HEMA LAB

• Hemoglobin


  The main content of the red blood cells which is responsible for transporting oxygen from the lungs to the tissues and CO2 from the tissues to the lungs to be eliminated to the outside

  
• Hemoglobin


  A conjugate protein that consists of 2 portions, the globin (a simple protein) and heme (organic compound in iron). Each hemoglobin molecule consists of the one molecule of globin (four globin chains) and four molecules of heme
• Hemoglobinemia


  Condition where hemoglobin is found in the blood plasma

  
• Hemoglobinuria


  Condition where the free hemoglobin in the plasma reaches a concentration between 30 and 300 mg/100mL of blood, and hemoglobin is detected in the urine

  
• Normal hemoglobin levels


  • MALE: 13.5-17.5 g/dL
  • FEMALE: 12.0-16.0 g/dL

  
• Hyperchromia


  Increased hemoglobin value, found in polycythemia, dehydration and in changing from low to high altitude

  
• Hypochromia


  Decreased hemoglobin value, seen in anemia

  
• Methods for hemoglobin determination

  • Van Slyke method
  • Kennedy's and Wong; Assendelt method
  • Physical method
  • Copper sulfate method
  • POCT (Point of Care Testing Devices)
  • Direct methods (Acid hematin, Alkali hematin)
  • Indirect methods (Oxyhemoglobin, Cyanmethemoglobin)

  
• Van Slyke method


  Gasometric method that determines hemoglobin and oxygen saturation. 1 gram of hemoglobin carries 1.34 mL of oxygen.

  
• Kennedy's and Wong's/Assendelt method

  Chemical method that measures iron bounded to hemoglobin. 1 gram of hemoglobin = 3.47mg of iron.
• Physical method (Rule of Three)

  Applicable only to patients with normocytic, normochromic RBCs. Hemoglobin = RBC count x 3, Hematocrit = Hemoglobin x 3.

  
• Copper sulfate method


  Gravimetric method based on the specific gravity of copper sulfate (CuSO4) - 1.053. Semi-quantitative, for mass assays. Drop of blood is placed into the copper sulfate solution and observed whether it will sink within 15 seconds.
• Direct methods


  Colorimetric methods using comparator block with an end color of yellow brown. Acid hematin method uses 0.1N HCl, Alkali hematin method uses 0.1N NaOH.
• Indirect methods


  Spectrophotometric methods. Oxyhemoglobin method uses aqueous ammonium hydroxide, Cyanmethemoglobin method is the gold standard.
• Acid hematin method


  1. Introduce 0.1N HCl solution up to 2 mark of Sahli graduated tube

  2. Draw blood to 0.02 mL or 20 uL mark using Sahli pipette

  3. Dispense blood into graduated tube, mix

  4. Allow to stand for 5 minutes

  5. Add distilled water drop by drop, stir after each addition, compare with color standard in comparator block

  6. Read result from grams or percentage scale
• Cyanmethemoglobin (HiCN) method


  Hemoglobin iron is converted from ferrous to ferric state to form methemoglobin by the action of ferricyanide. Methemoglobin then combines with potassium cyanide to produce the stable cyanmethemoglobin which is measured spectrophotometrically at 540 nm.
• Cyanmethemoglobin method procedure


  1. Place 5 mL of Drabkin's reagent in test tube

  2. Draw blood to 0.02 mL or 20 uL mark using Sahli pipette, dispense into test tube, mix

  3. Let stand for 5 minutes

  4. Transfer mixture to cuvette

  5. Set spectrophotometer to 100% transmittance at 540 nm using reagent as blank

  6. Read patient's sample, obtain hemoglobin concentration from calibration curve

  
• Components of Modified Drabkin's Reagent

  Potassium ferricyanide - converts hemoglobin to methemoglobin<|>Potassium cyanide - combines with methemoglobin<|>Dihydrogen potassium phosphate - replaces potassium carbonate for faster reaction<|>Non-ionic detergents/surfactants - improve cell lysis and decrease turbidity
• Cyanmethemoglobin is the method of choice for hemoglobin determination because it is stable in dilutions, standards are readily available, and all hemoglobin derivatives except sulfhemoglobin are measured

  
• Errors in cyanmethemoglobin method are usually caused by turbidity, and can be addressed by centrifuging the sample, using a blank, or diluting the sample
• Hematocrit


  Also referred to as the packed cell volume, volume of erythrocytes or reading of packed cells
• Hematocrit test


  Measures the proportion of red cells to plasma in the peripheral blood but not in the entire circulation
• Hematocrit


  Gives the number of millimeters of packed red blood cells/100 millimeter of blood or simply expressed as volume percent
• Hematocrit reporting


  Reported as: PCV (%) or EVF (L/L)
• Normal hematocrit values


  • At birth: 45-60%
  • Females: 36-48%
  • Males: 40-55%
• Methods for hematocrit determination

  • Macrohematocrit method
  • Microhematocrit method
  • Automated method

  
• Macrohematocrit method


  • Less commonly used; time consuming
  • Required increased blood volume; higher amount of trapped plasma
  • Centrifugation: 30 minutes at 2,000- 2,300 x g
• Trapped plasma


  Plasma trapped in the RBC portion after centrifugation, increases hematocrit by: 1-2 or 1-3%
• Anticoagulants used in macrohematocrit methods

  • Wintrobe's: Double oxalate
  • Van Allen's: Sodium oxalate
  • Sanford Magath: Sodium oxalate
  • Haden's: Sodium oxalate
  • Bray's: Heparin
• Microhematocrit method


  • Requires small amount of blood; more commonly used
  • Aka: ADAM's method
  • Specimen: Whole blood, Capillary blood
  • Sealer/Clay: 4-6mm long
  • Centrifugation: 5 mins for 10,000-15,000 x g
• Layers of the spun hematocrit

  • Fatty layer (topmost)
  • Plasma (second)
  • Buffy coat (third)
  • PRBC's (fourth layer)
• Fatty layer


  Barely visible, increased in lipidemia
• Plasma layer


  Normal: pale yellow<|>Pink: slight hemolysis<|>Red: gross hemolysis<|>Yellow-brown: increased bilirubin (hemolysis)
• PRBC's


  PCV (hematocrit)
• Errors in hematocrit determination


  • Technical errors (increased): Prolonged tourniquet application, Insufficient mixing (RBC first), Inadequate centrifugation, Prolong standing of the tube, Reading of the buffy coat
  • Technical errors (decreased): Excessive anticoagulant (RBC shrinkage), Insufficient mixing (plasma first), Improper flushing of IV catheter
  • Physiologic errors (increased): Hemoconcentration, Dehydration, Abnormal RBC shape, Trapped plasma, Sickle cell anemia, Spherocytosis, Thalassemia, Macrocytic anemias
  • Physiologic errors (decreased): Blood loss (plasma replaced faster than RBC's), Tissue fluid, Hemolysis
• Reticulocyte count


  The last immature red blood cell stage
• Objectives for reticulocyte count

  • Know the different methods for Reticulocyte counting
  • Know the proper procedure of Reticulocyte counting
  • Identify Reticulocytes properly in a PBS
  • Know the different formulas in relation to Reticulocyte counting

  
• Reticulocyte


  Also known as: Polychromatic RBC, Polychromato philic RBC, Diffusely basophilic RBC
• Reticulocyte


  • Normally spends 2 days in the bone marrow and 1 day in the peripheral blood before developing into a mature red blood cell
  • Contains remnant cytoplasmic ribonucleic acid (RNA) and organelles such as the mitochondria and ribosomes

  
• Reticulocyte staining


  Whole blood is incubated with new methylene blue to detect the presence of RNA