Flow Cytometry in Detection of Fetal Red Blood Cells and Maternal F Cells to Identify Fetomaternal Hemorrhage.

Accurate detection and quantitation of fetomaternal hemorrhage (FMH) is critical to the obstetric management of rhesus D alloimmunization in Rh-negative pregnant women. The flow cytometry is based on the detection of fetal red blood cells using a monoclonal anti-HbF antibody, and is the method most indicated for this estimation. The objective of this study was to quantify fetal red blood cell levels of pregnant women using flow cytometry. We analyzed 101 peripheral blood samples from Rh-negative and Rh-positive women, whose mean age was 24 years (20-32 years), after vaginal delivery or cesarean section. Our study showed that 53% of pregnant women had fetal red blood cells levels <2.0 mL, 31% between 2.0-3.9 mL, 16% between 4.0-15.0 mL, and 1% >15.0 mL. Accurate quantitation of fetal red blood cells is necessary to determine the appropriate dose of anti-D (RHD) immunoglobulin to be administered to pregnant or postpartum women.

Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients.

INTRODUCTION: Infection and sepsis are major health problems. Therefore, the need for improved diagnostic indicators, as well as for better therapeutic monitors in the treatment of infection, remains, since the current diagnostic tools have low specificity and passed through minimal changes in the last two decades. OBJECTIVE: The aim of this study was to establish the correlation of neutrophil CD64 with indicators of infection and sepsis. METHODS: We established the correlation of the neutrophil CD64 expression with the following variables: complete white blood count, band count, neutrophils, C-reactive protein (CRP), cultures, flags released by automated hematology analyzers and clinical groups. Accordingly clinical groups were divided into two: patients "without clinical or laboratory evidence of infection or inflammatory process" and "clinical or laboratory evidence of a systemic inflammatory response (SIRS) and systemic sepsis" based upon identification of organisms by culture. We analyzed 93 whole blood samples anticoagulated with K3EDTA of patients admitted in the Intensive Care Unit (ICU) of a community hospital. RESULTS: The expression CD64 was statistically significant with clinical groups, flags, and neutrophils and was not significantly correlated with total count of white blood cells and cultures. CONCLUSION: Our results indicate that high expression of CD64 is an indicator important in the diagnosis of infection and sepsis.

Detection of minimal residual disease by flow cytometry for patients with multiple myeloma submitted to autologous hematopoietic stem cell transplantation.

The treatment strategy in multiple myeloma (MM) is to get complete remission followed by high-dose chemotherapy and autologous Hematopoietic Stem Cell Transplantation (HSCT). Neoplastic Plasma Cells (NPCs) are CD45(-/dim), CD38(+high), CD138(+), CD19(-), and CD56(+high) in most cases. The description of this immunophenotype is of major importance as it leads to the correct identification of minimal residual disease (MRD). Samples from 44 Patients were analyzed prospectively in this study. We analyzed if the presence of MRD at three months after HSCT was predictive of relapse or death. There were 40 evaluable patients of whom 16/40 patients had MRD at three moths after HSCT and there were none in cytological relapse. The mean overall survival (OS) was 34 months and disease-free survival (RFS) was 28 months after HSCT. There was no significant difference in the log rank analysis comparing OS and the presence of MRD (P = 0,611) and RFS (P = 0,3106). Here, we demonstrate that three color flow cytometry (FCM) is more sensitive for MDR evaluation than cytological analyzes. However, based in our data we can not affirm that MRD is a good predictor of MM relapse or death. In conclusion, our results could be attributed to a short followup, small sample size, and over most to the inability of a three-color FCM to detect the NPC population.

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.

Definition of reference ranges for the platelet distribution width (PDW): a local need.

BACKGROUND: Automated hematological analyzers have contributed to more precise and faster results. They also make it possible to measure several blood cell parameters automatically. Among the parameters provided, platelet indices are probably the most ignored by clinical laboratories due to the difficulty of standardization, as well as being affected by a range of methodological problems. It has been suggested that each laboratory determines its own reference intervals with the equipment used. METHODS: Our goal was to determine the reference range of platelet distribution width (PDW) in venous blood samples from 231 patients using the Pentra 120 ABX hematology analyzer. RESULTS: The PDW median was 13.3%, with a reference range of 10.0%-17.9% for the 5th-95th percentiles, with a confidence interval of 95%. CONCLUSIONS: Among all indices, the PDW has been receiving attention due to its usefulness for distinguishing between reactive thrombocytosis and thrombocytosis associated with myeloproliferative disorder. Determination of the PDW reference range is fundamental, and the association of this parameter with the platelet number and mean platelet volume may be used for the diagnosis and differentiation of several pathologies.

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.

Determinação da acurácia do método qualitativo da medida da atividade da glicose-6-fosfato desidrogenase / Determination of the accuracy of the measurement method for dehydrogenase activity

A deficiência de glicose-6-fosfato desidrogenase (G6PD) é um problema de saúde pública que afeta aproximadamente 400 milhões de pessoas no mundo. No mercado, existem vários métodos que medem a atividade da G6PD. Os objetivos deste estudo foram determinar a acurácia do método de Brewer frente a um padrão de referência e estimar a prevalência de deficiência de G6PD na amostra. Foi realizado um estudo transversal de grupo de pacientes internados no HCPA com icterícia a esclarecer, no período de junho de 2004 a maio de 2005. Amostras foram processadas pelo método de Brewer e pelo método de Normalização da Hemoglobina, o qual foi usado como padrão ouro. Foi analisado para atividade da G6PD um total de 173 pacientes. A idade variou de 1 dia a 82 anos, sendo que 66 por cento da amostra possuía até 15 dias de vida. A atividade média e o desvio padrão da G6PD na amostra analisada foi de 17.67± 5,66 U/gHb. A freqüência estimada, pelo padrão ouro, da deficiência de G6PD, foi de 13 (7,7 por cento) pacientes com deficiência parcial ou total, e pelo método de Brewer foi de 14 (8,67 por cento). A sensibilidade do método de Brewer comparada com o método quantitativo da Normalização da Hemoglobina foi de 92,8 por cento e a especificidade foi de 98,7 por cento. A deficiência de G6PD é prevalente em nosso meio. Testes de baixo custo, tais como o teste de Brewer, podem ser utilizados como testes de triagem desta deficiência, principalmente no monitoramento de recém-nascidos que estão sob o risco de desenvolver icterícia neonatal.

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is a public health problem which affects about 400 millions of people all over the world. Some methods that measure the activity of G6PD have already been developed. Thus, the aim of this study was to evaluate the accuracy of the Brewer's method compared with a standard reference and estimate the prevalence of G6PD deficiency in the sample. A cross-sectional study of a group of patients in HCPA presenting with jaundice was carried out from June 2004 to May 2005. Samples were processed by the metahaemoglobin reduction test (Brewer's method) and by the method of Haemoglobin Normalization, which was used as the standard reference. A total of 173 patients were analyzed for G6PD activity. The ages varied from one day to 82 years old with 66 percent of the sample being less than 16 days old. The mean activity and standard deviation of G6PD for the analyzed sample was 17.67 ± 5.66 U/gHb. The estimated frequencies of G6PD deficiency for the standard reference and Brewer's method were 13 (7.7 percent) subjects (total or partial deficiency) and 14 (8.67 percent), respectively. When the Brewer's method was compared with the quantitative method of Hemoglobin Normalization, it showed a sensitivity of 92.8 percent and specificity of 98.7 percent. As G6PD deficiency predominates in our society, low cost tests, such as the Brewer's test can be used for screening this deficiency, mainly to monitor newborn babies who are at risk of developing jaundice.

Infiltração cutânea na leucemia megacariocítica aguda com expressão de CD56 / Cutaneous infiltration in acute megakaryocytic leukemia with CD56 expression

A LMA-M7 é um subtipo raro de leucemia mielóide aguda (LMA). Está freqüentemente associada a mielofibrose e representa um subtipo de mau prognóstico. Raramente apresenta infiltração em sítios extramedulares. O aspirado de medula óssea ou biópsia mostra uma população de células pleomórficas e basofílicas, que podem apresentar projeções citoplasmáticas. A utilização da imunofenotipagem é essencial para o diagnóstico de LMA-M7. O imunofenótipo característico apresenta uma população de células leucêmicas com ausência da maioria dos marcadores linfóides e mielóides de superfície, porém com expressão para os antígenos da linhagem megacariocítica: CD41a (complexo glicoprotéico IIb/IIIa), CD42b (glicoproteína Ib) e/ou CD61 (glicoproteína IIIa), ou antígeno relacionado ao fator VIII. Freqüentemente, a coloração citoquímica Sudan Black para os blastos megacariocíticos é negativa; neste caso, foi positiva para 40 por cento das células analisadas. A presença de CD56, cuja expressão aberrante em algumas leucemias mielóides é indicativo de mau prognóstico, pode estar associada à infiltração da pele.

AML-M7 is a rare subtype of acute myeloid leukemia (AML). It is frequently associated with myelofibrosis and corresponds to a poor prognosis subtype. It rarely presents with infiltration at extramedullary sites. The bone marrow aspirate or biopsy identifies pleomorphic and basophilic cell populations that may present with cytoplasmatic projections. The use of immunophenotyping is essential for the diagnosis of AML-M7. The characteristic immunophenotype presents a leukemic cell population without most lymphoid and myeloid surface markers, but with an expression of the megakaryocytic antigens: CD41a (glycoprotein complex IIb/IIIa), CD42b (glycoprotein Ib) and/or CD61 (glycoprotein IIIa), or the factor VIII-related antigen. The cytochemical stain Sudan Black is frequently negative for megakaryocytic blasts; in this case, it was positive in 40 percent of the analyzed cells. The presence of CD56, whose aberrant expression in some myeloid leukemias indicates poor prognosis, might be associated with skin infiltration.