Evaluation of RET-He values as an early indicator of iron deficiency anemia in pregnant women

Abstract Introduction During pregnancy, women are at an increased risk of developing iron-deficiency anemia. Objective The objective of this study was to assess the diagnostic performance of the reticulocyte hemoglobin equivalent (RET-He) in the early detection of iron-deficiency anemia in a group of pregnant women and to establish a reference range for this parameter in a group of control individuals. Method: A total of 60 patients and 130 control subjects were included in the study. Blood samples collected from the subjects were submitted to a complete blood count and a serum ferritin test and the data were analyzed by comparing the groups and ROC curves. Results The reference range found for the RET-He was between 29.75pg and 38.24pg, with a median of 35pg. The receiver operating characteristic (ROC) curve analysis for the ferritin parameter showed an area under the curve of 0.732 for the RET-He, 0.586 for hemoglobin, 0.551 for the mean corpuscular hemoglobin concentration and 0.482 for the mean corpuscular volume. Conclusion Early diagnosis of iron deficiency anemia in pregnancy is essential to prevent damage to both maternal and fetal health. The RET-He presents an excellent potential as an auxiliary tool for the diagnosis of iron deficiency in pregnant women.

Hemophagocytosis by blasts in a child with acute monocytic leukemia after chemotherapy / Hemofagocitose por células blásticas em paciente pediátrico com leucemia monocítica aguda após quimioterapia

ABSTRACT Objective: To describe the case of a child who presented hemophagocytic lymphohistiocytosis (HLH) associated with acute monocytic leukemia after chemotherapy, with hemophagocytosis caused by leukemic cells. Case description: In a university hospital in Southern Brazil, a 3-year-old female was diagnosed with acute monocytic leukemia with normal karyotype. The chemotherapy regimen was initiated, and she achieved complete remission six months later, relapsing after four months with a complex karyotype involving chromosomes 8p and 16q. The bone marrow showed vacuolated blasts with a monocytic aspect and evidence of hemophagocytosis. The child presented progressive clinical deterioration and died two months after the relapse. Comments: HLH is a rare and aggressive inflammatory condition characterized by cytopenias, hepatosplenomegaly, fever, and hemophagocytosis in the bone marrow, lymph nodes, spleen, and liver. Although rare, malignancy-associated HLH (M-HLH) is fatal. The patient in this case report met five out of the eight established criteria for HLH. The evolution of the patient's karyotype, regardless of the diagnostic profile, seemed secondary to the treatment for acute monocytic leukemia. In this case, the cytogenetic instability might have influenced the abnormal behavior of leukemic cells. This is a rare case of HLH in a child with acute monocytic leukemia.

RESUMO Objetivo: Descrever um caso de um paciente pediátrico que apresentou linfo-histiocitose hemofagocítica (LHH) associada à leucemia monocítica aguda pós-quimioterapia, com hemofagocitose causada pelas próprias células leucêmicas. Descrição do caso: Em um hospital universitário do Sul do Brasil, uma menina de três anos foi diagnosticada com leucemia monocítica aguda com cariótipo normal. Após receber protocolo quimioterápico, atingiu remissão seis meses depois do início do tratamento, recaíndo quatro meses após com um cariótipo complexo envolvendo ambos os cromossomos, 8p e 16q. A medula óssea mostrava-se infiltrada por células blásticas vacuolizadas com aspecto monocítico, com evidências de hemofagocitose. A criança apresentou um declínio clínico progressivo e dois meses após a recaída foi a óbito. Comentários: A LHH é uma condição inflamatória rara e agressiva caracterizada por citopenias, hepatoesplenomegalia, febre e hemofagocitose na medula óssea, linfonodos, baço e fígado. A LHH associada a doenças malignas, embora seja uma condição rara, é potencialmente fatal. A paciente deste caso apresentou cinco dos oito critérios estabelecidos para o diagnóstico de LHH. A evolução do cariótipo do paciente, independentemente do perfil do diagnóstico, pareceu ser secundária ao tratamento da leucemia monocítica aguda, sendo que a instabilidade citogenética pode ter influenciado o comportamento atípico observado nas células leucêmicas. Este é um dos raros casos de LHH em uma criança com leucemia monocítica aguda.

Comparison of two techniques for assessing pancreatic islet viability: flow cytometry and fluorescein diacetate/propidium iodine staining

Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay. Methods: Viability was analyzed in islets isolated from 10 male Wistar rats. Upon FDA/PI staining, 50 islets from each animal were analyzed under fluorescence microscopy by two well-trained researchers. For flow cytometry, islets were dispersed and 100 000 single cells were incubated with the 7-amino-actinomycin D (7AAD) fluorophore (dyes necrotic and late apoptotic cells) and the Annexin V-APC antibody (marks early apoptotic cells). Results: A moderate correlation was found between techniques (r = 0.6; p = 0.047). The mean islet viability measured by flow cytometry was higher than that estimated using FDA/PI staining (95.5 ± 1.4% vs 89.5 ± 5.0%; p = 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique with greater reproducibility that is less subject to inter-observer variability than FDA/PI. Therefore, flow cytometry appears to be the technique of choice when aiming for a more precise determination of islet viability. (AU)

Influence of minimal residual disease by multiparametric flow cytometry at day 15 of induction in risk stratification of children with B-cell acute lymphoblastic leukemia treated at a referral hospital in southern Brazil

ABSTRACT Background: The minimal residual disease (MRD) is the most important prognostic factor for acute lymphoblastic leukemia (ALL) in children. This study aimed to investigate the influence of detecting the MRD by the multiparametric flow cytometry (MFC) at day 15 (D15) of the induction on the analysis of the risk group classifications of the different childhood ALL treatment protocols used in a referral hospital in southern Brazil. Method: We retrospectively reviewed the medical records of patients with B-cell ALL, aged 1 to 18 years, treated at a hospital from January 2013 to April 2017. Main results: Seventy-five patients were analyzed. Regarding the MRD by the MFC at D15, the analyses showed statistical significance when the MRD was grouped into three categories, < 0.1%, 0.1-10%, and > 10%, with the following distribution: 30.7%, 52.0%, and 17.3%, respectively. There was a significant association between D15 MRD-MFC < 0.1% and the likelihood of dying or relapsing and between D15 MRD-MFC > 10% and the likelihood of dying or relapsing. The cumulative hazard ratio for the relapse of patients with D15 MRD-MFC < 0.1%, 0.1-10%, and > 10% was 19.2%, 59.8%, and 80.1%, respectively. Conclusion: Our analysis suggests D15 MRD-MFC < 0.1% as a cut-off point for patients with more favorable outcomes and that the MRD at D15 in risk classifications is particularly useful for the stratification of patients with a more favorable prognosis.

Quantification of peripheral blood CD34+ cells prior to stem cell harvesting by leukapheresis: a single center experience

ABSTRACT Background: Due to laboratory logistic issues, our center has traditionally scheduled peripheral blood stem cell harvests based on timing from the start of mobilization. This has proved to be useful in some cases, but also resulted in many fruitless harvests due to poor mobilization. In order to improve the efficiency of collections and compare the effectiveness of peripheral blood CD34+ cells as a predictor with data from other reports, this study analyzed the implementation of this routine. Methods: Peripheral blood and leukapheresis samples were quantified by flow cytometry and the association between these parameters was assessed. Results: Sixty-six consecutive leukapheresis samples were collected from 34 patients after the collection of peripheral blood samples for CD34+ quantification. A moderate positive correlation was observed between peripheral blood CD34+ cell count and total CD34+ cell count/kg (r = 0.596; p-value < 0.001). A multivariable regression model also confirmed this association and allowed the estimation that for every increase in five CD34+ cells/µL in the peripheral blood, a mean increase of 0.38 × 106 CD34+ cells/kg could be predicted. Demographic characteristics, baseline comorbidities and mobilization regimen did not influence final CD34+ cell count in this sample. Conclusions: As observed in other centers, quantification of peripheral blood CD34+ progenitor cells is a strong predictor of effectiveness to guide stem cell harvesting. Due to the results of this study, a modification in the peripheral blood stem cell harvesting logistics was implemented at our center in order to incorporate this routine.

Cut-off value for absolute lymphocytes as an alternative for the immunophenotypic analysis of CD3+ T cells in the monitoring of immunosuppressive therapy with thymoglobulin / Valor de corte para linfócitos absolutos como alternativa para análise imunofenotípica de células T CD3+ no monitoramento de terapia imunossupressora com timoglobulina

Abstract Introduction: Immunosuppression of T lymphocytes is required for preventing acute rejection after transplantation and for the treatment of chronic autoimmune and inflammatory diseases. The laboratory monitoring for this therapy is the measurement of T cells by immunophenotyping, aiming the target value of less than 20 cells per µL. Objective: To establish a cut-off point for the total number of lymphocytes in the automated blood cell count that reflects less than twenty T cells µL by immunophenotyping. Methods: We studied and evaluated 242 kidney transplant patients that had results of automated blood cell count and quantification of T cells by immunophenotyping technique. The patients were divided into two groups, depending on the T lymphocyte immunophenotyping rates established by lower and higher than 20 cells per µL. After, we evaluated the cut-off point for lymphocytes in the blood cell count with a specificity of 100% to exclude patients with high levels of T lymphocytes. Results: We found that the cut-off point of 70 lymphocytes per µL obtained by automated blood cell count showed 100% of specificity to exclude patients with T-cell counts higher than 20 cells per µL by immunophenotyping. Conclusion: The results found in this study may be helpful to monitor the immunosuppressive therapy in kidney transplant patients in places where a flow cytometer is not available, or when this equipment is not present in the full routine.

Resumo Introdução: A imunossupressão de linfócitos T é necessária para a prevenção da rejeição aguda em transplantes e no tratamento de doenças autoimunes e inflamatórias crônicas. O seu monitoramento laboratorial consiste na quantificação dos linfócitos T realizada pela técnica de imunofenotipagem, na qual o valor preconizado é manter inferior a 20 células/µL. Objetivo: Estabelecer um ponto de corte para o número de linfócitos totais no hemograma automatizado que reflita uma contagem de linfócitos T inferior a 20 células/µL por imunofenotipagem. Métodos: Foram avaliados 242 pacientes transplantados renais que continham resultados do hemograma automatizado e quantificação de linfócitos T por imunofenotipagem. Os pacientes foram divididos em dois grupos, conforme os valores de linfócitos T estabelecidos pela imunofenotipagem: inferiores e superiores a 20 células/µL. A partir disto, foi avaliado o ponto de corte de linfócitos no hemograma com especificidade de 100% para excluir os pacientes com valores elevados de linfócitos T. Resultados: Este estudo evidenciou que o ponto de corte de 70 linfócitos/µL obtidos pelo hemograma automatizado apresentou especificidade de 100% para excluir os pacientes com contagens de linfócitos T superiores a 20 células/µL na imunofenotipagem. Conclusão: Esta pesquisa poderá auxiliar no monitoramento da terapia imunossupressora em pacientes transplantados renais em locais que não possuem um citômetro de fluxo disponível, ou ainda quando este equipamento não se faz presente na rotina integral.

Determination of reference ranges for immature platelet and reticulocyte fractions and reticulocyte hemoglobin equivalent

ABSTRACT Introduction: The immature platelet and immature reticulocyte fractions represent the ratios of platelets and reticulocytes recently released into the circulation and thus with higher RNA content. They are considered early indicators of bone marrow recovery. Objective: The aim of this study was to determine the reference ranges for the immature platelet and reticulocyte fractions of hematologically normal individuals in a university hospital. Methods: Venous blood samples collected in ethylenediaminetetraacetic acid K3 were analyzed using a Sysmex XE-5000™ analyzer. Individuals with platelet and reticulocyte counts within the reference ranges, and a blood count within the laboratory's screening criteria were included. Individuals with clinical conditions that could affect hematological results were excluded. The immature platelet fraction, high, medium and low fluorescence reticulocyte fractions and reticulocyte hemoglobin equivalent were evaluated. The reference ranges were determined according to the recommendations of the International Federation of Clinical Chemistry. Results: One hundred and thirty-two outpatients were evaluated. The mean age was 44 years (range: 13-80 years), 72 (54.5%) were women treated in a university hospital. The mean platelet count was 250.8 × 109/L and the mean reticulocyte count was 0.052 × 109/L. The following reference ranges were obtained: immature reticulocyte fraction 1.6-12.1%, the high, medium and low fluorescence reticulocyte fractions were 0.0-1.7%, 1.6-11.0% and 87.9-98.4%, respectively, the reticulocyte hemoglobin equivalent was 30.0-37.6% and immature platelet fraction was 0.8-5.6%. There was a statistically significant difference (p-value = 0.006) between genders in respect to the immature platelet fraction with 0.8-4.7% for females and 0.7-6.1% for males. The immature reticulocyte fraction was directly correlated with the reticulocyte count. Conclusion: Determining the reference range is critical to the introduction of a new parameter. The reference ranges obtained herein corroborate those reported in previous publications and will contribute to the clinical and laboratory application of the indices.

Could CD64 expression be used as a predictor of positive culture results in children with febrile neutropenia?

Background: Early recognition of infectious processes in neutropenic patients is hampered by the fact that these processes may have dissimilar and non-specific clinical presentations. CD64 is a neutrophil surface marker that is not expressed in non-sensitized neutrophils. When the neutrophil is exposed to tumor necrosis factor-alpha it is activated and is measured via the CD64 index. Methods: This paper evaluated the relationship between the index value of CD64 on the first day of febrile neutropenia and a positive blood culture. The correlations with white blood count, C-reactive protein and erythrocyte sedimentation rate were also evaluated. This case-control, prospective, diagnostic study included 64 episodes of neutropenia. Case group (n = 14) comprised positive blood cultures, and the control group (n = 50), negative blood cultures. Results: The median rates of CD64 were 2.1 (a ± 3.9) in the case group and 1.76 (a ± 5.02) in the control group. There was no correlation between the value of the CD64 index and blood cultures. The CD64 index was also not correlated with C-reactive protein positivity. Further- more, the CD64 index was not able to predict blood culture positivity. The sensitivity was 64.3%, the specificity was 42%, the positive predictive value was 23.7% and the negative predictive value was 80%. For C-reactive protein, the sensitivity, specificity, positive predictive value, and negative predictive value were 71.4%, 32%, 22.7%, and 80%, respectively. Conclusion: The CD64 index is not suitable for predicting the positivity of blood cultures in this specific population of patients with febrile neutropenia.

Importância clínica e laboratorial do volume plaquetário médio / The clinical and laboratory importance of mean platelet volume

Os índices plaquetários fornecidos pelos analisadores hematológicos são provavelmente os parâmetros mais ignorados pela maioria dos laboratórios clínicos, em virtude da dificuldade de sua padronização. Desses índices, o volume plaquetário médio (VPM) vem merecendo destaque por sua grande utilidade, não só em casos de trombose e hemostasia, mas também em uma série de patologias, como diabetes, doenças da tireoide, doenças vasculares, entre outras. O VPM é um parâmetro plaquetário fornecido no hemograma que não gera custos adicionais para o laboratório. Junto com a contagem de plaquetas, ele é um sensível indicador de desordens plaquetárias in vivo, mas pode ser tecnicamente difícil de analisá-lo in vitro por causa dos interferentes pré-analíticos, como tempo de armazenamento da amostra e artefatos gerados pelos anticoagulantes. Neste artigo descrevemos as principais metodologias e seus interferentes na determinação da contagem plaquetária e do VPM, destacando a importância do laboratório de análises clínicas em validar esse parâmetro, proporcionando sua utilização no diagnóstico de desordens hematológicas e de outras patologias.

The platelet indices provided by the hematological analyzers are probably the parameters mostly ignored by most clinical laboratories due to standardization difficulties. Among those indices, the mean platelet volume (MPV) has stood out due to its usefulness not only in cases of thrombosis and hemostasy, but also in a series of pathologies, such as diabetes, thyroid and vascular diseases, among others. MPV is a platelet parameter provided by the hemogram, which does not represent additional costs to the laboratory. Along with platelet count, it is a sensitive indicator of platelet disorders in vivo. However, it may be technically difficult to analyze it in vitro owing to preanalytical interferences, such as sample storage time and anticoagulant artifacts. In this article we described the main methodologies and the interfering factors in determining platelet count and MPV, highlighting the importance of the clinical laboratory analyses to validate this parameter, allowing its use in the diagnosis of hematological disorders and other pathologies.

Volume plaquetário médio e doença cardiovascular / Mean platelet volume and cardiovascular disease

As plaquetas têm importante papel no desenvolvimento do trombo intravascular, a maior causa de síndrome coronariana aguda (SCA). Após a erosão ou ruptura da placa aterosclerótica, a ativação das plaquetas é crucial nos eventos pró-trombóticos que levam ao infarto do miocárdio (IM). O aumento da reatividade plaquetária está associado à evolução do volume plaquetário. Plaquetas grandes são enzimática e metabolicamente mais ativas e apresentam alto potencial trombótico. O volume plaquetário médio (VPM) é um marcador da função plaquetária. Níveis elevados de VPM têm sido identificados como fatores de risco independentes para o IM em pacientes com doença cardíaca coronariana. No entanto, os valores biológico e prognóstico de níveis elevados de VPM ainda são controversos. Os novos analisadores hematológicos fornecem esse índice plaquetário como parte integrante do hemograma, não havendo custos adicionais para o laboratório, podendo ser utilizado como marcador precoce de risco de eventos cardiovasculares, associado a marcadores tradicionais. Neste estudo descrevemos o valor clínico do VPM na aterosclerose coronariana e seu papel como fator de risco para SCA.

Platelets play an important role in intravascular thrombus development, a major cause of acute coronary syndrome (ACS). After erosion or rupture of atherosclerotic plaque, platelets activation plays a crucial role in prothrombotic events leading to myocardial infarction. Increased platelets reactivity is associated with increased platelet volume. Large platelets are enzymatically and metabolically more active and have high thrombotic potential. Mean platelet volume (MPV) is a marker of platelet function. Elevated levels of MPV have been identified as an independent risk factor for myocardial infarction in patients with coronary cardiac disease. However, the biological and prognostic value of increased levels of MPV is still controversial. The new hematological analyzers provide the platelet index as part of the hemogram, without additional costs to the laboratory, and it may be used as an early risk marker of cardiovascular events in association with traditional markers. In this study we describe the clinical value of MPV in coronary atherosclerosis and its role as a risk factor for ACS.

Determinação do intervalo de referência para o volume plaquetário médio (VPM) utilizando o analisador hematológico Pentra 120 ABX / Determining the reference interval for the mean platelet volume (MPV) making use of the hematology analyzer Pentra 120 ABX

De todos os parâmetros fornecidos pelos analisadores hematológicos, os índices plaquetários, certamente, são os mais ignorados pela maioria dos laboratórios clínicos, devido à dificuldade de padronização e por serem afetados por uma série de problemas metodológicos. Recomenda-se, portanto que cada laboratório determine seu próprio intervalo de referência. Destes índices, o volume plaquetário médio (VPM) vem merecendo destaque pela sua grande utilidade, além de trombose e hemostasia, em uma série dedoenças como, diabetes, doenças da tireóide, doenças vasculares, etc. Neste estudo foram analisadas 227 amostras de indivíduos adultos, ambulatoriais, do Hospital de Clínicas de Porto Alegre (HCPA), executadas no analisador hematológico Pentra 120 ABX, com afinalidade de determinar o intervalo de referência para este parâmetro, permitindo assim, sua utilização no hemograma e sua aplicação clínica. O valor de referência para VPM encontrado neste estudo foi de 6,5 - 9,5 fL, sendo calculado a partir dos percentis 2,5-97,5 e determinado conforme recomendação do international Federation of Clinical Chemistry (IFCC). O valor obtido foi corresponde aos encontrados em estudos anteriores, os quais também utilizaram a metodologia de impedância volumétrica, para a realização dascontagens plaquetárias.

Among all the parameters provided by the haematology analysers, the platelet rate is surely, the least acknowledged one, by most of the clinic laboratories, due to the standardization difficulty and also for being affected by a series of methodologic problems. Therefore, every laboratory would be in charge to determine its own reference interval. Among those rates, the mean plateletvolume (MPV) should be emphasized due to its high level of importance in a series of pathologies; such as thrombosis and hemostasy, diabetes, thyroid and vascular diseases, etc. In this study, 227 adult outpatients samples from Hospital de Clínicas de Porto Alegre (HCPA) have been analysed and with the Pentra 120 ABX hematological analyser, in order to determine the reference interval for thisstandard, thus allowing for its utilization in the hemogram as well as its clinical application. The MPV reference interval found in this study was 6,5 – 9,5 fL, according to percentile 2.5-97.5 and determined according to recommendation of the International Federation of Clinical Chemistry (IFCC). The value obtained corresponds to the ones found in previous studies, which have also used the volumetric impedance methology for platelet count.

Diagnóstico laboratorial das leucemias linfóides agudas / In vitro diagnosis of acute lymphoid leukemias

A leucemia linfóide aguda (LLA) é a forma mais comum de câncer na infância, compreendendo 70 por cento dos casos; em adultos a incidência é de apenas 20 por cento. A abordagem inicial do diagnóstico consiste no exame citomorfológico do sangue periférico e da medula óssea. O estudo imunofenotípico eleva para 99 por cento o percentual de casos corretamente classificados, permitindo identificar a linhagem celular (T ou B) e os diferentes estágios de maturação da célula. Aproximadamente 20 por cento dos casos são de origem de célula T; 75 por cento, precursores de célula B; e 5 por cento, de célula B madura. As técnicas citogenéticas têm contribuído de maneira fundamental para a compreensão da biologia molecular e do tratamento da LLA. As anormalidades cromossômicas, quando associadas ao painel de imunofenotipagem, constituem o parâmetro mais importante para a classificação das leucemias, e, juntamente com outros fatores clínicos e laboratoriais, possibilitam a estratificação dos pacientes em diferentes grupos de risco, tendo importância fundamental para determinar o prognóstico e estabelecer o tratamento adequado. O objetivo deste trabalho é fazer uma revisão bibliográfica dos métodos laboratoriais através dos critérios morfológicos, citoquímicos, imunológicos, citogenéticos e de genética molecular, que são úteis para a classificação e o diagnóstico das leucemias linfóides agudas.