Abstract:
Testing and screening for factor deficiency using one-stage clotting
assays is the most prevalent methodology, but it is susceptible to
misestimating a patient’s true coagulable state due to either the clot
detection methodology or the aPTT reagents involved. In our case report,
a 16-year-old with mild hemophilia B had a normal aPTT and factor IX
activity using a new platform in the clinical laboratory. We evaluated
the impact of instrument and aPTT reagents that differed in clot
detection methodology, the activator and the source of phospholipid in
one stage assays (OSA) for FIX:C and aPTT using plasma from this
patient.
Introduction:
Laboratory testing for bleeding disorders includes two widely available
screening assays for coagulation factor deficiencies, activated partial
thromboplastin time (aPTT) and prothrombin time
(PT).1-3 Clinicians must understand the potential
limitations of screening and specific factor activity testing, as
performed in their laboratory, to accurately reflect their patient’s
hemostatic status. The aPTT independently tests coagulation factors
VIII, IX and XI, but also assesses contact factors XII, prekallikrein
and high molecular weight kininogen. The aPTT shares screening of
“common pathway” factors V, X, II and I with the PT, which
independently screens factor VII.3 An isolated
prolongation of the aPTT (with normal PT) would prompt the clinician to
evaluate factors XI, IX and VIII for deficiency that might be associated
with a bleeding phenotype. Normal PT and aPTT may provide reassurance
against a severe deficiency of the screened coagulation factors. We
report on a failure of the screening aPTT and a specific factor activity
assay to detect mild factor IX deficiency.
A variation of the aPTT utilizing specific factor-deficient plasma can
estimate the patient’s factor VIII, IX or XI activity to detect
deficiency or monitor coagulation factor infusions.3-5Variant aPTT testing can monitor unfractionated heparin effect or screen
for a lupus anticoagulant.2 When screening or testing
for hemophilia is desired, the aPTT should be free from heparin
contamination and sensitive to low factor XI, IX or VIII activity, but
have low sensitivity to factor XII deficiency or lupus
anticoagulants.2 Most laboratories use a one-stage
assay (OSA) for the aPTT. Patient plasma is mixed with an aPTT reagent,
incubated briefly at 37 oC, and recalcified. Time to
fibrin clot formation is measured. For specific factor assays, patient
plasma is diluted with stock plasma deficient in the factor of interest,
and the correction of the aPTT is compared to a standard curve of serial
dilutions with known factor activity.1
Coagulation instruments differ in their methods for clot detection.
Mechanical detection senses cessation of movement of a metal ball
between two magnets. Photo-optical detection registers a change in
optical density or turbidity. Various manufacturers offer aPTT reagents
that differ in the contact activator (silica, ellagic acid, or kaolin)
and phospholipid source (animal brain, plant-based or a combination).
Clinicians must understand the strengths and pitfalls of their
laboratory’s platform (instrument + aPTT reagent), particularly when
results deviate from expectations.
Case Description: A 16-year-old with mild hemophilia B (HB), baseline
factor IX activity (FIX:C) 0.14-0.20
IU mL-1, had normal
FIX:C and aPTT from annual comprehensive visit testing, despite no
recent factor IX product infusion. Review of recent annual testing
revealed FIX:C had been higher for the past few years, temporally
corresponding with a change in the laboratory coagulation platform. The
patient’s de novo factor IX gene (F9) missense mutation in
exon 8, c.1174 A>G (p.Asn392Asp), impacts subdomain 2 of
the C-terminal catalytic protease domain6,7 and is
consistent with type 2 HB.
Methods: Assent and consent had been given by the patient and his
guardian, respectively, for participation in the institutional,
IRB-approved tissue and data repository for bleeding disorders. Residual
3.2% sodium citrate buffered platelet-poor plasma (PPP) samples frozen
at -70oC were available for testing. The PPP was
obtained from citrated whole blood samples via double centrifugation at
4440 × g for 3 minutes. Samples from several visits were assayed for
FIX:C and aPTT on four coagulation instruments, using various aPTT
reagents (Table 1).
Results:
The most disparate results of a normal range aPTT and increased FIX:C
relative to historical values were generated using the clinical
laboratory’s platform which utilizes photo-optical detection of clot
formation and an aPTT reagent with a purified soy-based phosphatide
lipid. Other platforms generated prolonged aPTT and FIX:C levels in
range for a diagnosis of mild hemophilia B (Table 2).
Discussion:
For the detection, classification and treatment of hemophilia,
clinicians require laboratory tests with acceptable accuracy and
precision.5 Historically the OSA aPTT should be
sensitive to FIX:C of <30 IU
mL-1.8 We demonstrate that
coagulation test results are highly dependent upon the testing platform,
which is a combination of the instrument’s method of clot detection as
well as the phospholipid source and activator in the aPTT reagent.
The OSA has been reported to misestimate activity in a subset of FVIII
deficient patients/factor products, but this is less well-appreciated
for other contact sequence factors, including factor IX. Over-estimation
of FIX:C has been reported in HB,3 particularly type 2
HB.4 In physiologic coagulation, activated FIX
interacts with coagulation cofactor VIIIa via its protease and
epidermal-growth-factor-like 2 domains on the platelet surface.7 Our patient’s protease domain mutation likely
affects binding to the FVIII A2 subunit.6,7 Abnormal
interaction of type 2 HB plasma with soy phospholipids has been
hypothesized based upon reduced sensitivity to FIX deficiency in type 2
HB plasmas tested with plant-based
phosphatides.8,11,12
Our coagulation testing platforms varied in clot detection method, as
well as both aPTT reagent phospholipid source and activator. Clot
detection (mechanical vs. optical) seems of less impact than differences
in fatty acid content and source of the aPTT reagent (Table
1).9,10 The historical concern about plant-based
phosphatides is not widely known.11 In a three-way
laboratory comparison of plasma FIX:C in 40 HB patients, Pouplard et al.
(2009)8 reported FIX:C overestimation in type 2, but
not type 1, HB plasmas assayed with a soy phosphatide-containing aPTT
reagent. An abnormal interaction of the mutated FIX with soy
phospholipids was hypothesized.8 The effect of
variation in contact activators is less clear, since phospholipid source
and activators differ by reagent. Touri and Peerschke
(1986)12 examined three activators in (older) aPTT
reagents and ranked kaolin less sensitive than ellagic acid or silica
for deficiencies in factors VIII, IX and XII, although factor XI
deficiency was equally detectable by all. Silica was more sensitive than
ellagic acid to factor IX deficiency. 12
Ultimately an aPTT reagent change was implemented.
Conclusion
For over 20 years, imperfect sensitivity has been described for some OSA
platforms, not only for FVIII, but also for FIX and FXI8,11,12; however, clinicians may be less aware of
potential differences for assays other than FVIII:C.11Serious consideration should be given to selection of the coagulation
platform for screening and factor activity testing. Differential
sensitivities, at least in part attributed to the phospholipid source in
aPTT reagents, could adversely affect identification and management of
patients with hemophilia. While chromogenic assays do not suffer from
this problem, they are not common as front-line testing.
We report a patient with known mild HB, attributable to rarer type 2 HB,
who had a normal FIX:C and aPTT result in a recently-acquired
coagulation platform employing photo-optical clot detection and a soy
phospholipid aPTT reagent. Back-up testing on a different platform
should be considered if OSA results are inconsistent or unexpected. More
importantly, clinicians should work with their laboratories both on the
choices for coagulation testing platform and in understanding the
potential for over- or under-estimating results using OSAs.
Conflict of Interests
The authors claim no conflict of interests.
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