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Preanalytical variables in coagulation testing
This document discusses pre-analytical variables that can affect coagulation testing. It emphasizes the importance of proper sample collection, including using the correct anticoagulant (sodium citrate), collection technique (slow draw with mixing), and storage requirements. Errors in collection such as short draws, improper mixing or storage, excessive mixing, or hemolysis can falsely affect test results. Proper centrifugation is also needed to obtain platelet-poor plasma for certain tests.
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Overview of preanalytical steps crucial for accurate coagulation test results, focusing on sample collection, storage, and common errors.
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Preanalytical variables in coagulation testing
- 1. Preanaly(cal Variables in Coagula(on Tes(ng • Pre-‐analy(cal variables in coagula(on tes(ng 1. Sample Collec(on 2. Site Selec(on 3. Storage Requirements 4. Transporta(on of Specimen 5. Venipuncture vs line draws 6. Tube sequence 7. Unintended drug effects 1
- 2. • Ideally Sample Collec(on the results of blood tests should accurately reflect the values in vivo • Many misleading results in blood coagula(on arise not from errors in tes(ng but from carelessness in the preanaly)cal phase. – Labeling – Improper collec(on – Fas(ng/non-‐fas(ng – Sample collec(on • Slow draw, vigorous mixing, an(coagulant • When blood is withdrawn from a vessel, changes begin to take place in the components of blood coagula(on • Some occur almost immediately, such as platelet ac(va(on and the ini(a(on of the cloTng mechanism dependent on surface contact
- 3. Sample Collec(on •An(coagulant of choice – 3.8% or 3.2% Sodium Citrate • 3.2 % Preferred as the standard measure due to stability and closeness to the plasma osmolality – An(coagulant/blood ra(o is cri(cal (1:9) • Exact amount of blood must be drawn. No short draws are acceptable, this will falsely increase results due to presence of too much an(coagulant • CLSI guideline is +/-‐ 10 % of fill line – Purpose of the an(coagulant is to bind or chelate calcium to prevent cloTng of specimen
- 4. • Other Sample Collec(on an(coagulants, including oxalate, heparin, and EDTA, are unacceptable. • The labile factors (factors V and VIII) are unstable in oxalate, whereas heparin and EDTA directly inhibit the coagula(on process and interfere with end-‐point determina(ons • Addi(onal benefits of trisodium citrate are that the calcium ion is neutralized more rapidly in citrate, and APTT tests are more sensi(ve to the presence of heparin
- 5. Storage Requirements —Prothrombin Time: PT ◦ Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 2-‐4 oC or 18-‐24 oC must be tested within 24 hours of collec(on — Ac(vated Par(al Thrombin Time: APTT ◦ Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 2-‐4 oC or 18-‐24 oC must be tested within 4 hours of collec(on ◦ Other assays – Fibrinogen, Thrombin Time, Factor Assays – Centrifuged with plasma remaining on top of cells in unopened tube kept at 2-‐4 oC or 18-‐24 oC must be tested within 4 hours of collec(on
- 6. • Other Storage Requirements general notes – Perform coagula(on tests ASAP • Specimen may deteriorate rapidly (especially factors V and VIII) – If the tes(ng is not completed within specified (mes, plasma should be removed from the cells and placed in a frost free freezer • -‐ 20 oC for two weeks • -‐70 oC for six months
- 7. • Platelet Centrifuga(on –Poor plasma (PPP) – <10 x 10 9 /L – Specimen has been centrifuged for 15 minutes @ 2500 x g – Why is PPP essen(al? 1. Contains platelet factor 4(heparin neutralizer) 2. Contains phospholipid (affects lupus an(coagulant and factor assay tes(ng) 3. Contains proteases (affect tes(ng for vWF)
- 8. Common Collec(on Problems Error Consequence Comment Drawing coagula(on tube PRIOR to other an(coagulant tubes PT/PTT falsely affected Contamina(on Probing the vein PT/PTT falsely shortened Tissue thromboplas(n is released ac(va(ng coagula(on Heparin contamina(on from line draw PTT falsely prolonged Heparin keeps the blood from cloTng Lipemia Test may not work Photo-‐op(cal methods affected
- 9. Common Collec(on Problems Error Consequence Comment Short draw <2.7 mL PT/PTT falsely prolonged An(coagulant to blood ra(o exceeds 1:9 Failure to mix specimen aher collec(on PT/PTT falsely prolonged Blood clots form when an(coagulant & blood do not mix Excess vigorous mixing PT/PTT falsely shortened Hemolysis and platelet ac(va(on cause start of cascade Hemolysis PT/PTT falsely shortened Reject specimen Improper storage: wrong temperature or held too long PT/PTT falsely prolonged Must follow storage requirements Chilling in refrigerator or placing on ice PT falsely shortened Chilling to 4 oC ac(vates factor VII. Inadequate centrifuga(on PTT loses sensi(vity for lupus an(coagulants and heparin. Factor assays inaccurate Desire platelet poor plasma Prolonged tourniquet applica(on Falsely elevates vWF, factor VIII Tourniquet causes venous stasis,
- 10. Analy(cal variables •Instrumenta(on – Op(cal versus mechanical clot detec(on • Affected by interferences – lipemia, bilirubinemia, etc. • Reagents – PT source (recombinant, brain extract, etc) – aPTT • ac(vator • phospholipid source and concentra(on • designed for heparin, factor VIII & IX, possible LA