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  • 1. Peripheral blood smear examination Dr Hemang Mendpara DNB pediatrics Choithram Hospital & Research Centre Indore www.dnbpediatrics.com
  • 2. • Hemogram: measured and calculated parameters • Histograms: size distribution of WBC, RBC and Plt • Cytogram: WBC differential CBC on automated analyzers Flagging for abnormalities necessitates a manual PBS reviewwww.dnbpediatrics.com
  • 3. A well made peripheral smear is thick at one end and progressively thinner at the opposite end. The "zone of morphology" (area of optimal thickness for light microscopic examination) should be at least 2 cm in length. The smear should occupy the central area of the slide and be margin-free at the edges www.dnbpediatrics.com
  • 4. Slide fixation and staining 1. Romanowsky staining Leishman's stain : a polychromatic stain •Methanol : fixes cells to slide •methylene blue stains RNA,DNA blue-grey color •Eosin stains hemoglobin, eosin granules orange-red color •pH value of phosphate buffer is very important www.dnbpediatrics.com
  • 5. PBS examination requires a systematic approach in order to gather all possible information. In addition, all specimens must be evaluated in the same manner, to assure that consistent information is obtained. www.dnbpediatrics.com
  • 6. • 1. Macroscopic view : quality of the smear • 2.The microscopic analysis • begins on lower power (10x), • to assess cellular distribution, staining quality, and to select an area where the RBCs are barely touching each other. •On hi-dry (40x), to obtain a WBC estimate. All of the detailed analysis of the cellular elements using high power or oil immersion. PBS examination - preliminary www.dnbpediatrics.com
  • 7. (a) Ten microscopic fields are examined in a vertical direction from bottom to top or top to bottom (b) slide is horizontally moved to the next field (c) Ten microscopic fields are counted vertically. (d) procedure is repeated until 100 WBCS have been counted (zig zag motion) Scanning technique for WBC differential count and morphologic evaluation www.dnbpediatrics.com
  • 8. 1. RBC • Size • Shape • Color • Arrangement • Inclusions • Young RBCs 2. WBC • Total counts • Differential counts • I:T ratio • Abnormal WBC 3. Platelets • Counts • Abnormality 4. Parasites Evaluation of PBS www.dnbpediatrics.com
  • 9. •A fairly accurate estimate of the WBC count (cells/mL) can be obtained by counting the total number of leukocytes in ten 40X microscopic fields, dividing the total by 10, and multiplying by 3000. These estimates should approximate that obtained by the cell analyzer. WBC estimation on peripheral smear www.dnbpediatrics.com
  • 10. Morphologic Evaluation of Red Blood Cells Biconcave disc Diameter : 7 ~ 8 μm Central pallor occupy 1/3 rd of total Size : approx. same as nucleus of mature lymphocyte www.dnbpediatrics.com
  • 11. Microcytic hypochromic red cells Decreased size and Hb content (MCH) and conc (MCHC). Expanded central zone of pallor Iron deficiency, thalasemia trait Anemia of chronic disease www.dnbpediatrics.com
  • 12. Iron deficiency anemia www.dnbpediatrics.com
  • 13. Thalassemia trait www.dnbpediatrics.com
  • 14. Megaloblastic anemia (PS) Macrocyte Large RBCs • size > 8.5 mm, • MCV > 95 fL • Normal MCH •Normal newborn •Chromosomal disorders (e.g., Trisomy 21) •Drug associated anticonvulsants, antidepressants, sulpha, chemotherapeutic agents, estrogen and antiretroviral agents) •Dyserythropoiesis •Myelodysplasia •Preleukemia •Hypothyroidism •Liver disease •Folate deficiency •BI2 deficiency www.dnbpediatrics.com
  • 15. Elliptocytes or ovalocytes Ovalocytes are due to abnormal membrane cytoskeleton found in hereditary elliptocytoisis www.dnbpediatrics.com
  • 16. seen when there is extramedullary erythropoiesis Tear drop cells / dacrocytes • Osteopetrosis • Myelofibrosis • Bone marrow infiltrated with hematological or non-hematological malignancies • Iron deficiency anemia • Pernicious anemia www.dnbpediatrics.com
  • 17. Polychromasia Blue-gray coloration of RBCS. Due RNA remnants Increased - Increased erythropoietic activity. Decreased - Hypoproliferative states. Hemolytic anemias •Blood loss anemias •Recovering anemia www.dnbpediatrics.com
  • 18. Sickle cell anemia Irregular, curved cells with pointed ends Hb S hemoglobinopathies (sickle cell anemia, Hb SC disease, Hb S-beta-thalassemia, Hb SD disease, hb Memphis /S disease) * Don’t be confused with fragmented RBC www.dnbpediatrics.com
  • 19. Spherocytosis Hereditary spherocytosis •ABO incompatibility •Autoimmune hemolytic anemia (warm antibody type) •Infections (e.g., EBV, CMV, E. coli, Sepsis/Urosepsis) •Severe burns •DIC and HUS www.dnbpediatrics.com
  • 20. Acanthocytes or spur cells, are spherical cells with blunt-tipped or club-shaped spicules of different lengths projecting from their surface at irregular intervals. Acanthocytes Acanthocytes are seen in •Hereditary abetalipoproteinemia •Hereditary acanthocytosis •End stage liver disease •Anorexia nervosa •Malnutrition •Post splenectomy •Intravenous hyperalimentation particularly with intralipid infusion www.dnbpediatrics.com
  • 21. Echinocytes "Sea urchin cells, crenated cells, burr cells" Post-splenectomy, uremia, hepatitis of the newborn, malabsorption states, after administration of heparin, pyruvate kinase def phosphoglycerate kinase deficiency, uremia, HUS. Crenated / Burr cells / Echinocytes (Echinocytes, or burr cells or crenated red cells, in contrast, have shorter, sharp to blunt spicules of uniform length which are more evenly spaced around their periphery). www.dnbpediatrics.com
  • 22. Mechanical damage to RBCs from fibrin deposits  DIC  MicroAngiopathic HA  TTP/HUS  prosthetic heart valves  severe valvular stenosis  malignant hypertension  March hemoglobinuria  myelofibrosis  hypersplenism Schistocyte – fragmented RBC  normal newborns  bleeding peptic ulcer  Aplastic Anemia  pyruvate kinase def  Vasculitis  Glomerulonephritis  renal graft rejection  severe burns  iron deficiency, thalassemia www.dnbpediatrics.com
  • 23. hemolyic anemias Hallmark: Presence of schistocytes , fragmented RBC www.dnbpediatrics.com
  • 24. Uniconcave RBC, slitlike area of central pallor Hereditary or acquired hemolysis. Hereditary stomatocytosis, alcoholic cirrhosis, acute alcoholism, obstructive liver disease, malignancy, severe infection, treated acute leukemia, artifact. Stomatocyte – fish mouth cell www.dnbpediatrics.com
  • 25. HA due to red cell enzyme defects – bite or blister cells • Glucose 6 phosphate dehydrogenase (G-6-PD) deficiency • Unstable hemoglobin variants • Congenital Heinz body anemia Suggest oxidative stress www.dnbpediatrics.com
  • 26. Target cell Peripheral rim of pallor surrounding central hyperchromia Target cells are commonly seen in •Hemoglobin C •Sickle cell disease •Hemoglobin E •Hemoglobin H disease •Thalassemias •Iron deficiency anemia •Liver disease •Target cells are seen with most of the hemoglobinopathies www.dnbpediatrics.com
  • 27. Irregular RBC agglutination/ clumping Anti-RBC antibody, paraprotein. Cold agglutinin disease, autoimmune hemolytic anemia, macroglobulinemia, hypergammaglobinemia RBC autoagglutination www.dnbpediatrics.com
  • 28. Roulex formation Seen in case of high level of fibrinogen, immunoglobulins, intra venous administration of plasma volume expanders like dextran www.dnbpediatrics.com
  • 29. • multiple blue-purple inclusions attached to the inner surface of the red cell membrane. visible in supravitally stained smears. • are precipitated normal or unstable hemoglobin usually secondary to oxidant stress. • G6PD deficiency • Unstable hemoglobinopathy • Cong. Bite cell anemia Heinz body www.dnbpediatrics.com
  • 30. Small (1 mm), round, dense, basophilic bodies in RBCs. Splenectomized patients, Functional asplenia, Anatomical absence of spleen Howell Jolly bodies Howell-Jolly bodies are small round bodies composed of DNA, about 1 µm in diameter, usually single and in the periphery of a red cell. They are readily visible on the Wright- Giemsa-stained smear. The spleen is responsible for the removal of nuclear material in the red cells, so in absence of a functional spleen, nuclear material is removed ineffectively. Howell-Jolly bodies are seen in •Post splenectomy •Functional asplenia •Anatomical absence of spleen www.dnbpediatrics.com
  • 31. Basophillic strippling • Lead poisoning • Iron deficiency anemia • Thalassemia Are abnormal aggregrates of ribosome and polyribosomes www.dnbpediatrics.com
  • 32. • Smaller then Howell jolly body • Stain with Prussian blue stain • Suggest iron over load www.dnbpediatrics.com
  • 33. WBC Morphology www.dnbpediatrics.com
  • 34. www.dnbpediatrics.com
  • 35. Manual differential counts • These counts are done in the same area as WBC and platelet estimates with the red cells barely touching. • This takes place under 100 (oil) using the zigzag method. • Count 100 WBCs including all cell lines from immature to mature. Reporting results • Absolute number of cells/µl = % of cell type in differential x white cell count www.dnbpediatrics.com
  • 36. •If 10 or more nucleated RBC's (NRBC) are seen, correct the • White Count using this formula: •Corrected WBC Count = WBC x 100/( NRBC + 100) •Example : If WBC = 5000 and 10 NRBCs have been counted •Then 5,000 100/110 = 4545.50 •The corrected white count is 4545.50 www.dnbpediatrics.com
  • 37. • Left-shift: non-segmented neutrophil > 5% – Increased bands Means acute infection, usually bacterial www.dnbpediatrics.com
  • 38. • Basophils are increased in the blood in – Myeloproliferative disorders (e.g., chronic myelogenous leukemia) – Hypersensitivity reactions – Mastocytosis – Xeroderma pigmentosa – Hypothyroidism www.dnbpediatrics.com
  • 39. • Morphologically abnormal eosinophils are seen in – Myelodysplastic syndrome – Megaloblastic anemias • Eosinophils are increased in the following conditions:  Allergies  Parasitic infestations  Infections  Acute leukemia  Myeloproliferative diseases  Hypereosinophilic syndrome  Drug-associated www.dnbpediatrics.com
  • 40. Band cells www.dnbpediatrics.com
  • 41. Leukemic myeloblast www.dnbpediatrics.com
  • 42. Leukemic myeloblast stained with peroxidase Note the AUER ROD www.dnbpediatrics.com
  • 43. Burkitt lymphoma www.dnbpediatrics.com
  • 44. Large, coarse, dark purple, azurophilic granules that occur in the cytoplasm of most granulocytes. These are characteristically found in the Alder-Reilly anomaly and in patients with mucopolysaccharidoses Alder-Reilly anomaly www.dnbpediatrics.com
  • 45. Chédiak-Higashi granules are very large red or blue granules that appear in the cytoplasm of granulocytes, lymphocytes, or monocytes in patients with the Chédiak- Steinbrinck-Higashi syndrome. It is a rare autosomal recessive disorder Chédiak-Higashi www.dnbpediatrics.com
  • 46. Variably sized (0.1 to 2.0 um) and shaped, blue or grayish- blue cytoplasmic inclusions usually found near the periphery of the cell. Dohle bodies are lamellar aggregates of rough endoplasmic reticulum, which appear in the neutrophils, bands, and metamyelocytes of patients with infection, burns, uncomplicated pregnancy, toxic states, or during treatment with hematologic growth factors - G-CSF. Döhle bodies www.dnbpediatrics.com
  • 47. May-Hegglin anomaly Neutrophils contain small basophilic cytoplasmic granules which represent aggregated ribosomes. Leukopenia and large platelets are also found. An autosomal dominant trait, the May-Hegglin anomaly is associated with a mild bleeding tendency, but not by an increased susceptibility to infection www.dnbpediatrics.com
  • 48. Neutrophilic toxic granulation Small dark blue to purple granules resembling primary granules in the cytoplasm of metamyelocytes, bands, and segmented neutrophils during inflammatory states, burns, and trauma, and upon exposure to hematopoietic growth factors. It is usually accompanied by a shift to the left and vacuolations in the cytoplasm (toxic vacuolations) and Dohle bodies. www.dnbpediatrics.com
  • 49. Platelets Neubars chamber : count platelets in 64 small squares Counts * 250 = total platelets Normal counts 4.5 to 5.5 lakh Common Causes of Thrombocytopenia •Decreased production −Aplastic anemia −Acute leukemia −Viral infections *Parvovirus *CMV −Amegakaryocytic thrombocytopenia (AMT) •Increased destruction −Immune thrombocytopenia *Idiopathic thrombocytopenic purpura (ITP) *Neonatal alloimmune thrombocytopenia (NAITP) −Disseminated intravascular coagulation (DIC) −Hypersplenism Thrombocytosis • Reactive thrombocytosis  Post infection  Inflammation  Juvenile rheumatoid arthritis  Collagen vasvular disease • Essential thrombocythemia www.dnbpediatrics.com
  • 50. www.dnbpediatrics.com
  • 51. :Giant plateletsPlatelet morphology www.dnbpediatrics.com
  • 52. Platelet satellitism www.dnbpediatrics.com
  • 53. Macrocytosis with giant platelets (MDS, 5q- syndrome) www.dnbpediatrics.com
  • 54. Disadvantages of the Peripheral Blood Smear Provides information that cannot be obtained from automated cell counting. However, some limitations are: • Experience is required to make technically adequate smears. • There is a non-uniform distribution of white blood cells over the smear, with larger leukocytes concentrated near the edges and lymphocytes scattered throughout. • There is a non-uniform distribution of RBCs over the smear, with small crowded red blood cells at the thick edge and large flat red blood cells without central pallor at the feathered edge www.dnbpediatrics.com
  • 55. Merozoits www.dnbpediatrics.com
  • 56. Schizonts are commonly seen in P. vivax infection and appear as large bodies containing 12 to 24 nuclei and a loose pigmented body. This photograph shows an early schizont of P. vivax on the left and mature schizonts www.dnbpediatrics.com
  • 57. Schuffer’s dots seen in plasmodium vivex www.dnbpediatrics.com
  • 58. Cresent shaped gametocyte charectaristiclly seen in p.falciparum malaria www.dnbpediatrics.com
  • 59. www.dnbpediatrics.com
  • 60. www.dnbpediatrics.com
  • 61. Eucheria bancrofti www.dnbpediatrics.com
  • 62. www.dnbpediatrics.com
  • 63. www.dnbpediatrics.com
  • 64. OSMOTIC FRAGILITY TEST • Defination: • it is a test that measures the resistance to hemolysis of red blood cells (RBC) by osmotic stress created by hypotonic solutions • RBC are exposed to a series of saline (NaCl) solutions with increasing dilution • The sooner hemolysis occurs, the greater is osmotic fragility of RBC www.dnbpediatrics.com
  • 65. • Isotonic (physiological) solution – 0.9 % NaCl • RBC burst in hypotonic (< 0.9 % NaCl), and shrink (crenate) in hypertonic solutions (> 0.9 % NaCl) • Red cells are suspended in a series of tubes containing hypotonic solutions from 0.9 to 0 % NaCl. Degree of hemolysis measured for each NaCl concentration. www.dnbpediatrics.com
  • 66. • NORMAL RANGE: • - hemolysis onset at: 0.45-0.5 % NaCl • - hemolysis complete at: 0.3-0.33 % NaCl • FACTORS AFFECTING OSMOTIC FRAGILITY • - cell membrane permeability • - surface-to-volume ratio www.dnbpediatrics.com
  • 67. - Hereditary spherocytosis - Acquired spherocytosis - Hemolytic anemia (HDN) - Malaria - Severe pyruvate kinase deficiency www.dnbpediatrics.com
  • 68. www.dnbpediatrics.com
  • 69. • Thalassemia • Sickle cell anemia (hemoglobinopathy) • Iron deficiency anemia • Asplenia • Liver disease www.dnbpediatrics.com
  • 70. www.dnbpediatrics.com
  • 71. www.dnbpediatrics.com
  • 72. introduction www.dnbpediatrics.com
  • 73. Principle of test • Deoxygenated Hb-S is insoluble in the presence of a concentrated phosphate buffer solution and forms a turbid suspension that can be easily visualized. • Normal Hemoglobin A and other hemoglobins remain in solution under these conditions. These different qualitative outcomes allow for the detection of sickle cell disease and its traits. • SICKLEDEX® uses Saponin to lyse the red blood cells. Sodium Hydrosulfite then reduces the released hemoglobin. Reduced Hb-S is insoluble in the concentrated phosphate buffer and forms a cloudy, turbid suspension. Thus give a positive result. www.dnbpediatrics.com
  • 74. Procedure • 1. sodium diethanoid 200mg+10 ml distilled water • 2. sickling buffer solutions • Take 2 part of 1st solution and 3 part of 2nd solution • Have one drop of blood on slide and put single drop of mixed solution • Wait for 30 mins • Watch under microscopewww.dnbpediatrics.com
  • 75. Result www.dnbpediatrics.com
  • 76. www.dnbpediatrics.com

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