Laboratory Testing in Coagulation

MLAB 1227- CoagulationKeri Brophy-Martinez

Lab Testing For Primary Hemostasis DisordersPurpose

Evaluate platelet concentration and function

TestsBleeding time: discussed in lab

PFA-100Platelet aggregometry

Lab Testing For Secondary Hemostasis Disorders

PurposeEvaluates coagulation factorsDetects inhibitors

Screening TestsPTAPTT Discussed in lab FibrinogenThrombin Time

Thrombin Time

QualitativeUseful to detect parameters not detected by PT

and aPTT; such as heparin, presence of FDPs, presence of dys/hypofibrinogenemiaThrombin time is prolonged with these

parametersMeasures conversion of fibrinogen to fibrinThrombin cleaves fibrinogen in undiluted

plasmaNormal reference range 10-16 seconds

Lab Testing For Secondary Hemostasis Disorders

If a screening test is prolonged, further testing must be performed to locate the specific cause of the abnormality

2 Possible CausesFactor DeficiencyCirculating Inhibitors

Factor Deficiency Evaluation

First ConsiderationsPT and/or APTT must be prolongedPatient history and symptoms must be

consideredBleeding tendency important to note

Reflex Testing (follow-up tests)Mixing Study

Will determine whether a factor deficiency is present or a circulating inhibitor

Mixing StudyAlso referred to as a circulating inhibitor

screenPrinciple

Patient plasma is diluted with normal pooled plasma to demonstrate factor levels

The normal plasma provides the missing factor in the patient plasma

50% activity is generally ample to produce a normal PT or APTT

Clotting times tend to increase with time and incubation due to the loss of labile factors, so it is important to compare the results of the patient’s diluted sample with the normal pooled plasma

Mixing Study

If the procedure corrects the prolonged PT or APTT, the defect is in the procoagulant family. Mixing study corrects=Factor deficiency

If the procedure does not correct the PT or APTT, the defect is due to an inhibitorMixing study does NOT correct=Inhibitor

Mixing Study

Factor AssaysPrinciple

Ability of the patient’s plasma to correct a prolonged PT or APTT of a known factor deficient plasma

Normal activity range is 50-150% or 50% factor activity

Determines type of factor deficiency and activity

Targets either PT: Factors VII, X,V, III and II APTT: Factors XII, XI,IX and VIII

Factor Assay ProcedureHow is it done?

1.Commercially prepared Factor deficient plasmas are used that contain 100% of all factors except the one in question. A series of these plasmas is usually used which contain various levels of the factor. For example 0%, 10%, 20%, 30%, 40%, 50%, etc.

2.As a control to compare results to, normal plasma (containing 100% of all factors) is added to the commercially prepared factor deficient plasma in the same way.

Factor Assay Procedure, cont’d.

3. The patient mixture results and the normal control mixture results are then compared to quantitate the factor level in the patient plasma.

4. A factor assay curve is the basis for plotting patient clotting times at various dilutions

5. Results of the patient are expressed as a percent of the normal plasma. A patient with a normal factor level should be 50%-150% of the normal control plasma.

Inhibitor Screens

When a PT or PTT is prolonged it must first be determined if the defect is due to a true factor deficiency or if it may be due to an abnormal circulating inhibitor (autoantibody to a factor). An inhibitor screen will rule out one or the other.

Inhibitor Screen Procedure Based on the fact that if a specimen

contains at least 50% of a normal level of factors, the PT or APTT will give a normal result.

Normal plasma (containing 100% of all factors and giving a normal APTT) is added in equal proportion to the unknown plasma (which has already given us an abnormal APTT result).

This 1:1 mixture we know contains at least 50% of all factors (because we added it) so we expect the APTT on this mixture to be normal.

Inhibitor Screen Procedure, cont’d.

If the APTT on the 1:1 mixture results in a “corrected” APTT (the patient sample was abnormal before but is normal now that we added normal plasma to it), then this indicates a factor deficiency was present in the original patient sample. The problem is not an autoantibody.

If the APTT on the 1:1 mixture does not correct the APTT, (the APTT is still abnormal even after normal plasma was added), then this indicates there is an autoantibody present. This antibody is not only binding the patient's factors, but the factors in the normal plasma that was added to the mixture as well.

Lupus Inhibitor Screen

Lupus inhibitor should be suspected in a patient with markedly prolonged PT and APTT, but no clinical symptoms or bleeding problems.

The abnormal antibody reacts mildly in vivo with thrombotic events, but reacts stronger in vitro by binding to the phospholipids in the reagents used for coag testing. Commercially prepared reagents are available that do not contain phospholipids and should be used to perform the APTT on these patients. APTT results will then be normal

Factor Deficiency vs. Circulating Inhibitor

Deficiency or Inhibitor

Immediate PT or APTT after Mixing

Study

Mixing study following 2 hour

incubation

Factor deficiency Correction Correction

Lupus-like anticoagulant

No correction No correction

F-VIII inhibitor CorrectionNo correction

Anti Xa Assay

Used to monitor LMWHChromogenic assayProcedure

Excess FXa is added to patient PPPHeparin/AT complexes in patient sample will

bind FXa Chromogenic substrate is added and any

unbound FXa cleaves the chromogenProduces a yellow color, read

spectrophotometricallyResults read off a standard curve

Factor XIII

Necessary for the formation of a stable fibrin clot

Cross-linking by Factor XIII does not affect PT and APTT

F-XIII deficiency test indicated if screening tests are NORMAL

Patient exhibits delayed bleeding, poor wound healing, or bruising

F-XIII TestPrinciple

Based on the observation that the fibrin clot has increased solubility because of the lack of cross-linking of the fibrin polymer in the absence of F-XIII

Patient PPP mixed with calcium chloride and allowed to clot for an hour at 37°C.

The clot is removed and placed in another tube containing urea

If the clot dissolves in less than 24 hours, there is less than 2% F-XIII activity

Lab Testing of the Fibrinolytic System

D-Dimer --Discussed in lab

FDP Detects fibrin/fibrinogen degradation

productsRequires a special collection tube that

contains thrombin and a fibrinolytic inhibitor

Patient serum is mixed with latex particles that detect FDPs and slide is observed for agglutination

Activated Clotting Time= ACT

Developed to monitor coagulation status and heparinization in immediate need situations.

Bedside test (POC) The ACT uses tubes containing a negatively

charged particulate activator of coagulation, such as kaolin.

When whole blood is drawn into the tube, the contact system is activated and clotting occurs.

This assay is particularly sensitive to heparin, but is affected by platelets, coagulation factors, and inhibitors.

Referenceshttp://labmed.yale.edu/education/c

me/casestudies/6/7.aspxMcKenzie, Shirlyn B., and J. Lynne.

Williams. "Chapter 40." Clinical Laboratory Hematology. 2nd ed. Boston: Pearson, 2010. Print.