Peripheral smear – RBC

PERIPHERAL SMEAR – RBC

 

  • What are the uses of peripheral smear examination
    • Peripheral smear provides information regarding
      • morphology of RBC’s (size, shape and appearance) to evaluate anemia
      • Differential leukocyte count
      • identification of abnormal cells (immature cells)
      • Morphology of platelets and platelet count 
      • Presence of parasites 

 

  • What is the normal RBC morphology
    • RBC’s are flexible oval biconcave discs without nucleus
    • They have peripheral hemoglobin and central pallor of less than 1/3rd of total volume of RBC
    • Measurements
      • Diameter – 7- 8µm
      • Maximum thickness of 2–2.5 µm and a minimum thickness in the centre of 0.8–1 µm
      • Volume – 80 – 100fL

 

  • What is Anisocytosis
    • Smears with RBC’s of different sizes both microcytes (MCV<80ft) and macrocytes(MCV>100ft)

 

  • What is poikilocytosis?
    • Variation in shape of RBC’s is called poikilocytosis

 

  • Mention the conditions of microcytic hypochronic anaemia 
    •  Size of RBC’s will be – less than 7µm
    • MCV – less than 80fL
    • PCV, MCH and MCHC reduced below normal limits
    • RBC’s appear small and pale with increased central pallor (hypochromic
    • Seen in conditions like
      • Iron deficiency anemia
      • Thalassemia

 

  • How to differentiate iron deficiency anemia and thalassemia
    • In iron deficiency anema, serum ferritin is very low and B.M iron is nil while in thalassemia serum ferritin is very high, B.M iron is increased

 

  • Conditions with microcytic normochromic anemia
    • MCV and MCH – reduced
    • MCHC – normal
    • Conditions associated 
      • Anemias of chronic disease

 

  • In which conditions macrocytic normochromic anemia is seen
    • Size of RBC – more than 8µm
    • MCV is elevated – morethan 100fL
    • MCH – is elevated
    • MCHC – normal
    • Conditions associated – 
      • Vitamin B12 or folic acid deficiency

 

  • What is dimorphic anaemia and mention the conditions
    • Presence of two populations of cells like normocyte and microcytes, or microcytes and macrocytes
    • Conditions associated are
      • After iron therapy (normocytes and microcytes)
      • After blood transfusion (normocytes and microcytes)
      • Combined deficiency of vitamin B12, folic acid and iron deficiency (microcytes and macrocytes)

 

  • Name the conditions in which macrocytes appear in peripheral smear
    • Megaloblastic anaemia
    •  Myelodysplastic syndrome
    • Alcoholism
    • Liver disease

 

  • What are polychromatophils
    • Polychromasia means RBC’s stained in many colors
    • These cells are larger than mature RBC and have bluish grey tint on leishmans stain.
    • These are usually reticulocytes

 

  • What are red cell inclusions
    • Howel Jolly bodies –
      • These are nuclear chromatin remnants which appear as small rounded structures.
      • Seen in megaloblastic anemia, hemolytic anemia and after splenectomy
    • Pappenheimer bodies –
      • abnormal ferritin aggregates in RBC appearing as basophilic granules at the periphery of the cell.
      • Seen in sickle cell anemia, Sideroblastic anemia, Myelodysplastic syndrome, hemolytic anemia and lead poisoning
    • Cabot rings –
      • They are threadlike, red to violet rings or “figure 8” shaped RBC inclusion.
      • They are remnants of the mitotic spindle
      • Seen in myelodysplasia, megaloblastic anemia, medication effect,and in dyserythropoiesis.
    • Basophilic stippling –
      • also called punctate basophilia is due to precipitation of RNA in RBC in which numerous basophilic granules are distributed through out the cell.
      • Presence of basophilic stippling indicates disturbed erythrypoiesis. Basophilic granules are negative with pearls stain incOntrast to pappenheimer body
      • Seen in conditions like thalassemia, megaloblastic anemia, infections and liver disease
    • Heinz bodies –
      • are inclusions within RBC which are composed of denatured hemoglobin.
      • They cannot be seen in routine blood staining techniques, but can be seen with reticulocyte staining

 

  • What is reticulocyte? What stain is used to demonstrate reticulocytes?
    • They are juvenile RBC’s which contain remnants of ribosomes and RNA
    • Stain used to demonstrate reticulocytes are Brilliant Cresyl blue or New methylene blue

 

  • What is another name for sickle cells?
    • Drepanocytes

 

  • What is the difference between intravascular and extravascular hemolysis?
    • In extravascular hemolysis , e.g in thalassemia and sickle cell anemia red cells are lysed by the reticulonedothelial cells of spleen and other reticuloendothelial organs
    • In intravascular hemolysis, red cells in circulation are lysed resulting in hemoglobinemia and hemoglobinuria e.g in PNH and PCH.

 

  • What anemias are included in deficiency anemia
    • Anemias occuring due to deficiency iron, folic acid, B12,  vitamin C, protein and zinc

 

  • Which parasites cause deficiency anemias
    • Hook worm – causes iron deficiency anemias
    • Diphyllobothrium latum – B12 deficiency

 

  • What is aplastic anemia?
    • Aplastic anemia is a condition where there is bone marrow failure of producing sufficient number of blood cells due to decrease in hematopoietic precursors. It can be acquired or inherited.

 

  • What is confirmatory test for diagnosing sickle cell anemia
    • Sickle cell anemia can be diagnosed by Sickling test
    • Sickling of RBC’s develop under low oxygen tension
    • Sickling depends upon amount of HbS and degree of low oxygen ension
    • SIckling develops within 1/2 hour in Sickle cell disease where as with in 4 hours in trait

 

  • What is the method of sickling test
    • Prepare 2% of sodium metabisulfite solution
    • Take 2 slides and mark them as control and test and keep one drop of solution on both slides
    • Add 1/2 drop of patients blood to the test slide and normal blood to the control slide
    • mix the solutions
    • Put a cover slip on each slide and seal the edges with DPX
    • Examine the slide immediately and atimely intervals  i,e. 15min, 1/2 hour, 1 hour, 2 hours, 4 hours and 12 hours for development of sickle cells

 

  • What other tests (except sickling test) can be done to diagnose sickle cell disase
    • Hb electrophoresis can be used
    • During electrophoresis at pH 8.6 HbS interacts with reducing agent in the gel and forms sickle cells which are inflexible and cannot move through the gel. Hence they are trapped in gel and form red line
    • Whereas normal Hb forms slowly moving Hb band

 

  • How does spherocyte appear in blood
    • Spherocytes are smaller than RBC, and are binconvex with no central pallor

 

  • What are the causes of spherocytosis
    • Herditary spherocytosis
    • Autoimmune hemolytic anemia
    • Hemolytic episode in G-6-PD

 

  • What test is used to demonstrate the spherocytosis
    • Osmotic fragility test – SPherocytes are osmotically fragile ( target cells are osmotically resistant)

 

  • What is osmotic fragility test
    • This test is used for measuring erythrocyte resistance to hemolysis when exposed to various levels of dilutions of saline solution
    • When erythrocytes are exposed to a hypotonic solution,  water enters the cell causing swelling and further lysis
    • Susceptibility of osmotic lysis of erythrocytes is a function of surface area to volume ratio.

 

  • Describe the Osmotic fragility test
    • Principle- This test measures the ability of red cells to swell in different grades hypotonic solution. 
    • Reagents Buffered stock solution of buffered sodium chloride which is osmotically equivalent to 10% NaCl ia prepared
    • preparation of stock solution (10% NaCl)
      • NaCl – 90 gm
      • Na2HPO4 – 13.65gm
      • Na2HPO4 ,2H2O – 2.43gm
      • Distilled water – 1 litre
    • Take 10 test tubes and add 1% buffered saline solution, distilled water and NaCl solution in different grades as described below

    • Add 0.1ml of blood to each test tube and mixed well. This is allowed to stand for 30 minutes at room temperature 
    • Again mix and centrifuge fro 5 minutes at 2000rpm
    • Amount of RBC lysis in each test tube is measured by using colorimetre
    • The results of hemolysis are plotted against concentration of saline and osmotic fragility curve will be obtained
    • Interpretation of result
      • When RBC’s are normal hemolysis begins at 0.5% of saline and 100% lysis occurs at 0.3% of saline solution
      • In increased osmotic fragility lysis begins at 0.75% saline and complete lysis occurs at 0.45% saline  

 

  • Mention the conditions with increased and decresased osmotic fragility
    • Conditions associated with increased osmotic fragility
      • Hereditary spherocytosis
      • Severe burns
      • Poisoning
    • Conditions associated with decreased osmotic fragility
      • Iron deficiency anemia
      • Thalassemias
      • Sickle cell anemia

 

  • How to differentiate between the acquired spherocytosis and hereditary spherocytosis
    • Osmotic fragility curve shows shift to right in both types of spherocytosis
    • Coomb’s test is positive in acquired spherocytosis

 

  • What is the use of Coomb’s test
    • In immune hemolytic anemias, RBC are lysed from the binding of autoantibodies or complement components on the RBCmembrane
    • IgM antibodies cause agglutination and lysis at room temperature where as IgG antibodies do not agglutinate
    • Direct Coom’s test demonstrate antibodies present on the RBC membrane Eg. Examination of cord blood in erythroblastosis foetalis
    • Indirect Coom’s test detects serum antibodies capable of attaching to normal RBC Eg. Autoimmune hemolytic anemia
References
  1. P.Chakraborthy, Gargi Chakraborthy. Practical Pathology
  2. S.Sanyal. Pathology Practical book, 4th edition
  3. Dr .Tejindar Singh,  Dr .K. Uma  Chaturvedi.Practical pathology for undergraduate and post graduate students-First edition
  4. Ganga S. pilli. Viva in pathology, First edition

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