BLOOD COLLECTION FOR VARIOUS LABORATORY

TESTS, ANTICOAGULATED VACUTAINERS AND

PREANALYTICAL ERRORS

Presented by : Dr. Utkarsh Sharma Under the guidance of : Dr. Ashish Bagaria

swab

• Sterile disposable syringes and needles should be used for venepuncture.

• Needle size should be 19 to 21 gauge in adults and 23-gauge in children.

• Tourniquet should be released as soon as the blood begins to flow into the syringe

• The needle is detached from the syringe after withdrawal of blood to deliver it into appropriate anticoagulant tube.

• Blood is mixed with the anticoagulant in the container thoroughly by gently inverting the container several times.

SEQUENCE OF FILLING OF TUBES

• First Container : blood culture

• First tube : Plain tube [serum]

• Second tube : Tube containing anticoagulant citrate for PT.

• Third tube : anticoagulant like EDTA or Heparin• Fourth tube : Tube containing additional stablilizing

agent like fluoride.

ANTICOAGULANT VACUTAINERS• Anticoagulants used for hematological investigations

are EDTA [ Ethylene diamine tetra-acetic acid ], heparin, double oxalate, and trisodium citrate.

• EDTA [1.5mg/ml], dried: also called SEQUESTRENE or VERSENE. This is the recommended anticoagulant for routine hematological investigations. EDTA is used for CBC, blood smear, Hb electrophoresis, sickling test. However it cannot be used for coagulation studies. EDTA can cause pseudothrombocytopenia on hematology analyser. Its mechanism of action is by chelation of Calcium. Erythrocytes, leukocytes (white blood cells) and thrombocytes (platelets) are stable in EDTA anticoagulated blood for up to 24 hours. Preparation of blood smears should be done within 3 hours after blood collection.

PCV AND MEAN CELL VOLUME INCREASE DUE TO RED CELL SWELLING OSMOTIC FRAGILITY INCREASES TLC AND PLATELET COUNTS SHOW PROGRESSIVE DECREASE

• HEPARIN• ACTS BY INHIBITION OF THROMBIN

ACTIVITY• USED IN CONCENTRATION 15U/ml• MAINLY USED IN OSMOTIC FRAGILITY TEST

AND IMMUNOPHENOTYPING• HEPARIN CAUSES LEUCOCYTE CLUMPING

AND BLUE BACKGROUND OF SMEARS

• DOUBLE OXALATE [WINTROBE MIXTURE]CONTAINS AMMONIUM OXALATE AND POTASSIUM OXALATE IN 3:2 PROPORTION.ACTS BY REMOVING CALCIUM.IT IS USED FOR ROUTINE HEMATOLOGICAL TESTS AND FOR ESTIMATION OF ESR BY WINTROBE METHOD. IT CAUSES CRENATION OF RED CELLS AND MORPHOLOGIC ALTERATIONS IN WBC ,IT CANNOT BE USED FOR MAKING BLOOD FILMS.

TRISODIUM CITRATE [3.2 %]

• ANTICOAGULANT OF CHOICE FOR COAGULATION STUDIES AND FOR ESTIMATION OF ESR BY WESTERGREN METHOD

• 1:9 [ANTICOAGULANT TO BLOOD ] IS RECOMMENDED FOR COAGULATION STUDIES, AND FOR ESR 1:4 PROPORTION IS RECOMMENDED.

• ESR SHOULD BE MEASURED WITHIN 4 HOURS AND COAGULATION STUDIES SHOULD BE PERFORMED WITHIN 2 HOURS OF COLLECTION OF BLOOD.

• PLAIN TUBES ARE USED FOR CHEMISTRY STUDIES AFTER SEPERATION OF SERUM.

• FLUORIDE BULB IS USED FOR COLLECTION OF WHOLE BLOOD FOR ESTIMATION OF BLOOD GLUCOSE. ADDITION OF SODIUM FLUORIDE MAINTAINS STABLE GLUCOSE LEVEL BY INHIBITING GLYCOLYSIS.

Becton Dickinson's Vacutainer® Serum Separator Tubes (SST) contain spray-coated silica to aid in clotting and a polymer gel for serum separation. Samples processed in these Venous Blood Collection Tubes are used for serum determinations in chemistry, blood donor screening, and infectious disease testing.

Potassium Oxalate and Sodium FluorideSodium fluoride/Na2 EDTASodium Fluoride (no anticoagulant, will result in serum sample)The tube must be inverted 4-5 times after collection to allow adequate mixing of the blood with the additive

• The whole testing process can be divided into 3 stages

• Pre-analytical : test request, patient and specimen identification, specimen collection ,transport , accessioning and processing.

• Analytical: specimen testing• Post analytical: reporting test results,

interpretation , follow up, storage, retesting if needed.

Types of preanalytical errors• Patient misidentification [ incorrectly labelled tubes or

incorrectly filled forms ] . Most common causes are :1] inadequate data on test requisition form2] missing patient identifiers3] labelling specimen container away from bedsidePossible consequences include wrong blood transfusion leading to acute hemolytic reaction.Specimen collection from wrong patient leading to delayed diagnosis or misdiagnosisTo minimize such errors, atleast 2 patient identifiers should be used, specimen should be labelled immediately after specimen collection

• Lipemic specimenCauses:*Test collection after heavy meals*Pre-existing metabolic disordersPossible consequences include:*interference of fat with optical reading of instruments, wrong electrolyte values

Hemolysis

• CAUSES• Forcing blood through needle of syringe• Collecting blood through IV line• Vigorous shaking of specimen• Centrifuging specimen before clotting*TOURNIQUET SHOULD NOT BE APPLIED FOR MORE THAN 1 MINUTE AS IT CAN CAUSE LOCAL STASIS AND RUPTURE OF RBC

Clotted plasma specimen

• Results from inappropriate mixing of tubes• Results in false leucopenia and aberrant red

cell indices.• Manufacturers guidelines for tube mixing

should be followed.

THANK YOU