RESUMO
von Willebrand disease (VWD) and haemophilia represent common inherited or acquired bleeding disorders, but many laboratories and clinicians continue to struggle with their diagnosis or exclusion. Difficulties in achieving a correct diagnosis or exclusion of VWD or haemophilia might be due to analytical issues. Sometimes assays may generate a wrong result (ie an analytical error) or may have limitations in their dynamic range of measurement and/or their level of low analytical sensitivity. Less well recognized is the influence of preanalytical issues on the diagnosis of VWD or haemophilia. Therefore, this narrative review aims to provide an overview of some important preanalytical aspects that may affect the diagnosis of VWD or haemophilia, as well as a range of solutions that may help in mitigating or abrogating their influence. The review includes discussion of the more commonly noted preanalytical issues, such as haemolysis/icterus/lipaemia, and sample collection, processing and transport. However, we also extensively discuss other less well-recognized preanalytical issues, including clinical requests, anticoagulants and anticoagulant therapy, and laboratory test choices to name a few.
Assuntos
Erros de Diagnóstico/tendências , Hemofilia A/diagnóstico , Doenças de von Willebrand/diagnóstico , Hemofilia A/patologia , Humanos , Doenças de von Willebrand/patologiaRESUMO
INTRODUCTION: von Willebrand disease (VWD) reflects a loss or dysfunction in von Willebrand factor (VWF), while haemophilia represents a loss or dysfunction of clotting factors such as factor VIII (FVIII) or FIX. Their diagnosis requires laboratory testing, with this potentially compromised by preanalytical events, including poor sample quality. This study assessed the effect of inadequate mixing as a potential cause of VWD and haemophilia misdiagnosis. METHODS: After completion of requested testing, 48 consecutive patient samples comprising separate aliquots from single collections were individually pooled, appropriately mixed, then frozen in separate aliquots, either at -20°C or -80°C for 2-7 days. Each sample set was then thawed and the separate aliquots subjected to separate mixing protocols (several inversions, blood roller, vortex) vs a non-mix sample, and all aliquots then tested for various VWF and factor assays. RESULTS: Non-mixing led to substantial reduction in VWF and factors in about 25% of samples, that in some cases could lead to misdiagnosis of VWD or haemophilia. Interestingly, there were also some differences observed with respect to different mixing protocols. CONCLUSIONS: Our study identified ineffective or variable mixing of thawed plasma samples as potential causes of misdiagnosis of VWD or haemophilia. Further education regarding the importance of appropriate mixing appears warranted.
Assuntos
Testes de Coagulação Sanguínea/normas , Hemofilia A/sangue , Hemofilia A/diagnóstico , Doenças de von Willebrand/sangue , Doenças de von Willebrand/diagnóstico , Fatores de Coagulação Sanguínea , Testes de Coagulação Sanguínea/métodos , Erros de Diagnóstico , Fator VIII , Humanos , Estudos Prospectivos , Estudos Retrospectivos , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Fator de von WillebrandRESUMO
INTRODUCTION: Appropriate diagnosis of von Willebrand disease (VWD), including differential identification of qualitative vs. quantitative von Willebrand factor (VWF) defects has important management implications, but remains problematic. AIM: The aim of the study was to assess whether 2M VWD, defining qualitative defects not associated with loss of high molecular weight (HMW) VWF, is often misidentified, given highly variable reported frequency ranging from 0 to ~60% of all type 2 VWD. METHODS: A comparative evaluation of laboratory ability to appropriately identify 2M VWD (n = 4) vs. HMW VWF reduction (n = 4), as sent to participants of an international external quality assessment programme. RESULTS: Laboratories had considerably greater difficulty identifying type 2M VWD, correctly identifying these on average only 29.4% of occasions, with the 70.6% error rate representing use of insufficient test panels (41.7%), misinterpretation of test results (10.0%) and analytical errors (13.3%). One type 2M case, giving a median of 49 U dL(-1) VWF:Ag, was more often misidentified as type 2A/2B VWD (46.7%) than 2M (34.8%). Another 2M case, giving a median of 189 U dL(-1) VWF:Ag, was instead often misidentified as being normal (non-VWD) (36.4%), with identifications of type 2A/2B VWD (13.6%) also represented. In comparison, errors in identification of HMW VWF reduced samples only averaged 11.5%, primarily driven by use of insufficient test panels (6.3%) or misinterpretation of results (4.2%) and infrequently analytical errors (1.0%). CONCLUSION: Type 2M VWD is more often misidentified (70.6%) than correctly identified as 2M VWD (29.4%), and potentially explaining the relative under-reported incidence of 2M VWD in the literature.
Assuntos
Proteínas Sanguíneas/análise , Erros de Diagnóstico/estatística & dados numéricos , Doença de von Willebrand Tipo 2/diagnóstico , Fator de von Willebrand/análise , Austrália , Coagulação Sanguínea/genética , Plaquetas/fisiologia , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Técnicas de Laboratório Clínico/normas , Diagnóstico Diferencial , Erros de Diagnóstico/prevenção & controle , Humanos , Incidência , Mutação/genética , Nova Zelândia , Valores de Referência , Doença de von Willebrand Tipo 2/epidemiologia , Fator de von Willebrand/química , Fator de von Willebrand/genéticaRESUMO
We investigated whether the contamination of samples with glucose subsequently tested for haemostasis affected the results, including prothrombin time, activated partial thromboplastin time and fibrinogen concentration. Venous blood was collected from 12 healthy subjects and divided into four aliquots, which were subjected to different degrees of contamination with standard glucose solution (0%, 5%, 10%, 20%). With increasing glucose contamination, prothrombin time increased from mean (SD) 11.0 (0.7) s to 11.2 (0.7) s, 11.5 (0.7) s and 12.2 (0.8) s, all p < 0.001. Activated partial thromboplastin time decreased from 32.3 (0.9) s to 30.9 (0.8) s, 30.8 (0.8) s, and 29.7 (0.7) s, all p < 0.001. Fibrinogen concentration decreased from 3.8 (0.7) g.l(-1) to 3.7 (0.6) g.l(-1), 3.6 (0.6) g.l(-1), and 3.4 (0.6) g.l(-1), all p < 0.001. Bias was clinically meaningful from 5% contamination for activated partial thromboplastin time, 10% contamination for prothrombin time and 20% contamination for fibrinogen concentration. We conclude that if glucose contamination of haemostasis samples is suspected or has occurred, the specimens should not be analysed.
Assuntos
Glicemia/análise , Testes Hematológicos , Hemostasia , Hiperglicemia/sangue , Fibrinogênio/análise , Voluntários Saudáveis , Humanos , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Valores de Referência , Reprodutibilidade dos TestesRESUMO
Inhibitor assays are performed when patients present with unexplained prolonged routine coagulation test times and unexpected and/or unusual bleeding (potential for acquired haemophilia) as well as being a part of normal congenital haemophilia management and monitoring, particularly when bleeding occurs on therapy, or when increments in factor levels post-factor replacement remain lower than expected. In this article, we will describe the assays used, as well as their development, pitfalls in testing such as inter-laboratory variability and false negative/positive results, as well as some strategies for overcoming these pitfalls and potential alternative test approaches. The inter-laboratory coefficient of variation often approaches (and sometimes exceeds) 50%, as evidenced by various external quality assessment groups, and this variability has not improved over recent years. Additional important considerations include appropriate interpretation of test results, repeat testing for confirmation, and assessment of recovery as part of the diagnostic process.
Assuntos
Testes de Coagulação Sanguínea/métodos , Fator VIII/imunologia , Isoanticorpos/imunologia , Testes de Coagulação Sanguínea/normas , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The diagnosis and management of bleeding disorders is made difficult by the complexity and variety of disorders, clinical symptoms and bleeding type and severity. von Willebrand disease (VWD) and platelet disorders are disorders of primary haemostasis and together represent the most common inherited bleeding disorders. In this article, we describe the diagnosis of VWD and platelet disorders and the treatment options for VWD.
Assuntos
Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/terapia , Doenças de von Willebrand/diagnóstico , Doenças de von Willebrand/terapia , Testes de Coagulação Sanguínea/métodos , Testes de Coagulação Sanguínea/normas , Hemostasia/efeitos dos fármacos , Hemostáticos/farmacologia , Hemostáticos/uso terapêutico , Humanos , Pré-MedicaçãoAssuntos
Fator VIII/genética , Mutação de Sentido Incorreto/genética , Complicações Hematológicas na Gravidez/diagnóstico , Doença de von Willebrand Tipo 2/diagnóstico , Adolescente , Fator VIII/metabolismo , Feminino , Hemorragia , Humanos , Linhagem , Gravidez , Complicações Hematológicas na Gravidez/terapia , Ligação Proteica , Adulto Jovem , Doença de von Willebrand Tipo 2/terapia , Fator de von Willebrand/metabolismoRESUMO
von Willebrand disease (VWD) is the most common inherited bleeding disorder, but variable severity and several classification types mean that diagnosis is often not straightforward. In many countries, the assays are not readily available and/or are not well standardized. The latest methods and the basis of VWD are discussed here, together with information from the international quality assessment programme (IEQAS). Factor XIII deficiency is a rare, but important bleeding disorder, which may be missed or diagnosed late. A discussion and update on this diagnosis is considered in the final section of our review.
Assuntos
Técnicas de Laboratório Clínico/normas , Deficiência do Fator XIII/diagnóstico , Doenças de von Willebrand/diagnóstico , Colágeno , Hemaglutininas , Hemofilia A/diagnóstico , Humanos , Agregação Plaquetária , Controle de Qualidade , Ristocetina , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: The antiphospholipid syndrome (APS) is an autoimmune condition characterised by vascular thromboses and/or pregnancy morbidity. Diagnosis of APS typically requires laboratory evidence of antiphospholipid antibodies (aPL). Depending on their clinical presentation, affected individuals might be seen by a variety of clinical specialities. AIM: To evaluate clinical ordering patterns for aPL/APS at a tertiary level public facility. METHODS: We performed an audit of internal clinical requests for aPL tests at our institution for a 6-month period. RESULTS: We identified a wide variety of clinical ordering background for aPL, of predominantly obstetric (72/268; 26.9%) or thrombophilic (78/268; 29.1%) patients. Only 11/268 samples (4.1%) were positive for lupus anticoagulant (LA) and 14/268 (5.2%) were positive for anticardiolipin antibody (aCL). The percentage of aCL positivity in the LA-positive group was 46% (5/11). None of the 72 obstetric patients tested was identified to have aPL. Of the 11 LA-positive patients, the reasons identified for testing comprised: prolonged Activated Partial Thromboplastin Time (assay) (n= 3), thrombosis (n= 3), APS (n= 2), systemic lupus erythematosus (n= 2), vasculitis (n= 1). CONCLUSION: We determined a wide variety of clinical ordering background for aPL at a tertiary level institution, with an overall low rate (<10%) of aPL positivity among a hospital population of predominantly obstetric or thrombophilic patients. That no positive obstetric aPL cases were identified suggests local clinical ordering guidelines may need review, as also potentially practised at other institutions. We also observed a moderate rate (46%) of coincidence of aCL and LA, in agreement with guidelines indicating that multiple tests are required to identify APS.
Assuntos
Anticorpos Anticardiolipina/sangue , Anticorpos Antifosfolipídeos/sangue , Síndrome Antifosfolipídica/diagnóstico , Trombofilia/diagnóstico , Síndrome Antifosfolipídica/imunologia , Auditoria Clínica , Feminino , Humanos , Masculino , Gravidez , Estudos Retrospectivos , Trombofilia/imunologiaRESUMO
As a result of incessant genetic discoveries and remarkable technological advancements, the availability and the consequent consumer's request for genetic testing are growing exponentially, leading to the development of a 'parallel' market, i.e. the direct-to-consumer (DTC) testing, also known as 'direct access testing' (DAT). Analogous to the traditional laboratory diagnostics, drawbacks of DTC testing might arise from any step characterising the total testing process, and include poor control of both appropriateness and preanalytical requirements, potential operation outside national or international regulation for in vitro diagnostic testing, little evidence of quality as well as the risk of transfer of genetic materials from the companies to other entities. Another important issue is the test panels offered to consumers, which are often based on preliminary, speculative or unsupported scientific information. Finally, the potential of this type of testing to generate anxiety or false reassurance should also be carefully considered. Although DTC testing carries some theoretical advantages (e.g. greater consumer autonomy and empowerment), solid clinical studies and costs vs. benefit analyses are needed to definitely establish whether DTC testing might be effective for decreasing the burden of diseases, delay their onset or modify their progression and therefore the clinical outcome.
Assuntos
Acesso à Informação , Testes Genéticos/normas , Marketing de Serviços de Saúde/organização & administração , Comércio , Marcadores Genéticos , Testes Genéticos/métodos , Humanos , Aceitação pelo Paciente de Cuidados de Saúde , Sistemas de Identificação de Pacientes/normas , Controle de Qualidade , Projetos de Pesquisa , Manejo de Espécimes/normas , Trombofilia/genéticaRESUMO
GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.
Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/química , Glicoproteínas da Membrana de Plaquetas/química , Sequência de Aminoácidos , Antígenos CD/química , Humanos , Dados de Sequência Molecular , Selectina-P , Fragmentos de Peptídeos/química , Ativação Plaquetária , SolubilidadeRESUMO
The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titres during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either using the original or Nijmegen-modified Bethesda assay. In view of previously demonstrated high variability in laboratory results for inhibitor assays, we have more extensively examined laboratory performance in the identification of FVIII inhibitors. Over the past 3 years, we conducted two questionnaire-based surveys and two wet-challenge surveys utilizing eight samples comprising no FVIII inhibitor (n = 1), or low-titre (n = 2), medium-titre (n = 3) or high-titre (n = 2) FVIII inhibitor. Four samples were tested by 42 laboratories in 2007, and four by 52 laboratories in 2009. High inter-laboratory variation was evident, with CVs around 50% not uncommon, and some 10% of all laboratories (or around 15% of laboratories using Bethesda method) failed to detect low-level inhibitors of around 1 BU mL(-1). Laboratories using the Nijmegen method appeared to perform better than those using a standard Bethesda assay, with lower evident assay variation and no false negatives. There was a wide variety of laboratory practice, with no two laboratories using exactly the same process for testing and interpretation of factor inhibitor findings. In conclusion, our study indicates that there is still much need for standardization and improvement in factor inhibitor detection, and we hope that our findings provide a basis for future improvements in this area.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/análise , Testes de Coagulação Sanguínea/normas , Fator VIII/antagonistas & inibidores , Australásia , Humanos , Garantia da Qualidade dos Cuidados de Saúde , Inquéritos e QuestionáriosAssuntos
Hemostasia/efeitos dos fármacos , Cloreto de Sódio/administração & dosagem , Adulto , Relação Dose-Resposta a Droga , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/efeitos dos fármacos , Fibrinogênio/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Reprodutibilidade dos TestesRESUMO
Consensus guidelines on anti-beta 2 glycoprotein I (anti-beta2GPI) testing have been developed to help minimise laboratory variation in the performance and reporting of assays for these antibodies. These guidelines include minimum and optional recommendations for the following aspects of anti-beta2GPI testing and reporting: (1) isotype of anti-beta2GPI tested; (2) specimen type; (3) controls and assay precision; (4) calibrators; (5) patient samples; (6) rheumatoid factors and IgM anti-beta2GPI testing; (7) reporting of results; (8) cutoff values; and (9) interpretative comments. Issues related to inter-kit/assay standardisation and the manufacturing process of commercial anti-beta2GPI kits/assays have not been addressed in the current guidelines.
Assuntos
Anticorpos Anti-Idiotípicos/sangue , Síndrome Antifosfolipídica/diagnóstico , beta 2-Glicoproteína I/imunologia , Anticorpos Anti-Idiotípicos/imunologia , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/imunologia , Australásia , Cardiolipinas/imunologia , Técnicas de Laboratório Clínico , Humanos , Sensibilidade e EspecificidadeRESUMO
von Willebrand disease (VWD) is a bleeding disorder caused by inherited defects in the concentration, structure, or function of von Willebrand factor (VWF). VWD is classified into three primary categories. Type 1 includes partial quantitative deficiency, type 2 includes qualitative defects, and type 3 includes virtually complete deficiency of VWF. VWD type 2 is divided into four secondary categories. Type 2A includes variants with decreased platelet adhesion caused by selective deficiency of high-molecular-weight VWF multimers. Type 2B includes variants with increased affinity for platelet glycoprotein Ib. Type 2M includes variants with markedly defective platelet adhesion despite a relatively normal size distribution of VWF multimers. Type 2N includes variants with markedly decreased affinity for factor VIII. These six categories of VWD correlate with important clinical features and therapeutic requirements. Some VWF gene mutations, alone or in combination, have complex effects and give rise to mixed VWD phenotypes. Certain VWD types, especially type 1 and type 2A, encompass several pathophysiologic mechanisms that sometimes can be distinguished by appropriate laboratory studies. The clinical significance of this heterogeneity is under investigation, which may support further subdivision of VWD type 1 or type 2A in the future.