Factor

Deficiencies
and Inhibitor
Studies

SHERWIN B. TORIANO, RMT, MSMT

 Factor Deficiencies and
Inhibitor Studies
Routine Screening Tests of Coagulation
Test Interpretation
• Platelet count Platelet number
• Peripheral blood smear Cellular morphology and count
• Bleeding time (BT) Platelet & vessels function
• Prothrombin time (PT) Extrinsic and common pathway
• APTT Intrinsic and common pathway
• Thrombin time (TT) Fibrinogen conversion to fibrin
Factor Deficiencies and
Inhibitor Studies
Interpretation
After performing the routine tests, one may consider the
following situations, in which the deficiency, together with
possible causes are presented:

 PT high and APTT normal

• Deficiency: low VII
• Causes: early liver disease, vitamin K deficiency,
warfarin or dicoumarol treatment , factor VII inhibitor
Factor Deficiencies and
Inhibitor Studies

 PT high and APTT high

• Deficiency: low I,II,V,X (fibrinogen group)
• Causes: single or multiple factor deficiency, e.g. DIC,
liver failure, vitamin K deficiency, factor inhibitors,
improper anticoagulant ratio, high doses of heparin or
warfarin effect, and low fibrinogen (<80 mg/dl),
dysfibrinogenemia and afibrinogenemia.
Factor Deficiencies and
Inhibitor Studies

 PT normal and APTT high

• Deficiency: low VIII, IX, XI, XII (contact factors)
• Causes: hemophilia A or B, von Willebrand disease,
single or multiple deficiency, heparin, non-specific
inhibitors of these factors (e.g. lupus anticoagulants),
antiphospholipid antibodies
Factor Deficiencies and
Inhibitor Studies
 PT normal and APTT normal
• Deficiency: low XIII
• Causes: normal patient, platelet abnormality,
single deficiency of factor XIII, LMW heparin.

Thrombin time (TT) high:

• DIC (FDPs interfere with polymerization)
• low fibrinogen levels
• dysfibrinogenemia
• presence of heparin or direct thrombin
inhibitors (very sensitive).
 Factor Deficiencies and
Inhibitor Studies
Correlation
• In Disseminated Intravascular coagulation (DIC):
Platelet count low, fibrinogen low, TT high, FDP high.
• In Vit. K deficiency:
PT high, assay for II, VII, IX, X low.
Treatment:
Give vit K and repeat after 48 hrs. If PT will not correct
to normal with vit K treatment, it means liver disease.
If PT returns to normal, it means vit. K deficiency.
Factor Deficiencies and
Inhibitor Studies

• If BT, APTT, PT are all normal, in the presence of

bleeding, one or more of the following must be true:
 surgical problem (suture deficiency)
 patient is hypothermic (APTT, PT run at 37C)
 laboratory test error
Factor Deficiencies and
Inhibitor Studies

Evaluation of an Elevated Prothrombin time (PT):

• If an elevated PT is the only laboratory abnormality, this
indicates a factor VII deficiency and usually confers no
additional risk of bleeding since one needs only 5 -10 %
of normal factor VII levels to support hemostasis.
• Congenital factor VII deficiency is very rare and presents
with childhood bleeding. Heterozygotes for factor VII
deficiency present with no bleeding but an elevated
prothrombin time (INR 1.5–2.0).
 Factor Deficiencies and
Inhibitor Studies

• The most common acquired etiology of an elevated PT is

vitamin K deficiency due to warfarin use or inadequate
vitamin K intake.
• Liver disease is the next most common acquired cause.
Since factor VII has the shortest half-life, its synthesis
(and levels) will drop first with liver disease.
 Factor Deficiencies and
Inhibitor Studies
Evaluation of Combined elevations of the PT and aPTT

• Combined elevations of the PT and aPTT, the cause is

either the rare factor V, X, or II deficiency or multiple
acquired defects such as those occurring with DIC.
• In very sick patient’s levels of factor VII often fall, causing
a modest prolongation of the PT (INR up to 3.0).
 Factor Deficiencies and
Inhibitor Studies

Evaluation of an elevated Activated Partial Thromboplastin

Time (APTT)
There are four etiologies to consider when the aPTT is elevated:
• Factor deficiency
• Factor inhibitors
• Lupus inhibitors (antiphospholipid antibodies, APLA)
• Heparin or direct anticoagulation
Factor Deficiencies and
Inhibitor Studies

• Factor deficiency
The aPTT does not rise until the plasma level of a single
coagulation factor is below 30–40 %. However, only mild
decrements (60–70% range) in multiple factors will
prolong the aPTT.
Factor Deficiencies and
Inhibitor Studies

• Factor inhibitors
These are antibodies to specific coagulation factors
such as factor VIII. These inhibitors are usually found in
people with hemophilia, or may be acquired in the
elderly, or after pregnancy. The presence of these
inhibitors is usually associated with severe bleeding,
often with large ecchymoses.
Factor Deficiencies and
Inhibitor Studies

• Lupus inhibitors (antiphospholipid antibodies)

Antiphospholipid antibodies (APLA) are antibodies that
react with certain phospholipids in the body. They will also
react with the phospholipid in the test reagent for the aPTT.
Thus, they will artifactually prolong the aPTT. The presence
of these antibodies may indicate a higher risk of thrombosis
and not bleeding.
Factor Deficiencies and
Inhibitor Studies

They may be a part of an autoimmune disease, found

after infections, with intake of certain medicines, and
can occur in low titers in up to 30% of the population.

• Heparin or direct anticoagulation

Heparin, even in minute amounts, can prolong the
aPTT. This most often occurs when blood for the aPTT
is drawn from catheter lines.
MIXING STUDIES
 MIXING STUDIES

Mixing studies are tests performed on blood plasma used

to distinguish:
• factor deficiencies from factor inhibitors, such as lupus
anticoagulant
• specific factor inhibitors, such as antibodies directed
against factor VIII.
Mixing studies take advantage of the fact that factor levels
that are 50% of normal should give a normal activated
Partial thromboplastin time (APTT) result.
 MIXING STUDIES

• If the problem is a simple factor deficiency, mixing the

patient plasma 1:1 or 50:50 with plasma that contains
100% of the normal factor level results in a level ≥50% in
the mixture (say the patient has an activity of 0%; the
average of 100% + 0% = 50%). The APTT will be normal
(the mixing study shows correction).
• Correction with mixing indicates factor deficiency; failure
to correct indicates an inhibitor.
• Performing a thrombin time on the test plasma can
provide useful additional information for the
interpretation of mixing tests.
 MIXING STUDIES

• Some inhibitors are time dependent. In other words, it

takes time for the antibody to react with and inactivate
the added clotting factor. The clotting test performed
immediately after the specimens are mixed may show
correction because the antibody has not had time to
inactivate its target factor.
• A test performed after the mixture is incubated for 1 to 2
hours at 37°C will show significant prolongation over the
clotting time obtained after immediate mixing.
 MIXING STUDIES

• Many specific factor inhibitors are time dependent, and

the inhibitor will not be detected unless the test is
repeated after incubation (factor VIII inhibitors are
notorious for this).
• Nonspecific inhibitors, like the lupus anticoagulant
usually, are not time dependent; the immediate mixture
will show prolongation.
 MIXING STUDIES

The Four causes of elevated APTT and the response to

50:50 Mixing studies:
1. Factor deficiency - corrects
2. Anti-phospholipid antibodies - do not fully correct
3. Factor inhibitors - correct at time 0, and then prolonged
4. Heparin, direct anticoagulants - does not correct (usually
obvious from history)
 MIXING STUDIES

Patient’s APTT 50 (in seconds)

Results after mixing patient’s plasma with normal plasma (1:1):
Incubation Time 0 30 60 120 (in minutes at 37C)
(1) Corrected 30 32 33 34
(2) Uncorrected 40 38 42 39
(3) Corrected at 0 30 40 45 65 (prolonged up to 2 hrs)

Interpretation: 1. Factor deficiency

2. Antiphospholipid or Lupus antibodies
3. Factor inhibitor (to VIII)
 MIXING STUDIES

1. Factor deficiency
• An initially elevated aPTT will correct to normal at time 0
and stays in the normal range on each of the time points.
Since it takes only 30–40 % of normal coagulation
factors to normalize the aPTT, even with a complete lack
of a factor, the mixing of the normal pool will raise this
level to 50 % and normalize the aPTT.
 MIXING STUDIES

2. APLA (Antiphospholipid antibodies)

• The aPTT does not correct to normal at time 0 or any time
point. The aPTT may actually prolong further with the
addition of patient’s plasma (lupus cofactor effect). The
crucial point is that the aPTT will not fully correct with the
50:50 mix.
 MIXING STUDIES

3. Factor inhibitors
• The aPTT may correct to normal at time 0 but then
prolongs with further incubation. This demonstrates the
importance of the incubation step in performing the
mixing test. Really strong inhibitors may prolong the
50:50 mix aPTT even at time 0, but the aPTT will be
more prolonged with longer incubation.
 Coagulation Factor Inhibitors

Inhibitors are antibodies that in coagulation are usually

targeted against either:
- Specific clotting factors, e.g. FVIII
- Phospholipids, i.e. a lupus anticoagulant (most lupus
anticoagulants are directed against a protein-phospholipid
combination)

Circulating inhibitors or anticoagulants that target clotting

factors may be either time-dependent e.g. FVIII inhibitors, or
immediately acting e.g. FIX inhibitors.
 Coagulation Factor Inhibitors

• An inhibitor is usually suspected from the clinical

history or the finding of a prolonged clotting test that
does not correct in a 50:50 mix with normal plasma.
The most frequently seen inhibitors are targeted
against phospholipids – the lupus anticoagulant.
• Lupus anticoagulant (also known as lupus antibody,
LA, LAC, or lupus inhibitors) is an immunoglobulin
that binds to phospholipids and proteins associated
with the cell membrane.
 Coagulation Factor Inhibitors

• Lupus anticoagulant is a misnomer, as it is actually a

prothrombotic agent. Lupus anticoagulant antibodies in
living systems cause an increase in blood clotting. The
name is derived from their properties in vitro, since in
laboratory tests, these antibodies increase aPTT.
• In vivo, the antibodies are thought to interact with platelet
membrane phospholipids, increasing adhesion and
aggregation of platelets, which accounts for the in vivo
prothrombotic characteristics.
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