The screening of rare blood donors in a highly admixed population: A new approach for Holley and Diego genotyping and impact of genomic and self-reported ancestry.

OBJECTIVES: The present study aimed to develop strategies for genotyping DO*HY (Dombrock system) and DI*A/DI*B (Diego system) alleles and to evaluate the impact of genomic and self-declared ancestry on rare donor screening in admixed populations. BACKGROUND: The antigens Hy and Dib demonstrate clinical importance. The lack of antisera for the serological evaluation of these antigens makes it necessary to develop molecular methods. In addition, considering that some rare red blood cell phenotypes present differences in frequency between ethnic groups, it is important to assess the applicability of self-declared ancestry in the search for rare donors in admixed populations. METHODS: DO*HY and DI*A/DI*B genotyping based on real-time polymerase chain reaction (PCR) was standardised. A total of 457 blood donors clustered by self-defined skin colour/race categories were genotyped. Furthermore, individual genomic ancestry was used in the analyses. RESULTS: The assays developed are reproducible and provide satisfactory results even at low concentrations of DNA, which make them useful in situations where the DNA is scarce, such as dried blood spots on filter paper, or when screening for pooled samples. No significant difference was observed in the frequencies of the DI*A, DI*B and DO*HY, comparing the self-declared White (branco) donors with those who are Black (preto) and Brown (pardo). CONCLUSION: Real-time PCR, especially using pooled samples, is a promising strategy to screen rare blood donors. Although both self-reported race/colour and some blood group phenotypes are associated with ancestry, the results point to a greater complexity in the application of self-declared race/colour in the screening of rare donors in admixed populations.

A new index to discriminate between iron deficiency anemia and thalassemia trait.

BACKGROUND: The most common microcytic and hypochromic anemias are iron deficiency anemia and thalassemia trait. Several indices to discriminate iron deficiency anemia from thalassemia trait have been proposed as simple diagnostic tools. However, some of the best discriminative indices use parameters in the formulas that are only measured in modern counters and are not always available in small laboratories. The development of an index with good diagnostic accuracy based only on parameters derived from the blood cell count obtained using simple counters would be useful in the clinical routine. Thus, the aim of this study was to develop and validate a discriminative index to differentiate iron deficiency anemia from thalassemia trait. METHODS: To develop and to validate the new formula, blood count data from 106 (thalassemia trait: 23 and iron deficiency: 83) and 185 patients (thalassemia trait: 30 and iron deficiency: 155) were used, respectively. Iron deficiency, ß-thalassemia trait and α-thalassemia trait were confirmed by gold standard tests (low serum ferritin for iron deficiency anemia, HbA2>3.5% for ß-thalassemia trait and using molecular biology for the α-thalassemia trait). RESULTS: The sensitivity, specificity, efficiency, Youden's Index, area under receiver operating characteristic curve and Kappa coefficient of the new formula, called the Matos & Carvalho Index were 99.3%, 76.7%, 95.7%, 76.0, 0.95 and 0.83, respectively. CONCLUSION: The performance of this index was excellent with the advantage of being solely dependent on the mean corpuscular hemoglobin concentration and red blood cell count obtained from simple automatic counters and thus may be of great value in underdeveloped and developing countries.

Effect of acetylsalicylic acid on platelet activation and oxidative profile in a set of Brazilian patients with type 2 diabetes mellitus.

Type 2 diabetes mellitus (DM2) is a metabolic disorder associated with hyperactivation of platelets, increased formation of platelet microparticles (PMPs) and oxidative stress that are related to cardiovascular complications. Acetylsalicylic acid (ASA) is an antiplatelet agent used in the prevention of atherothrombosis. The aim of this study was to evaluate the effect of ASA by means of platelet activation and oxidative profile. We collected blood samples of 81 patients with DM2 before and during ASA treatment. These samples were analyzed to determine the levels of 2,3-dinor thromboxane-B2 (2,3-dinor-TXB2), PMPs, thiobarbituric acid reactive species (TBARS) and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT). Moreover, the relationship between the levels of 2,3-dinor-TXB2 with some clinical and laboratory variables such as glycated hemoglobin, platelet count, D dimer, low-density lipoprotein cholesterol and glycoprotein IIb/IIIa and cyclooxygenase-1 polymorphisms was evaluated. ASA intake did not change the levels of PMP, TBARS and MTT. Although a significant decrease in the levels of 2,3 dinorTXB2 (P < 0.001) in patients under ASA has been observed, an equal and satisfactory response to this drug was not found. However, the presence of PIA2 allele in GPIIIa gene may be associated with a better response to ASA intake in these patients, whereas other clinical and laboratory variables showed no association with this drug use. These findings are consistent with previous reports in the literature that patients with DM2 do not benefit in an equal way from the use of ASA for primary prevention of atherothrombotic events.

Association of microparticles and preeclampsia.

Preeclampsia (PE) is a syndrome characterized by poor placentation and endothelial dysfunction. The diagnosis for this syndrome is based in hypertension and proteinuria presented after the 20th week of pregnancy. Despite intensive research, PE is still one of the leading causes of maternal mortality, although reliable screening tests or effective treatments of this disease have yet to be proposed. Microparticles (MPs) are small vesicles released after cell activation or apoptosis, which contain membrane proteins that are characteristic of the original parent cell. MPs have been proven to play key role in thrombosis, inflammation, and angiogenesis, as well as to mediate cell-cell communication by transferring mRNAs and microRNA from the cell of origin to target cells. Placenta-derived syncytiotrophoblast MPs are one of the most increased MPs during PE and may play an important role in the pathogenesis of this syndrome. Therefore, a better overall understanding of the role of MPs in PE may be useful for new clinical diagnoses and therapeutic approaches.

Circulating microparticles in severe preeclampsia.

The present study aimed to evaluate microparticles (MPs) from different sources in women with severe preeclampsia (PE) compared with normotensive pregnant women and non-pregnant women. This case-control study evaluated 28 pregnant women with severe PE, 30 normotensive pregnant women, and 29 non-pregnant women. MPs from neutrophils, endothelial cells, monocytes, platelets, leukocytes, erythrocytes, and syncytiotrophoblast were evaluated using flow cytometry. A higher total number of MPs were observed in women with severe PE compared with normotensive pregnant women and non-pregnant women (P=0.004 and P=0.001, respectively). MPs derived from erythrocytes were increased in women with severe PE compared with normotensive pregnant women (P=0.002). A trend towards association was observed between platelet count and the number of MPs derived from platelets (P=0.09) in severe PE group. A positive correlation was also found between the number of endothelial cell-derived MPs and the number of platelet-derived MPs, leukocyte-derived MPs, neutrophil-derived MPs, and lymphocyte-derived MPs (P<0.05) in severe PE pregnant women. MP counts can be increased in severe PE, and erythrocyte and endothelial cell-derived MPs seem to be associated to severe PE.

ADAMTS13 and Von Willebrand factor in patients undergoing hemodialysis.

Hemodialysis (HD) is associated with increasing thrombotic trend. Vascular access thrombosis (VAT) increases morbidity in HD patients. The aim of this study was to evaluate ADAMTS13 and VWF plasma levels from patients undergoing HD as putative biomarkers of the hypercoagulability state, as well the association between these markers and VAT occurrence. This study included 195 patients on HD for more than 6 months. HD patients were allocated into two groups according to the occurrence or not of previous episode of VAT; HD with VAT (N = 46) and HD without VAT (N = 149). ADAMTS13 and VWF were performed by ELISA. There was no significant difference between HD patients with and without VAT for ADAMTS13 and VWF levels. However, VWF levels were higher (P < 0.001) and ADAMTS13 were lower (P < 0.001) in HD patients, comparing to the control group composed by healthy subjects without kidney disease, age and sex-matched (N = 80). Taken together our data suggest a potential role of the kidneys function compromised on ADAMTS13 synthesis or metabolism, regardless other known sources of ADAMTS13. The imbalance between ADAMTS13 and VWF levels does not explain the development of VAT in HD patients by itself, although it should contribute for the hypercoagulability state. Therefore, additional studies to identify other risk factors are warranted and essential for better management of HD patients.

Hemodialysis vascular access thrombosis: The role of factor V Leiden, prothrombin gene mutation and ABO blood groups.

BACKGROUND: Vascular access thrombosis increases morbidity in hemodialysis (HD) patients. The aim of this study was to investigate the association between HD vascular access thrombosis and mutations in the prothrombin and factor V Leiden (FV) genes and ABO blood system. METHODS: This cross-sectional study included 195 patients with end stage renal disease (ESRD) on HD for more than six months. HD patients were allocated into two groups according to the occurrence (cases, N=46) or not (controls, N=149) of previous vascular access thrombosis. FV and prothrombin gene mutations were investigated by polymerase chain reaction and ABO blood group phenotyping was performed by the indirect technique. Univariate analysis detected the variables with a trend to be associated with thrombosis and was followed by multivariate analysis to define independent predictors of vascular access thrombosis. RESULTS: FV Leiden mutation and ABO blood group were not associated with vascular access thrombosis, whereas G20210A mutation in the prothrombin gene was significantly higher in patients with vascular access thrombosis and independently associated with this complication (OR=12.0; CI 95%=1.8-83.5; p=0.012). CONCLUSIONS: G20210A mutation emerges as an important genetic factor predisposing to vascular access thrombosis. The definition of risk factors for thrombosis will certainly enable a rational approach for HD patients.

Púrpura trombocitopênica trombótica: o papel do fator von Willebrand e da ADAMTS13 / Thrombotic thrombocytopenic purpura: the role of von Willebrand factor and ADAMTS13

A púrpura trombocitopênica trombótica (PTT) instala-se de modo abrupto e é caracterizada pela oclusão difusa de arteríolas e capilares da microcirculação, levando à isquemia de tecidos. A oclusão é causada por microtrombos compostos basicamente de plaquetas e fator von Willebrand (FvW). O FvW é uma glicoproteína de estrutura multimérica sintetizada exclusivamente por células endoteliais e megacariócitos. Este fator promove a adesão das plaquetas ao endotélio lesado, participa do processo de agregação plaquetária e é a proteína carreadora do fator VIII na circulação. Em condições fisiológicas, os grandes multímeros do FvW encontram-se dentro das células endoteliais e nas plaquetas e não estão presentes no plasma. Tão logo estes grandes multímeros são liberados da célula endotelial, são clivados e removidos da circulação pela enzima ADAMTS13 (A Desintegrin And Metalloprotease with eight Thrombo Spondin-1-like). A deficiência funcional ou quantitativa de ADAMTS13 resulta no acúmulo de grandes multímeros de FvW no plasma, propiciando a agregação das plaquetas e oclusão difusa das arteríolas e capilares. A maioria dos casos de PTT está associada à deficiência da ADAMTS13 e já estão disponíveis no mercado internacional conjuntos diagnósticos para a determinação dos níveis de antígenos desta enzima, da de sua atividade e dos anticorpos anti-ADAMTS13. A avaliação laboratorial da ADAMTS13 parece constituir um avanço para o diagnóstico precoce da PTT. No entanto, a interpretação dos resultados exige cautela e um conhecimento do princípio do método, bem como das etapas das reações envolvidas.

Thrombotic thrombocytopenic purpura (TTP) starts abruptly and is characterized by diffuse occlusion of microcirculation arterioles and capillaries, leading to ischemia of tissues. Occlusion is caused by microscopic clots primarily composed of platelets and von Willebrand factor (VWF). VWF is a multimeric glycoprotein synthesized exclusively by endothelial cells and megakaryocytes. This factor promotes adhesion of platelets to injured endothelium, participates in the process of platelet aggregation and is the carrier protein of factor VIII in the circulation. In physiological conditions, large VWF multimers are present in endothelial cells and platelets and are not present in plasma. As soon as these large multimers are released from the endothelial cell, they are cleaved and removed from circulation by the ADAMTS13 enzyme. A quantitative or functional deficiency of ADAMTS13 results in the accumulation of large VWF multimers in the plasma and may result in the aggregation of platelets and diffuse occlusion of arterioles and capillaries. Most cases of PTT are associated with ADAMTS13 deficiency. The levels of antigens, activity and antibodies of MTS13 can be evaluated using internationally manufactured kits. The laboratory evaluation of ADAMTS13 appears to be a useful tool for the early diagnosis of PTT. However, interpretation of the results requires caution, as well as knowledge of the principles of the method and the steps of the reactions involved.

Polymorphisms in exons 6 and 7 of the ABO locus and their association with venous thrombosis in young Brazilian patients.

The A1 and B alleles of the ABO blood system have been associated with high levels of both factor VIII and von Willebrand factor and with a predisposition to venous thromboembolism (VTE). In this study, genotypes of the ABO system were determined by PCR-restriction fragment length polymorphism for 148 young VTE Brazilian patients and 233 unrelated control individuals. The blood group O was more frequent among the controls [odds ratio (OR), 0.21; 95% confidence interval (CI), 0.13-0.34; P = 0.000) and groups A and B (OR, 2.24; 95%, CI, 1.46-3.42; P = 0.000 and OR, 2.52; 95% CI, 1.42-4.48; P = 0.002, respectively) among patients. The patients' group was under Hardy-Weinberg equilibrium, whereas the control group was not (P < 0.0051), suggesting that natural selection might be acting in favor of carriers of the O blood group. When the allelic frequencies were compared through multivariate logistic regression analysis for adjustments of covariates, the alleles A1 (OR, 1.69; 95% CI, 1.17-2.45; P = 0.006), A2 (OR, 2.19; 95% CI, 1.24-3.87; P = 0.010), and B (OR, 2.65; 95% CI, 1.64-4.26; P = 0.000) were independently associated with VTE and may represent important risk factors to the development of VTE among young Brazilian patients. Thus, the inclusion of ABO blood group determination may be helpful to discriminate individuals with high risk for VTE allowing target intervention as well as to manage VTE in young patients.

O papel do RDW, da morfologia eritrocitária e de parâmetros plaquetários na diferenciação entre anemias microcíticas e hipocrômicas / The role of RDW, erythrocyte morphology and platelet parameters in the differentiation between microcytic and hypochromic anemias

As anemias microcíticas e hipocrômicas atingem grande parcela da população mundial. Entre estas, significativa porcentagem de casos se deve à deficiência do ferro, enquanto em algumas regiões a frequência de talassemia menor se torna importante. Por outro lado, a anemia de doença crônica é a causa mais comum de anemia em pacientes hospitalizados. O diagnóstico diferencial destas doenças é clinicamente importante, e é atualmente realizado através dos exames padrão ouro envolvendo a avaliação do metabolismo do ferro e dosagem de HbA2. Embora dotados de grande utilidade, estes testes podem apresentar uma metodologia mais demorada e onerosa que, em casos de concomitância de doenças, comuns na prática clínica, não conseguem proporcionar um correto diagnóstico. Na tentativa de otimizar e direcionar o diagnóstico destas anemias, o uso de alguns parâmetros derivados dos modernos contadores automáticos tem sido sugerido. Neste estudo, o papel do RDW, parâmetros plaquetários (número de plaquetas, PDW, VPM) e morfologia eritrocitária como parâmetros diferenciadores, foi avaliado em um grupo de 159 pacientes portadores de anemia microcítica e hipocrômica devido à deficiência do ferro, anemia de doença crônica e talassemia beta menor, comprovado pelos exames padrão ouro. Foi possível observar que o RDW não se mostrou um bom discriminante, enquanto o índice plaquetário PDW pode ser um parâmetro auxiliar no diagnóstico diferencial das anemias microcíticas e hipocrômicas. Com relação às alterações morfológicas dos eritrócitos, o pontilhado basófilo foi um achado bastante comum apenas em pacientes portadores de talassemia beta menor, com indícios de potencial utilidade na elucidação de casos de microcitose.

Microcytic and hypochromic anemias affect many people worldwide. A significant percentage of cases are due to iron deficiency, while in certain regions the frequency of thalassemia minor is important. On the other hand, anemia of chronic disease is the most common cause of anemia in hospitalized patients. Differential diagnosis between these anemias, currently established by using gold standard tests involving evaluation of iron metabolism and measurement of HbA2, is of clinical importance. Although very useful, these tests are time consuming and onerous. In cases of concomitant diseases, a common finding in the clinical practice, these tests are unable to provide a correct diagnosis. In an attempt to optimize diagnosis of these anemias, the use of some parameters derived from modern automated blood count analyzers has been suggested. In this study, the role of RDW, platelet parameters (platelet number, PDW, MPV) and erythrocyte morphology as differentiating parameters were evaluated in a group of 159 patients diagnosed as carriers of microcytic or hypochromic anemias due to iron deficiency, anemia of chronic disease and â-thalassemia minor, as confirmed by gold standard tests. The RDW did not prove to be a good discriminator, while the platelet index, PDW, may be helpful in the differential diagnosis of microcytic and hypochromic anemias. Regarding the red cell morphologic alterations, basophilic stippling was a quite common finding just in patients with â-thalassemia minor, suggesting it to be a potential marker for elucidating cases of microcytosis.

Índice de anisocitose eritrocitária (RDW): diferenciação das anemias microcíticas e hipocrômicas / Red blood cell distribution width (RDW): differentiation of microcytic and hypochromic anemias

A anemia ferropriva, talassemia menor e anemia de doença crônica são as anemias microcíticas e hipocrômicas mais comuns em nosso meio. O diagnóstico diferencial das referidas anemias é de grande importância clínica; contudo, muitas vezes é complexo em virtude de concomitância de doenças, além de demandar tempo e apresentar custos significativos. Com o propósito de conferir maior simplicidade e eficiência ao diagnóstico diferencial destas anemias, o uso de índices derivados de modernos contadores automáticos tem sido sugerido. Entre estes, pode ser citado o índice de anisocitose eritrocitária (RDW), que indica o grau de variabilidade do tamanho das hemácias. Neste estudo, o poder de discriminação deste índice quanto ao tipo de anemia microcítica e hipocrômica foi avaliado em um grupo de 159 pacientes sabidamente portadores de um quadro de anemia causado por deficiência de ferro, beta talassemia menor ou uma anemia de doença crônica. Não foi observada diferença significativa para o RDW entre os três grupos de anemias microcíticas, indicando não ser este índice uma ferramenta útil para a diferenciação entre anemia ferropriva, beta talassemia menor e anemia de doença crônica.

Iron deficiency anemia, the thalassemia trait and chronic disease anemia are the most common microcytic and hypochromic anemias in the Brazilian population. Differential diagnosis of these anemias is of great clinical importance however, frequently, it is complex due to coexistence of diseases, as well as being time consuming and expensive. In order to simplify and increase efficiently of checking the differential diagnoses of these anemias, the use of indexes derived from modern blood cell counters has been suggested. Among them, is the index called red blood cell distribution width which indicates the variability in red blood cell size. In this study, the discriminative power of the red blood cell distribution width in differentiating microcytic and hypochromic anemias was evaluated in a group of 159 patients diagnosed as carriers of either iron deficiency anemia, â-thalassemia minor or chronic disease anemia. No difference was found for the red blood cell distribution width among the three groups of microcytic and hypochromic anemias indicating that this index is not a useful tool to distinguish among iron deficiency, â-thalassemia minor and chronic disease anemia.

Monocytes and plasma tissue factor levels in normal individuals and patients with deep venous thrombosis of the lower limbs: potential diagnostic tools?

INTRODUCTION: Tissue factor (TF) is the main physiological initiator of blood coagulation; it is membrane-bound on monocytes (mTF) and free in plasma (pTF). Abnormal expression of TF by monocytes has been implicated in various diseases. We therefore quantified monocytes expressing TF and pTF levels in patients with lower-limb deep venous thrombosis (DVT). MATERIALS AND METHODS: DVT was confirmed by Duplex Scan. Blood mTF levels under resting condition (baseline), after incubation without (unstimulated) and with (stimulated) lipopolysaccharide (LPS), and total mTF levels were determined by flow cytometry using two analytical methods (Histogram and Quadrant-Statistics). Plasma TF levels were measured using an enzyme-linked immunoabsorbent assay (ELISA). Results were compared with age-matched controls. RESULTS: Histogram analysis in patients with DVT showed significantly elevated mTF levels for baseline, unstimulated and total mTF over controls. For Quadrant-Statistics, DVT patients also showed significantly raised baseline, unstimulated, stimulated and total mTF. Similarly, pTF levels were significantly raised in subjects with DVT compared to controls. Baseline mTF levels correlated with pTF levels by Histogram and Quadrant-Statistics analysis. Using the relative operating characteristic (ROC) curve, baseline mTF and pTF assays displayed sensitivity and specificity in detecting DVT. Quadrant-Statistics baseline mTF and pTF gave the best discrimination. CONCLUSIONS: The TF assays used in this study showed acceptable sensitivity and specificity and are cost-effective and practical. Therefore, they should be considered in patients with, or at risk of, DVT.

Avaliação da incidência das mutações G1691A no gene do fator V (fator V Leiden) e G20210A no gene da protrombina em pacientes com diabetes mellitus tipo 2 / Incidence of G1691A factor V gene (factor V Leiden) and G20210A prothrombin gene mutations in type 2 diabetes patients

Em virtude da alta prevalência de Diabetes mellitus tipo 2 (DM2) na população mundial e da alta taxa de mortalidade decorrente de eventos trombóticos, é de extrema importância o conhecimento das alterações no sistema hemostático em pacientes portadores deste distúrbio. A mutação no gene do fator V (G1691A - fator V Leiden) em heterozigose ou homozigose confere aos portadores o fenótipo de resistência à proteína C ativada, situação que aumenta em sete vezes o risco de desenvolver uma trombose. A mutação G20210A no gene da protrombina resulta no quadro de hiperprotrombinemia, aumentando o risco de trombose em três vezes. A pesquisa dessas mutações de interesse em trombofilia é de grande relevância considerando que a presença das mesmas pode exacerbar o estado de hipercoagulabilidade acelerando as complicações no diabetes. O presente estudo teve como objetivo avaliar a incidência dessas mutações em indivíduos hígidos (Controle, n=16), pacientes com DM2 (n=7), com hipertensão (HAS, n=12) e com DM2+HAS (n=18), através da técnica de PCR-RFLP. As freqüências encontradas nos grupos estudados foram baixas e similares àquelas observadas na população brasileira em geral. Não foi possível estabelecer correlação entre a presença da mutação e características específicas de cada grupo. Dessa forma, ainda não está claro se há ou não uma maior prevalência dessas mutações em indivíduos diabéticos e se a presença das mesmas contribui para o aumento do risco de desenvolver trombose nesses indivíduos, sendo necessário estudos mais amplos para a elucidação da questão.

Because of the high prevalence of type 2 diabetes mellitus (DM2) worldwide and the high mortality rate due to thrombotic events, it is extremely important to know about changes in the hemostatic system of such patients. Factor V mutation (G1691A - factor V Leiden) in either heterozygosis or homozygosis confers the activated protein C resistant phenotype, which increases the risk of thrombotic events by a factor of seven. The G20210A mutation of the prothrombin gene results in hyperprothrombinemia, increasing the risk of thrombotic events by a multiple of three. These mutations are of great relevance considering that the presence of one or both can contribute to a hypercoagulability state accelerating complications in diabetes. The aim of this study was to evaluate the incidence of these mutations in controls (n=16), DM2 subjects (n=7), hypertensive subjects (HAS) (n=12) and DM2+HAS subjects (n=18). The frequencies found were low and similar to those observed in the Brazilian population in general. It was not possible to establish any correlation among mutations and specific features of each group. It is still not clear if there is or not a higher prevalence of these mutations in diabetic individuals or if the mutation contributes to an increase in the risk of thrombotic events in these individuals. Further studies involving a larger number of patients are necessary in order to answer these questions.