ADLM 2025 Special Edition Summer Newsletter - Flipbook - Page 10
Science of Sysmex - Hemostasis
Laboratory Testing of Hemophilia
Hemophilia is a bleeding disorder caused by the
de昀椀ciency of either coagulation factor VIII (FVIII) or
factor IX (FIX), called hemophilia A and B respectively.
Prolongation of the baseline coagulation screening tests
would identify a patient with a clotting factor de昀椀ciency.
Speci昀椀c clotting factor assays are needed to identify the
speci昀椀c factor that is de昀椀cient. The expected coagulation
test result 昀椀ndings in a patient with hemophilia are shown
in Table 1.
Laboratory testing for hemophilia
When the APTT is prolonged, correction studies should
be performed by mixing the patient’s plasma with normal
pooled plasma in a ratio of 1:1. This mixing study is used to
determine whether the prolongation of the APTT is due
to a factor de昀椀ciency – which would correct the APTT
– or due to an inhibitor such as heparin – which would
not correct the APTT. Figure 1 shows the coagulation
pathway and the roles played by FVIII and FIX. A
de昀椀ciency of either of these clotting factors will prolong
the clotting time of the APTT to above the normal range.
Individual factor assays must be performed to con昀椀rm
a diagnosis of hemophilia. Because hemophilia A is far
more common than hemophilia B, it is typical to 昀椀rst
perform a FVIII assay, followed by a FIX assay if the
FVIII level is normal. The test principle and method are
essentially the same for both factors, but this section will
describe testing for FVIII only.
Prothrombin time (PT)
Normal
Activated partial thromboplastin
time (APTT)
Prolonged
Thrombin time (TT)
Normal
Fibrinogen
Normal
Platelet count
Normal
Table 1: Expected 昀椀ndings for baseline laboratory tests in hemophilia
a)Test principle of the FVIII assay
The activated partial thromboplastin time (APTT)
assesses the collective function of coagulation factors
that make up the intrinsic pathway and common
coagulation pathway (FXII, FXI, FIX, FVIII, FX, FV, FII,
昀椀brinogen). For the APTT to be within the normal range,
all the participating factors need to be present in normal
or near normal quantities. As all factors are essential for
the ultimate generation of a 昀椀brin clot, a de昀椀ciency of
any one clotting factor will result in a prolonged APTT.
The clotting time of the APTT will be prolonged in direct
proportion to the extent of the de昀椀ciency—i.e., the lower
the level of factor, the longer the clotting time. Therefore,
if the only variable altering the APTT of a patient sample
is the level of a speci昀椀c coagulation factor, then the value
of the APTT can be used to calculate the concentration of
the missing factor.
In the FVIII assay, the patient sample with an unknown
FVIII level is mixed with a reference plasma which
contains normal quantities of all clotting factors except
factor VIII—this plasma is referred to as FVIII de昀椀cient
plasma. By performing this mix, all clotting factor levels
are normalized except for FVIII which is the unknown
quantity under investigation. Any prolongation of
the APTT can therefore be solely attributed to the
concentration of FVIII in the patient sample.
Fig. 1: Clotting factors measured by the PT and APTT.
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