hema notes

• Whole blood


  Uses both the liquid portion of the blood (plasma) and the cellular components (red blood cells, white blood cells, and platelets)
• Primary venipuncture site


  • Antecubital fossa
• Primary veins for venipuncture

  • Median Cubital
  • Cephalic
  • Basilic vein
• Most preferred vein


  Median Cubital Vein
• Most preferred vein for overweight or obese patients

  Cephalic vein
• Least preferred vein


  Basilic Vein due to increased risk of injury to the median nerve and/or accidental puncture of the brachial artery
• Other venipuncture sites


  • Veins on the dorsal surface of hands and feet
  • Veins on the heel should not be punctured in adults
  • Veins on the feet should not be used without physician's consent (same with ankle and leg veins)
• Routine Venipuncture needle


  21-gauge needle (1-1.5" long) for adults; 23-gauge needles for pediatric patients
• Capillary blood


  Preferred specimen for newborn screening tests and for POCT
• Capillary blood values has a lower RBC count, Hematocrit, Hemoglobin, and Platelet count and higher glucose concentration and WBC count, compared to venous blood

  
• Order of draw for venipuncture procedure

  • Yellow top tube – Sodium Polyanethol Sulfonate (SPS)
  • Light Blue top tube – Sodium Citrate
  • Red top tube
  • Green top tube - Heparin
  • Lavender top tube – Ethylene Diamine Tetra-acetic Acid
  • Gray top tube – Sodium Fluoride
• Order of draw for capillary puncture procedure

  • Blood Gas Analysis
  • Slides
  • EDTA Micro collection tubes
  • Other anticoagulated micro collections
  • Serum micro collection tubes

  
• Serum


  Remaining liquid portion of the clotted blood (does not contain Fibrinogen due to clotting and has a higher potassium level due to release from platelets)
• Centrifugation of whole blood and its components

  Approximately 10 minutes at an RCF of 1000 to 2000 x g

  
• Arterial Blood


  Samples used to measure blood gases and pH. Syringes containing heparin anticoagulant are used instead of evacuated tubes because of the pressure in an arterial blood vessel

  
• Primary arterial sites


  • Radial
  • Brachial
  • Femoral arteries
• Must perform the Modified Allen's Test first before puncturing an artery
• Arterial blood samples should be analyzed within 4 hours
• Hemolysis


  Due to mechanical destruction of red blood cells causing the release of hemoglobin in the serum/plasma

  
• Icterus


  Increased bilirubin pigment in the serum/plasma
• Lipemia


  Increased lipid content in the blood; usually due to improper fasting prior to testing
• Complications in blood collection

  • Vascular
  • Infection
  • Cardiovascular
  • Anemia
  • Neurological
  • Dermatological

  
• Vascular complications


  • Ecchymosis (Bruise)
  • Pseudoaneurysm
  • Thrombosis
  • Reflex arteriospasm
  • Arteriovenous fistula
  • Hematoma
  • Syncope (Fainting)
• Evacuated tube colors


  • Red (Glass)
  • Red (Plastic)/ Gold
  • Lavender (Glass)
  • Lavender (Plastic)
  • Pink
  • White
  • Light Blue (9:1)
  • Black (4:1)
  • Light Green/ Black
  • Green
  • Royal Blue
  • Gray
  • Yellow
  • Yellow
  • Tan (Glass)
  • Tan (Plastic)
  • Yellow/Gray and Orange
  • Red/Gray and Gold
• Anticoagulant/additive and mechanism of action for evacuated tube colors

  • None
  • Silica Particles - Clot Activator
  • K3 EDTA in liquid form - Chelates/ Binds Calcium
  • Spray dried K2 EDTA - Chelates/ Binds Calcium
  • EDTA and gel
  • Na Citrate – 3.2% (Preferred) - Chelates/ Binds Calcium
  • Na Citrate - Chelates/ Binds Calcium
  • Lithium heparin and gel - Inhibits thrombin
  • Sodium Heparin, Lithium Heparin - Inhibits thrombin
  • Sodium heparin, K2 EDTA - Heparin inhibits thrombin, EDTA binds calcium
  • Sodium fluoride/ Potassium Oxalate - Inhibits glycolysis
  • Sodium polyanetholesulfonate - Inhibits complement, phagocytes, and certain antibiotics
  • Acid Citrate Dextrose - WBC Preservative
  • Thrombin - Clot Activator
  • Silica particles and a separation gel - Clot Activator

  
• Physiologic factors affecting test results

  • Posture
  • Diurnal rhythm
  • Exercise
  • Stress
  • Diet
  • Smoking
• Complete Blood Count


  Primary screening test for diagnosis of certain diseases. Consists of WBC Count, RBC Count, Hemoglobin, Hematocrit, and WBC Differential. Platelet Count is not part of routine Complete Blood Counting
• Hemoglobin


  Comprised of heme (iron in ferrous form + protoporphyrin —> 4 pyrole rings) and 4 globin chains (1:1 ratio with heme)
• Normal hemoglobin values


  • At birth: 15-20 g/dL
  • Adult Women: 12-16 g/dL
  • Adult Men: 13-18 g/dL
• Hemoglobin fluctuations


  • Diurnal Variation: increased in AM, decreased in PM
  • Position/ Posture: decreased when lying down
  • Strenuous Muscular Activity: increased
  • Smokers: increased
  • High altitude: increased
  • Increased also in Polycythemia vera and dehydration (burns/diarrhea)
  • Decreased in Anemias, Leukemias, and slightly after 50 years old

  
• Methods of hemoglobin determination

  • Cyanmethemoglobin Method
  • Blood Oxygen Capacity (Gasometric/ Van Slyke Method)
  • Blood Iron Content
  • Copper Sulfate Method (Gravimetric Method)
  • Colorimetric Method - Acid Hematin (Sahli's Method)
  • Colorimetric Method - Alkali Hematin

  
• Cyanmethemoglobin Method


  Measured spectrophotometrically (absorbance) —> adopted by Automated Hematology Analyzers. Most accurate method for hemoglobin determination. Uses the Modified Drabkin's Reagent
• Reagents for Cyanmethemoglobin Method

  • Potassium cyanide – donates cyanide to Hemoglobin (KCN)
  • Potassium Ferricyanide – converts Fe2+ to Fe3+ (K3Fe[CN]6)
  • KH2PO4 – replaced the sodium bicarbonate in the original drabkin's reagent, shortens the reaction time from 15 minutes to 3 minutes
  • Non-ionic detergent (Sterox/Triton) – decreases amount of turbidity resulting from abnormal proteins and improves RBC lysis (liberates hemoglobin)
• Principle of Cyanmethemoglobin Method


  a. Potassium ferricyanide converts the hemoglobin iron from the ferrous state to ferric state to form methemoglobin (Hi = Hemiglobin)

  b. Hi then combines with potassium cyanide to form a stable pigment Cyanmethemoglobin (HiCN)

  c. The color intensity of this mixture is measured in a specctrophotometer at a wavelength of 540nm

  d. The optical density of the solution is proportional to the concentration of hemoglobin, all forms of hemoglobin are measured except Sulfhemoglobin (limitation of cyanmethemoglobin method)
• Sources of error in Cyanmethemoglobin Method

  • High WBC count (>20 x 109/L) or High Platelet count (>700 x 109/L) – can cause turbidity and a falsely high result

  Hemoglobin S and Hemoglobin C

  Lipemic Blood – can cause increased Hb and MCH

  
• Blood Oxygen Capacity (Gasometric/ Van Slyke Method)


  Measures functional hemoglobin only. Based on the fact that 1g of Hb carries 1.34mL of Oxygen

  
• Blood Iron Content


  Hemoglobin iron content = 100g of Hb = 0.347g of Fe2+
• Copper Sulfate Method (Gravimetric Method)


  Used for blood donor screening. The density of the drop of blood is directly proportional to the amount of Hb. If the hemoglobin is equal to or more than 12.5 g/dL, the drop of blood will sink within 15 seconds and the donor is accepted. The specific gravity of copper sulfate solution is 1.053. The drop of blood should be added from the height of about 1 cm

  
• Colorimetric Methods


  • Acid Hematin (Sahli's Method)

  Alkali Hematin
• Acid Hematin (Sahli's Method)


  Reagent: 0.1 N HCl. A comparator block is used to compare the brownish-yellow color of the resulting solution