Detection of alloimmunization in Glanzmann Thrombasthenia and Bernard-Soulier Syndrome: Data from a Brazilian Center

ABSTRACT Introduction: The Glanzmann Thrombasthenia (GT) and Bernard-Soulier Syndrome (BSS) are rare hereditary disorders of platelet function. Their treatment often requires platelet transfusion, which can lead to the development of alloantibodies. Objective: In this study, we aim to develop a strategy for alloantibody detection and to describe the frequency of alloimmunization in a patient population from a single center in southeastern Brazil. Methods: Samples from patients with GT or BSS were tested using the Platelet Immunofluorescence Test (PIFT). If a positive result was obtained, a confirmatory step using the Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) and Luminex bead-based platelet assay (PAKLx) was executed. Main results: Among 11 patients with GT, we detected the presence of alloantibodies in 5 using PIFT, with confirmation through MAIPA and PAKLx in 2 (1 anti-HLA and 1 anti-HPA), resulting in a frequency of 18.1%. Among 4 patients with BSS, PIFT was positive in 3, with confirmation by MAIPA and PAKLx in 1 (anti-HLA), showing a frequency of 25%. The two patients with anti-HLA antibodies exhibited a panel reactive antibody (PRA-HLA) testing greater than 97%. Conclusion: Our study highlights the importance of identifying platelet alloimmunization in this patient population. The proposed algorithm for platelet alloantibodies detection allows resource optimization.

Association between FY* 02N. 01 and the severity of COVID-19: initial observations

ABSTRACT Introduction The pro-inflammatory immune response underlies severe cases of COVID-19. Antigens of the Duffy blood group systems are receptors for pro-inflammation chemokines. The ACKR1 c.-67T>C gene variation silences the expression of Duffy antigens on erythrocytes and individuals presenting this variant in homozygosity have impaired inflammatory response control. Our aim was to evaluate the association between the ACKR1 c.-67T>C and the severity of COVID-19. Methods This was a retrospective single-center case-control study, enrolling 164 participants who were divided into four groups: 1) Death: COVID-19 patients who died during hospitalization; 2) Hospital Discharge: COVID-19 patients who were discharged for home after hospitalizations; 3) Convalescent Plasma Donors: COVID-19 patients who were not hospitalized, and; 4) Controls: patients with diagnosis other than COVID-19. Patients were genotyped for the ACKR1 c.-67T>C (FY*02 N.01 allele) and the frequency of individuals presenting the altered allele was compared between the groups. Results The groups significantly differed in terms of the percentage of patients presenting at least one FY*02N.01 allele: 36.8% (Death group), 37% (Hospital Discharge group), 16.1% (Convalescent Plasma group) and 16.2% (Control group) (p= 0.027). The self-declared race (p < 0.001) and the occurrence of in hospital death (p= 0.058) were independently associated with the presence of the FY*02N.01 allele. Hypertension (p < 0.001), age (p < 0.001) and the presence of at least one FY*02N.01 allele (p= 0.009) were independently associated with the need for hospitalization. Conclusion There is a suggestive association between the presence of the FY*02N.01 and the severity of COVID-19. This may be a mechanism underlying the worse prognosis for Afro-descendants infected with SARS-CoV-2.

Transfusion of ABO non-identical platelets increases the severity of trauma patients at ICU admission

ABSTRACT Background: Transfusion of ABO-compatible non-identical platelets (PTLs), fresh plasma (FP) and red blood cells (RBCs) has been associated with increased morbidity and mortality of recipients. Trauma victims are frequently exposed to ABO non-identical products, given the need for emergency transfusions. Our goal was to evaluate the impact of the transfusion of ABO non-identical blood products on the severity and all-cause 30-day mortality of trauma patients. Methods: This was a retrospective single-center cohort, which included trauma patients who received emergency transfusions in the first 24 h of hospitalization. Patients were divided in two groups according to the use of <3 or ≥3 ABO non-identical blood products. The patient severity, measured by the Acute Physiology and Chronic Health Evaluation (APACHEII) score at ICU admission, and the 30-day mortality were compared between groups. Results: Two hundred and sixteen trauma patients were enrolled. Of these, 21.3% received ≥3 ABO non-identical blood products (RBCs, PLTs and FP or cryoprecipitate). The transfusion of ≥3 ABO non-identical blood products in the first 24 h of hospitalization was independently associated with a higher APACHEII score at ICU admission (OR = 3.28 and CI95% = 1.48-7.16). Transfusion of at least one unit of ABO non-identical PTLs was also associated with severity (OR = 10.89 and CI95% = 3.38-38.49). Transfusion of ABO non-identical blood products was not associated with a higher 30-day mortality in the studied cohort. Conclusion: The transfusion of ABO non-identical blood products and, especially, of ABO non-identical PLTs may be associated with the greater severity of trauma patients at ICU admission. The transfusion of ABO non-identical blood products in the trauma setting is not without risks.

Padrozação de estratégia molecular custo-efetiva para rastreamento de fenótipos eritrocitários e plaquetários raros em doadores de sangue visando à organização de banco de doadores raros no estado de São Paulo / Cost-effective molecular strategy standardization for screening of erythrocyte phenotypes and rare platelet in blood donors aiming at the organization of a rare donor bank in the state of São Paulo

OBJETIVOS: 1- Padronizar a genotipagem em larga escala para determinação de antígenos eritrocitários e plaquetários pela plataforma de OpenArray®em doadores de sangue. 2- Elaboração de software para registro destes doadores, com interface com o equipamento de genotipagem. METODOLOGIA: Extração automatizada de DNA e genotipagem através demicroarranjos líquidos (OpenArray®) para 32 alelos codificantes de antígenos eritrocitários e plaquetários. RESULTADOS: Foi realizada a genotipagem de 5487 doadores para os antígenos propostos, de forma completamente interfaceada e automatizada. O ensaio customizado de Open Array® mostrou-se acurado e de rápida execução. Elaborou-se software próprio para interfaceamento dos resultados da genotipagem e busca dos genótipos. CONCLUSÃO: Padronizou-se estratégia efetiva para rastreamento de doadores de sangue com fenótipos raros. A automação de todas as etapas experimentais e o interfaceamento completo dos dados minimizaram os erros humanos e aumentaram a rapidez do processo descrito, que pode ser aplicado como estratégia de genotipagem de doadores de todo o Estado de São Paulo.

Transfusion of older red blood cell units, cytokine burst and alloimmunization: a case-control study

BACKGROUND: Experimental data have shown that the transfusion of older red blood cell units causes alloimmunization, but the clinical applicability of this statement has never been properly assessed in non-sickle cell patients. It has been hypothesized that older units have higher numbers of cytokines, increasing the risk of alloimmunization related to antigen-presenting events. The goal of this study was to evaluate the association between the transfusion of older red blood cell units subjected to bedside leukodepletion and alloimmunization.METHODS: All patients submitted to transfusions of bedside leukodepletion red blood cell units proven to have become alloimmunized in one oncologic service between 2009 and 2013 were enrolled in this study. A control group was formed by matching patients without alloimmunization in terms of number of transfusions and medical specialty. The median age of transfused units, the percentage of transfused red blood cell units >14 days of storage in relation to fresher red cell units (≤14 days of storage) and the mean age of transfused units older than 14 days were compared between the groups.RESULTS: Alloimmunized and control groups were homogeneous regarding the most relevant clinical variables (age, gender, type of oncological disease) and inflammatory background (C-reactive protein and Karnofsky scale). The median age of transfused red blood cell units, the ratio of older units transfused compared to fresher units and the mean age of transfused units older than 14 days did not differ between alloimmunized and control patients (17 vs. 17; 68/32 vs. 63.2/36.8 and 21.8 ± 7.0 vs. 21.04 ± 7.9; respectively).CONCLUSION: The transfusion of older red blood cell units subjected to bedside leukodepletion is not a key risk factor for alloimmunization. Strategies of providing fresh red cell units aiming to avoid alloimmunization are thus not justified.

Prophylactic strategies for acute hemolysis secondary to plasma-incompatible platelet transfusions: correlation between qualitative hemolysin test and isohemagglutinin titration

Brazilian legislation has recently suggested the use of the qualitative hemolysin test instead of isohemagglutinin titers as prophylaxis for acute hemolysis related to plasma-incompatible platelet transfusions. The efficacy of this test in preventing hemolytic reactions has never been evaluated while isohemagglutinin titers have been extensively studied. The main objective of this study was to evaluate the correlation between the results of these two tests. The impact of each type of prophylaxis on the platelet inventory management and the ability of the qualitative hemolysin test to prevent red cell sensitization after the transfusion of incompatible units were also studied.METHODS: A total of 246 donor blood samples were evaluated using both isohemagglutinin titers and the qualitative hemolysin test, and the results were statistically compared. Subsequently, 600 platelet units were tested using the hemolysin assay and the percentage of units unsuitable for transfusion was compared to historical data using isohemagglutinin titers (cut-off: 100). Moreover, ten patients who received units with minor ABO incompatibilities that were negative for hemolysis according to the qualitative hemolysin test were evaluated regarding the development of hemolysis and red cell sensitization (anti-A or anti-B).RESULTS: Isohemagglutinin titration and the results of qualitative hemolysin test did not correlate. The routine implementation of the qualitative hemolysin test significantly increased the percentage of platelet units found unsuitable for transfusions (15-65%; p-value <0.001)...

Estudo das propriedades mecânicas das células de músculo liso vascular em situações fisiológicas e patológicas / Study of the mechanical properties of vascular smooth muscle cells under physiological and pathological situations

Introdução: As células do músculo liso vascular (CMLV) são quiescentes nos vasos adultos, com baixa capacidade de migração e de secreção de matriz extracelular, caracterizando fenótipo contrátil. Evidências apontam para a heterogeneidade fenotípica das CMLV ao longo da árvore arterial: há distribuição heterogênea de doenças e de resposta a determinadas drogas nos diferentes vasos, além de variabilidade de expressão dos genes de proteínas contráteis de músculo liso entre eles. O papel das CMLV, em fase adulta, é classicamente descrito como restrito à regulação do tônus de pequenos vasos, sendo insignificante a contribuição da mecânica das CMLV para a complacência das artérias elásticas. Existe a hipótese de que a viscoelasticidade das CMLV contribua para a mecânica final das artérias, sendo o enrijecimento dessas células associado à rigidez arterial. Objetivo: Estudar a variabilidade das propriedades mecânicas e de expressão proteica das CMLV, ao longo da árvore arterial, buscando identificar moduladores regionais para esse fenótipo. Avaliar se situações clínicas sabidamente associadas à rigidez arterial (envelhecimento, sexo feminino pós-menopausa, ancestralidade genética africana, diabetes mellitus e tabagismo) cursam com enrijecimento de CMLV. Métodos: 1) Estudou-se a composição e a organização da camada média de diferentes artérias. As CMLV desses vasos foram avaliadas quanto à viscoelasticidade de citoplasma (G), por meio do ensaio de Citometria Magnético Ótica de Oscilação e, quanto à expressão proteica global, usando cromatografia multidimensional e espectrometria de massas em tandem de alta resolução (Proteômica Shotgun). Os dados mecânicos obtidos foram correlacionados com as características da matriz extracelular (MEC) dos vasos de origem (porcentagem de elastina e quantidade de MEC). Em paralelo, foi realizado experimento de estiramento cíclico (10%/1Hz) das CMLV das diferentes artérias por 24 e 48h, seguido pela mensuração de rigidez de...

Rational: Vascular smooth muscle cells (VSMC) lose their ability to migrate and secrete extracellular matrix (ECM) with the end of vascular development, condition known as contractile phenotype and reversible in the presence of vascular injury. There is evidence of heterogeneity of VSMC phenotype along arterial tree, as the distribution of diseases (atherosclerosis) and the response to drugs vary between different vessels, as well as the expression of smooth muscle-contractile protein genes. The role played by VSMC mechanics on determining large arteries' compliance was always considered irrelevant. It has been hypothesized that the VSMC mechanical properties are important for vascular mechanics, especially in the pathological scenario, where VSMC stiffening may be associated with arterial rigidity. Goals: Study the variation of VSMC mechanics and protein expression along arterial tree, identifying regional modulators of this phenotype. Evaluate if clinical situations associated with arterial rigidity (ageing, post-menopausal women, African ancestry, diabetes mellitus and smoking) concur with VSMC stiffening. Methods: 1) Different arteries were studied in terms of composition and organization of their media layer. VSMC isolated from these arteries were evaluated regarding cytoplasm viscoelasticity, measured using Optical Magnetic Twisting Cytometry Assay (OMTC), and protein expression, using two-dimensional liquid chromatography and tandem mass spectrometry (Shotgun Proteomics). Mechanical data were correlated with ECM characteristics (percentage of elastin and ECM amount) of the vessels of origin. In parallel, VSMC of different arteries were subjected to cyclic stretching (10%/1Hz) during 24 and 48h, followed by the measurement of their cytoplasm rigidity. 2) VSMC were isolated from fragments of mammary artery of 80 patients subjected to coronary bypass surgery and evaluated regarding their viscoelasticity (G, G' e G''). A statistic model was elaborated to...

Study of possible clinical and laboratory predictors of alloimmunization against red blood cell antigens in cancer patients

Background: The inflammatory background of patients influences the process of alloimmunization against red blood cell antigens. Proof of this statement to clinical practice is still lacking. Objective: The aim of this study was to verify whether factors related to disease severity and inflammatory status of cancer patients can predict alloimmunization. Methods: This was a case-control study in which alloimmunized oncologic patients treated between 2009 and 2012 were compared with a non-alloimmunized control group regarding the severity of the disease (metastasis/performance status/body mass index) and C-reactive protein levels. Results: The groups did not differ significantly in terms of C-reactive protein, Eastern Cooperative Oncology Group (ECOG)/Karnofsky performance status, presence of metastasis and body mass index. Conclusion: It is not possible to predict alloimmunization in cancer patients based on severity of illness and inflammatory markers. Strategies of screening patients by phenotyping blood based on these criteria are not justified. .

Non-HFE hemochromatosis

Hereditary hemochromatosis (HH) is an autosomal recessive disorder classically related to HFE mutations. However, since 1996, it is known that HFE mutations explain about 80% of HH cases, with the remaining around 20% denominated non-HFE hemochromatosis. Nowadays, four main genes are implicated in the pathophysiology of clinical syndromes classified as non-HFE hemochromatosis: hemojuvelin (HJV, type 2Ajuvenile HH), hepcidin (HAMP, type 2B juvenile HH), transferrin receptor 2 (TFR2, type 3 HH) and ferroportin (SLC40A1, type 4 HH). The aim of this review is to explore molecular, clinical and management aspects of non-HFE hemochromatosis.

Immunoglobulin: production, mechanisms of action and formulations

Human immunoglobulin (Ig) began to be applied in the clinical practice with the treatment of primary immunodeficiencies. Quickly, applications of Ig increased, as its anti-inflammatory and immunomodulatory functions were elucidated. Currently, Ig is the most commonly used blood product. Ig is obtained by processing plasma; methods, in particular, techniques to reduce plasma viral loads have been evolving over the years and include: pasteurization, solvent/ detergent treatment, caprylic acid treatment and nanofiltration. These methods contribute to increased safety and quality of blood products. The mechanisms of action of Ig not only involve the blockade of Fc receptors of phagocytes, but also control complement pathways, idiotype-anti-idiotype dimer formation, blockage of superantigen binding to T cells, inhibition of dendritic cells and stimulation of regulatory T cells (Tregs). There are several formulations of Ig available, each one with its own peculiar characteristics. In Brazil, there is stringent legislation regulating the quality of Ig. Only Ig products that completely fulfill the quality control criteria are released for use. These standards involve different tests from visual inspection to determination of anti-complementary activity. This paper will further review the history and current status of Ig, including its production and mechanisms of action. The formulations available in Brazil and also the criteria of quality control currently applied will be presented.

Clinical applications of immunoglobulin: update: [review]

Human immunoglobulin is the most used blood product in the clinical practice. Immunoglobulin applications have increased quickly since the elucidation of its immunomodulatory and antiinflammatory properties which turned this blood product into a precious tool in the treatment of numerous diseases that present with humoral immune deficiency or that cause immune system dysfunction. Currently, the approved indications for Ig are: primary immunodeficiencies, secondary immunodeficiencies (multiple myeloma or chronic lymphoid leukemia), Kawasaki syndrome, immune thrombocytopenic purpura, Guillain Barré syndrome, graft-versus-host disease following bone marrow transplantation and repeat infections in HIV children. On the other hand, there are numerous "off-label" indications of immunoglobulin, which represent 20-60 percent of all clinical applications of this drug. It is important to study all these indications and, above all, the scientific evidence for its use, in order to provide patients with a new therapeutic option without burdening the health system. This review results from a wide selection of papers identified in the Pubmed and Lilacs scientific electronic databases. A group of descriptors were used from human immunoglobulin to the names of each disease that immunoglobulin is clinically applied. Our main objective is to list the numerous indications of immunoglobulin, both authorized and "off-label" and to analyze these indications in the light of the most recent scientific evidence.

Porfirias: quadro clínico, diagnóstico e tratamento / Porphyrias: clinical presentation, diagnosis and treatment

As porfirias são doenças incomuns e de herança genética na maior parte doscasos. As porfirias são divididas em eritropoiéticas, hepáticas agudas e hepáticas crônicas. Os subtipos de maior relevância clínica são a porfiria cutânea tarda e a porfiria intermitenteaguda. O diagnóstico das porfirias pode ser bastante difícil, dada a sobreposição de quadros clínicos e achados bioquímicos. A precisão do diagnóstico depende da dosagem de porfirinasurinárias e fecais, da análise da atividade enzimática de eritrócitos e, eventualmente, da pesquisa de mutações. O objetivo do presente artigo é realizar revisão literária das porfirias,com ênfase no diagnóstico e tratamento de seus diversos subtipos...

Porphyrias are uncommon diseases that have genetic inheritance in the majorityof the cases. Porphyrias are divided in: erythropoietic porphyria, acute hepatic porphyria and chronic hepatic porphyria. The subtypes with major clinical relevance are porphyria cutaneatarda and acute intermittent porphyria. Diagnosing porphyrias may be quite difficult, as there is significant overlapping between clinical and biochemical findings. The diagnosis depends on the measurement of urinary and fecal porphyrins, enzymatic analysis of erythrocytes and, eventually, analysis of mutations. The main purpose of this article is to make a review of porphyrias, with emphasis on diagnosis and treatment of its several subtypes...

Talidomida no tratamento de angiodisplasias intestinais em paciente com síndrome CREST: relato de caso e revisão da literatura / Thalidomide as a treatment of intestinal angiodisplastic lesions in a patient with CREST syndrome: case report and literature review

A anemia ferropriva grave secundária à hemorragia digestiva por angiodisplasias intestinais representa um grande desafio terapêutico. Comumente, as ectasias vasculares são múltiplas e dispersas ao longo do intestino, limitando a eficácia do tratamento hemostático local. Nos últimos anos houve significativo avanço no tratamento anti-angiogênico sistêmico das angiodisplasias intestinais, sendo talidomida a droga mais empregada para tal fim. Relatamos o caso de uma paciente de 49 anos com angiodisplasias intestinais secundárias a síndrome CREST (Calcinose, Raynaud, Dismotilidade Esofágica, Esclerodactilia e Telangiectasias). A paciente apresentava quadro de melena recorrente e alta necessidade transfusional, e não obteve resposta clínica após realização de enteroscopia e eletro-coagulação das lesões com plasma de argônio. Após a introdução de talidomida 100mg ao dia, a paciente evoluiu de forma bastante satisfatória. O caso apresentado neste texto, além de demonstrar sucesso da talidomida no tratamento de angiodisplasias intestinais refratárias à eletro-coagulação com plasma de argônio, também revela eficácia da droga na situação específica da síndrome CREST. Tal fato pode ser de grande valia quando da abordagem de hemorragia intestinal por angiodisplasias nesses pacientes, representando nova opção terapêutica...

The severe ferropenic anemia secondary to digestive bleeding due to intestinal angiodisplastic lesions represents a great challenge. Commonly, angiodisplastic lesions are multiples and disperse through the intestine and that fact limits local treatments. Over the last years, there was a great advance in the antiangiogenic treatment of intestinal angiodisplastic lesions and thalidomide was the most employed drug for this purpose. We report a case of a 49 year-old patient with intestinal angiodisplastic lesions due to CREST syndrome (Calcinosis, Raynaud phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia). The patient presented repeated episodes of digestive bleeding and did not achieve clinical improvement after enteroscopy and argon plasma coagulation. The treatment consisting of the introduction of thalidomide 100mg per day demonstrated success. The case presented in this text reveals success in the use of thalidomide in the treatment of intestinal angiodisplastic lesions, probably representing a new therapeutic option...