Welcome to the latest issue of Seminars in Thrombosis & Hemostasis, the second to be devoted to the concept of “quality” within the field of thrombosis
and hemostasis.[1] This series of issues is intended to thoughtfully cover several clinical and laboratory
issues related to diagnosis, management, and specific testing in the field of hemostasis.
The concept of quality in health care is often misinterpreted. According to the US
Institute of Medicine, it should be intended as the extent to which health care services
are capable of providing the best outcomes.[2] Pursuing total quality in health care should therefore be seen as an attitude, an
inclination that permeates an entire organization. The setting of thrombosis and hemostasis
is not an exception to this rule, inasmuch as clinical, diagnostic, and therapeutic
efforts should be directed toward the best possible outcome in patients with either
bleeding or thrombotic disorders.
The first contribution of this issue is by the Guest Editors,[3] and it covers the regulation of diagnostic products, termed “in vitro diagnostic
devices” or IVDs by the regulators. The diagnostic or test-performance process, as
associated to the evaluation or diagnosis of hemostasis and thrombosis and related
disorders, is influenced or controlled by the activity of a large number of professional,
expert, and government organizations. These may be involved in driving standardization
or harmonization, providing professional or expert guidelines, or in regulation of
therapeutic products. Although all organizations involved in this activity would propose
an intention to drive improvements in diagnostics and human health, and although there
are benefits to the overall process of standardization and regulation for tests of
hemostasis and thrombosis, it should also be recognized that there are several specific
problems and limitations to this process. As highlighted in this report, which specifically
relates to the regulation of IVDs, several case studies are used as examples to show
that regulation, aimed to reduce risks associated with the implementation of diagnostic
testing, may instead encourage the adverse outcomes of locking out clinically useful,
new, and improved technologies, and locking in old and outdated technologies. This
has potential for significant adverse outcomes related to the clinical diagnosis and
management of hemostasis- and thrombosis-related disorders. This article represents
an extension and update of previous reports from these authors.[4]
[5]
[6]
[7] Another related article (Part 2) by these authors, and devoted to regulation of
therapeutic agents in the field of thrombosis and hemostasis, is planned for a future
issue of Seminars in Thrombosis & Hemostasis.
Arterial and venous thromboses represent the most prevalent disorders worldwide, the
former ranked first among the causes of death and disability in western countries[8] and the latter being the most frequent complication of prolonged hospitalization.[9] The second article of this quality issue, by Simoens and Huys,[10] is therefore devoted to an intriguing, clinically significant and “quality” topic
related to the therapy of thrombotic disorders, and in part, also related to regulatory
considerations of drug compounds, that is, the market entry of biosimilar low-molecular-weight
heparin (LMWH) formulations in Europe. The article contains a comprehensive analysis
of regulatory requirements, pricing, reimbursement, prescribing, and dispensing of
LMWHs. Among the leading issues currently driving the market, the authors identified
some regulatory requirements such as quality dossier, biological and clinical investigations,
comparability exercises, along with costs, prices, and the adverse impact of the heparin
contamination crisis. Even more interestingly, the authors concluded that demand-side
incentives for biosimilar LMWHs are largely lacking, whereas several issues persist
about the interchangeability and substitution between originator and biosimilar LMWHs,
all of which impact the potential differential “quality” of LMWH therapy, depending
on the compounds used.
The extra-analytical phases of testing (i.e., all those “preanalytical” activities
that anticipates testing and those “postanalytical” issues related to reporting of
test results to the stakeholders—namely clinicians and patients) are the leading source
of vulnerability in diagnostic testing,[11] obviously also affecting the quality output of the hemostasis laboratory.[12]
[13]
[14] Although due to dissimilar mechanisms and to differential extents, test results
of clotting assays may be strongly influenced by the presence of common interfering
substances, especially spurious (i.e., “in vitro”) hemolysis, hyperbilirubinemia (i.e.,
icterus), and turbidity (i.e., lipemia). In the article by Lippi and colleagues,[15] the impact on hemostasis testing of the presence of these substances is comprehensively
discussed, including the leading mechanisms of interference, their impact on the specific
clotting assays, as well as the recommended approaches to limit the likelihood of
producing unsuitable test results and thus jeopardize patient safety.[16] It is noteworthy that the type and degree of interference found in the different
types of unsuitable specimens is remarkably different, so that distinctive approaches
are specifically required to manage hemolyzed, icteric, and turbid samples.
The term lupus anticoagulants (LA) include a class of antiphospholipid antibodies that show marked interference
with in vitro phospholipid-dependent clotting tests, although being paradoxically
associated with a kaleidoscope of thrombotic manifestations, including recurrent venous
and/or arterial thrombosis, as well as complications of pregnancy.[17] The current diagnostic testing for LA is an essential part of diagnosis and management
of patients with antiphospholipid syndrome (APS) and other hypercoagulable states,[18] although there are several issues that make the pertinent investigations challenging
and highly vulnerable to preanalytical, analytical, as well as postanalytical problems.[19]
[20]
[21]
[22] The leading issues in APS testing include the heterogeneous sensitivity of tests
and reagents, the clinically meaningful rate of false-negative and false-positive
results, an absence of consensus for the use of mixing tests,[23]
[24] and a well-known lack of compliance with current guidelines.[25] In the article by Adams,[26] these issues are each reviewed according to the perspective of updated guidelines
and according to results of recent external quality assessment (EQA) surveys.
In the next article, Duncan and colleagues[27] provide a timely update about the well-known issue of factor VIII (FVIII) assessment
in routine and specialized laboratories.[28] Basically, several different methods are used for measuring the plasma concentration
of FVIII; these entail one-stage or two-stage clotting assays, chromogenic tests,
and immunoassays. The chromogenic tests are a form of two-stage assay and use activated
factor X (FXa) enzymatic activity as a surrogate measure of FVIII cofactor activity
on factor IX (FIX). The one-stage clotting assays, which are mostly based on a modification
of the activated partial thromboplastin time (aPTT), represent the methodology used
by most laboratories. Interestingly, one-stage clotting assays may provide FVIII levels
that are up to 50% lower than those of the two-stage clotting, chromogenic and immunological
assays when used in patients with hemophilia A undergoing treatment with B-domain-deleted
recombinant FVIII or full-length recombinant FVIII.[28] It is noteworthy, however, that a subgroup of patients with mild hemophilia A (up
to 40%) display method-related biases in FVIII:C results, where the one-stage clotting
assay instead provides significantly higher results than the two-stage clotting or
the chromogenic assays.[27] With the aim of identifying these patients, Duncan et al recommend that automated
chromogenic assays should replace the challenging, manual two-stage clotting methods,
and provide a comprehensive set of suggestions for obtaining accurate results, especially
in the abnormal range of values.[27] This may be critical to reliably reflect the clinical scenario and thereby prevent
misdiagnosis or misclassification of this otherwise clinically significant bleeding
disorder. The authors also endorsed that due to the limited array of FVIII gene (F8) missense mutations observed in these subset of mild hemophilia A patients, molecular
analysis of the A1, A2, or A3, domains of FVIII should be advisable for guiding diagnostic,
clinical, and therapeutic decision making.
In the subsequent article, Kershaw and Orellana discuss a foremost topic for the coagulation
laboratory, that is, the issue of mixing tests.[29] Prolonged values of prothrombin time (PT), aPTT, or both, are a rather common occurrence,
which may reflect clinically threatening or “benign” deficiencies of clotting factors,[30] other bleeding disorders such as acquired inhibitors of coagulation factors (i.e.,
acquired hemophilia),[31]
[32]
[33] the presence of LA,[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26] as well as other less frequent physiological or pathological conditions.[34] Basically, a mixing test is based on the combination of an abnormal patient sample
with “normal” pooled plasma (NPP), followed by duplication of the abnormal screening
test on this mixture to establish as to whether the clotting time normalizes (“factor
deficiency”) or instead remains abnormal (“factor inhibitor”). Therefore, the inherent
aim of this relatively simple and inexpensive process is to obtain suitable information
about the potential source of prolongation of PT, aPTT, or both ([Fig. 1]). As noted earlier, a normalization of the clotting time on mixing usually reflect
an acquired or inherited clotting factor deficiency(s), whereas incomplete normalization
is always suggestive for the presence of inhibitory substance(s). In the latter case,
high dose heparin or antibodies (i.e., LA, specific coagulation factor inhibitors,
or another type of inhibitor)—that ultimately interfere with the clotting process,
by prolongation of the clotting time(s)—could be present, and this would need to be
specifically evaluated by additional testing. In this article, the authors thereby
discuss the leading objective triggering mixing test(s), the pertinent technical features
(e.g., sensitivity of reagents, composition of the NPP, different formulas for expressing
test results), as well as the recommended interpretative criteria and the subsequent
diagnostic actions.[29]
Fig. 1 Troubleshooting abnormal results of prothrombin time (PT) and activated partial thromboplastin
time (aPTT). Tissue factor pathway = “extrinsic” in older terminology; contact factor
pathway = “intrinsic” in older terminology.
The next article by Bunimov and colleagues outlines the genetic loci associated with
platelet traits and platelet disorders.[35] Genetic investigations have led to important advances in our knowledge of genes,
proteins, and micro ribonucleic acid that influence various aspects of circulating
platelet counts, size, and function. The application of genome wide association studies
(GWAS) to platelet traits has also identified multiple loci with significant association
to platelet number, size, and function in aggregation and granule secretion assays.
Moreover, the genes altered by disease causing mutations have now been identified
for several platelet disorders, including X-linked recessive, autosomal dominant,
and autosomal recessive platelet disorders. Nevertheless, the genetic mutations that
cause common inherited platelet disorders, and impair platelet aggregation and granule
secretion, remain largely unknown. This review summarizes current knowledge on the
genetic loci that influence platelet traits, including the genes with well characterized
mutations in certain inherited platelet disorders.
The last three articles in this issue comprise original articles. In the first of
these, Fuchs and colleagues investigate the distinct role of von Willebrand factor
(VWF) triplet bands in glycoprotein Ib–dependent platelet adhesion and thrombus formation
under flow.[36] These authors found that under high-shear flow conditions, a VWF preparation enriched
in intermediate triplet bands was less active in recruiting platelets to collagen
type III than a VWF preparation with similar multimeric distribution but prevalently
containing larger triplet bands. It was also observed that defective thrombus formation
was less restored when the former preparation was tested with blood from patients
with von Willebrand disease (VWD). It was therefore concluded that VWF forms that
lack larger size triplet bands seem to display a lowered ability to recruit platelets
to collagen-bound VWF under arterial flow conditions, so that variations in triplet
band distribution observed in patients with VWD may be associated with abnormal platelet
adhesiveness under high-shear flow conditions.
In the second of the original articles, McGlasson and Fritsma return us to the issue
of screening and diagnostic testing in hemostasis, and namely assessment of LA.[37] McGlasson and Fritsma assessed six dilute Russell viper venom time (DRVVT) LA screen
and confirm assays to test intermethod consistency using a large number of normal
and LA positive plasmas. Reagents were obtained from Diagnostica Stago Inc (Parsippany,
NJ), Precision BioLogic Inc (Dartmouth, Nova Scotia, Canada), Siemens Healthcare (Malvern,
PA), T Coag (Bray Co. Wicklow, Ireland), Instrumentation Laboratory (Bedford, MA),
and Sekisui Diagnostics (Framingham, MA); and all were assayed an STA-R Evolution-automated
coagulometer following manufacturer's instructions. It is noteworthy that DRVVT screen/confirm
ratios for all but one manufacturer product were found able to discriminate between
LA-positive and LA-negative samples, providing satisfactory imprecision (i.e., typically
lower than 6%) and acceptable agreement among the various assays, although a certain
degree of variability was observed in the different reagent-instrument combinations.
It was also interestingly found that normalization was virtually ineffective in improving
the clinical interpretation of test results at the local level. Nevertheless, normalization
of test results is still considered important in the context of harmonization of test
results across methodologies and across laboratories, and analogous to the international
normalized ratio test system for monitoring of vitamin K antagonist therapy.
The final article in this issue is by Bonar and colleagues.[38] As noted earlier, inhibitors to coagulation factors cause prolongation of routine
hemostasis laboratory test results, such as PT and aPTT. Inhibitors of FVIII have
particular clinical relevance in the treatment and management of hemophilia patients,
and can be either alloantibodies (as in hemophilia A), or autoantibodies (as in acquired
hemophilia).[31]
[33] The most commonly used assays for detecting these inhibitors are the classical Bethesda
assay or a modified (Nijmegen) method. Previous laboratory assessments from EQA programs
have shown a wide variability in FVIII inhibitor results and methods performed, as
well as a significant degree of false positive and false negative interpretations.
The Bethesda assay is still the primary assay used in laboratories for detecting the
presence and strength of a FVIII inhibitor. Therefore, it is of utmost importance
that this assay is appropriately performed. The current report reviews the most recent
findings from the Royal College of Pathologists of Australasia Quality Assurance Program
Haematology, which show there is still a need for better standardization and improvement
in the detection of low-level FVIII inhibitors to ultimately provide better clinical
management of affected patients in the future.
We wish, as usual, to congratulate and thank all authors to this issue of Seminars in Thrombosis & Hemostasis for their unique and comprehensive contributions, and we hope that our readership
will find interest in the issue's contents.
