MLAB 1227- Coagulation
Keri Brophy-Martinez
Laboratory Testing in Coagulation
Lab Testing For Primary Hemostasis
Disorders
 Purpose
 Evaluate platelet concentration and
function
 Tests
 Bleeding time: discussed in lab
 PFA-100
 Platelet aggregometry
Lab Testing For Secondary Hemostasis
Disorders
 Purpose
 Evaluates coagulation factors
 Detects inhibitors
 Screening Tests
 PT
 APTT
 Fibrinogen
 Thrombin Time
Discussed in lab
Thrombin Time
 Qualitative
 Useful to detect parameters not detected by PT and aPTT;
such as heparin, presence of FDPs, presence of
dys/hypofibrinogenemia
 Thrombin time is prolonged with these parameters
 Measures conversion of fibrinogen to fibrin
 Thrombin cleaves fibrinogen in undiluted plasma
 Normal 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 Causes
 Factor Deficiency
 Circulating Inhibitors
Factor Deficiency Evaluation
 First Considerations
 PT and/or APTT must be prolonged
 Patient history and symptoms must be considered
 Bleeding tendency important to note
 Reflex Testing (follow-up tests)
 Mixing Study
 Will determine whether a factor deficiency is present or a
circulating inhibitor
Mixing Study
 Also referred to as a circulating inhibitor screen
 Principle
 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 inhibitor
 Mixing study does NOT correct=Inhibitor
Mixing Study
Factor Assays
 Principle
 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 Procedure
 How 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
Correction
No correction
Anti Xa Assay
 Used to monitor LMWH
 Chromogenic assay
 Procedure
 Excess FXa is added to patient PPP
 Heparin/AT complexes in patient sample will bind
FXa
 Chromogenic substrate is added and any unbound FXa
cleaves the chromogen
 Produces a yellow color, read spectrophotometrically
 Results 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 Test
 Principle
 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 products
 Requires 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.
References
 http://labmed.yale.edu/education/cme/casestu
dies/6/7.aspx
 McKenzie, Shirlyn B., and J. Lynne. Williams.
"Chapter 40." Clinical Laboratory Hematology. 2nd
ed. Boston: Pearson, 2010. Print.