En hématologie, des tensioactifs tels que les saponines sont utilisés comme agent érythrolytique afin de compter et identifier les différentes populations de globules blancs, qui sont mille fois moins abondants que les érythrocytes. Ces saponines sont utilisées pour leur lyse sélective des globules rouges dans les réactifs d’HORIBA Médical. Au cours de cette thèse, nous avons étudié l’action de différents tensioactifs sur les cellules sanguines afin d’avoir une meilleure compréhension des interactions tensioactifs/membrane. Le premier projet a porté sur l’observation et la caractérisation des effets des saponines sur les érythrocytes par des méthodes microscopiques et spectrophotométriques, dans le but d’obtenir une meilleure compréhension de leur sélectivité vis-à-vis des globules rouges. Nous avons ainsi démontré la participation du cholestérol, des transporteurs tels que la bande 3 et du cytosquelette lors de la lyse par les saponines. Dans une seconde étude, des recherches ont été entreprises afin d’identifier les propriétés requises d’un tensioactif pour obtenir un agent lytique efficace. Nous avons sélectionné deux types de familles de tensioactifs, l’une non-ionique de type éther d’alcool polyoxyéthylèné, et l’autre anionique à base d’acides aminés. Ces études se sont concentrées sur la relation entre le pouvoir érythrolytique et certains paramètres physico-chimiques des tensioactifs tels que la concentration micellaire critique (CMC), la balance hydrophile-lipophile (HLB), le coefficient de partage entre la membrane et le milieu aqueux (Kb), et le packing parameter (P). Nous avons montré qu’une considération globale de l’ensemble de ces paramètres était nécessaire pour pouvoir prédire les interactions des tensioactifs avec la membrane des érythrocytes. Enfin, l’effet lytique de ces tensioactifs sur les leucocytes a été étudié en cytométrie en flux afin de vérifier l’intégrité des globules blancs lors des différentes lyses. Les résultats obtenus au cours de ces travaux de thèse rendent possible le développement de futurs réactifs hématologiques pour la différenciation leucocytaire. / Surfactants such as saponins are employed, in the haematology field, as erythrolytic agent in order to count and to identify different leukocyte populations, which are typically thousand times less abundant than erythrocytes. These saponins are used for erythrocytes selective lysis in HORIBA Medical reagents. This thesis aimed to study the action of different surfactants on red blood cells in order to have a better understanding of surfactants/membrane interactions. The first project involved the observation and the characterization of saponins effects on erythrocytes by microscopic and spectrophotometric methods, in order to understand saponins selectivity regarding erythrocytes. We have thus demonstrated the participation of cholesterol, transporters such as band 3 and erythrocyte cytoskeleton during saponins lysis. In a second study, investigations have been performed in order to identify surfactants properties required to get a suitable erythrocyte lysing agent. We have chosen two surfactants families: non-ionic surfactants of the polyoxyethylene alkyl ethers series and anionic amino acid-based surfactants. These studies are focused on the relation between surfactants erythrolytic potency and some physico-chemical parameters such as the critical micellar concentration (CMC), the hydrophile-lipophile balance (HLB), the surfactants membrane / water partition coefficient (Kb) and the packing parameter (P). We have thus shown that the global consideration of all these physico-chemical parameters was necessary to predict surfactants interactions with the erythrocytes membrane. Finally, the lytic effects of surfactants on leukocytes were studied in flow cytometry to verify leukocytes integrity during different lysis. The results obtained in this thesis make possible the future development of hematological reagent for leukocytes differentiation.

Orientador: Albetiza Lobo de Araujo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T13:35:19Z (GMT). No. of bitstreams: 1 Martins_LucimaraJulio_M.pdf: 11295607 bytes, checksum: 8492693ea03dda6f72a75c0c23ee0d9c (MD5) Previous issue date: 2006 / Resumo: O veneno de Bothrops lanceolalus exerce várias atividades biológicas, dentre elas a atividade hemolítica indireta. Neste trabalho verificamos algumas das características desta atividade in vitro. O veneno induziu hemólise indireta em eritrócitos de cavalo, boi, rato e carneiro, sendo que, dentre essas espécies, a primeira foi mais sensível; não houve atividade hemolítica direta. A hemólise foi concentração-dependente e atenuada em temperaturas >40°C. O tratamento do veneno com brometo de p-bromofenacil aboliu a atividade da fosfolipase A2 (PLA2) do veneno e impediu a hemólise. Corroborando estes achados, uma PLA2 ácida purificada deste veneno também induziu hemólise. Esta PLA2 reagiu contra o anti-soro do veneno de B. lanceolatus. Estes resultados indicam que atividade hemolítica do veneno de B. lanceolatus é mediada pela PLA2. A reação cruzada da PLA2 com o anti-soro sugere que a atividade desta enzima pode eficazmente ser neutralizada durante a terapia sorológica / Abstract: Bothrops lanceolatus venom exerts a variety of biological activities, including indirect hemolysis. In this work, we examined some of the characteristics of this activity in vitro. The venom caused indirect hemolysis in horse, ox, rat and sheep erythrocytes, with the first of these species being the most sensitive; there was no direct hemolysis. The hemolysis was concentration-dependent and was markedly attenuated at >40°C. Treatment of the venom with />-bromophenacyl bromide abolished the phospholipase A2 (PLA2) activity of the venom and prevented the hemolysis. In agreement with this finding, an acidic PLA2 purified from this venom also caused hemolysis. This PLA2 reacted with antivenom against B. lanceolatus venom. These results indicate that the hemolytic activity of B. lanceolatus venom is mediated by PLA2. The cross-reactivity of the PLA2 with antivenom suggests that the activity of this enzyme may be effectively neutralized during antivenom therapy / Mestrado / Mestre em Farmacologia

O avanço da bioengenharia atual tem sido motivado pela crescente necessidade da humanidade em se buscar formas para amenizar o sofrimento, garantir tempo para um tratamento, melhorar a qualidade de vida ou sanar quadros clínicos de pacientes. Assim, a crescente necessidade de órgãos artificiais impulsiona a área de bioengenharia para que seja dado um suporte no desenvolvimento destes mecanismos. Neste contexto se encontra o presente trabalho. Aqui, apresenta-se uma metodologia numérica para que seja aplicada a desenvolvimento e testes de Dispositivos de Assistência Cardíaca, de forma a otimizar o processo de desenvolvimento e estimar os possíveis problemas inerentes ao seu funcionamento. Compondo a metodologia, uma ampla discussão de cada etapa numérica foi elaborada, contribuindo para uma metodologia flexível para uma ampla variedade de aplicações. A hemólise também foi investigada através dos principais modelos da literatura, bem como foram propostos modelos adaptados para tentar estimar a hemólise verificada experimentalmente. Além das metodologias para cada etapa, uma metodologia geral utilizando Sistemas de Referências Múltiplas (SRM) compõe os resultados, para uma sequência de passos numéricos que possa obter resultados satisfatórios em comparação com resultados de bancada por loop test. Outros resultados encontrados e devidamente discutidos foram inerentes a uma densidade de malha a ser utilizada para que ocorra simulações com independência de malha, na qual uma densidade a partir de 318,43 elementos/mm³ para referenciais não-inerciais é proposta. O mapeamento da turbulência por seis modelos que aplicam a média de Reynolds também é discutido, indicando os melhores modelos a serem empregados para um número de Reynolds relativo (Re*) que relaciona a influência dos contornos nos resultados numéricos obtidos. Além disso, uma nova abordagem por tensão fisiológica é proposta para o cálculo da hemólise, sendo comparada aos modelos clássicos adotados. Os resultados para hemólise indicam um bom ajuste para o novo modelo proposto, bem como indica o correto tratamento de unidades para os modelos tradicionais. Por fim, as análises recaem na melhoria do dispositivo e conclui a metodologia numérica a ser empregada, preenchendo lacunas em cada etapa e determinando uma maneira de análise numérica cujos resultados são confiáveis. / The advance of current bioengineering has been driven by the increasing need of humanity to seek ways to alleviate the suffering, ensure time to treatment, improve the quality of life or cure medical conditions of patients. Thus, the growing need for artificial organs boosts bioengineering area to be given a support in the development of these mechanisms. In this context is the present work. Here, we present a numerical methodology to be applied to development and Cardiac Assist Devices tests in order to optimize the development process and estimate the potential problems inherent in their operation. Compounding the methodology, a comprehensive discussion of each numerical step was developed, contributing to a flexible methodology for a wide variety of applications. Hemolysis was also investigated through the main models of literature, and have been proposed models adapted to try to estimate hemolysis verified experimentally. In addition to the methodologies for each step, a general methodology using Multiple Reference Systems (MRF) makes up the results for a sequence of numeric steps you can get satisfactory results compared to bench test results for loop. Other findings were discussed and properly attached to a mesh density being used for simulations occurring independently mesh in which a density from 318.43 elements/mm³ Non-inertial frames is proposed. The mapping of turbulence six models applying Reynolds medium is also discussed, indicating the best designs to be employed for a number of relative Reynolds (Re*) that relates the influence of the contours of numerical results obtained. In addition, a new approach by physiological stress is proposed for the calculation of hemolysis, being compared to classic models adopted. The results for hemolysis indicate agreement with the proposed new model, and indicates the correct treatment units to the traditional models. The final analyses fall into improved device and completes the numerical methodology to be used by filling gaps in each step and determining a way to numerical analysis results which are dependable.

Introdução: Anemias hemolíticas induzidas por fármacos (AHIF) são caracterizadas por uma anemia hemolítica imune após exposição a um fármaco. Testes sorológicos têm o potencial de confirmar a presença de anticorpo contra fármacos, mas a realização destes testes ainda é um desafio. Os anticorpos IgM e IgG envolvidos na AHIF são principalmente de dois tipos: fármaco-dependentes e fámaco-independentes. Os anticorpos fármaco-independentes reagem com os eritrócitos \"in vitro\" sem a presença do fármaco, e os resultados imunohematológicos são idênticos aos encontrados nas anemias hemolíticas autoimunes idiopáticas. Anticorpos fármaco-dependentes resultam em teste da antiglobulina direto (TAD) positivo e eluato negativo, os anticorpos só reagem \"in vitro\" com os eritrócitos na presença do fármaco. Indivíduos saudáveis podem desenvolver anticorpos contra fármacos, mesmo na ausência de anemia hemolítica. Os indivíduos podem ser sensibilizados durante exposição prévia a fármacos, exposição a antibióticos usados em ração animal, tanques de piscicultura, berçários de criação de suínos ou tratamento de mastite em bovinos. Estes indivíduos apresentam um maior risco de desenvolvimento de AHIF grave na ocasião de uma segunda exposição ao fármaco. Objetivos: Padronizar testes imunohematológicos para a detecção de anticorpos dirigidos contra fármaco-dependentes. Materiais e métodos: Amostras de 162 doadores de sangue foram submetidas a pesquisa de anticorpos fármaco-dependentes anti-cefalexina, anti-rifampicina e anti-diclofenaco de sódio. Amostra de um doador de plaquetas colhidas por aférese com TAD positivo foi investigada para detecção de anticorpos fármaco-dependentes anti-valsartana. Amostras de 8 pacientes com suspeita diagnóstica de AHIF foram investigadas para a presença de anticorpos contra os fármacos que foram prescritos ao paciente no período de duas semanas prévias ou até o momento em que a hemólise aguda foi constatada. Anticorpos fármaco-dependentes que reagem na presença do fármaco ligado covalentemente à membrana da hemácia, ou na presença do fármaco em solução, foram investigados em doadores de sangue (técnicas em gel) e em pacientes (técnicas em tubo e em gel). Resultados: Os testes laboratoriais detectaram a presença de anticorpo anti-valsartana no eluato de um doador de plaquetas colhidas por aférese com TAD positivo e a presença de anticorpo anti-ceftazidima no soro de um paciente com suspeita clínica de AHIF. A investigação de anticorpo anti-cefalexina e anti-rifampicina foi negativa em amostras de doadores de sangue. O teste laboratorial para investigação de anticorpo anti-diclofenaco de sódio não foi finalizado devido a presença de hemólise em hemácias sensibilizadas com o fármaco e após a adição do fármaco na solução de hemácias. Conclusão: A detecção de anticorpo anti-valsartana em um doador de plaquetas colhidas por aférese evidenciou a presença de anticorpo anti-fármaco na ausência de hemólise. A detecção de anticorpo anti-ceftazidima em amostra de um paciente com anemia hemolítica confirmou a suspeita clínica de AHIF. Reconhecer os sinais clínicos da AHIF precocemente e a disponibilidade de testes sorológicos é essencial para confirmação diagnóstica, orientação terapêutica e prevenção de uma hemólise potencialmente fatal. / Introduction: Drug-induced immune hemolytic anemia (DIHA) is characterized by immune hemolytic anemia following exposure to a drug. Serological testing has the potential to confirm the presence of anti-drug antibodies, but the testing is still a challenge. The IgM and IgG antibodies involved in DIHA comprise mainly two types: drug-dependent and drug-independent. Drug-independent antibodies react with erythrocytes in vitro without the presence of the drug, and the immunohematologic results are identical to those found in idiopathic autoimmune hemolytic anemias. Drugdependent antibodies induce positive direct antiglobulin tests (DAT) and negative eluates, and in vitro antibody reactions with erythrocytes are only detected in presence of the drug. Healthy individuals can develop antibodies against drugs even in the absence of hemolytic anemia. Individuals may be sensitized during previous exposure to drugs, exposure to antibiotics used in animal feed, fish culture ponds, swine nurseries or mastitis treatment in bovines. These individuals present higher risk of developing severe DIHA on the second exposition. Aims: To standardize immunohematological tests for detection of anti-drug dependent antibodies. Materials and methods: Samples from 162 blood donors were tested for anti-cephalexin, anti-rifampicin and antidiclofenac antibodies. Sample from one platelet apheresis donor with positive DAT was investigated for anti-valsartan drug-dependent antibody. Samples from 8 patients with suspected DIHA were investigated for the presence of antibodies directed against the drugs that were prescribed to the patient in the period of two weeks prior to or until the time when acute hemolysis was detected. Drug-dependent antibodies that react in presence of the drug covalently attached to the erythrocyte membrane, or in presence of the drug in solution were investigated in samples from blood donors (gel techniques) and from patients (tube and gel techniques). Results: Laboratory tests detected antivalsartan antibody in the eluate of an platelet apheresis donor with positive TAD and anti-ceftazidime antibody in the serum of a patient with clinical suspicion of DIHA. Investigation of anti-cephalexin and anti-rifampicin antibody was negative in blood donor samples. The laboratory test for anti-diclofenac antibody was not possible due to the presence of hemolysis in erythrocytes sensitized with the drug and when the drug in solution was added to the erythrocyte. Conclusion: The detection of anti-valsartan antibody in an platelet apheresis donor revealed the presence of an anti-drug antibody in the absence of hemolysis. Detection of anti-ceftazidime antibody in a patient with hemolytic anemia confirmed the clinical suspicion of DIHA. It is essential to recognizing early clinical signs of DIHA and have serological tests available for diagnostic confirmation, therapeutic guidance and prevention of potentially fatal hemolysis.

Vibrio parahaemolyticus (Vp) infection is commonly caused by the consumption of raw or undercooked shellfish. Raw oysters are associated with most Vp outbreaks. Pathogenic Vp express thermostable-direct hemolysin (tdh) and to some extent thermostable-related hemolysin (trh). Additionally, some pathogenic Vp express urease (uh). The objectives of this work were to discern any relationships between urease (uh) expression, tdh/trh expression, and hemolytic activity in pathogenic and non-pathogenic clinical strains, and to compare urease, motility, tdh/trh expression, and hemolytic activity in raw oyster isolates. This information would determine if urease could be used as a biomarker to detect pathogenic Vp. About 80% of pathogenic strains were uh+ and all non-pathogenic strains were uh-. Two oyster samples were uh+ and no tdh or trh was detected in raw oyster strains.

No description available.

Neuronspecifikt enolas (NSE) är en viktig biomarkör för att diagnostisera t.ex. neuroendokrina tumörer, särskilt småcellig lungcancer (SCLC) och neuroblastom. NSE används även som en del i utredning av hjärnskada vid hjärtstopp. Eftersom NSE finns i höga koncentrationer i erytrocyter, kan hemolys i blodprovet orsaka falskt förhöjda NSE-nivåer i serum utan hjärnskada. Syftet med studien var att utvärdera hur hemolys påverkar NSE-analysen på Cobas, ett helautomatiserat analysinstrument. Mätning av NSE-koncentration utfördes på Cobas 8000 från Roche Elecsys, baserad på immunokemisk sandwich-metod med ElectroChemi-LuminiscenceImmunoassay (ECLI) detektionsteknik. För att studera hemolysens inverkan, tillverkades hemolysat från 20 patientprover. Dessa hemolysat tillsattes till poolat serum, med NSE-nivåer inom referensintervallet (< 17 µg/L). Även graden av hemolys bestämdes på Cobas 8000. Resultatet visade ett linjärt samband mellan de uppmäta hemolysindex (HI) värden och S-NSE värden. Variationen i NSE-tillskott på individnivå undersöktes och resulterade i slutsatsen att varje hemolysenhet motsvarar ett NSE-tillskott på 0,33 ± 0,07 µg/L som frigörs från erytrocyter. Ett förslag för att lösa problemet med hemolys vid analys av S-NSE är att använda en kompenserande faktor för att korrigera NSE-koncentrationen. Kompensering kan utföras med hjälp av det erhållna sambandet i studien (1 HI = 0,33 ± 0,07 µg/L NSE-tillskott) genom att subtrahera tillskottet från den uppmätta NSE-koncentrationen. / Neuron Specific Enolase (NSE) is an important biomarker for diagnosing e.g. neuroendocrine tumors, especially small cell lung cancer (SCLC) and neuroblastoma. NSE is also used as a part of the investigation of brain damage in cardiac arrest. Because NSE is present in high concentrations in erythrocytes, hemolysis in the blood sample can cause falsely elevated NSE levels in serum without brain damage. The purpose of this study was to evaluate how hemolysis affects NSE analysis on Cobas, a fully automated analytical instrument. Measurement of NSE concentration was performed on Cobas 8000 from Roche Elecsys, based on immunochemical sandwich method with ElectroChemi-Luminescence Immunoassay (ECLI) detection technique. To study the effect of hemolysis, hemolysates were prepared from 20 patient samples. These hemolysates were added to pooled serum, with NSE levels within the reference range (<17 μg/L). The degree of hemolysis was also determined on Cobas 8000. The result showed a linear relationship between the measured hemolysis index (HI) values and S-NSE values. The variation in NSE contribution at the individual level was examined with the result that each hemolysis unit corresponds to an NSE contribution of 0.33 ± 0.07 µg/L, which is released from erythrocytes. A suggestion to solve the problem of hemolysis relating to NSE analysis is to use a compensatory factor to correct the NSE concentration. Compensation can be performed by using the relationship obtained in the study (1 HI = 0.33 ± 0.07 µg/L NSE contribution) and subtracting the contribution from the measured NSE concentration.

Erythrocyte pyruvate kinase (R-PK) is a key glycolytic enzyme catalyzing the transphosphorylation of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP respectively3,4. The substrate PEP and product pyruvate of this reaction are involved in a number of energetic and biosynthetic pathways; hence a tight regulation of R-PK activity is crucial not only for glycolysis, but also for the entire cellular metabolism. Deficiency of R-PK is one of the most common enzymatic defects of RBC, and may be caused by mutations of the PK-LR (pyruvate kinase liver red blood cell) gene31, 32. Clinically, R-PK deficiency manifests itself as a chronic life-long hemolysis ranging from very mild or fully compensated anemia to life-threatening neonatal anemia and pronounced jaundice. Current treatment options are limited to continuous blood transfusions and splenectomy. Thus, there is an urgent need for medications to counter R-PK deficiency without resorting to these complicated procedures. Our aim is to identify novel allosteric modifiers of R-PK using a combination of computational studies and enzyme activity assays. Such compounds could be of medical interest. Human R-PK was expressed in DH-5α cells and was purified by the procedure reported by Wang et al57. However, this method gave a very low yield of R-PIS (5mg/L). In an attempt to increase the yield, we expressed R-PK in Rosetta strain cells. Further, addition of His-tag to the protein's N-terminus simplified purification to a one step Ni-NTA (Nickel- nitrilotriacetic acid) column resulting in a 6-fold increase in the yield. Computational methods were applied to identify small molecules that bind to the allosteric activator fructose 1,6-bisphosphate (FBP) binding site of R-PK to identify compounds that could interact with the protein. The software UNITY, as present in the molecular modeling software Sybyl was used to perform 3D searches against the National Cancer Institute (NCI) chemical database. From these searches we obtained 29 hits that were subjected to further computational analysis. The small molecules were docked into the FBP binding site of R-PK with different docking methods includingFlexX, GOLD and energy minimization. The energy scoring function of HINT was then applied to analyze the interactions between the docked molecules and R-PK. Compounds with highest HINT score were requested fiom NCI and were subjected to further kinetic analysis to identify possible allosteric effectors of R-PK.In the kinetic analysis, we employed a lactate dehydrogenase (LDH) coupled spectrophotometric assay to determine the activity of R-PK in the presence of these compounds. The steady state kinetics of R-PK gave a typical S-shaped curve that fitted a signloidal function indicative of allosterism. All the kinetic parameters of our enzyme were in excellent agreement with native R-PK activity as previously reported5, 57. R-PK activity in the presence of the analyzed compounds revealed both activators and inhibitors of R-PK. X-ray crystallographic analysis of R-PK in the presence of FBP and the identified small molecule effectors are currently in progress. These experiments were initiated to reveal the binding site of the compounds in R-PK, allowing for further optimization of the starting phannacophores and syntheses of new molecular entities for enhanced allosteric activity.In conclusion, we have developed a simple and efficient method for the expression and purification of R-PK. Using computational screening and HINT analysis we have also identified several compounds that interact with R-PK and kinetic analysis revealed both activators and inhibitors of the protein. Crystals of R-PK in the presence of effectors have been obtained and identification of the binding site on R-PK is under investigation. R-PK effectors discovered in this study could prove to be lead compounds for developing medications for the treatment of anemia and other disorders arising from R-PK malfunction.

Segundo a legislação brasileira e normas internacionais, os concentrados de hemácias (CH) devem ser mantidos sob controle rigoroso de temperatura, armazenados de 2 a 6°C e transportados de 1 a 10°C, por até 24 horas. Entretanto, não existem evidências científicas de que estes valores sejam relevantes para manutenção de qualidade, segurança e viabilidade dos CH. O objetivo deste trabalho foi monitorar parâmetros laboratoriais in vitro em dois tipos de CH (CPDA-1, preparado pela metodologia do plasma rico em plaquetas e em SAGM preparado pelo buffy-coat), no decorrer de condições normais de armazenamento e em temperaturas de transporte inadequadas. O trabalho foi executado em três etapas. Na primeira etapa, foram estabelecidos os valores de referência para os dois tipos de CH armazenados em condições normais, através de testes laboratoriais semanais para determinação de hematócrito, hemoglobina total, hemoglobina plasmática, grau de hemólise, glicose, lactato, desidrogenase lática e potássio. Na segunda etapa, amostras de CH foram submetidas a temperaturas entre -2°C e +1°C, ou entre 11 e 15°C, por um período de 6 horas ou 24 horas, no 14º dia de armazenamento. Na terceira etapa os CH foram expostos a condições de estresse extremo, submetendo-os a temperaturas entre -2°C e +1°C ou entre 22°C e 25°C, por período de 48 horas. Os resultados dos testes laboratoriais das segunda e terceira etapas foram comparados com controles e aos valores de referência da primeira etapa. Nas unidades de CH em CPDA-1 foram encontrados valores significativamente mais elevados de potássio, hemoglobina livre, grau de hemólise, lactato e LDH do que nos CH em SAGM, enquanto que a concentração de glicose foi muito mais baixa em CH-CPDA1. Na 2ª etapa, exposições por até 24 horas em temperaturas de até - 2°C ou até 15°C não causaram diferenças significativas nas dosagens de hemoglobina livre, grau de hemólise, glicose, lactato, LDH e potássio quando comparados aos controles. Amostras de CH-CPDA1, quando avaliadas imediatamente após período de 48 horas de exposição, apresentaram resultados de glicose superiores aos controles quando submetidas a -2°C e inferiores ao controle quando a 25°C. Exposições por até 48 horas em temperaturas de até -2°C ou até 25°C não causaram diferenças significativas na hemoglobina livre, grau de hemólise, lactato, LDH e potássio quando comparados aos controles, nos dois tipos de CH. Nesse trabalho foram elaborados perfis padrão de parâmetros laboratoriais para dois tipos de CH. Estes perfis padrão constituirão uma ferramenta de grande utilidade para controle de qualidade e monitoramento das lesões de armazenamento em bolsas de CH produzidas na Fundação HEMOMINAS. Nossos resultados demonstraram que bolsas dos dois tipos de CH submetidas a temperaturas de - 2°C ou 14°C por períodos inferiores a 24 horas não apresentam alterações significativas nos parâmetros laboratoriais avaliados in vitro e permitiram algumas elaboramos recomendações práticas e importantes para serviços de hemoterapia. Entretanto, ainda não existem ainda evidências suficientes na literatura para modificação das normas de temperatura e tempo de transporte atualmente preconizadas pela legislação nacional e internacional. / The Brazilian legislation and international guidelines require that red blood cell concentrates (RBC) are kept under a strict temperature control, stored between 2 and 6°C and transported between 1 and 10°C for up to 24 hours. Nevertheless, there is no scientific evidence that these values are relevant to maintain RBC quality, safety and viability. This study was performed in order to monitor in vitro laboratorial variables of two different RBC types (CPDA-1, prepared using the platelet-rich plasma methodology, or SAGM, prepared using the buffy-coat) stored under normal conditions or under inadequate temperatures during transportation. The study had three phases. In the first phase, weekly laboratorial tests were performed to establish the reference values of hematocrit, total hemoglobin, plasma hemoglobin, rate of hemolysis, glucose, lactate, lactic acid dehydrogenase (LDH) and potassium, for each of the RBC types stored under normal conditions. In the second phase, RBC samples were submitted to temperatures between -2°C and +1°C, or between 11 and 15°C, for 6 hours or 24 hours, on the 14th storage day. Laboratorial test results were compared to a control group (2-6°C) and to the reference values established in the 1st phase. In the third phase, RBCs were exposed to extreme stress, i.e., temperatures between -2°C and +1°C or between 22°C and 25°C, for 48 hours, and the laboratory test results were compared to a control group. CPDA1-RBC had higher levels of potassium, free hemoglobin, rate of hemolysis, lactate and LDH compared to SAGM-RBC, whereas glucose was significantly lower in CPDA1. In the second phase, exposure for up to 24 hours in temperatures until -2°C or 15°C had no effect on free hemoglobin, rate of hemolysis, glucose, lactate, LDH and potassium when compared to control. CPDA1 samples right after the 48-h exposure had higher glucose levels than controls when kept at -2°C and lower than control if exposed to 25°C. Exposures up to -2°C or 25°C for up to 48 hours had no effect on free hemoglobin, rate of hemolysis, lactate, LDH and potassium when compared to control groups, both for CPDA1-RBC and SAGM-RBC. In this study, it was established standards for laboratory analyses for two different RBC types. Such standards will comprise valuable and useful tools for the quality control and monitoring of storage lesions of RBC units produced by Fundação HEMOMINAS. Our results demonstrate that units from both RBC types submitted to -2°C or 14°C for up to 24 hours had no significant changes in in vitro laboratory variables and allow some practical and important recommendations for hemotherapy services. Nevertheless, there are not enough evidences in the literature to support changes in the current guidelines for transportation recommended by national and international legislation.

O grau de hemólise (GH) é um dos parâmetros de qualidade de concentrados de hemácias (CH). Conforme a Portaria 158/2016, ao menos 1% da produção mensal de CH deve ser controlada para o GH e 75% desta parcela deve apresentar resultado inferior a 0,8% de hemólise em relação à massa eritrocitária no último dia de validade do CH. O GH é definido como a porcentagem de hemoglobina livre (HbL) em relação à hemoglobina total (HbT) com a devida correção do volume globular do CH. O método analítico utilizado para a dosagem da HbL pela maioria dos hemocentros da rede pública nacional não referencia a fonte bibliográfica consultada. A hemoglobina liberada dos eritrócitos devido à hemólise é tóxica e desencadeia reações fisiopatológicas, resultando em implicações clínicas com gravidade que varia em função do grau de hemólise e do volume de hemácias transfundidas. O objetivo deste estudo foi avaliar os resultados de três metodologias analíticas espectrofotométricas para a determinação da HbL. Foram comparados o método de espectrofotometria direta de Harboe, o método de espectrofotometria direta de Cinco comprimentos de onda (5CO) e o método espectrofotométrico de Primeira derivada (1ªD). Os métodos de Harboe e de 5CO utilizam fórmulas matemáticas que convertem diretamente as absorbâncias lidas no espectrofotômetro UV/Visível em concentração de HbL. O método de 1ªD requer espectrofotômetro de varredura para a visualização dos espectros e a concentração da HbL é dada pelo valor referente à distância entre o vale e o pico de absorção da hemoglobina e do fator de correção, resultante de curva de calibração. Nesse estudo foram testados os sobrenadantes de 187 CH com CPDA-1 e CH com solução aditiva de SAG-Manitol. As amostras foram diluídas segundo o aspecto visual de hemólise do sobrenadante. A cada corrida analítica foram incluídas amostras controle preparadas in-house a partir de CH com concentração de HbT conhecida. O método de Harboe emprega leituras espectrofotométricas em 380, 415 e 450 nm. Para o método de 5 CO as absorbâncias são lidas em 370, 415, 510, 577 e 600 nm. O método da 1ªD utiliza o espectro de absorção analisado em 568 nm e 580 nm. A correlação entre as três metodologias testadas foi considerada ótima, evidenciando a equivalência entre os métodos. O método de Harboe mostrou-se compatível para a dosagem de baixas e de altas concentrações de HbL. O intervalo de linearidade espectrofotométrica oscilou de 0,00041 a 0,06075 g/dL. Entretanto, para resultados confiáveis de HbL, é imprescindível que o espectrofotômetro tenha especificação de largura da banda espectral igual ou inferior a 5 nm. O método de Harboe é embasado cientificamente, de fácil execução e baixo custo. Este método proporciona resultados fidedignos, reprodutíveis e padronizados, além de dispensar o uso de substâncias químicas perigosas. Complementarmente, disponibilizou-se um Procedimento Operacional Padrão para a determinação da HbL pelo método de Harboe e um Guia visual de hemólise para assessorar os profissionais que atuam na área de controle de qualidade de hemocomponentes em hemocentros nacionais. / The hemolysis is one of the parameters of the red cell concentrate (RCC) control quality. According to the Brazilian Ordinance 158/2016, at least 1% of the monthly production of RCC should be controlled regarding hemolysis and 75% of this amount should present below than 0.8% of hemolysis in relation to the red cell mass at end of RCC storage. The hemolysis is defined as the percentage of free hemoglobin (FHb), relative to the total hemoglobin (THb), with the appropriate correction of the RCC hematocrit. The analytical method used by most the national public healthcare blood centers to dosage FHb does not reference the bibliography that has been consulted. Hemoglobin released from the erythrocytes due to hemolysis is toxic and triggers pathophysiological reactions, resulting in clinical implications whose severity varies depending on the hemolysis grade and the amount of transfused RCC. The aim of this study was to evaluate the results of three spectrophotometric analytical methodologies for the determination of FHb. The Harboe spectrophotometry method, the method of five wavelengths direct spectrophotometry (5Wa) and the first derivative spectrophotometric method (1stD) were compared to each other. The Harboe and the 5Wa methods use mathematical formulas that directly convert the absorbance read from the UV/visible spectrophotometer in FHb concentration. The 1stD method requires scanning spectrophotometer to visualize the spectra and concentration of the FHb is given by the value calculated from the distance between the valley and the peak absorption of hemoglobin and a correction factor, resulting from the calibration curve. One hundred eighty-seven (187) RCC supernatants with CPDA-1 and RCC with SAG-mannitol additive solution were tested in this study. The samples were diluted according to the visual appearance of supernatants hemolysis. For each analytical run in-house control samples, prepared from RCC with known THb concentration, were included. The Harboe method employs spectrophotometric readings at 380, 415 and 450 nm. Absorbance is read at 370, 415, 510, 577 and 600 nm with the 5 Wa method. In the 1stD method the absorption spectrum is analyzed at 568 and 580 nm. There was correlation between the three tested methodologies that were tested, demonstrating equivalence between the methods. The Harboe method was compatible for dosage of low and high FHb concentrations. The spectrophotometer linear range varied from 0.00041 to 0.06075 g/dL. However, in order to achieve reliable FHb dosages it is imperative that the spectrophotometer has a spectral bandwidth equal to 5 nm or below. The Harboe method is scientifically based, easy to perform and inexpensive. It provides reliable, reproducible and standardized results, dismissing the use of dangerous chemical substances. In addition, this study has provided an Operational Procedure to determine FHb by the Harboe method and a Visual Guide of hemolysis to assist professionals working in the quality control of blood products.