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PERIPHERAL SMEAR REPORTING ppt.pptx path
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- 2. INDICATIONS • Diagnose adisease • Monitor response • Detect pre-analytical or analytical error in output of automated analyzer • Correlate instrument flag
- 3. • Macroscopic examination 1.Assess whether the spreading technique was satisfactory 2. Judge its staining characteristics 3. Presence of any abnormal particles- Large platelet aggregates Cryoglobulin deposits • Clumps of tumour cells
- 5. • Well preparedand well stained smear • Tongue shaped smear • No irregularities • No serrations at tail end • No air bubbles
- 6. • Low power10 x: 1)Quality of film and staining 2) Location and selection of proper field 3)Approx. no. of RBCs 4)Microfilaria like parasites
- 7. 5) Degree ofrouleaux or RBC agglutination 6)Platelet clumps and Platelet satellitism 7)Fibrin strands
- 8. Best field-Area betweenbody and tail Rbcs just become separate / no overlapping 1.RBCs are uniformly and singly distributed 2.Few RBCs are touching or overlapping 3.Normal biconcave RBC appearance 4. 200 to 250 RBC per 100x
- 9. • Thin area-Flattenedred cells • Thick area-Rouleaux normal in such areas
- 10. • Tailing • LowWBC count Look at tail end and edges of smear
- 11. HIGH POWER 40x •RBC -Morphology • WBC-Estimate of TLC, DC • Choose a portion- only slight overlapping of the RBCs • Count 10 fields, take average number of WBC/ hpf • Multiply the average number of white cells by 2500
- 12. Normal -4000-11000 Leucocytosis – Mild->11000 Moderate->50000 Severe ->1,00,000
- 13. • Detect Toxicgranulation • Howell–Jolly bodies • Pappenheimer bodies
- 14. OIL IMMERSION 100x •RBC, WBC, Platelet morphology • RBC inclusions • Hemoparasites • Estimate of platelet count 1.Estimate the number of platelets per oil immersion field. (Normal-10 platelets in each field) 2.Take an average of at least 10 fields 3.Multiply the average by 15000
- 15. RBC MORPHOLOGY 1.Size (Anisocytosis) 2.Shape(Poikilocytosis) 3.Color (Relative hemoglobin content) 4.Polychromatophilia 5.Rouleaux formation or agglutination 6.Inclusions
- 16. • NORMOCYTIC (6-8μm) •MICROCYTIC • MACROCYTIC
- 17. ANEMIA-MORPHOLOGY Microcytic hypochromic • IronDeficiency (IDA) • Anemia of chronic disease • Chronic Infections • Thalassemias • Hemoglobino-pathies • SideroblasticAnemia Normocytic normochromic • C/c kidney liver, endocrine ds • Early IDA • Marrow infiltration • Marrow hypoplasia • Haemolytic anemias • Macrocytic normochromic • Megaloblastic anemia • Liver disease/alcoholism • Hypothyroidism • MDS • Myelophistic anaemias • Aplastic anaemia • Congenital dyserythropoietic anemia
- 18. D/D of MHA •IRON DEFICIENCY ANEMIA • Moderate anisopoikilocytosis • Pencil cells, elongated cells • Thrombocytosis+/-
- 19. • THALASSEMIA TRAIT •Mild Anisocytosis, • Target cells
- 20. • Homozygous Thalassemia(Thal major , intermedia)
- 21. • Sideroblastic anemia •Dimorphic anemia • Lead poisoning • Basophilic stippling
- 22. DIMORPHIC ANEMIA • Twodifferent population of RBC –Macrocytes + normocytes –Microcytes with normocytes –Macrocytes with microcytes •MCV –low, normal or high
- 23. Causes of Dimorphicanemia • Response to iron or vitamin therapy • Blood transfusion in a pt. with microcytic/macrocytic anemia • Myelodysplastic syndromes • Sideroblastic anemias
- 24. MEGALOBLASTIC ANEMIA • Pancytopenia •Macrovalocytes • Hypersegmented neutrophils • Cabot rings • nRBCs with megaloblastic maturation
- 25. • Well-made films •<10% are definitely oval shaped • Percentage of ‘pyknocytes’ (irregularly contracted cells) and schistocytes : • Adults does not exceed 0.1% • Full-term infants - Higher-0.3–1.9% • Premature infants still higher- up to 5.6%
- 26. HEMOLYSIS • Polychromasia • n-RBC •Specific morphologic • abnormalities
- 28. SPHEROCYTES Diameter-less Thickness-greater • Spherocyticanaemia - Coombs test, History •Hereditary Spherocytosis Uniform sized spherocytes •Auto Immune Hemolytic Anemia Variable sphero, nRBCs, Agglutination+/- •Hemolytic Ds of Newborn Many nRBCs •MAHA Microspherocytes, Schistocytes •Burns Spherocytes, Smaller RBC fragments Delayed transfusion reactions Bacterial toxins- Clostridium perfringens
- 30. SPHERO-ECHINOCYTES • Crenated spheres •Artefact-Prolonged standing of blood • Post transfusion smear transfused with stored blood • Sodium chlorate poisoning
- 31. IRREGULARLY CONTRACTED CELLS •Smaller than normal (appear contracted) • Margins are slightly irregular • May be partly concave • Densely stained • Drug- or chemical-induced haemolytic anaemias • Unstable haemoglobinopathies Hb Koln or Hb St Mary’s and in Hb E homozygosity , HbE heterozygosity
- 32. ELLIPTOCYTES • Heriditary Elliptocytosis(>25%) • Thalassemia trait • IDA • Megaloblastic anemia • Post splenectomy • Myelofibrosis
- 33. STOMATOCYTES • Artifact • Liverdisease / Alcoholism • South east Asian stomatocytosis
- 34. BLISTER CELLS ANDBITE CELLS • G6PD deficiency • Oxidant drugs, chemicals • Dapsone induced hemolysis • Chronic liver disease • Wilson’s disease • Post splenectomy
- 35. TARGET CELLS Microcytic • SevereIDA • Thalassemia trait • Hb E ds • Hb C ds Normocytic Sickle cell anemia •Sickle thalassemia Macrocytic Liver disease •Post splenectomy •Preterm infants
- 37. SCHISTOCYTES • Smaller than normalRBC • Have sharp angles • or spines (spurs) , sometimes round in contour, • Stained deeply but occasionally palely • Result of loss of Hb at the time of fragmentation TTP HUS Thalassemias Pregnancy and postpartum period HELLP syndrome TTP/HUS Direct thermal injury DIC MAHA
- 38. KERATOCYTES • Pair ofspicules, one pair or two pairs • Formed either by removal of a Heinz body • by the pitting action of the spleen • or by mechanical damage • Also called ‘helmet cell’ and ‘bite cell’
- 39. SPUR CELLS (ACANTHOCYTE) Smallnumber of spicules of inconstant length, thickness and shape
- 40. BURR CELL (ECHINOCYTE) •Artefact • Anemia of renal disease • Premature infants after exchange transfusion or transfusion of normal RBCs Numerous short, regular projections from their surface
- 41. TEAR DROP CELL(DACRYOCYTE) • >4% is abnormal • Leucoerythroblastic picture • Fibrosis in marrow • Thalassemia • Liver disease
- 42. LEPTOCYTES • Abnormally thinred cells • Stain as rings of membrane with a • little attached haemoglobin with large, almost unstained, central areas • Defect in haemoglobin synthesis -Thalassaemias and Iron deficiency • Liver disease
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- 44. BASOPHILIC STIPPLING/ PUNCTATE BASOPHILIA-Abnormally aggregated ribosomes Coarse stippling Fine stippling • Thalassaemia Associated with increased RBC pdn • Megaloblastic anaemia • Infections • Chronic liver disease • Lead and other heavy metal poisoning • Unstable hemoglobins • Pyrimidine-5’ nucleotidase deficiency
- 45. HOWELL JOLLY BODIES •Nuclear remnants • Small, round cytoplasmic inclusions • Stain purple on a Romanowsky stain • Hyposplenic states • Post splenectomy • Megaloblastic anemia • Follow up of post splenectomy refractory ITP/ AIHA
- 46. PAPPENHEIMMER BODIES • Smallperipherally sited basophilic • (almost black) erythrocyte inclusions • Smaller than Howell–Jolly bodies • Composed of haemosiderin • Sideroblastic erythropoiesis • Hyposplenism • Confirmed by Perls’ stain-Remain blue
- 47. AGGLUTINATION • Cold agglutinindisease- • Tight clusters, polychromatophils, • Spherocytes, n-RBCs
- 48. Rouleaux Formation • Positivelycharged molecules, Igs • reduce zeta potential between RBCs • RBCs aggregate on top of each other • Plasma cell neoplasms • Chronic liver disease with • Hypergammaglobulinemia • Chronic infections • Chronic infammation
- 49. nRBC Correction Hemolytic anemia,LEB picture
- 51. NEUTROPHIL ABNORMALITIES • Nucleus 1.Pelger-Huet anomaly • Heriditary-Hyposegmentation, • 2 discrete equal sized lobes • connected by thin chromatin bridge • chromatin coarsely clumped, • granule content normal • Acquired-MDS, AML-Hypogranular , • irregular nuclear pattern 2. Hypersegmentation • Megaloblastic anemia
- 52. • Cytoplasm 1. Hypogranulation •MDS 2.Toxic granule Larger and more basophilic • Bacterial infection • Burns • Malignancies
- 53. • Pregnancy • Drugreactions • Aplastic anemias • Hypereosinophilic syndrome 3.Vacuolisation • Bacterial infection • Chanarin-Dorfman syndrome • Lipid storage disease
- 54. • Chediak–Higashi syndrome- • Giant but scanty azurophilic granules • Alder–Reilly anomaly • granules are very large, are discrete, • stain deep red and • may obscure the nucleus
- 55. BACTERIA • Septicemia-(e.g. meningococcalor pneumococcal) • Bacteria may be seen within vacuoles or • free in the cytoplasm of neutrophils
- 56. DOHLE BODIES • Skyblue inclusions in cytoplasm of neutrophils • Infections • Burns • Myleproliferative disorders • Pregnancy • Composed of RER and glycogen granules
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- 59. PLATELETS • Estimate count •Compare with counter value- spurious low or high count • Morphological abnormalities
- 60. Spurious low counts 1.EDTAinduced pseudothrombocytopenia 2.Platelet satellitism- EDTA
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- 62. 5. Large platelets •ITP • Bernard Soulier syndrome • May Hegglin anomaly • Immature reactive platelets- ITP on steroids Post chemotherapy recovery
- 63. D: Giant platelets-Primary myelofibrosis, cellular phase E: Bizarre platelets-Essential thrombocythemia F: Giant platelets and a megakaryoblast -Acute megakaryoblastic leukemia G: Stripped megakaryocyticnucleus. H: Bernard-Souliersyndrome I: May-Hegglin anomaly- Large Döhle-like inclusions and giant platelets
- 64. SPURIOUS HIGH COUNTS •MAHA • Burns patient • Cryoglobulins • Bacterial overgrowth in stored sample
- 65. • Microcytic redcells –HbH • Fragmented red cells –TTP, DIC • Leukemic fragments, apoptotic cells -TLS • Cryoglobulinemia • Hyperlipidemia • Bacteria, fungi, parasitised red cells • Inadvertant heating of sample
- 66. HEMOPARASITES • THICKSMEARS • 2-3drops of blood • Increased sensitivity • Decreased specificity • All parasites extracellular • THINSMEARS • 1 drop of blood • Decreased sensitivity • Increased specificity • Intracellular forms also well made out
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- 70. Malaria other findings •Monocytosis • Pigments in monocytes, neutrophils - peripheral smear or marrow • Features of hemolysis
- 71. Wuchereria bancrofti-Microfilaria • Directwet mount with no staining • Giemsa or Leishman stained thick blood smear • Post DEC provocation test • Examine the extreme tail end of a smear under scanner view to screen
- 72. Features of awell stained smear • Macroscopically-Colour pink to purple • Microscopically- • RBCs-Orange to salmon pink • WBC-Nuclei-purple to blue Cytoplasm-pink to tan Granules –lilac to violet • Eosinophil Granules-Orange • Basophil Granules-Dark blue to black
- 73. Artefacts and Troubleshooting
- 74. Pale Staining • TooAcidic Stain: • RBC pale, WBC barely visible 1.Insufficient staining time 2.Prolonged buffering or washing 3.Old stain Correction: 1)Lengthen staining time 2)Check stain and buffer pH 3)Shorten buffering or wash time
- 75. Dark Staining • TooAlkaline Stain: • RBC gray, WBC too dark, Eo granules gray 1.Thick blood smear 2.Prolonged staining 3.Insufficient washing 4.Alkaline pH of stain components 5.Heparinized sample Correction : 1.Check pH 2.Shorten stain time 3.Prolong buffering time
- 76. Water Artifact • Motheaten RBC, •Heavily demarcated central pallor •Crenation •Refractory shiny blotches on the RBC What contributes to the problem: 1.Humidity in the air during air drying 2.Water absorbed from the humid air into the alcohol based stain Solution: 1.Drying the slide as quickly as possible. 2.Fix with pure anhydrous methanol before staining. 3.Use of 20% v/v methanol
- 77. Storage induced Artefacts •Loss of central pallor- simulate spherocytosis • Crenation or echinocytic changes in RBCs • Degeneration of neutrophils • Lobulation of some lymphocytic nuclei
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