiderin – bright blue – green
granulesgranules
42.
43.
44.
45.
46. PS reportingPS reporting
Size of the RBC is compared to smallSize of the RBC is compared to small
lymphocyteslymphocytes
Microcytes –MCV<80 flMicrocytes –MCV<80 fl
Macrocytes – MCV> 96 flMacrocytes – MCV> 96 fl
AnisocytosisAnisocytosis
PoikilocytosisPoikilocytosis
47. SpherocytesSpherocytes
Dark stained central pallor region –Dark stained central pallor region –
hyperchromasiahyperchromasia
1.1. Heriditary SpherocytosisHeriditary Spherocytosis
2.2. AIHAAIHA
3.3. Chemical & Bacterial ToxinsChemical & Bacterial Toxins
Editor's Notes
AML M0- MPO negative ,,,Neutrophil is Positive
AML M 1 – strongly MPO positive
AML M2 – MPO positive
ALL L 3…………Burkkitt’s Lymphoma
AML M 5
Erythrophagocytosis
Hemosiderin – Bright blue green granules – Hemochromatosis in cardiac muscle
Erythroleukemia
Sickle cells / drepanocytes
Target cells
Acanthocytes
Burr
Schistocytes
Pencil / cigar shaped cells
Blister cells –single or multiple vacoules at the periphery
Keratocytes / Horn cells
Reticulocytes / Polychromatic cells
Nucleated RBC
Schuffner’s dots
Maurer’s dot
Howell Jolly bodies – Nuclear remennts
Cabot ring
Basophilic stippling – aggregated ribosomes
Heinz bodies – denatured hb bound to cell membrane
Pappenheimer bodies – ferric iron complexed with Protein
Siderocytes
Toxic granules
Dohle or Amato bodies – blue staining cytoplasmic inclusions
Alder Rielly anomaly – course dark azurophilic granules
Chediak Higashi – giant lysosomes
May Hegglin –irregular sized inclusions
Smudge cells – degenerated lymphocytes
Jordan’s anomaly Vacoulation
Pelger Huet Anomaly – bilobed nucleated beutrophil
