This document describes the different types of peripheral blood smears including wedge, spun, thick, buffy layer, and wet films. It discusses staining techniques like Leishman, Wright, and May-Grunwald Giemsa stains. Special stains are also covered such as peroxidase, Sudan Black B, PAS, and iron stains which can identify characteristics of cells and diseases. Proper preparation, fixation, and interpretation of peripheral blood smears is essential for hematological diagnosis and identification of abnormalities.

1. Peripheral smearPeripheral smear

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

3. Wedge filmWedge film
 Spreading slide 30 – 45 degreeSpreading slide 30 – 45 degree
 Severe anemia - > 60 degreeSevere anemia - > 60 degree
 Polycythemia -< 15 degreePolycythemia -< 15 degree

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

9. Biological causes of poor filmBiological causes of poor film
1.1. Cold agglutininCold agglutinin
2.2. LipemiaLipemia
3.3. RouleauxRouleaux

10. FixativesFixatives
1.1. Methyl alcohol – Fixative Of ChoiceMethyl alcohol – Fixative Of Choice
2.2. Ethyl alcoholEthyl alcohol

11. Staining of peripheral smearStaining of peripheral smear
 Romanowsky type dyesRomanowsky type dyes
1.1. LeishmanLeishman
2.2. WrightWright
3.3. May Grunwald GiemsaMay Grunwald Giemsa
4.4. JennerJenner
 Compound dyesCompound dyes
 Neutral dyesNeutral dyes
 Field stain rapid stain for parasitesField stain rapid stain for parasites

12. EosinEosin
 Cytoplasm , collagen ,muscle fibersCytoplasm , collagen ,muscle fibers
 AcidicAcidic
 H & E staining –H & E staining –
 Cytoplasm pink –orangeCytoplasm pink –orange
 Nuclei – blue / purpleNuclei – blue / purple
 Eosin stains red blood cells intenslyEosin stains red blood cells intensly
redred
 Haemetoxylin – nuclear stainHaemetoxylin – nuclear stain

13. Methylene blueMethylene blue
 Component of Gram stain .WrightComponent of Gram stain .Wright
stain ,Jenner’s stainstain ,Jenner’s stain

14. May Grunwald Giemsa stainMay Grunwald Giemsa stain
 Histopathological diagnosis forHistopathological diagnosis for
Malaria and other parasitesMalaria and other parasites
 Fixative – acetone free methylFixative – acetone free methyl
alcoholalcohol

15. Jenner’s stainJenner’s stain
 Methylene blue eosinateMethylene blue eosinate

16. Wright stainWright stain
 Eosin + MBEosin + MB

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

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

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

36. Non specific EsteraseNon specific Esterase
 Positive for Monocytic SeriesPositive for Monocytic Series
 Diagnosis of AML M5Diagnosis of AML M5

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

41.  Nucleus and Cytoplasm pinkNucleus and Cytoplasm pink
 Hemosiderin – bright blue – greenHemosiderin – bright blue – green
granulesgranules

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