ROUTINE HEMATOLOGIC TESTS

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  • ROUTINE HEMATOLOGIC TESTS
    • Manual
    • Semi-automated
    • Automated
  • LEUKOCYTE DIFFERENTIAL COUNT
    • A stained smear is examined to determine the percentage of each type of leukocyte present
    • Two categories of WBCs:
    • Typical
    • Atypical
  • Typical
    • refers to WBCs with normal morphology
  • Atypical
    • includes immature cells that are indicative of hematologic diseases
  • LEUKOCYTE DIFFERENTIAL COUNT
    • A stained smear is examined to determine the percentage of each type of leukocyte present
    • Two categories of WBCs:
    • Typical
    • Atypical
    • Specimen: Peripheral Blood, bone marrow, or body fluid sediments.
    • reference values differ between specimens

    • 8 to 20 platelets should be present per oil immersion field.
    • Examine at least 10 different fields, the release the estimated total platelet concentration using Average platelets per field x 20,000
  • Differential count is performed to correlate, study, and analyze the machine-generated results that require confirmation, especially when flagging occurs.
  • Flagging refers to abnormalities detected by the machine, where it either fails to read the specimen and produce results or generates inaccurate results.
  • Nucleated erythrocytes (nRBCs)
    • are recorded separately per 100 WBCs but not included in the total WBC count
    • are immature erythrocytes that are larger in size and can be miscounted as WBCs due to their nucleus, potentially leading to a false increase in the WBC count.
  • LEUKOCYTE DIFFERENTIAL COUNT
    • Count at least 100 leukocytes and express results as a percentage of total leukocytes counted.
  • Distinguishing Factors of WBCs:
  • Reference Ranges:
  • RED BLOOD CELL COUNT
    • Principle: A specimen containing formed cellular elements, such as erythrocytes, is diluted in an isotonic fluid (maintains the shape and morphology of RBCs) to facilitate enumeration.
    • RBCs shrink in Hypertonic solutions.
    • Specimen: Anticoagulated whole blood or capillary blood can be used. EDTA is the preferred anticoagulant.
    • Diluents:
    • Gower’s Solution
    • Hayem’s Solution
    • Dacie’s Fluid
    • Normal Saline (0.85% NaCl)
    • Standard Dilution: 1:100 (Blood-todiluent).
    • RBCs are counted in five small squares of the large central square (0.2 mm2).
    • RBC Pipette:
    • 0.5 mark for blood and 101 mark for diluent.
    • Bulb with a red bead for mixing.
  • LEUKOCYTE COUNT
    • Principle: WBC (leukocyte) count represents the number of WBCs per liter (L) or milliliter (mL) of blood.
    • Specimen: Whole blood anticoagulated with EDTA or capillary blood.
    • Diluents: The purpose is to lyse RBCs to prevent them from interfering with the manual WBC count.
    • 1% buffered ammonium oxalate.
    • Weak acid solutions (3% acetic acid or 1% hydrochloric acid).
    • Standard Dilution: 1:20 (Blood-todiluent).
    • WBCs are counted in the four large corner squares (4 mm2).
    • WBC Pipette:
    • 0.5 mark for blood, and 11 mark for diluent.
    • Bulb with white bead for mixing.
  • PLATELET COUNT
    • Principle: Platelet count measures the number of PLTs per liter (L) or milliliter (mL) while of blood.
    • Specimen: Whole blood anticoagulated with EDTA.
    • Diluents:
    • 1% ammonium oxalate.
    • Standard Dilution: 1:100 (Blood-todiluent).
    • PLTs are counted in the 25 small squares within the large center square (1 mm2).
  • HEMACYTOMETER
    • Levy chamber with improved Neubauer ruling.
    • Two raised surfaces.
    • Each with a 3 mm x 3 mm square counting area (total area 9 mm2).
    • Raised surfaces are separated by an Hshaped moat.
    • Coverslip is placed on top of the counting surfaces.
  • CALCULATIONS FOR CELL COUNTING
    • One mm3 is equivalent to 1 microliter (ul).
    • The count per ml is converted to the count per liter (L) by multiplying by a factor of 106.
  • for cell counting
    A) 1% ammonium oxalate
    B) 3% acetic acid
    C) 1% hydrochloric acid
    D) Isotonic saline
    E) 1% ammonium oxalat
    F) 1:20
    G) 1:100
    H) 1:100
    I) 1:100
    J) 10X
    K) 10X
    L) 40X
    M) 40X
    N) 4 mm2
    O) 9 mm2
    P) 0.2 mm2
    Q) 1 mm2
  • HEMOGLOBIN CONCENTRATION
    • Hemoglobin within RBCs primarily functions to:
    • Transport oxygen to tissues.
    • Carry carbon dioxide away from tissues.
    • Reference Method: CYANMETHEMOGLOBIN (HEMOGLOBINCYANIDE) METHOD
    • Principle of Cyanmethemoglobin Method:
    • Blood is diluted in an alkaline Drabkin solution (stored in amber/brown bottle) containing:
    • Potassium ferricyanide (K3Fe(CN)6)
    • Potassium cyanide (KCN)
    • Sodium bicarbonate
    • Surfactant
  • HEMOGLOBIN CONCENTRATION
    • Chemical Reactions:
    • Potassium ferricyanide oxidizes hemoglobin (Fe2+) to methemoglobin (Fe3+).
    • Potassium cyanide then converts methemoglobin to cyanmethemoglobin:
    • Measurement:
    • Cyanmethemoglobin absorbs light at 540 nm, with absorbance directly proportional to hemoglobin concentration through spectrophotometry
  • HEMOGLOBIN CONCENTRATION
    • Limitations:
    • Sulfhemoglobin is not converted to cyanmethemoglobin and cannot be measured using this method.
    • Sulfhemoglobin fractions above 0.05 g/dL are rarely encountered in clinical practice.
  • SOURCES OF ERROR
    • Light Sensitivity
    • Cyanmethemoglobin reagent is light sensitive and should be stored in a brown bottle or dark place.
    • Turbidity due to High WBC or PLT Count
    • A high WBC count (>20 x 109 /L) or high PLT count(>700 x 109 /L) can cause turbidity and falsely high results.
    • Solution: Centrifuge the reagent specimen solution and measure the supernatant.
  • MICROHEMATOCRIT DETERMINATION
    • Packed Cell Volume (PCV) measures the ratio of RBC volume to whole blood volume in a capillary or venous blood sample.
    • Clinical Uses of PCV:
    • Detects anemia, polycythemia, hemodilution, and hemoconcentration.
    • Used with the erythrocyte count to calculate Mean Corpuscular Volume (MCV).
    • Used with hemoglobin concentration to calculate Mean Corpuscular Hemoglobin Concentration (MCHC)
  • RULE OF THREE
    • Quick visual check used when analyzing hemoglobin (HGB) and hematocrit (HCT) results.
    • Performed when there are discrepancies or abnormalities in the results that do not align with those provided by the machine. •
    • Applies only to normocytic, normochromic RBCs.
    • Formula:
    Hematocrit (HCT) = Hemoglobin (HGB) x 3 [±] (or HGB x 3 = HCT ± 0.03 L/L)
    • RBC x 3 = HCT
    • HCT x 3 = HGB
    • A discrepancy may indicate abnormal RBC morphology or analytical error.
    • Not applicable and not used as a basis for HCT and HGB results.
  • RED BLOOD CELL INDICES
    • RBC indices include:
    • Mean Cell Volume (MCV)
    • Mean Cell Hemoglobin (MCH)
    • Mean Cell Hemoglobin Concentration (MCHC).
    • Calculated parameters to determine the average RBC volume, hemoglobin content, and hemoglobin concentration.
    • Used for:
    • Quality control check.
    • Initial classification of anemias
  • MEAN CELL VOLUME
    • Average volume of the RBC.
    • Femtoliters (fL) or 10^-15 L
    • An MCV of less than 80 fL may indicate microcytosis, while an MCV greater than 100 fL indicates macrocytosis.
  • MEAN CELL HEMOGLOBIN
    • Average weight of hemoglobin in all RBCs.
    • Picograms (pg) or 10^-12 g.
  • MEAN CELL HEMOGLOBIN CONCENTRATION
    • Average concentration of hemoglobin in each individual RBC.
    • Grams per deciliter (g/dL) or prev. %.
    • An MCHC of less than 32 g/dL indicates hypochromic RBCs, while an MCHC greater than 36 g/dL indicates hyperchromic RBCs.
  • RETICULOCYTE COUNT: NEW METHYLENE BLUE METHOD
    • Reticulocyte:
    • Last immature stage of RBCs.
    • 2 days in the bone marrow and 1 day in PBS.
    • Contain remnant cytoplasmic RNA, mitochondria, and ribosomes. o
    • Used to assess erythropoietic activity in the bone marrow.
    • Not seen in Wright-Giemsa stain.
    • 0.5 – 2.5 % in PBS.
  • RETICULOCYTE COUNT: NEW METHYLENE BLUE METHOD
    Principle:
    • Sample: Whole blood anticoagulated with EDTA.
    • Viability of EDTA is 2 hours, once it goes beyond it is non-viable because of biological changes.
    • Stain: Supravital stain (e.g., new methylene blue).
    • Procedure:
    • One drop of blood is added to a test tube along with one drop of supravital stain, then mixed, set aside for 10 minutes, incubated, and smeared onto a glass slide.
    • This process allows the supravital stain to penetrate the cells. RBCs that still contain mitochondria, ribosomes, and RNA will show blue-stained granulo-filamentous structures.
  • RETICULOCYTE COUNT: NEW METHYLENE BLUE METHOD
    Calculation:
    • Count 1000 RBCs under the oil immersion objective lens (1000 x total magnification).
    • Reticulocytes are included in the total RBC count.
  • MILLER DISC
    • Improve precision in reticulocyte counting by reducing the laborintensive process of counting large number of RBCs.
    • Structure:
    • The disc has two squares:
    • Smaller square: 1/9 the area of the larger square, used for counting RBCs.
    • Larger square: Used for reticulocyte counting.
  • MILLER DISC
    Procedure:
    • The Miller disc is inserted into the microscope eyepiece.
    • RBCs are counted in the smaller square.
    • Reticulocytes are counted in the large square.
    • Minimum Cell Count:
    • At least 112 RBCs must be counted in the smaller square.
    • This is equivalent to 1000 RBCs in the larger square, meeting CAP (College of American Pathologists) hematology standards for manual reticulocyte count (>/= 1000 RBCs).
  • ABSOLUTE RETICULOCYTE COUNT
    • Principle: The absolute reticulocyte count (ARC) is the actual number of reticulocytes in 1 L or 1 mL of blood.
  • CORRECTED RETICULOCYTE COUNT
    • Principle: Applied for samples with low hematocrit, which leads to falsely elevated count.
    • 45 is the representation of normal average hematocrit.
  • RETICULOCYTE PRODUCTION INDEX
    • Principle: Shift reticulocytes are release of prematurely from the bone marrow.
    • Shift reticulocytes take up 2 -3 days to mature.
  • ERYTHROCYTE SEDIMENATION
    • Principle: Anticoagulated blood (3.8 Sodium Citrate) is allowed to stand at RT undistributed for 1 hour and RBC fall distance is measured in millimeters.
    • Normal RBCs have a relatively small mass and settle slowly.
    • Methods:
    • Modified Westergren Erythrocyte Sedimentation Rate
    • Wintrobe Erythrocyte Sedimentation Rate
  • Modified Westergren ESR
    Column Height: 200 mm (taller column); more column height, the more ESR is measured.
    Bore Diameter: 2.55 mm
    Anticoagulant Used: EDTA or citrate (diluted with saline)
    Advantages: Detects highly elevated ESRs due to taller column.
    Sensitivity: More sensitive to highly elevated ESRs
    Recommended By: ICSH and CLSI (International Council for Standardization in Hematology and Clinical and Laboratory Standards Institute); Standard.
  • Wintrobe ESR
    Column Height: 100 mm (shorter column)
    Bore Diameter: 3.0 mm
    Anticoagulant Used: EDTA or citrate (undiluted)
    Advantages: None mentioned
    Sensitivity: More sensitive to highly elevated ESRs
    Recommend ed By: Not Widely Recommended
  • Phases of ESR:
    1. Rouleaux formation – First 10 minutes
    2. Settling/Sedimentation – 40 minutes
    3. Packing – Last 10 minutes