Peripheral Smear technique and reporting

Peripheral smear presentation including techniques and reporting of peripheral smear

1. PERIPHERAL SMEAR Moderator- Dr. Meena Mittal Speaker: Dr. Upma Gupta

2. 1 . Specimen Collection 2. Peripheral Smear Preparation 3. Staining of Peripheral Blood Smear 4. Peripheral Smear Examination Step-by-Step

3. Venipuncture Should be collected in an EDTA (disodium or t ripotassium ethylene diamine tetra-acetic acid) tube Chelates calcium. 1.2 mg anhydrous EDTA salt/ml of blood used. EDTA effect Specimen Collection

4. Wedge method Cover glass method Spinner method Specific methods Buffy coat smear for WBC < 1.0 x10^9/L Thick blood smear for blood parasites PERIPHERAL SMEAR PREPARATION

5. WEDGE METHOD A. Hold spreader slide at correct angle B. Blood spread across width of slide C. Completed wedge smear

6. WEDGE METHOD Place a drop of blood in the centre of a clean glass slide 1cm away from the end. Place the spreader in front of the drop at an angle of 30 degrees to the slide and move it back to make contact with the drop. The drop should spread quickly along the line of contact, with a steady movement of the hand spread the drop of blood along the slide. Smear should be 3 cm in length, smooth,tongue shaped. Air dry and stain.

7. COVER GLASS METHOD Take 22 mm clean cover glass,touch it on drop of blood and place it on another cover glass in cross wise direction with slide containing drop of blood facing down.

8. SPINNER METHOD Automated method-place a drop of blood in the centre of the glass slide and spin at a high speed in a centrifuge cytospin . Blood spread uniformly, dry it and stain.

9. FEATURES OF AN IDEAL Peripheral smear Does not cover the entire area of slide. Should be tongue shaped, ie broad at starting point (head.) & taper towards end. Has both thick and thin areas with gradual transition. Does not contain any lines or holes. Smooth margins. Tail Head

10. Labelling of PS- The film should be labelled immediately after spreading with lab reference number or the patients name. Fixing - To preserve the morphology of cells ,films must be fixed without delay. Take methanol in a coplin jar ,give 4 to 6 dip to the smear in methanol and air dry. Methanol is used as a fixative & must be free from water. Methanol precipitates the plasma proteins, which then acts as a glue to fix the cells to the slide.

11. Romanowsky stains – Differentially stain the blood cell components on the basis of their pH. Standardized stain is composed of: Azure B- trimethylthionine -cationic dye Eosin Y- tetrabromofluoroscein -anionic dye Polychrome methylene blue and eosin are the outgrowths of the original method. Methylene blue and azure B are basic dyes that stains acidic components of the cell like nucleic acids, granules of basophil . The eosin is acidic or anionic dye that stain basic components like hemoglobin & granules of eosinophils . STAINING OF PERIPHERAL SMEAR

12. Stains include- AzureB EosinY stain-highly purified standard. May- Grunwald - Giemsa stain(used in Europe) Wright’s stain(used in N. America) Leishman’s stain Field stain( Rapid)- used in our department. Giemsa stain JSB stain- for malarial parasite Automated stains

13.       MAY-GRUNWALD-GIEMSA METHOD   1.  Air dry slides. 2.  Fix in Methanol for 2-3 minutes at room temperature. 3.  Stain for 15  mins  in May- Grunwald  stain freshly diluted with an equal volume of buffered distilled water, pH=6.8 4. Stain for 10 minutes in  Giemsa  stain freshly diluted with buffered distilled water, pH=6.8 (1/9) 5.  Wash in running tap-water and then leave for 3-4 minutes in buffered distilled water, pH 6.8  

14. It consists of methylene blue and eosin dissolved in absolute methyl alcohol. Weigh 0.2 gm of powdered dye in conical flask & add 100 ml methanol & warm the mixture to 50 C for 15 min. Allow to cool & filter. METHOD: 1.Air dry the film & flood the slide with stain for 2 min. 2.Add double volume of water & stain the film for 5-7 min. 3.Wash in a stream of buffered water & dry it. LEISHMAN METHOD

15. RAPID STAINING METHOD: Field’s method (45 sec) Tap water (3 sec) (1 min 15 sec) Wash gently to remove excess stain, under tap water. Drain and dry. Methanol Eosin Polychromed methylene blue

16. Cell Component Staining Colour Chromatin(including Howell-Jolly body) Purple Promyelocyte granules & Auer rods Purplish-red Cytoplasm of lymphocytes Blue Cytoplasm of monocytes Blue-grey Cytoplasm rich in RNA(i.e. Basophilic cytoplasm) Deep blue Dohle bodies Blue-grey Specific granules of neutrophils , granules of lymphocytes, granulomere of platelets Light purple or pink Specific granules of basophils Deep purple Specific granules of eosinophils Orange Red cells Pink STAINING FEATURES OF CELL COMPONENTS WITH ROMANOWSKY STAIN

17. PERIPHERAL SMEAR EXAMINATION

18. OPTIMAL ASSESSMENT AREA TOO THICK TOO THIN RBCs are uniformly and singly distributed Few RBC are touching or overlapping Normal biconcave appearance UNDER THE MICROSCOPE

19. Observations Under 10X Assess the overall quality of the smear. 2.Find an area where red cells are evenly distributed and not distorted. 3.Check to see if there are good counting areas available free of ragged edges and cell clumps. 4.Check the number,distribution and staining of leukocytes. 5.Assess whether red cell agglutination, platelet aggregation or fibrin strands are present.

20. WBC estimation Under 40X Choose a portion of the peripheral smear where there is only slight overlapping of the RBCs. Count 10 fields, take the total number of white cells and divide by 10. To do a WBC estimate by taking the average number of white cells and multiplying by 2000 . WBC:RBC::1:500 (normal leucocrit approx.)

21. Under 100X A)Manual Differential Counts

22. Reporting results Absolute number of cells/µl = % of cell type in differential x white cell count If 10 or more nucleated RBC's ( nRBC ) are seen, correct the leucocyte Count using this formula: Corrected WBC Count = WBC x 100/( nRBC + 100) B) Platelet estimation

23. NORMAL HAEMATOPOEISIS

24. RBC distribution Morphology Colour Size Shape Structure Inclusions Young rbcs RED BLOOD CELLS

25. CAUSE RESULTANT ABNORMALITY Abnormal Erythropoeisis -Effective/Ineffective Anisopoikilicytosis,Basophilic stippling,dimorphism (sometimes) Inadequate Hb synthesis Hypochromia / Anisochromia / Dimorphism Damage to normal cells after leaving bone marrow/ Hyposplenism / Splenectomy Poikilocytosis - Specific/Non specific; Red cell inclusions Compensatory increased erythropoeisis by bone marrow Less mature cell release: Polychromasia,Erythroblastemia MECHANISM OF RBC ABNORMALITY & RESULTANT FEATURES

26. ABNORMAL ERYTHROPOEISIS A) ANISOCYTOSIS & POIKILOCYTOSIS: Increased RDW Non specific Seen in various disorders of diverse etiology .

27. B) MACROCYTOSIS With reticulocytosis Megaloblastic erythropoeisis Associated with- Megaloblastic OR macronormoblastic erythropoeisis Macronormoblastic erythropoeisis Uncertain mechanism

28. C) MICROCYTOSIS - Decreased MCV Inherited Acquired

29. D ) BASOPHILIC STIPPLING : Indicates disturbed rather than increased erythropoeisis Basophilic Inclusions; RNA CAUSES Thalassemia trait and major Haemolytic Anemia Myelodysplastic syndrome/ Sideroblastic anemia Megaloblastic Anemia Pyrimidine 5’ nucleotidase deficiency Heavy metal poisoning(Coarse basophilic stippling) Lead, Zinc, Arsenic, Silver, Mercury

30. INADEQUATE HAEMOGLOBIN FORMATION A) HYPOCHROMIA-

31. B) ANISOCHROMASIA- Increased variation in degree of haemoglobinization of RBC Seen in development/resolution of iron deficiency anaemia; anaemia of chronic disease

32. C) DIMORPHIC CELL POPULATION Seen after response to iron therapy in iron deficiency anemia ; macrocytic anemia post blood transfusion; sideroblastic anaemia; Post stem cell transplantation

33. DAMAGE TO RED CELLS AFTER RELEASE FROM MARROW A) HYPERCHROMASIA In presence of macrocytes Abnormally round cells Seen in:

34. B) SPHEROCYTOSIS CAUSES Warm autoimmune hemolytic anaemia ABO hemolytic disease of newborn/ Rh hemolytic disease of newborn Cold autoimmune hemolytic anemia /paroxysmal cold hemoglobinuria Hereditary Spherocytosis Clostridium sepsis IV water infusion or fresh water drowning Bartonellosis Snake bite Hyposplenism Hypophosphatemia Rh -null phenotype

35. ELLIPTOCYTOSIS and OVALOCYTOSIS Large number of elliptocytes Hereditary Elliptocytosis Small number of elliptocytes Iron deficiency Thalassemia trait and major Megaloblastic anaemia Myelodysplastic syndrome Myelofibrosis

36. C) IRREGULARLY CONTRACTED CELLS CAUSES Oxidative hemolysis (Glucose-6-phosphate deficiency, glutathione synthetase deficiency, drugs, toxins) Unstable haemoglobins Hb C homozygous Hb SC disease Wilson’s disease Zieve’s syndrome

37. D)SPICULATED CELLS and RED CELL FRAGMENTATION SCHISTOCYTE ECHINOCYTE ACANTHOCYTE KERATOCYTE On basis of electron microscopy [ Bessis ] SCHISTOCYTE

38. CAUSES Artifact Uremia and chronic renal disease Hypophosphatemia Disseminated malignancy Liver disease Vitamin E deficiency Pyruvate kinase deficiency Phosphoglycerate kinase deficiency Early post transfusion of RBC Hyperlipidemia Myeloproliferative disorders ECHINOCYTE

39. ACANTHOCYTES Large numbers of acanthocytes Advanced liver disease Spur cell hemolytic anaemia Abetalipoproteinemia Hypobetalipoproteinemia , homozygous McLeod phenotype In(Lu phenotype) Choreoacanthycytosis Small numbers of acanthocytes Post splenectomy Hypothyroidism Panhypopituitarism Vitamin E deficiency Malnutrition Thalassemia Iron deficiency Psoriasis

40. Glucose-6 Phosphate Dehydrogenase deficiency KERATOCYTE: ‘Helmet’/Bite cell’ Haemolytic Anemia by Dapsone CAUSES Oxidative hemolysis (Glucose-6-phosphate deficiency, glutathione synthetase deficiency, drugs, toxins) Unstable haemoglobins

41. MISCELLANOUS ERYTHROCYTE ABNORMALITIES LEPTOCYTES : Unsually thin RBCs Seen in severe iron deficiency anaemia, thalessemia , liver disease

42. STOMATOCYTES CAUSES Artifact Alcoholism Alcoholic liver disease Obstructive liver disease Hereditary stomatocytosis Hereditary xerocytosis South east Asian ovalocytosis Tangier disease Rh -null phenotype Drugs ( hydroxy urea)

43. TARGET CELLS CAUSES Microcytic Hb E heterozygous+ homozygous B- thalassemia trait+major Alpha- Thalassemia trait Hb H disease Hb C trait+ disease Hb Lepore heterozygous +homozygous Hb O Arab disease Hb D disease Iron deficiency Hb Lepore trait Normocytic or Macrocytic Obstructive jaundice Liver disease Lecithin Cholesterol Acyl Transferase deficiency Hb SC disease Hyposplenic disease Hb O Arab disease

44. Primary Myelofibrosis DACRYOCYTES Megaloblastic Anaemia Also seen in bone marrow infiltration, Thalessemia major, some cases of hemolytic anemia

45. SICKLE CELLS (DREPANOCYTE) Sickle cell anaemia:homozygous for HbS

46. ERYTHROCYTE INCLUSIONS Pappenheimer Body Cabot Ring Howell Jolly Body Heinz Body

47. Howell Jolly body DNA Basophilic Stippling RNA Pappenheimer body Iron containing inclusions Heinz body Denatured Haemoglobin Hb H disease beta- globin tetramer Fessus body Alpha- globin tetramer Crystal Hb C Cabot Ring Mitotic Spindle RED CELL INCLUSIONS CONTENT

48. ROULEAUX and AUTOAGGLUTINATION ROULEAUX - due to- Increased plasma proteins (polyclonal or other proteins) Inflammatory state Infection Increased plasma proteins (monoclonal) MGUS Myeloma Amyloidosis Lymphoma RED BLOOD CELL AGGLUTINATION Cold agglutinins Cold autoimmune hemolytic anaemia Paroxysmal cold hemoglobinuria Ig M paraproteinemia

49. CHANGES WITH COMPENSATORY INCREASE IN ERYTHROPOESIS Polychromasia Nucleated RBCs Leukoerythroblastic blood picture

50. RETICULOCYTOSIS They are juvenile red cells containing remnants of ribosomal ribonucleic acid. They react with a basic dye to form a blue or purple precipitate of granule or filaments. This reaction needs supravital staining and unfixed preparation. Dye used-brilliant brilliant cresyl blue, new methylene blue, azure B. Normal count—in adults and children-0.5%-2.0%. In infants( cordblood )-2%-5%

51. Most immature:Grade I Most mature:Grade IV

52. MORPHOLOGY OF LEUCOCYTES

53. POLYMORPHONUCLEAR NEUTROPHILS Granules Vacuoles Bacteria Nuclei Hypersegmentation Pyknotic Neutrophils (Apoptosis)

54. B. Toxic Neutrophilia with toxic vacuolation A. Toxic granulation IN SEVERE SEPSIS~ Azurophilic cytoplasmic granules seen in severe infections, other toxic and reactive conditions Seen in infections, indicating phagocytosis

55. C.Overwhelming Septicemia:Bacterial inclusion Dohle Body in a neutrophil Pale blue, oval cytoplasmic remnants of ribosomes seen in infection and other toxic condition.

56. Alder-Reilly anomaly FEW INHERITED ANOMALIES~ May – Hegglin anomaly Chediak -Higashi syndrome Autosomal recessive disorder with giant granules,likely representing giant fused lysosomes & abnormal leukocyte function

57. Inherited Pelger-Huet anomaly Bilobed or rounded nuclei with pince-nez shape nucleus Pseudo Pelger-Huet anomaly Hypogranular neutrophils with irregular nuclear pattern

58. D. NUCLEI

59. E.Hypersegmented Neutrophil Segmentation Index=Number of neutrophils with 5-more lobes ___________________________________ Number of neutrophils with 4 lobes

60. F. Pyknotic neutrophils : Undergoing apoptosis. May normally be found in infection. Cells have round, dense, featureless nuclei and cytoplasm is dark pink.

61. EOSINOPHILIC GRANULOCYTE MATURE EOSINOPHIL HYPEREOSINOPHILIC SYNDROME

62. MATURE BASOPHIL Marked BASOPHILIA in case with Philadelphia chromosome positivity BASOPHILIC GRANULOCYTE

63. IMMATURE GRANULOCYTES Myeloblast : Large cell 10-18um. Round oval nucleus occupying most cell area. -- Nucleoli are typically prominent (2-5) -- Deep blue scanty cytoplasm no granules.

64. Promyelocyte Slightly larger 12-20um Shares same features with myeloblast . Less prominent nucleoli. Cytoplasm contains blue purple primary azurophilic granules. Myelocyte : Size 12-18 um Nucleus is oval or slightly indented, fine chromatin. No nucleoli are seen or very rare. Cytoplasmic granules now acquire specific characters. Both primary and secondary granules seen.

65. Metamyelocyte : Size 10-18  m Indentation less than ½ diameter of nucleus Condensed chromatin.No nucleoli. Cytoplasm is abundant with secondary granules only . Band cells : 10-16 micron. Indentation more than ½ diameter of nucleus. Cytoplasm is abundant with secondary granules only.

66. Lymphoblast Myeloblast Nuclear chromatin Coarse Fine Nucleoli 1-2 3-5 N:C ratio High High Auer rod Absent Present Accompanying cells Lymphocytes Myeloid precursors

67. SMALL LYMPHOCYTES Size-6-10 microns High nucleocytoplasmic ratio Scant amount of slightly basophilic cytoplasm. Smooth contour with mature “closed” chromatin and absent nucleoli.

68. LARGE LYMPHOCYTE Size- 12-15 micron Moderate nuclear- cytoplasmic ratio Oval nucleus Slightly open chromatin Pale cytoplasm About 1/3 rd may contain azurophilic granules; larger than neutrophilic granules

69. NORMAL CLL ALL L1 type of lymphoblast shown here usually has a high nuclear:cytoplasmic (N:C) ratio with an immature or “open” chromatin CLL cells appear as slightly larger versions of normal small lymphocytes. CLL cells can display clumped or “soccer ball” chromatin. Small lymphocyte

70. Large size, abundant basophilic cytoplasm at periphery Often vacuolated. Nucleus- Large; irregular Partially condensed chromatin Nucleoli present ACTIVATED LYMPHOCYTES ‘Atypical mononuclear cell’

71. ‘TURK CELL’; IMMUNOBLAST 10-15 um size. Round nucleus, abundant deeply basophilic cytoplasm Seen in severe bacterial and viral infection

72. PLASMA CELL Larger than small lymphocyte Oval in shape with eccentric round nucleus with clumped nuclear chromatin. Moderate amount of basophilic cytoplasm with prominent nuclear hof Plasma cell

73. MATURE MONOCYTE Size : 14-20 um Irregular, lobulated nucleus Has the most delicate nuclear chromatin pattern. Abundant light gray cytoplasm with fine granularity and vacuolation seen Deeply indented nucleus

74. Figure 33-24 Promonocyte ( center ) shows immature chromatin and finely convoluted nuclear folds in comparison with monoblasts .

75. Degenerative changes Broken cells Contracted cells Endothelial cells Radial segmentation of the nuclei Vacuolation Pseudophagocytosis ARTIFACTS

76. Ruptured Leukocyte Basket Cell Broken cell of myeloid series Smudge cell

77. Normal platelets are 2-4 um in diameter. Irregular outline with fine red granules . Normal peripheral count 1.0 -5 lac /mm3 Remain viable in circulation for 10 days. Large Platelets seen in– hyposplenism , Bernard- Soulier syndrome, May- Hegglin anomaly. MORPHOLOGY OF PLATELETS

78. PLATELET AGGREGATES PLATELET SATTELITISM False Thrombocytopenia:

79. Hyposplenism in coeliac disease: GIANT PLATELETS GRAY PLATELET SYNDROME ESSENTIAL THROMBOCYTOSIS MISCELLANEOUS PLATELET ABNORMALITIES

80. 1. MALARIA PARASITE : Plasmodium Blood is collected at or just after the height of a febrile paroxysm. One thick & one thin smear should be prepared . Procedure -Take a large of drop of blood in the centre of a clean glass slide & spread it in an area of 1.5 cm with a slide. Dry and stain by any Romanowsky combination. Leishman , Giemsa method are suitable. In P. falciparum often only early trophozoite (ring form) +/- gametocytes are found. In P. vivax , all stages of life cycle usually present. IDENTIFICATION OF PARASITES IN P.S.

81. Male gametocyte: P.falciparum Ring form trophozoite : P. falciparum Ring form: P.vivax Early schizont

82. 2.MICROFILARIA: W.bancrofti Blood is collected at midnight between 10 p.m.-4a.m. Blood concentration of microfilaria is higher in capillary blood than in venous blood. Morphology : When stained with Romanowsky stain: A hyaline sheath seen projecting beyond the body. Nuclei appear as granules in central axis of body extend from head to tail, but not to the tip of tail.

83. 3. TRYPANOSOMA BRUCEI (African sleeping sickness) Trypomastigotes can be found in peripheral blood,lymph node aspirate and CSF. When stained with Leishman stain, cytoplasm & the undulating membrane appear pale blue and the nucleus reddish purple or red. Kinetoplast & flagellum dark red.

84. 4. TRYPANOSOMA CRUZI – ( Chagas disease) Two forms are found a. Trypomastigote form can only be found in blood in acute form of Chagas disease. b. Amastigote form (in striated muscles) Trypomastigote form appear as C or U shaped .

85. 5. LEISHMANIA DONOVANI Diagnosed by direct visualization of amastigote ( LD bodies ) Causes kala azar . In aspirate smears amastigote forms are seen in groups inside macrophages or lying free between the cells. In buffy coat smears they are seen within monocytes or neutrophils in peripheral blood. Small round bodies 2-4 micron in diameter, indistinct cytoplasm, a nucleus, a small rod shaped kinetoplast .

86. PS Reporting: Summary Red Blood Cells, Examine for : Size and shape ( Anisocytosis , Poikilocytosis ) Relative hemoglobin content. Polychromatophilia. Inclusions.   Rouleaux formation or agglutination White Blood Cells Check for even distribution and estimate the number present (also, look for any gross abnormalities present on the smear). Perform the differential count. Examine for morphologic abnormalities and immature cells. Platelets. Estimate number present.  Examine for morphologic abnormalities, aggregates. Comment on any haemoparasites , if present.

87. THANK YOU!~