Hema

• 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
• Blood sample collection methods

  • Venipuncture
  • Skin puncture
  • Capillary puncture
• Primary sites for capillary puncture

  3rd or 4th finger or outer area of the bottom of the foot (infants)
• 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)
• Serum samples should be allowed to completely clot (20 minutes) before being centrifuged
• Centrifugation of whole blood and its components should be for 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


  Mechanism of action
• 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. Automated: RBC Indices. 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). Measurement of hemoglobin is one of the several tests used to diagnose and follow treatment of anemia

  
• 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, 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 in 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+