Tuesday, August 5, 2014

Pre-Analytical variables: Sample Collection (Part 1)

Errors during collection, processing and transport of biological specimens are common (Ref: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th Ed).

    • Common samples collected are
    • Whole blood
    • Serum
    • Plasma
    • Saliva
    • Pleural, pericardial, ascitic fluid
    • Various types of solid tissues
    • Spinal, Synovial, amniotic fluid.

 Blood Collection
  • Sources – Artery, veins, capillaries
  • Venous blood – Venipuncture
  • Arterial blood puncture – Arterial blood gas analysis
  • Capillaries – Skin puncture, in young children and for point-of-care testing

Venipuncture - Steps

  • Confirm identity of the patient – Name, MRD, ward or room number
  • Minimum 3 items of identification to be used (International regulations)
  • Phlebotomist dressing – Protective equipment, impervious gown and gloves.
  • Face mask and goggles needed for patient in isolation rooms.
  • Extent of protection varies with type of illness patient has.
  • Verify patient condition – Example whether fasting, wherever needed.
  • Patient should be comfortably seated or supine for 20 minutes before blood draw. This reduces effects due to hemoconcentration or hemodilution.
  • Either arm should be in straight line from shoulder to wrist.
    • Contraindications – Indwelling IV line, extensive scarring, hematoma, hand on the side mastectomy was done.
  • Estimate volume of blood needed and appropriate number and types of tubes needed, appropriate needles.
  • Choice of needles – Adult – G20, collapsable veins – G21, 30-50 ml blood – G18, children G22 or less.
  • Sterile, sharp and without barbs.
  • For trace elements – Stainless steel. Acid washed apparatus without contamination.
  • Location – Median cubital vein in antecubital fossa, crook of elbow preferred site.
  • Veins on back of hand, ankle – Next choice, but avoided in diabetics and patients with poor circulation.
  • Blood from cannula may be used.
  • Any fluid should be shut for 3 minutes before blood draw.
  • Blood draw facilitated by palpation of vessel.
  • Cleaned with prepackaged alcohol swab or gauze pad saturated with 70% isopropanol.
  • Cleaning – Circular motion, site outwards.
  • Should be dried in air. Complete drying reduces hemolysis !
  • Providone iodine can interfere with biochemistry results and to be avoided !
  • For alcohol estimation – Benzalkonium chloride used for cleaning.
  • Don’t touch site after cleaning.
  • Timing of specimen to be noted.
  • Corticosteroids, iron – Diurnal variation
  • Monitoring drug therapy.
  • Alcohol, drug measurement – Medicolegal considerations.
  • Blood pressure cuff ( to 60 mm Hg) or tourniquet (pre-cut soft rubber or Velcro bands) applied 10-15 cm (4-6 inches) above intended site.
  • Distends veins making collection easier.
  • Changes after 1 min and unacceptable changes after 3 min of tourniquet application.
  • First drawn specimen should be used as far as possible, most representative of circulating blood.
  • Second tube – 5% and third tube – 10%.
  • Prolonged stasis – More than 15% variation.
  • If volume is small (large volumes not obtained), prioritize tests.
  • Trauma also affects values.
  • Pumping of fists to be avoided for
    • Potassium
    • Phosphate
    • Lactate
  • Stress can affect values in all ages, especially children, struggling, frightened and physically held back !
    • Cortisol
    • Growth hormone
  • Evacuated blood tubes – Less expensive, more convenient, identified by color code.
  • Glass tubes (siliconized) – Reduces hemolysis.
  • Blood from one tube not to be transferred to another tube, whatever be the reason.
  • Needle gently guided into patient’s vein.
  • One needle in place, tube pressed forward to puncture stopper and release vacuum.
  • Once blood flows, tourniquet to be released immediately.
  • Multiple tubes may be filled with one puncture as needed.
  • Shut-off valve may be used if needed.


Special Tubes

  • Gel separation tubes
    • Polymer gel/silica/lithium heparin
  • Serum tubes
    • Non-additive, additive
  • Whole blood/plasma tubes
    • EDTA, citrate, NaF, heparin, oxalate, iodoacetate
  • Special chemistry tubes
    • Lead, trace elements, stat chemistry
  • Plasma preparation tubes
    • K-EDTA, polymer gel, silica activator
  • Plastic tubes have the advantage of being non-breakable.
  • Problems with evacuated tube – Expiry date, short draw related problems.
  • Back flow – Sterile tubes to be used, arm to held downwards.
  • Blood to be drawn in the order – (1) Blood cultures (2) Non-additive tubes (3) Citrate tubes (4) Serum separator tubes (5) Heparin tubes (6) EDTA and oxalate fluoride tubes.

Tube Codings :

  • Non-additive tubes
  • Coagulation or citrate containing tube
  • Serum separator tube
  • Heparin tube
  • EDTA
  • Oxalate-fluoride
  • Red stopper
  • Blue
  • Red with black flecks
  • Green
  • Lavender
  • Gray stopper

Blood Collection with Syringe

  • Needle placed firmly over nozzle of syringe, cover removed.
  • Bevel of needle upwards, nozzle downwards.
  • Aligned with the vein, 15 degree angle.
  • After entering vein, pressure released and blood entered by gently pulling plunger.
  • Tube capped with required anti-coagulant.
  • Hemolysis – Vigorous suction, forceful transfer from syringe, large-bore needle.
  • Patient given dry gauze pad over puncture site, arm raised a bit.
  • Needle disposed in sharps container.
  • Gloves etc to be disposed in hazardous waste receptacle.
  • Venipuncture in children -Similar technique.
    • 21G to 23G needle or 20G - 23G butterfly used.

Skin Puncture

  • Open collection technique – Skin punctured by lancet.
  • Small volume collected into microdevice, capillary tube.
  • Indications – (1) Pediatric cases, (2) Severe vein damage, (3) Burns, bandaged patients, (4) POCT cases.
  • Sites – Tip of finger, ear lobe, heel or big toe of infants, lateral or medial plantar surface of foot.
  • Skin cleaned with gauze pad soaked in 70% isopropanol.
  • After drying, quickly punctured by sharp stab with lancet.
  • Different site to be used each time.
  • Massage to be avoided, as it causes tissue debris accumulation.
  • Finger may be warmed, if needed, 3 min before by war, wet cloth.
  • First drop discarded. Subsequent drops collected.
  • To be rapidly done to avoid clotting. Air bubbles to be avoided.
  • Drop by drop collection increases hemolysis.
  • Filter paper – Gently touched against large drop of blood, soaked into the paper to fill the marked circle.
  • Only single application per circle.
  • Complete penetration of paper important.
  • Filter papers air dried. Not to be transferred from capillary tubes.

Arterial Puncture

  • Requires physicians or trained nurses.
  • Preferred sites – (1) Radial artery of wrist, (2) Brachial artery of elbow, (3) femoral artery in groin.
  • Neonate – Indwelling catheter in umbilical artery.
  • Older child, adult – Capillary puncture to obtain arterialized capillary blood.
  • Good for pH, pCO2, but not for pO2.
  • Older child, adult – Earlobe, young child or infant – Heel.
  • Capillary puncture for arterial blood should be avoided wherever possible.
  • Contraindications – Reduced cardiac output, hypotension, vasoconstriction.
  • Heparinized capillary tubes containing small metal bar used.
  • Tubes filled quickly, contents mixed well by magnet to move metal bar up and down to get uniform specimen.

Anti-coagulants and Preservatives

  • Heparin – Least interference except for PCR. High cost, temporary action. Unsuitable for Ca, T3, T4, ACP.
  • EDTA – Hematology. Inhibits ALP, CK, LAP, Ca, iron. Reduces cholesterol by 3-5%.
  • Sodium fluoride – Glucose. Weak. Inhibits enzymes. Larger amounts needed.
  • Citrate – Coagulation studies. Affects enzymes and phosphates.
  • Oxalates – Reduces hematocrit, electrolytes, enzymes.
  • Iodoacetate – Glucose, urea. Inhibits CK. No effect on other tests.

Effect of Sites

  • Skin specimen similar to arterial, dissimilar from venous blood.
  • Can be contaminated.
  • Central venous catheter – Composition may be affected by infused fluids.
  • Blood drawn from central venous catheter and peripheral vein can have different values even if properly collected.


  • Serum shows visible evidence of hemolysis if concentration exceeds 200 mg/L.
  • Slight lysis has no effect.
  • Severe hemolysis can affect aldolase, ACP, LDH, ICD, K, Mg, phophate in particular.
  • Serum protein electrophoresis gives additional band if hemolysis is present.
  • Correction for hemoglobin in lysed samples is not accurate for any parameter, hence lysis is undesirable.
(Source: supplementary information by Textbook of Biochemistry for Medical Students 7th Edn.)

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