Hemostasis Testing

Specimen Requirements (Preanalytical)

Coagulation testing is exquisitely sensitive to collection technique, and the MLS exam tests this heavily. The anticoagulant is 3.2% buffered sodium citrate in the light blue-top tube, drawn at a 9:1 ratio (9 parts blood to 1 part citrate). Citrate chelates calcium reversibly; calcium is added back during testing.

Common collection errors and their effects:

Specimens are spun to platelet-poor plasma. Order of draw places the blue top after any sterile/blood-culture tube but before tubes with other additives, to avoid additive carryover. The classic teaching order of draw is: blood culture, then light blue (citrate), then red/serum, then green (heparin), then lavender (EDTA), then gray (oxalate/fluoride). Drawing the citrate tube before EDTA prevents potassium-EDTA contamination that would chelate calcium and falsely prolong results. Specimens are generally stable about 4 hours at room temperature for PT/aPTT; cold storage activates factor VII and platelets and is avoided.

The hematocrit correction matters because the volume of citrate is fixed for a normal plasma fraction; for a hematocrit above 55%, the citrate volume must be reduced using the formula citrate (mL) = 0.00185 x blood volume x (100 - Hct).

The Core Screening Tests

• Prothrombin time (PT): thromboplastin + calcium added to plasma; measures the extrinsic and common pathways. Reported as the INR = (patient PT / mean normal PT)^ISI, which standardizes warfarin monitoring across reagents.
• Activated partial thromboplastin time (aPTT): measures the intrinsic and common pathways; monitors unfractionated heparin.
• Thrombin time (TT): prolonged by low/abnormal fibrinogen and by heparin; a normal reptilase time with a prolonged TT specifically implicates heparin contamination.
• Fibrinogen (Clauss method): functional assay; reference roughly 200-400 mg/dL. Fibrinogen is also an acute-phase reactant, so it rises with inflammation and falls with consumption (DIC) or liver failure.

Reporting the INR rather than raw PT seconds is essential for warfarin. Because different thromboplastin reagents have different sensitivities, two labs could report different PT seconds for the same plasma; the INR normalizes this using each reagent's international sensitivity index (ISI) against the WHO reference, so a target INR of 2-3 means the same thing everywhere. The INR is only validated for stable warfarin therapy, not for liver disease or DIC, a subtlety the exam sometimes probes.

Mixing Studies

When the PT or aPTT is prolonged, a 1:1 mix of patient plasma with normal plasma sorts out the cause:

• Corrects: points to a factor deficiency (the normal plasma supplies the missing factor).
• Does not correct: points to an inhibitor such as a lupus anticoagulant or a specific factor inhibitor.

This logic is a frequent exam item - tie "corrects = deficiency, fails to correct = inhibitor" to memory. Some inhibitors are time- and temperature-dependent (notably a factor VIII inhibitor), so a properly performed mixing study includes an incubated phase at 37 C for 1-2 hours: a result that corrects immediately but prolongs after incubation suggests a slow-acting factor VIII inhibitor rather than a lupus anticoagulant. A lupus anticoagulant is phospholipid-dependent and is confirmed by phospholipid-neutralization tests; despite the prolonged aPTT in vitro, it paradoxically increases thrombosis risk in vivo.

D-dimer And Disseminated Intravascular Coagulation

D-dimer is a fibrin degradation product released when cross-linked fibrin is broken down by plasmin. It is elevated in venous thromboembolism, DIC, and recent surgery; its high negative predictive value lets a normal D-dimer help rule out acute thrombosis.

Disseminated intravascular coagulation (DIC) is simultaneous widespread clotting and bleeding. The classic lab picture:

• Prolonged PT and aPTT
• Low fibrinogen (consumed)
• Low platelets (consumed)
• High D-dimer / FDPs
• Schistocytes on the smear (microangiopathic fragmentation)

The exam may contrast DIC with TTP/HUS, which also produce schistocytes and thrombocytopenia but show normal PT, aPTT, and fibrinogen because they are platelet-driven microangiopathies rather than consumptive coagulopathies. This coagulation-test pattern is the discriminator: abnormal PT/aPTT and low fibrinogen point to DIC, whereas normal coagulation tests with schistocytes and low platelets point to TTP/HUS. Liver disease can mimic DIC on screening tests but typically spares factor VIII, which is made outside the liver, so a normal factor VIII level favors liver disease over DIC.

Worked Example

A patient on warfarin has a PT/INR of 5.0 (target 2-3). The INR is far above the therapeutic window, indicating over-anticoagulation and a bleeding risk; the lab should ensure the result is verified and critical-value protocols followed. Because warfarin affects the vitamin-K factors, the PT/INR - not the aPTT - is the monitored test. A second scenario: a patient on unfractionated heparin has an aPTT at the upper end of the therapeutic range while the PT is only mildly affected - expected, because heparin potentiates antithrombin and is monitored by the aPTT (or anti-Xa assay), not the PT.

Low-molecular-weight heparin generally needs no routine monitoring but, when measured, uses an anti-factor-Xa assay rather than the aPTT, a distinction the exam likes to test.

Common Traps

• Accepting a short-draw blue top - underfilling falsely prolongs results.
• Reporting raw PT seconds for warfarin instead of the INR.
• Confusing the heparin monitor (aPTT) with the warfarin monitor (PT/INR).