Models for releasing the lupus anticoagulant test / Modelos de liberação do teste anticoagulante lúpico

ABSTRACT Introduction: Thrombophilia is a thrombosis susceptibility of genetic, acquired or mixed nature. Among acquired causes, the antiphospholipid syndrome (APS) stands out as an autoimmune disease characterized by antiphospholipid antibodies, thrombotic events or recurrent gestational loss. Laboratory diagnosis is based on the detection of lupus anticoagulant (LAC), anti-β2-glycoprotein 1 and anticardiolipin; however the determination of LAC still demands uniformity. The last guideline published by the Clinical and Laboratory Standards Institute (CLSI) prioritizes the screening and confirmatory steps, to the detriment of the mixing phase. Objectives: To compare the forms of releasing the LAC and to adopt an investigation protocol in agreement with the international guidelines. Methods: Thirty-six samples with prolonged results in the screening step by the dilute Russell viper venom time (dRVVT) or activated partial thromboplastin time (APTT) were subjected to the mixing steps (1:1) and to the confirmatory steps with high concentrations of phospholipids. Results: For APTT, values whose indexes of circulating anticoagulant (ICA) were greater than 15% were considered positive. For dRVVT, the ratio between screening and confirmation was also used. Of the 36 tested samples, 14 showed correction in the mixing step, but only one resulted negative. Conclusion: ICA aided in identifying the weak antibodies that were probably diluted in the mixing step. There is no gold standard test for the diagnosis of APS, and LAC detection still requires standardization of technique and interpretation.

RESUMO Introdução: Trombofilia é a suscetibilidade à trombose, de natureza genética, adquirida ou mista. Entre as causas adquiridas, destaca-se a síndrome do anticorpo antifosfolípide (SAF) - doença autoimune caracterizada por anticorpos antifosfolípides, eventos trombóticos ou perda gestacional recorrente. O diagnóstico laboratorial baseia-se na detecção do anticoagulante lúpico (ACL), do anti-β2-glicoproteína 1 e da anticardiolipina; entretanto a execução do ACL ainda demanda uniformização. A última diretriz publicada pelo Clinical and Laboratory Standards Institute (CLSI) prioriza as etapas de triagem e confirmatória, em detrimento da mistura. Objetivos: Comparar as formas de liberação do ACL e adotar um protocolo de investigação em anuência às normas internacionais. Métodos: Trinta e seis amostras com resultados prolongados na etapa de triagem pelo ensaio do tempo do veneno da víbora de Russel (dRVVT) ou tempo de tromboplastina parcial ativada (TTPA) foram submetidas às etapas de mistura (1:1) e confirmatórias com altas concentrações de fosfolipídios. Resultados: Para o TTPA, foram considerados positivos os valores cujo cálculo do índice de circulação de anticoagulante (ICA) resultasse superior a 15%. Para o dRVVT, utilizou-se também o valor da razão entre triagem e confirmatória. Das amostras testadas, 14 revelaram correção na etapa da mistura, mas somente uma resultou em pesquisa negativa. Conclusão: O cálculo do ICA auxiliou na identificação dos anticorpos fracos que possivelmente sofreram diluição na etapa da mistura. Não há um exame padrão-ouro para o diagnóstico da SAF, e a pesquisa do ACL ainda demanda uniformização da técnica e da interpretação.

Early increase in autoantibodies against human oxidized low-density lipoprotein in hypertensive patients after blood pressure control.

BACKGROUND: Oxidized lipoproteins and antioxidized low-density lipoprotein (anti-oxLDL) antibodies (Abs) have been detected in plasma in response to blood pressure (BP) elevation, suggesting the participation of the adaptive immune system. Therefore, treatment of hypertension may act on the immune response by decreasing oxidation stimuli. However, this issue has not been addressed. Thus, we have here analyzed anti-oxLDL Abs in untreated (naive) hypertensive patients shortly after initiation of antihypertensive therapeutic regimens. METHODS: Titers of anti-oxLDL Abs were measured in subjects with recently diagnosed hypertension on stage 1 (n = 94), in primary prevention of coronary disease, with no other risk factors, and naive of antihypertensive medication at entry. Subjects were randomly assigned to receive perindopril, hydrochlorothiazide (HCTZ), or indapamide (INDA) for 12 weeks, with additional perindopril if necessary to achieve BP control. Abs against copper-oxidized LDL were measured by enzyme-linked immunosorbent assay. RESULTS: Twelve-week antihypertensive treatment reduced both office-based and 24-h ambulatory BP measurements (P < 0.0005). The decrease in BP was accompanied by reduction in thiobarbituric acid-reactive substances (TBARS) (P < 0.05), increase in anti-oxLDL Ab titers (P < 0.005), and improvement in flow-mediated dilation (FMD) (P < 0.0005), independently of treatment. Although BP was reduced, we observed favorable changes in anti-oxLDL titers and FMD. CONCLUSIONS: We observed that anti-oxLDL Ab titers increase after antihypertensive therapy in primary prevention when achieving BP targets. Our results are in agreement with the concept that propensity to oxidation is increased by essential hypertension and anti-oxLDL Abs may be protective and potential biomarkers for the follow-up of hypertension treatment.

High circulating autoantibodies against human oxidized low-density lipoprotein are related to stable and lower titers to unstable clinical situation.

BACKGROUND: Oxidized lipoproteins and antibodies anti-oxidized low-density lipoprotein (anti-oxLDL) have been detected in human plasma and in atherosclerotic lesions. However, the role of these autoantibodies in the maintenance of vascular health or in the pathogenesis of acute vascular insults remains unclear. We examined the relationship of human immunoglobulin G (IgG) anti-oxLDL antibodies with cardiovascular disease risk markers in stable subjects and in patients after an acute coronary syndrome (ACS). METHODS: Titers of human anti-oxLDL antibodies were measured in hypertensive subjects in primary prevention (n=94), without other risk factors, and in individuals after a recent ACS event who also had metabolic syndrome (n=116). Autoantibodies against copper ion oxidized LDL were measured by enzyme-linked-immunosorbent assay. RESULTS: Anti-oxLDL titers were higher in hypertensive patients and these subjects presented lower high sensitivity C-reactive protein (hs-CRP) than those with ACS (p<0.0001). We found significant correlations between anti-oxLDL and hs-CRP (r=-0.284), body mass index (r=-0.256), waist circumference (r=-0.368), apolipoprotein B (r=-0.191), glucose (r=-0.303), systolic blood pressure (r=0.319), diastolic blood pressure (r=0.167), high-density lipoprotein cholesterol (r=0.224) and apolipoprotein A1 (r=0.257) (p<0.02 for all). After multiple linear regression hs-CRP, fasting glucose and waist circumference remained independently and inversely associated with anti-oxLDL. CONCLUSIONS: Acute inflammatory and metabolic conditions decrease titers of human antibodies of IgG class against oxidized LDL, and that circulating anti-oxLDL antibodies could be associated with a protective role in atherosclerosis.

Early glucometabolic profile in patients with acute coronary syndromes and metabolic syndrome.

BACKGROUND: Patients with metabolic syndrome (MetS) are at high coronary risk and beta-cell dysfunction or insulin resistance might predict an additional risk for early cardiovascular events. OBJECTIVE: This study aimed to evaluate early glucometabolic alterations in patients with MetS, but without previously known type 2 diabetes, after acute coronary syndrome. METHODS: A total of 114 patients were submitted to an oral glucose tolerance test (OGTT) 1-3 days after hospital discharge due to myocardial infarction or unstable angina. Based on the OGTT, we defined three groups of patients: normal glucose tolerance (NGT; n=26), impaired glucose tolerance (IGT; n=39), or diabetes (DM; n=49). The homeostasis model assessment (HOMA-IR) was used to measure insulin resistance; beta-cell responsiveness was assessed by the insulinogenic index at 30 min (DeltaI30/DeltaG30). RESULTS: Based on the HOMA-IR, patients with DM were more insulin-resistant than those with NGT or IGT (p<0.001). According to the insulinogenic index, the beta-cell responsiveness was also impaired in subjects with DM (p<0.001 vs NGT or IGT). CONCLUSION: High rates of glucometabolic alterations were found after acute coronary syndrome in patients with MetS. As these abnormalities markedly increase the risk for adverse outcomes, early OGTT among MetS patients might be used to identify those at the highest coronary risk.

Perfil glicometabólico inicial em pacientes com síndrome coronariana aguda e síndrome metabólica / Perfil glucometabólico inicial en pacientes con síndrome coronario agudo y síndrome metabólico / Early glucometabolic profile in patients with acute coronary syndromes and metabolic syndrome

FUNDAMENTO: Pacientes com síndrome metabólica (SM) têm alto risco coronariano e a disfunção da célula beta ou resistência à insulina pode prever um risco adicional de eventos cardiovasculares precoces. OBJETIVO: Avaliar as alterações glicometabólicas precoces em pacientes com SM, mas sem diagnóstico de diabete tipo 2, após síndrome coronariana aguda. MÉTODOS: Um total de 114 pacientes foi submetido ao teste oral de tolerância à glicose (TOTG), 1-3 dias da alta hospitalar, após infarto agudo do miocárdio ou angina instável. Baseado no TOTG, definimos três grupos de pacientes: tolerância normal à glicose (TNG; n=26), tolerância alterada à glicose (TAG; n=39) ou diabetes mellitus (DM; n=49). O Modelo de Avaliação da Homeostase (HOMA-IR) foi usado para estimar a resistência à insulina; a responsividade da célula beta foi avaliada através do índice insulinogênico de 30 minutos (ΔI30/ΔG30). RESULTADOS: Baseado no HOMA-IR, os pacientes com DM eram mais insulino-resistentes do que aqueles com TNG ou TAG (p<0,001). De acordo com o índice insulinogênico, a responsividade da célula beta também estava alterada em indivíduos com DM (p<0,001 vs TNG ou TAG). CONCLUSÃO: Altas taxas de alterações glicometabólicas foram encontradas após síndrome coronariana aguda em pacientes com SM. Como essas anormalidades acentuadamente aumentam o risco de desfechos adversos, o TOTG precoce pode ser utilizado em pacientes com SM para identificar aqueles que apresentam maior risco coronariano.

BACKGROUND: Patients with metabolic syndrome (MetS) are at high coronary risk and beta-cell dysfunction or insulin resistance might predict an additional risk for early cardiovascular events. OBJECTIVE: This study aimed to evaluate early glucometabolic alterations in patients with MetS, but without previously known type 2 diabetes, after acute coronary syndrome. METHODS: A total of 114 patients were submitted to an oral glucose tolerance test (OGTT) 1-3 days after hospital discharge due to myocardial infarction or unstable angina. Based on the OGTT, we defined three groups of patients: normal glucose tolerance (NGT; n=26), impaired glucose tolerance (IGT; n=39), or diabetes (DM; n=49). The homeostasis model assessment (HOMA-IR) was used to measure insulin resistance; beta-cell responsiveness was assessed by the insulinogenic index at 30 min (ΔI30/ΔG30). RESULTS: Based on the HOMA-IR, patients with DM were more insulin-resistant than those with NGT or IGT (p<0.001). According to the insulinogenic index, the beta-cell responsiveness was also impaired in subjects with DM (p<0.001 vs NGT or IGT). CONCLUSION: High rates of glucometabolic alterations were found after acute coronary syndrome in patients with MetS. As these abnormalities markedly increase the risk for adverse outcomes, early OGTT among MetS patients might be used to identify those at the highest coronary risk.

FUNDAMENTO: Pacientes con síndrome metabólico (SM) tienen alto riesgo coronario y la disfunción de la célula beta o la resistencia a la insulina puede prever un riesgo adicional de eventos cardiovasculares precoces. OBJETIVO: Evaluar las alteraciones glucometabólicas precoces en pacientes con SM, pero sin diagnóstico de diabetes tipo 2, tras el síndrome coronario agudo. MÉTODOS: Un total de 114 pacientes fue sometido a la prueba oral de tolerancia a la glucosa (POTG), de un a tres días tras el alta hospitalaria, y luego de infarto agudo de miocardio o angina inestable. Basado en el POTG, definimos tres grupos de pacientes: tolerancia normal a la glucosa (TNG; n=26), tolerancia alterada a la glucosa (TAG; n=39) o diabetes mellitus (DM; n=49). Se utilizó el Modelo de Evaluación de la Homeostasis (HOMA-IR) para estimarse la resistencia a la insulina; se evaluó la responsividad de la célula beta a través del índice insulinogénico de 30 minutos (ΔI30/ΔG30). RESULTADOS: Basado en el HOMA-IR, los pacientes con DM se mostraban más insulinoresistentes que los individuos con TNG o TAG (p<0,001). De acuerdo con el índice insulinogénico, la responsividad de la célula beta también estaba alterada en individuos con DM (p<0,001 vs. TNG o TAG). CONCLUSIONES: Se encontraron altas tasas de alteraciones glucometabólicas tras el síndrome coronario agudo en pacientes con SM. Como esas anormalidades incrementan acentuadamente el riesgo de desenlaces adversos, el POTG precoz se puede utilizar en pacientes con SM para identificar a los que presentan mayor riesgo coronario.

Association of lipoprotein lipase D9N polymorphism with myocardial infarction in type 2 diabetes: the genetics, outcomes, and lipids in type 2 diabetes (GOLD) study.

The association of polymorphisms affecting lipid metabolism with the risk of myocardial infarction (MI) in type 2 diabetes mellitus was investigated. The Genetics, Outcomes and Lipids in type 2 Diabetes (GOLD) Study is a prospective, multicenter study, conducted on 990 patients presenting diabetes and MI (n=386), or diabetes without previous manifestation of stroke, peripheral or coronary arterial disease (n=604), recruited from 27 institutions in Brazil. APO A1 (A/G -75 and C/T +83) and APO C3 (C/G 3'UTR) non-coding sequences, CETP (Taq 1B), LPL (D9N), APO E (epsilon2, epsilon3, epsilon4,), PON-1 (Q192R), and two LCAT variants Arg(147)-->Trp and Tyr(171)-->Stop were tested by PCR-RFLP. There was a higher prevalence of LPL DN genotype (19% vs.12%, p=0.03) and a higher frequency of the N allele (11% vs. 7%) among subjects with MI when compared to controls, with an odds ratio of MI for carriers of 9N allele of 2.46 (95% CI=1.79-3.39, p<0.0001). This association was present in men and women, in non-smokers and in hypertensive patients. A logistic regression model including gender, duration of diabetes, systolic blood pressure, HDL-C, left ventricle hypertrophy and D9N polymorphism showed that the latter still remained significantly associated with MI (OR=1.50, 95% CI=1.02-2.25, p=0.049). These findings suggest that D9N polymorphism can be a useful risk marker for myocardial infarction and that further potential candidate genes should be screened for exploratory analysis and for future therapeutic intervention in diabetes.

Prevalence of myocardial infarction is related to hyperhomocysteinemia but not influenced by C677T methylenetetrahydrofolate reductase and A2756G methionine synthase polymorphisms in diabetic and non-diabetic subjects.

BACKGROUND: Hyperhomocysteinemia has emerged as a novel risk factor for myocardial infarction (MI). Some mechanisms proposed to explain its relationship with coronary events are also shared by major coronary risk factors. We examined whether C677T methylenetetrahydrofolate reductase and A2756G methionine synthase polymorphisms could affect the relative risk for MI. METHODS: A sample of 196 individuals was divided into four groups (diabetics with MI, n=43; diabetics without MI, n=50; non-diabetics with MI, n=47; non-diabetics without MI, n=56) and compared regarding the prevalence of the polymorphisms, risk factors, and biochemical parameters. RESULTS: Higher prevalence of hyperhomocysteinemia was found in MI patients (p<0.05 vs. non-MI subjects), in males (p<0.001 vs. female) and in those > or = 65 years (p=0.01 vs. <65 years). Homocysteine was negatively associated with HDL-C (p<0.05) and glucose, although results did not reach significance (p=0.06). Similar distribution of studied polymorphisms was seen in all groups, which presented normal folate and vitamin B12 serum levels. CONCLUSIONS: Higher homocysteinemia was predominantly observed in men, presenting low HDL-C, and at advancing age. Methylenetetrahydrofolate reductase and methionine synthase polymorphisms did not contribute to risk assessment in diabetic and non-diabetic subjects presenting normal folate levels.

High glucose levels abolish antiatherosclerotic benefits of ACE inhibition in alloxan-induced diabetes in rabbits.

Renin-angiotensin system activation is recognized to play an important role in atherosclerosis. This study aimed to verify the antiatherosclerotic effects of ACE inhibition on an experimental model of diabetes and hypercholesterolemia. Diabetes was induced in New Zealand male rabbits with a single dose of alloxan (100 mg/kg, i.v.), and, according to plasma glucose levels obtained after 1 week, the animals were divided into 2 groups (> or =250 mg/dL or <250 mg/dL). Each group was randomly assigned to receive or not quinapril (30 mg/d) added to a 0.5% cholesterol-enriched diet. Animals with high glucose levels at 1 week and that remained high after 12 weeks presented higher triglyceride levels (P < 0.02 versus basal). Those initially hyperglycemic but presenting <250 mg/dL glucose at the end of study formed an additional group. Plasma ACE activity was lower in quinapril-treated animals (P < 0.01 versus untreated groups). However, aorta intima/media ratio and intima area were lower only in the subgroups of quinapril-treated animals with low glucose levels (P < 0.05). Our results support the hypothesis that high plasma glucose may abolish the antiatherosclerotic effect of ACE inhibitors.