(D) Lab Testing in Hematology

• Why Perform Manual Testing?

  • Proficiency samples
  • Testing location
  • Specimen type
  • Instrument problems
  • Specimen problems
• Tests That Can Be Performed Manually

  • MCV, MCH,MCHC (First three)
  • RBC count
  • Hemoglobin (HGB)
  • Hematocrit (HCT)
  • WBC count
  • PLT count
  • WBC Differential
• Leukocheck method

  • Consists of a reservoir and pipette with fixed volumes
  • Used with the Neubauer hemacytometer
• Dilutions


  • WBCs: 0.475 ml ammonium oxalate /25 ul pipette = 1:20 dilution
  • PLTs: 1.98 ml ammonium oxalate/20 ul pipette = 1:100 dilution
• Manual Cell Counts


  1. WBC Count = cells counted x dilution factor / area counted (mm2) x depth (mm)

  2. RBC Count = cells counted x 200 / 0.02mm3

  3. Platelet Count = cells counted x 100 / 0.1mm3

  4. WBC correction for nRBCs = WBC count x 100 / #nRBCs/100 WBCs + 100
• What is the Reticulocyte Count equation? used to assess? (4)

  • Used to assess erythropoietic activity of bone marrow
  • Whole blood in EDTA stained with supravital stain, e.g. new methylene blue
  • Retics are counted as both an RBC and a retic
  • % retics = # retics x 100 / 1000 (RBCs counted)
• Reticulocyte Counts: Miller Disc Method

  1. Reduces labor involved to count more RBCs needed for more accurate retic count

  2. Area of square B = 1/9 area of square A

  3. Count the # RBCs in square B (at least 112 which would be equivalent to 1008 in square A)

  4. Count the # retics in square A

  5. # retics = #retics in sq A x 100 / #RBCs in sq B x 9
• Reticulocyte Counts: Microhematocrit

  • To determine the percentage of whole blood that is composed of cellular components (mostly RBCs)
  • Supplies needed: Capillary tubes, Clay, Microcentrifuge, Reader, Patient whole blood
• Microhematocrit Procedure

  1. Specimen is collected in a capillary tube and sealed off on one side with clay

  2. The specimen is spun down in a microhematocrit centrifuge

  3. The specimen is read on the hematocrit reader in duplicate
• Erythrocyte Sedimentation Rate (ESR)


  • To detect inflammation. Does not indicate the source or the cause of the inflammation.
  • Anticoagulated blood is drawn up into a graded Westergren sedimentation tube. The blood is allowed to stand in the tube undisturbed for 1 hour. After 1 hour, the tube is read and that is the rate at which red cells settle out.
  • normal range vary between institutions. But in general a value <20mm/hr is considered normal, and values >20mm/hr should be further investigated
• Westergren Erythrocyte Sedimentation Rate

  • Increased in presence of increased serum proteins which can happen in response to almost anything
  • Directly proportional to RBC mass and inversely proportional to plasma viscosity
  • There is a LONG list of factors that affect the ESR
  • Should not be performed routinely
  • Results are mostly meaningless because it is so non-specific
• Complete Blood Count (CBC)


  • Analysis of the cellular components of blood is achieved through the complete blood count (CBC)
  • Enumeration, Morphology, and Distribution of all the cellular blood constituents
• What are the Components of the Complete Blood Count (CBC)

  • Erythrocytes (RBCs)
  • Leukocytes (WBCs)
  • Thrombocytes (PLTs)
• Erythrocytes (RBCs) Slide:Complete Blood Count, what is included in CBC


  • Red Blood Cell Count (RBC)
  • Hemoglobin (Hgb)
  • Hematocrit (Hct)
  • Mean Corpuscular Volume (MCV)
  • Mean Corpuscular Hemoglobin (MCH)
  • Mean Corpuscular Hemoglobin Content (MCHC)
  • Red Cell Distribution Width (RDW)
  • RBC morphology (part of diff)
  • Leukocytes (WBC) Total WBC Count
  • Thrombocytes (PLTs) Platelet Count and Mean Platelet Volume (MPV)
• MCV should be around 90 fl (range = 76-100 fl)
• MCH should be around 30 pg (range = 26-34 pg)
• MCHC should be around 33 g/dL (range 32-36 g/dL)
• MICROCYTIC (Mean Corpuscular Volume) Wintrobe Terminology


  MCV > 80 fl Means RBC size is smaller than usual
• NORMOCYTIC (Mean Corpuscular Volume) Wintrobe Terminology


  MCV 80-100 fl Means RBC is Normal size
• MACROCYTIC (Mean Corpuscular Volume) Wintrobe Terminology


  MCV < 100 fl Means RBC is larger than usual
• HYPOCHROMIC: Terminology for Mean Corpuscular Hemoglobin Concentration MCHC, Interpretation Indices.

  MCHC > 32%
• NORMOCHROMIC Terminology for Mean Corpuscular Hemoglobin Concentration MCHC, Interpretation Indices.

  MCHC 32-36%
• Clinical Correlation

  • NORMOCYTIC, NORMOCHROMIC: Hemoglobinopathies, Hemolytic Anemias
  • MICROCYTIC, HYPOCHROMIC: Thalassemias, Iron Deficiency Anemia
  • MACROCYTIC (NORMOCHROMIC): Megaloblastic Anemia, Pernicious Anemia
• Differential - Back to a Manual Method

  1. WBC Estimate: enumerate the average number of WBC in a peripheral blood smear and correlate it with the instrument counts

  2. 100 cell WBC differential/morphology: differentiate between the different WBC lineages and express them as percentages

  3. RBC morphology: identify and differentiate between morphologic changes of erythrocytes. Graded

  4. PLT morphology: identify and differentiate between morphologic changes of platelets. Graded

  5. PLT Estimate: enumerate the average number of PLTs in a peripheral blood smear and correlate it with instrument counts
• WBC Estimate: Manual Counting (3) about microscope, blood etc

  • Important to correlate amount of WBCs present in a peripheral blood smear with the instrument count
  • Done under a 40x High Dry objective, select 5 random fields, count WBCs, add them all and divide by 5, then multiply by 2000
  • Blood must be well mixed before making a smear to ensure a good dispersion of cells
• RBC Morphology -- Grading System

  • Cells seen 1-5 cells/hpf: Interpretation: Slight/few Grading: (1+)
  • Cells seen 6-15 cells/hpf: Interpretation: Moderate Grading: (2+)
  • Cells seen >15 cells/hpf: Interpretation: Many Grading: (3+)
  • High power Field
• RBC Morphology -- Nomenclature

  • Anisocytosis: Variations in size Measured by RDW (Normocytic, Microcytic, Macrocytic)
  • Poikilocytosis: Variations in shape (Spherocytes, Shistocytes, Acanthocytes, Echinocytes, Depanocytes, Elliptocytes, Codocytes, Dacryocytes, Stomaocytes, Bizzare shapes) (10)
  • Inclusions: Howell Jolly Bodies, Pappenheimer Bodies, Heinz Bodies, Cabot Rings, Basophilic Stippling, Parasites
• Platelet Phenomen: Explain Giant, Clumping, Satellitism

  • Giant Platelets: Young platelets, Poor division in the marrow, Abnormal division in the marrow
  • Platelet clumping: Poor specimen collection, Insufficient specimen collection
  • Platelet satellitism: Platelet response to anticoagulant (EDTA)
• Erythrocytes (RBCS) TESTING for Red Blood Cells

  Red Blood Cell (RBC): number of red blood cells
• Hemoglobin (Hgb)


  Concentration of hemoglobin in RBCs
• Hematocrit (Hct)


  Percentage of blood that is composed of blood cells (mostly RBCs)
• Mean Corpuscular Volume (MCV)


  Size of the RBC
• Mean Corpuscular Hemoglobin (MCH)


  Weight of Hemoglobin in RBC
• Mean Corpuscular Hemoglobin Concentration (MCHC)


  Percent of hemoglobin in RBCs
• Red cell distribution width (RDW)


  Quantified measure of the amount of variation in RBC morphology around a classification system
• Normocytic (MCV) Normochromic (MCHC) 


  MCV Normocytic(Normal) 76-100fl Normochromic (Normal) 32-36g/dL

  
• Microcytic (MVC) Hypochromic (MCHC)


  Microcytic less than 80fl, Hypochromic less than 32%

  
• Macrocytic(MCV) Normochromic (MCHC)

  Macrocytic: above 100fL Normochromic (Normal) 32-36g/dL

  
• Stain for WBC 


  Peroxidase (used by sysmex)
• Reticulocytes use what stain 


  Supravital stains (2) usual