Hematology Lab - 04 Hemoglobin, Hematocrit, and RBC Indices

HEMATOLOGY LABORATORY COMPLETE BLOOD COUNT: HEMOGLOBIN, HEMATOCRIT, & RBC INDICES OUTLINE • Complete Blood Count • Hemoglobin o Cyanmethemoglobin Method o Clinical Significance • Hematocrit o Clinical Significance o Methods o Sources of Errors • Red Blood Cell Indices o Mean Cell Volume (MCV) o Mean Cell Hemoglobin (MCH) o Mean Cell Hemoglobin Concentration (MCHC) COMPLETE BLOOD COUNT • Includes : o Red Blood Cell Count o Hemoglobin o Hematocrit o Red Blood Cell Indices o White Blood Cell Count o White Blood Cell Differential Count o Platelet Count HEMOGLOBIN • Gives red color to the blood o Heme part - provides red color ▪ Member of group of pigments called porphyrins • Carries oxygen from the lungs to the tissues and takes carbon dioxide o ↑ morning, ↓ evening o ↑ birth due to active red bone marrow o ↑ in high altitudes due to hypoxia ▪ Hypoxia - decreased oxygen in body ▪ Hgb compensates for decreased oxygen o ↑ in strenuous muscular activity due to androgen influence ▪ Androgen - indirect effect on bone marrow o ↑ in smokers due to formation of carboxyhemoglobin ▪ Carboxyhemoglobin - ferrous + CO ▪ CO is bound instead of O2 ▪ CO has more affinity to Heme o ↓ when in lying position due to the dilution of interstitial fluid CYANMETHEMOGLOBIN METHOD • aka “Hemoglobincyanide” • Reference method for hemoglobin determination • Standard approved by Clinical and Laboratory Standard Institute (CLSI) • Can measure all forms of hemoglobin except sulfhemoglobin o Shb cannot be convert to methemoglobin & cyanHi • Drabkin’s Reagent : o Potassium ferricyanide - oxidize Ferrous to Ferric; converting Hgb to methemoglobin o Potassium cyanide - convert methemoglobin to cyanmethemoglobin o Non-ionic detergent – improves RBC lysis o Dihydrogen potassium phosphate • Principle : o Absorbance of cyanmethemoglobin at 540 nm is directly proportional to the hemoglobin concentration ▪ Utilize spectrophotometer (set at 540 nm) • Procedure : 1. In 5mL of Drabkin’s reagent in a test tube, add 0.02cc/20uL of blood using sahli pipette. Rinse the pipette with reagent several times 2. Mix well and stand for 10 minutes 3. Read absorbance using spectrophotometer at 540 nm (perform reagent blank). ▪ Reagent blank - used to eliminate absorbance of reagent from total solution ▪ Drabkin’s reagent - has color which affect absorbance; its absorbance will be added to the absorbance of cyanmethemoglobin • Reference Ranges : g/dL g/L Female 12-15 120-150 Male 14-15 140-180 Newborn 16.5-21.5 165-215 CLINICAL SIGNIFICANCE • Increased Hemoglobin o Sickle cell anemia o Thalassemia o Transfusion reaction o Hemolysis o Dehydration o Polycythemia vera o High altitude • Decreased Hemoglobin o Anemia o Blood loss o Iron, folate, and vitamin B6, B12 deficiencies HEMATOCRIT • aka Packed Cell Volume (PCV), hct • Volume of packed RBC that occupies a given volume of whole blood. • Expressed in percentage (%) or in decimal fraction. • Most sensitive test when excess of anticoagulant is used during blood collection

• Reference Ranges : % Decimal Female 35 - 49 0.35 - 0.49 Male 40 - 54 0.40 - 0.54 Newborn 48 - 68 0.48 - 0.68 • Venous Hematocrit (VH) – proportion of RBC to plasma in peripheral blood (not the entire circulation) o Measured in hematocrit testing (since venous blood is collected) • Body Hematocrit (BH) – the total RBC mass occupying the total blood volume o Lower than venous hematocrit o Ratio : 1:0.9 (1 VH : 0.9 BH) CLINICAL SIGNIFICANCE • Rough estimate of RBC size and RBC concentration • Used to calculate red blood cell indices (MCV, MCHC) • Simple screening test for anemia, polycythemia, hemodilution and hemoconcentration o Iron Deficiency anemia - Decrease in RBC, Hgb counts = Decrease in Hct o Polycythemia - increase in blood cells ▪ High RBC = High Hct o Hemodilution - decrease in blood cells o Hemoconcentration - increase blood conc. = increase in blood cells • Increased : polycythemia, macrocytic anemia, hypochromic anemia, sickle cell anemia, spherocytosis, thalassemia • Decreased : anemia, age (>50 y.o.) METHODS • Macrohematocrit method • Microhematocrit method MACROHEMATOCRIT METHOD (WINTROBE METHOD) • Materials : o Centrifuge o Double Oxalate (anticoagulant) o Wintrobe tube stand o Wintrobe Tube : ▪ Length: 115 mm ▪ Diameter (bore): 3.0 mm ▪ Hematocrit marking (White): 10cm-0 (from top) ▪ ESR marking (Red): 0-10cm (from top) Wintrobe tube Wintrobe tube stand • Procedure : 1. Fill up the wintrobe tube up to 10 cm mark with oxalated blood avoiding bubble formation 2. Centrifuge at 2500 RPM for 30 mins ▪ Cotton is put around the tube beforehand to make it stable 3. Remove the tubes and carefully place in Wintrobe tube stand. 4. Measure the height of RBC layer ▪ Do not include white buffy coat; may cause false increase ▪ Measure below white buffy coat 5. Report in terms of volume percent MICROHEMATOCRIT METHOD • Uses capillary tubes/microhematocrit tube: o Length: 75 mm o Inner Bore: 1.2 mm o Volume: 0.05 mL • Two types of capillary tubes : Heparinized Plain Color of band Red Blue Additive Heparin None Uses Used when blood is directly collected from dermal puncture Used when blood is from a pre- collected EDTA blood • Materials : o Capillary tubes (red/blue) o Clay sealant o Microhematocrit centrifuge o Microhematocrit reader • Procedure : 1. Perform skin puncture and fill 2 heparinized capillary tubes (3/4) 2. Seal the end with the colored ring of the capillary tube with clay and wax (4-6mm) 3. Centrifuge for 5 minutes at 10,000-15,000 g 4. Read using microhematocrit reader (buffy coat should not be included)

• Layers of Microhematocrit Tube : o Descending Layer: (PBPS) ▪ First Layer : Plasma ▪ Second Layer : Buffy coat (WBC, plts) ▪ Third layer : Packed red blood cell ▪ Fourth layer : Seal/plug o Ascending Layer: (SPBP) ▪ First Layer : Seal/Plug ▪ Second Layer : Packed red blood cell ▪ Third layer: Buffy coat (WBC, platelets) ▪ Fourth layer : Plasma SOURCES OF ERROR • False Increase : o Insufficient centrifugation ▪ Macro - 10-30 mins ▪ Micro - 5 mins o Inclusion of buffy coat (may cause increase in Hct) o Hemoconcentration o Dehydration o Disorders such as Macrocytic anemia, Hypochromic anemia, and sickle-cell anemia o Trapped plasma in the packed RBC layer (~0.2 L/L) • False Decrease : o Improper sealing of capillary tubes o Increased concentration ofanticoagulant o Prolonged centrifugation o Acute blood loss o Hemolysis o Inclusion of interstitial fluid RED BLOOD CELL INDICES • Used to mathematically define RBC size and Hgb content within cells • Computed values • MCV - Mean Cell Volume • MCH - Mean Cell Hemoglobin • MCHC - Mean Cell Hemoglobin Concentration RDW - RBC Distribution Width MEAN CELL VOLUME (MCV) • Average size or volume of an individual RBC in femtoliter (fL) • Formula : • Reference value : 80-100 fL (normocytic) o Microcytic : <80 fL ▪ Iron deficiency anemia, sideroblastic anemia, thalassemia, and Lead poisoning o Macrocytic : >100 fL ▪ Megaloblastic anemia, Hemolytic anemia with reticulocytosis, liver disease and normal newborn, autoagglutination, severe hyperglycemia (<600 mg/dL), Leukocytosis ▪ Severe hyperglycemia - may result to osmotic swelling of RBC due to glucose entry to RBC MEAN CELL HEMOGLOBIN (MCH) • Average weight of hemoglobin in an individual RBC in picograms (pg) • Directly proportional to the size and hemoglobin concentration of an RBC • Not used in classification of anemia (MCV, MCHC only) • Formula : • Reference range : 26 – 32 pg / RBC MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC) • Average concentration of hemoglobin in each individual RBC • Ratio of hemoglobin weight to RBC volume • Formula : • Reference value : 32 – 36 g/dL or % (normochromic) o <32 g/dL : hypochromic cells (↓ Hgb) ▪ Thalassemia, Iron Deficiency Anemia o >36 g/dL : Hyperchromic (↑ Hgb conc.) ▪ Spherocytic cells, errors in RBC (hemolysis), presence of cold agglutinins. ▪ Spherocytes/Spherocytosis - new term