Skip to content
SearchProgress
Display

Display

Theme
Density
Text size

Sign in

Add your earlier progress to your account?

Study progress is waiting to be saved

Clot formation, fibrinolysis and the citrate tube

15 min

  • Trace thrombin generation from tissue factor and factor VIIa to the platelet surface
  • Trace how plasmin forms, is inhibited, and breaks cross-linked fibrin into D-dimer
  • Reject a coagulation specimen with the wrong fill, hematocrit, handling, or timing

Read the full reference

Try first

Try first

In the body, what starts coagulation at a site of vessel injury?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Where thrombin generation starts

Coagulation proceeds in three overlapping phases on cell surfaces. Tissue factor-bearing cells bind factor VIIa and generate small amounts of factors Xa and IXa and a small amount of thrombin. That initial thrombin activates platelets and cofactors V and VIII and supports factor XI activation. Intrinsic tenase and prothrombinase then assemble on activated platelet membranes and produce the thrombin burst that forms fibrin.1 Factors VIII and IX are required for enough thrombin to form a stable clot, which is why their deficiency causes bleeding. Factor XII, prekallikrein, and high-molecular-weight kininogen drive contact activation in the activated partial thromboplastin time (APTT) assay. Normal hemostasis in the body does not depend on them, so their deficiency can prolong the APTT markedly with intact clinical hemostasis.2

From cross-linked fibrin to D-dimer

Factor XIIIa cross-links fibrin as it forms. Tissue plasminogen activator, concentrated within the clot by fibrin binding, converts plasminogen to plasmin, and plasmin digests the cross-linked fibrin into fragments that include D-dimer. Plasminogen activator inhibitor-1 and α2-antiplasmin control the reaction so that lysis stays local to the clot.3 A raised D-dimer shows that cross-linked fibrin formed and was degraded. It does not confirm a thrombus or locate one, because inflammation, pregnancy, surgery, trauma, cancer, and age can all raise it.

The citrate tube that gives a true result

Plasma clotting assays need blood collected into buffered sodium citrate at a 9:1 blood-to-anticoagulant ratio, filled to the tube's validated volume.4 A short draw leaves extra citrate for the plasma present, and that excess citrate binds part of the calcium the assay adds back, falsely prolonging clotting times. Above a hematocrit of 55%, the smaller plasma volume does the same thing to a standard tube, so the citrate volume is adjusted for the hematocrit:

Citrate volume (mL) = [(100 − hematocrit %) ÷ (595 − hematocrit %)] × intended total tube volume (mL)

A clot in the tube, the wrong additive, and collection contamination make the specimen just as unusable as a bad fill.

References
  1. Hoffman M, Monroe DM III. A cell-based model of hemostasis. Thromb Haemost. 2001;85(6):958-965. doi:10.1055/s-0037-1615947
  2. Park S, Park JK. Back to basics: the coagulation pathway. Blood Res. 2024;59(1):35. doi:10.1007/s44313-024-00040-8
  3. Longstaff C, Kolev K. Basic mechanisms and regulation of fibrinolysis. J Thromb Haemost. 2015;13(suppl 1):S98-S105. doi:10.1111/jth.12935
  4. Clinical and Laboratory Standards Institute. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays. 6th ed. CLSI standard H21. Clinical and Laboratory Standards Institute; 2024. Accessed September 26, 2026. https://clsi.org/shop/standards/h21/

Watch one

A citrate tube arrives for coagulation testing. It is filled to its line, drawn 25 minutes ago, and not yet centrifuged. The requisition shows the patient's hematocrit from this morning's complete blood count (CBC) is 59%.

  1. Check the fill: the tube is filled to its line, so a short draw is not the problem here.

    A short draw leaves extra citrate acting on a smaller plasma volume, so fill is the first thing to check.

  2. Check the hematocrit: 59% is above the 55% threshold where a standard tube holds excess citrate for the plasma present.

    Above a hematocrit of 55%, the plasma volume itself is too small for the citrate in a standard tube, whatever the fill line shows.

  3. Recognize the effect: a standard tube at this hematocrit reads clotting times longer than the patient's true values.

    The excess citrate binds part of the calcium the assay adds back, which falsely prolongs clotting times.

  4. Decide: reject the standard tube for coagulation testing and request a specimen collected in a tube with citrate adjusted for a hematocrit of 59%.

    An adjusted tube, prepared and labeled under the laboratory's validated procedure, is the only way to remove the excess citrate before testing.

Reject the standard citrate tube and request recollection with the citrate volume adjusted for the patient's hematocrit.

Your turn

Problem 1 of 3

A patient with a deficiency of factor XII has a markedly prolonged APTT and no bleeding history at any age. Which statement fits the cell-based model of coagulation?

Tissue factor and factor VIIa start coagulation at the injury. Factor XII drives the APTT reagent's contact activation, a step normal hemostasis does not depend on.

The contact pathway is not the trigger the body uses at all, in any amount. The APTT's sensitivity to factor XII belongs to the reagent's own contact-activation step.

Treated the contact pathway as the in vivo trigger

In the body, tissue factor with factor VIIa on injured tissue starts coagulation, and factors VIII and IX amplify thrombin generation on activated platelet membranes. Factor XII, prekallikrein, and high-molecular-weight kininogen (HMWK) drive the APTT reaction, which is why their deficiency prolongs the APTT without a bleeding phenotype.

The PT evaluates tissue factor and factor VII. Factor XII deficiency prolongs the APTT through the contact system the reagent uses.

Hint
  1. Ask what starts coagulation in the body, apart from what the APTT reagent adds in the tube.
  2. The APTT reagent supplies a contact activator that factor XII responds to. The body does not rely on that same trigger.

Review Classic and cell-based models

Problem 2 of 3

A coagulation specimen is needed from a patient with a hematocrit of 60%. Using the total-volume formula for an intended total tube volume of 4.5 mL, how much citrate does the adjusted tube hold?

Correct. Citrate volume = [(100 − 60) ÷ (595 − 60)] × 4.5 mL = (40 ÷ 535) × 4.5 = 0.34 mL, and the rest of the tube is filled with blood under a validated, labeled procedure.

Incorrect. That is the standard 9:1 volume. Above a hematocrit of 55%, the smaller plasma volume leaves excess citrate that binds the calcium added in the assay and falsely prolongs clotting times.

Incorrect. This scales the standard 0.45 mL up by 60 ÷ 45. A high hematocrit leaves less plasma, so the tube needs less citrate.

Hint
  1. A higher hematocrit leaves less plasma in the tube, so the tube needs a smaller citrate volume.
  2. Apply the formula with the stated hematocrit and the intended total tube volume.

Review Citrated plasma

Problem 3 of 3

A postoperative patient has a D-dimer 6 times the assay's upper limit of normal. The surgical team asks whether this confirms a deep vein thrombosis. What do you report?

Surgery, like inflammation, pregnancy, trauma, cancer, and age, raises D-dimer on its own. The result shows that cross-linked fibrin formed and was degraded. It neither confirms a thrombus nor says where one would be.

D-dimer cannot confirm or locate a thrombus. Its main clinical use is helping exclude venous thromboembolism when a result falls below the cutoff in a patient with low pretest probability.

Read a raised D-dimer as evidence of thrombosis

D-dimer shows that cross-linked fibrin formed and plasmin digested it. Inflammation, pregnancy, surgery, trauma, cancer, and age also raise it, so a raised result neither confirms nor locates a thrombus. A result below the cutoff helps exclude venous thromboembolism when it is used within a validated algorithm for patients with low clinical probability.

A raised D-dimer shows that plasmin digested cross-linked fibrin. That is fibrinolysis working as expected.

Review Fibrinolysis

Use it

  • A patient with cirrhosis and a hematocrit of 61% (MRN 0092247) has a coagulation panel ordered before a procedure.
  • The citrate tube is filled to about three-quarters of its line.
  • The phlebotomist's note says the draw was difficult and took several attempts.
  • No clot is visible in the tube.
Decision 1 of 2

What is the first specimen problem to address?

The fill is also short of the line. Both problems affect this tube's citrate-to-plasma ratio.

Tested a high-hematocrit specimen in a standard citrate tube

Above a hematocrit of 55%, the plasma is too small a volume for the citrate in a standard tube, and substantial underfill does the same. The excess citrate binds calcium added by the reagent and falsely prolongs clotting times, so recollect an underfilled tube at full fill and a high-hematocrit specimen with citrate adjusted for the hematocrit.

A hematocrit of 61% already calls for an adjusted tube, even at a full fill. Two separate problems affect the citrate-to-plasma ratio here.

The tube is short of its line, and a hematocrit above 55% also leaves too little plasma for a standard tube's citrate. Either one alone would prolong clotting times falsely, and this specimen has both.

Review Citrated plasma

Decision 2 of 2

What do you do with this specimen?

A comment does not remove the excess citrate already acting on the plasma. The clotting times would still read longer than the patient's true values.

Both defects leave excess citrate for the plasma present, and only a correctly filled, hematocrit-adjusted tube removes that excess before testing.

An ad hoc dilution after the fact is not the validated way to correct citrate excess. The adjustment belongs in how the tube is prepared and filled before the draw.

Review Citrated plasma

The clue that settles this case is that two separate problems point the same way: the short fill and the high hematocrit both leave the plasma with more citrate acting on it than the assay expects. A hematocrit-adjusted tube, filled to its line, is the only specimen that removes both problems at once.

Keep

Sources checked