This document discusses blood collection for laboratory tests using anticoagulated vacutainers and sources of preanalytical errors. It describes the appropriate vacutainers and additives used for different tests like CBC, coagulation studies, chemistry etc. The sequence of filling different vacutainer tubes is explained. Common causes of preanalytical errors like hemolysis, lipemia, patient misidentification, and not following guidelines for mixing blood with additives are summarized. The importance of following standard operating procedures for collection, transport, and processing of specimens is highlighted to reduce preanalytical errors.

1. BLOOD COLLECTION FOR VARIOUS
LABORATORY TESTS, ANTICOAGULATED
VACUTAINERS AND PREANALYTICAL
ERRORS
Presented by : Dr. Utkarsh Sharma
Under the guidance of : Dr. Ashish Bagaria

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20. • 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.

24. 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.

25. 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.

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

27. • 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

28. • 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.

29. 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.

30. • 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.

31. 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 Fluoride
Sodium fluoride/Na2 EDTA
Sodium 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

32. • 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.

34. Types of preanalytical errors
• Patient misidentification [ incorrectly labelled tubes or
incorrectly filled forms ] .
Most common causes are :
1] inadequate data on test requisition form
2] missing patient identifiers
3] labelling specimen container away from bedside
Possible consequences include wrong blood transfusion
leading to acute hemolytic reaction.
Specimen collection from wrong patient leading to
delayed diagnosis or misdiagnosis
To minimize such errors, atleast 2 patient identifiers
should be used, specimen should be labelled immediately
after specimen collection

35. • Lipemic specimen
Causes:
*Test collection after heavy meals
*Pre-existing metabolic disorders
Possible consequences include:
*interference of fat with optical reading of
instruments, wrong electrolyte values

36. 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

37. 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.

39. THANK YOU