Preparation of Internal Quality Control Material for Lymphocyte Subset Analysis

Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20℃, -80℃, or -196℃. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80℃'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings.

Clinical Usefulness of Bronchoalveolar Lavage Cellular Analysis and Lymphocyte Subsets in Diffuse Interstitial Lung Diseases

BACKGROUND: Diffuse interstitial lung diseases (DILDs) form a part of a heterogeneous group of respiratory diseases. Bronchoalveolar lavage (BAL) analysis has been used for differential diagnosis of DILDs, but their clinical usefulness is controversial. The aim of this study was to investigate the clinical usefulness of BAL cellular analysis with lymphocyte subsets for the differential diagnosis of DILDs. METHODS: A total of 69 patients diagnosed with DILDs were enrolled. Basic demographic data, BAL cellular analysis with lymphocyte subsets, histology, and high resolution computed tomogram (HRCT) findings were analyzed and compared as per disease subgroup. RESULTS: Significant differences were found between groups in the proportion of neutrophils (P=0.0178), eosinophils (P=0.0003), T cells (P=0.0305), CD4 cells (P=0.0002), CD8 cells (P<0.0001), and CD4/CD8 ratio (P<0.0001). These findings were characteristic features of eosinophilic pneumonia and sarcoidosis. Other parameters were not significantly different between groups. At the cut-off value of 2.16 for sarcoidosis, CD4/CD8 ratio showed sensitivity of 91.7% (95% CI, 61.5-98.6%) and specificity of 84.2% (95% CI, 72.1-92.5%). CONCLUSIONS: Routine analysis of BAL lymphocyte subset may not provide any additional benefit for differential diagnosis of DILDs, except for conditions where BAL is specifically indicated, such as eosinophilic pneumonia or sarcoidosis.

Lymphocyte subsets in human immunodeficiency virus-unexposed Brazilian individuals from birth to adulthood

Ethnic origin, genetics, gender and environmental factors have been shown to influence some immunologic indices, so that development of reference values for populations of different backgrounds may be necessary. We have determined the distribution of lymphocyte subsets in healthy Brazilian individuals from birth to adulthood. Lymphocyte subsets were determined using four-colour cytometry in a cross-sectional study of 463 human immunodeficiency virus-unexposed children and adults from birth through 49 years of age. Lymphocyte subsets varied according to age, as previously observed in other studies. However, total CD4+ T cell numbers were lower than what was described in the Pediatric AIDS Clinical Trials Group P1009 (PACTG P1009), which assessed an American population of predominantly African and Hispanic backgrounds until the 12-18 year age range, when values were comparable. Naïve percentages and absolute values of CD8+ T cells, as assessed by CD45RA expression, were also lower than the PACTG P1009 data for all analysed age ranges. CD38 expression on both CD4+ and CD8+ T cells was lower than the PACTG P1009 values, with a widening gap between the two studies at older age ranges. Different patterns of cell differentiation seem to occur in different settings and may have characteristic expression within each population.

Establishing th reference range for t lymphocyes subpopulations in adults andd children from Brazil / Estabelecimento de valores de referencias para subpopulacoes de linfocitos T em adultos e criancas no Brasil

SUMMARY In Brazil, the existing reference values for T-lymphocytes subsets are based on data originated in other countries. There is no local information on normal variation for these parameters in Brazilian adults and children. We evaluated the normal variation found in blood donors from five large Brazilian cities, in different regions, and in children living in Salvador, and Rio de Janeiro. All samples were processed by flow cytometry. The results were analyzed according to region, gender, and lifestyle of blood donors. A total of 641 adults (63% males), and 280 children (58% males) were involved in the study. The absolute CD3+, and CD4+ cells count were significantly higher for females (adults and children). Higher CD4+ cell count in adults was associated with smoking, while higher CD8+ count was found among female children. Higher counts, for all T-cells subsets, were detected in blood donors from southeast / south regions while those living in the northern region had the lowest values. Individuals from midwestern and northeastern regions had an intermediate count for all these cells subsets. However, these differences did not reach statistical significance. In Brazil, gender and smoking, were the main determinants of differences in T-lymphocytes reference values. .

RESUMO Os valores de referências de linfócitos T existentes no Brasil são baseados em dados originados de outros países. Não existem dados locais da variação normal para estes parâmetros em adultos e crianças brasileiras. Avaliamos a variação normal encontrada em doadores de sangue de cinco grandes cidades brasileiras em diferentes regiões e em crianças residentes em Salvador e Rio de Janeiro. Todas as amostras foram processadas por citometria de fluxo. Os resultados foram analisados de acordo com região, gênero e estilo de vida dos doadores. Um total de 641 adultos (63% homens) e 280 crianças (58% meninos) participaram do estudo. Valores absolutos de CD3+ e CD4+ foram significantemente maiores no gênero feminino (adultos e crianças). Maiores valores de CD4+ em adultos foram associados com tabagismo, enquanto que maiores valores de CD8+ foram encontrados entre crianças do sexo feminino. Adultos das regiões sul e sudeste apresentaram maiores valores absolutos para todas as células T enquanto que adultos da região norte, apresentaram menores valores. Indivíduos residentes no nordeste e centro-oeste obtiveram contagens intermediárias para todas as populações de células T. Entretanto, estas diferenças entre as regiões, não demonstraram diferença estatística. No Brasil, gênero e tabagismo foram os principais determinantes para diferenças em valores de referências de linfócitos T. .

Lymphocyte and NK cell subpopulations in HIV seronegative Thais.

Lymphocyte subpopulations, i.e. T, B and natural killer (NK) cells including NK cell subsets which express CD16 molecules (with or without co-expression of CD56 molecules) and NK cell subsets which express CD56 molecules (with or without co-expression of CD16 molecules) were enumerated by two color-flow cytometry in a total of 125 HIV seronegative Thai adults. The study demonstrated relatively low CD4 counts in the subjects, i.e. 26.3% of them had a CD4 count of less than 500 cells/microl. In contrast, their NK cell counts were relatively high. Statistical analyses of the percentage values showed that females had significantly higher CD3 (total T cells), but lower NK cell counts as compared to males (p < 0.05). Regarding age variation, an increase of 1.1% of CD4 cells per decade was seen. It was roughly estimated that about 86% of NK cells harbored both CD16 and CD56 molecules. Collective data from several studies including the present one suggest that high NK cell counts may be a compensation for low CD4 cell counts in Mongoloid people. Thus, the role of NK cells in the defense cascade against viral infections, especially human immunodeficiency virus infections deserves further investigation.

Preliminary observations on lymphocyte subpopulations in HIV seropositive & HIV seronegative tuberculosis patients in Pune, India.

CD4 and CD8 lymphocyte counts were determined in 59 HIV seropositive and 41 HIV seronegative newly diagnosed tuberculosis patients in Pune. There were significant differences in the CD4 counts and CD4/CD8 ratios between HIV seropositive and HIV seronegative tuberculosis patients. Majority of the HIV seropositive patients had a CD4 count less than 500 cells/cu.mm, whereas among the HIV seronegative patients, majority had a CD4 count more than 500 cells/cu.mm. In HIV seropositive patients with extrapulmonary and pulmonary tuberculosis, the CD4 counts were lower than in those who had only pulmonary or extrapulmonary tuberculosis. There was no significant differences in the CD8 counts between HIV seropositive and HIV seronegative tuberculosis patients, except for patients with pulmonary cavity, where the CD8 counts were significantly higher in HIV seropositive tuberculosis patients. In HIV seropositive individuals with pulmonary tuberculosis, the CD8 counts in those with pulmonary cavity were higher than in those without any pulmonary cavity. Absence of cavitation and presence of pulmonary with extrapulmonary tuberculosis occurred when immune activation was at a lower level.

Citometría capilar volumétrica: Nueva alternativa en la determinación de subpoblaciones linfocitarias / Volumetric capillary cytometry: New alternative in determination of lymphocyte subpopulations

La determinación de células T CD4+ y CD8+ por citometría de flujo es uno de los métodos de laboratorio de mayor utilidad en el manejo de los pacientes infectados con los virus de la inmunodeficiencia humana; sin embargo, su complejidad técnica e instrumental y su elevado costo han limitado su empleo, lo que ha llevado a la búsqueda de tecnologías alternativas lo suficientemente precisas y costo-efectivas como para poder emplearlas en cualquier nivel de atención médica. En este trabajo, el estudio de una de estas tecnologías alternativas la citometría capilar volumétrica (Equipo IMAGN 2000, Imagin Co) comparada con la citometría de flujo (equipo Epics Profile II, Coulter Co), en la determinación de subpoblaciones linfocitarias CD4+ y CD8+ en 29 pacientes con SIDA, mostró en términos de precisión y reproducibilidad 100 por ciento de correlación en los resultados obtenidos con ambos métodos en los niveles de células CD4+ y CD8+. La citometría capilar volumétrica demostró ser una alternativa precisa, totalmente automatizada, sencilla en su manejo, sobre todo para clínicas y hospitales que habitualmente no realizan citometría de flujo

Carga viral y subpoblaciones linfocitarias en la infección con VIH-1 / Viral load and lymphocyte subpopulations in HIV-1 infection

Se determinaron simultáneamente los niveles plasmáticos de ARN del VIH-1 y las cuentas de células t CD4(+) en 64 pacientes infectados con VIH-1, en diferentes estadios de la infección. Los niveles basales de ARN VIH-1 plasmático (NASBA VIH-1 RNA QT, OT) fueron los mejores índices de predicción de la evolución clínica. Las cuentas de células T CD4(+) consideradas aisladamente, mostraron una gran variabilidad, un limitado rango dinámico y no fueron el mejor método con el cual estratificar a los pacientes

Introduccion de un ultramicrometodo inmunocitoquimico para la cuantificacion de subpoblaciones linfocitarias identificadas con anticuerpos monoclonales

Se introdujo en nuestro laboratorio un ulktramicrometodo inmunocitoquimico (UMICIQ) para la cuantificacion de las principales subpoblaciones de linfocitos T identificadas con los anticuerpos monoclonales anti-CD3, anti-CD4 y anti-CD8. Las deteminaciones se realizaron en celulas mononucleares de sangre periferica de donantes de sangre supuestamente sanos, con edades entre 21 y 47 anos. El estudio de los 15 primeros individuos se realizo tanto por el UMICIQ como por un micrometodo de inmunofluorescencia indirecta (IFI), y cuando se compararon los resultados, no se encontraron diferencias estadisiticamente significativas. Para establecer los valores de referencia para el UMICIQ se tomaron las muestras de 30 donantes. Con la aplicacion de este metodo se pueden evaluar las subpoblaciones linfocitarias con una sensibilidad y especificidad similares a las observadas con la microtecnica del IFI; sin embargo, se introducen una serie de ventajas como: uso de menores concentraciones de celulas y por ende menor volumen de sangre para cada determinacion, uso de diluciones mayores de los anticuerpos y conjugados, se hace innecesario el uso del microscopio de luz UV y de la centrifuga refrigerada, se pueden distinguir la morfologia y el linaje de las celulas simultaneamente con la reactividad inmunologica de sus antigenos

Valores de referencia de subpoblación de linfocitos T por citofluorometría en 120 sujetos sanos residentes de la Ciudad de México / Reference values of T-lymphocytes subpopulations measured by cytofkluorometry in 120 healthy subjects living in Mexico city

En la última década, el uso de la citometría de flujo ha hecho posible el estudio de marcadores de membranas sobre células con gran precisión e inclusive, en número de 2 ó 3 simultáneamente, lo cual ha permitido la identificación de subclases de linfocitos en sangre periférica, comprobándose que estas células están ligadas a funciones inmunes específicas y su número y proporciones, son mantenidas dentro de intervalos definidos. Actualmente, la cuantificación de estas células se ha convertido en una prueba rutinaria en el diagnóstico y evolución de los pacientes con SIDA, sin embargo, no se cuenta con valores de referencia para estos parámetros en nuestra población mestiza mexicana, determinados por esta metodología en donde existe posibilidad de variaciones debidas a altitud, clima, desnutrición, etnia y contaminación. En el presente trabajo, se establecen los valores de normalidad para estas células usando el método de inmunofenotipificación de doble color por lísis rápida de sangre periférica para inmunofluorescencia directa por citometría de flujo. La población de estudio fue de 120 sujetos mestizos mexicanos residentes permanentes del área metropolitana divididos en intervalos de edad. Los resultados obtenidos muestran que no existen variaciones importantes al compararlos con los reportes internacionales enfatizando, además, la necesidad de establecer valores de referencia por intervalos de edad en las poblaciones

SIDA y embarazo: un caso de transmissión madre-hijo / AIDS and pregnancy: a case of mother-child transmission

Se presenta el caso de un niño de cuatro meses de edad, nacido de una madre en un estado avanzavdo de infección por el HIV, en quien se desarrolló un cuadro clínico compartible con SIDA que cumplía con la definición de caso clínico del CDC. En lo que hace al mecanismo probable de transmisión de la enfermedad, este niño nacido por cesárea no recibió en ningun momento sangre ni hemoderivados; no fue amamantado ni estuvo expuesto a punciones accidentales con material contaminado por sangre materna, y se siguieron normas estríctas tendientes a evitar todo contacto con sangre o secreciones potencialmente infectantes. Consideramos que lo más probable haya sido la transmisión madre-hijo en la vida intrauterina