TESTING IN HEMA

• Reticulocyte Count


  A test that measures the number of immature red blood cells in the blood
• Principle


  1. Manual, Semiautomated, and Point-of-Care Testing in Hematology

  2. Modified Westergren Erythrocyte Sedimentation Rate

  3. Wintrobe Erythrocyte Sedimentation Rate

  4. Absolute Reticulocyte Count

  5. Corrected Reticulocyte Count

  6. Reticulocyte Production Index

  7. Automated Reticulocyte Counts

  8. Erythrocyte Sedimentation Rate

  9. Principle

  10. Disposable Kits

  11. Automated Erythrocyte Sedimentation Rate

  12. Additional Tests

  13. Point-of-Care Testing

  14. Point-of-Care Tests
• After studying the material in this chapter, the reader should be able to respond to the following case studies:
• Normocytic, normochromic red blood cells (RBCs)

  Red blood cells that are normal in size and hemoglobin content
• Rule of three


  A method to estimate the hematocrit value based on the hemoglobin concentration
• Hemoglobin concentration is falsely elevated

  Hematocrit is falsely low
• Correcting for interferences


  1. Identify the cause

  2. Take appropriate action to correct the issue
• Calculating red blood cell indices

  Use the provided RBC count, hemoglobin, and hematocrit values
• Red blood cell volume and hemoglobin concentration


  Described based on the calculated red blood cell indices
• Verifying the red blood cell volume and hemoglobin concentration


  Examine the peripheral blood film
• Conductivity method


  A point-of-care technique to measure the hematocrit
• Electrolyte concentration


  Can affect the hematocrit value
• Electrolyte concentration


  Falsely decreases or increases the hematocrit value
• Other factors


  Can decrease the hematocrit value using the point-of-care device
• Collection tubes must be filled to the proper level to maintain an appropriate blood-to-anticoagulant ratio
• Specimens for CBC testing should be analyzed within 6 hours of collection if stored at room temperature, and 24 hours if stored at 4°C
• Peripheral blood films should be prepared within 3 hours of collection to reduce cell deterioration and morphology artifacts
• Standard precautions and appropriate safety protocols must be followed for specimen collection and handling
• Published protocols for specimen collection are critically important for providing accurate laboratory test information
• Manual cell counts


  1. Use a hemacytometer and manual dilutions

  2. Principle is the same for WBCs, RBCs, and platelets

  3. Dilution, diluting fluid, and area counted vary
• Hemacytometer


  A counting chamber used for manual cell counts
• Levy chamber with improved Neubauer ruling


  The most common type of hemacytometer
• Calculating manual cell counts

  1. Total count = cells counted x dilution factor / area (mm²) x depth (0.1)

  2. Total count = cells counted x dilution factor x 10 / area (mm²)
• White blood cell (WBC) count

  1. Dilute blood 1:20 with 1% buffered ammonium oxalate or weak acid

  2. Count cells in 4 large corner squares of hemacytometer

  3. Calculate WBC count using the formula
• The difference between cell counts on the two sides of the hemacytometer should be less than 10%
• A moist chamber is used to allow the cells to settle before counting
• The four large squares on one side of the chamber yield counts of 23, 26, 22, and 21. The total count is 92. The four large squares on the other side of the chamber yield counts of 28, 24, 22, and 26. The total count is 100. The difference between sides is less than 10%
• The average number of cells of the two sides of the chamber is 96
• WBC count calculation


  Cells counted x dilution factor / area counted (mm²) x depth
• Using the average of 96 cells, the WBC count is 4800/mm3 or 4800/μL or 4.8 x 103/μL or 4.8 × 109/L
• Alternative WBC count calculation

  Cells counted x dilution factor / area counted (mm²) x depth
• Using an average of 54 cells counted in the entire counting area on both sides, the WBC count is 6000/mm3 or 6000/μL or 6.0 × 10³/μL or 6.0 × 10°/L
• General reference intervals for males and females in different age groups can be found on the inside front cover of this text. Reference intervals may vary slightly according to the population tested and should be established for each laboratory
• Sources of error and comments for manual WBC count

  • The hemacytometer and coverslip should be cleaned properly
  • The diluting fluid should be free of contaminants
  • If the count is low, a greater area may be counted to improve accuracy
  • The chamber must be charged properly
  • Allow the cells to settle for 10 minutes before counting
  • Nucleated red blood cells (NRBCs) present must be corrected for
  • The accuracy can be assessed by performing a WBC estimate on a Wright-stained peripheral blood film
• Platelet count


  The number of platelets in 1 L or 1 μL of whole blood
• Platelet count procedure


  1. Make a 1:100 dilution

  2. Mix the dilution and charge the chamber

  3. Allow the platelets to settle for 15 minutes

  4. Count the platelets in the 25 small squares in the center square

  5. Calculate the platelet count using the formula
• If 200 platelets were counted in the entire center square, the platelet count is 200,000/mm³ or 200,000/μL or 200 x 10³/μL or 200 × 10⁹/L
• Sources of error and comments for manual platelet count

  • Inadequate mixing and poor collection can cause platelets to clump
  • Dirt in the pipette, hemacytometer, or diluting fluid may cause inaccurate counts
  • If fewer than 50 platelets are counted, repeat with 1:20 dilution
  • If more than 500 platelets are counted, use a 1:200 dilution
  • Platelet satellitosis may occur with EDTA anticoagulant
• Manual RBC counts are rarely performed because of the inaccuracy of the count and questionable necessity
• Disposable blood cell count dilution systems

  Capillary pipette and diluent reservoir systems commercially available for WBC and platelet counts