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.

Hemolysis

• 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.)