Evaluation of flow cytometry as a diagnostic method for detection of Giardia lambila in comparison to IFAT and other conventional staining techniques in fecal samples

Giardia lamblia is one of the most common diarrhea-related parasites in humans, where infection ranges from asymptomatic to acute or chronic disease. Because of the non-characteristic symptoms of giardiasis, as well as significant prevalence of cyst carriers, it is necessary to upgrade parasitological techniques for examination of feces, in order to avoid false negative results. Accurate diagnosis is important to exclude other parasitic causes of diarrhea. The present study is designed to assess the efficacy of flow cytometry [FC] as a sensitive method for detection of G. lamblia cysts in stool samples in comparison with other standard conventional diagnostic methods. 70 patients [30 males and 40 females] ranging in age from 5 to 60 years were included in this study. Stool samples were taken from each patient on three successive days to detect Giardia cysts and evaluate the intensity of infection. Different methods were used that included concentration methods, permanent stained slides, immunofluorescent antibody test [IFAT] and FC. Giardia lamblia cysts were detected in 24 samples by IFAT with detection rate of 34.3%. Using trichrome stain 20 samples [28.6%] were positive, modified Ziehl- Neelsen Stain [MZN] detected 11samples [15.7%], while Acridine Orange [AO] detected 14 positive samples [20%]. Flow cytometry detected 18 samples with detection rate of 25.7%. Sensitivity and specificity of FC were 90% and 100%, respectively and IFAT sensitivity and specificity were100% and 92%, respectively. Results of the present study clearly demonstrated that incorporation of IFAT and FC can improve sensitivity of detection of Giardia cysts in stool samples. Although FC is more expensive than the other staining methods and IFAT, it is rapid, simple and accurate in estimating the quantity of parasites in each sample. Thus, FC can be recommended for detection of protozoa in stool

Expression of p-selectin [CD62p] on platelets as activated marker in preeclampsia

Pre-eclampsia is a medical condition in which hypertension arises in pregnancy [pregnancy-induced hypertension] in association with significant amounts of protein in the urine. Pre-eclampsia may develop from 20 weeks gestation [it is considered early onset before 32 weeks, which is associated with increased morbidity]. Platelets play an important role in the pathophysiologic mechanisms of preeclampsia. The aim of this work was to study the platelet activation state by flow cytometer analysis of platelet expression of CD62p in patients with preeclampsia. This study was conducted on ten cases of mild preeclampsia [group I], their ages range was 22- 36 years and ten cases of severe preeclampsia [group II] their ages range was 20-35 years .Also ten normotensive pregnant women were included as a control group [group III] . The percentage of platelets expression of the CD61, CD62p and MFI were analyzed by the flow- cytometr . The mean percentage of CD62p expression on platelets and MFI were 67. 3% and 6.5 respectively in mild preeclampsia compared with 3.7% and 1.5 in normotensive pregnant as control [p < 0.01 and p < 0.015 respectively]. Also the mean percentage of CD62p expression on platelets and MFI were 73.3% and 2.1 respectively in severe preeclampsia, they showed significant increase when compared with normotensive pregnant as control [p < 0.01 and p < 0.015 respectively]. There were a positive significant correlation between% of expression of CD 62p on platelets and SBP, DBP, protein in urine, and% CD61. While a negative significant correlation between% of expression of CD 62p on platelets and age, platelet count and CD62P MFI was found. High levels of platelet glycoprotein CD62p expressions in patients with mild and severe preeclampsia, could be a compensatory mechanism for the preeclampsia induced thrombocytopenia

Establecimiento de intervalos de referencia en subpoblaciones linfocitarias por citometría de flujo / Reference values in lymphocyte subpopulations using flow cytometry

La determinación de Subpoblaciones Linfocitarias Humanas (SLH) por Citometría de Flujo, Linfocitos CD3+ (LT), LTCD4+, LTCD8+ y la relación LTCD4+/LTCD8+, permiten monitorear pacientes infectados con virus de la inmunodeficiencia humana (VIH), los cuales son comparados con Valores de Referencia (VR). A su vez los VR informados por la bibliografía son variables debido a la falta de validación analítica y partición estadística de los datos de VR, entre otros. En este trabajo, utilizando un ensayo validado (ISO15189), se establecieron VR para SLH con criterios estadísticos de partición en individuos con serología negativa para VIH (397 mujeres (F) y 279 varones (M), edad: 17-83 años). Las SLH fueron medidas en un Citómetro de Flujo (Coulter Epics XL-MCL). Los datos fueron procesados empleando como criterio estadístico de partición el algoritmo de Martin Gellerstedt (AMG). Del análisis estadístico y del AMG se determinó la partición de los datos en 6 subpoblaciones definidas por edad (intervalo de años) y género (F y M): grupos I y IV: 17-35 (F y M); grupos II y V: 36-50 (F y M); y grupos III y VI: >50 años (F y M). Para los 6 grupos se definieron VR de linfocitos totales, LT, LTCD4+, LTCD8+ y LTCD4+/LTCD8+. Para cada límite inferior y superior de VR se estableció el intervalo de confianza al 90%. En conclusión, este estudio permitió establecer VR para SLH en un ensayo validado empleando un criterio estadístico de partición, lo cual permitiría incrementar la confiabilidad de los resultados y uniformar a nivel internacional los VR en SLH en diferentes poblaciones.

The determination of Human Lymphocyte Subpopulations (HLS) including Lymphocytes CD3+ (LT), LTCD4+, LTCD8+ and LTCD4+/LTCD8+ ratio by flow cytometry, makes it possible to monitor patients infected who HIV, which are compared against reference values (RV). However, RV reported by the international bibliography are variable due to the absence of two main factors: analytical validation procedures and partitioning statistical criteria. This study, using a validated method (ISO15189), RV were established for HLS in individuals with negative serology for HIV (397 females (F) and 279 males (M), age: 17-83 years) applying partitioning statistical criteria. The values of HLS were obtained by flow cytometry (Coulter Epics XL-MCL). The data were processed using partitioning statistical criteria based on Martin Gellerstedt's algorithm (MGA). From the statistical analysis and MGA the partition of the data was determined in 6 subpopulations defined by age (interval of years) and gender (F and M): groups I and IV: 17-35 (F and M); groups II and V: 36-50 (F and M); and groups III and VI: >50 years (F and M). Hence, RV of total lymphocytes, LT, LTCD4+, LTCD8+ and LTCD4+/LTCD8+ ratio were defined. For each limit (lower and upper) of reference values, the 90% confidence interval was determined. In conclusion, this study allowed establishing RV for HLS in a validated method using a partitioning statistical criterion, which would allow increasing the assurance results and establishing an international criterion for reference values in HLS in different populations.

T-lymphocyte subsets and thymic size in malnourished infants in Egypt: a hospital-based study

Thymus size was assessed ultrasonographically and correlated to the percentage of CD4 and CD8 T-lymphocytes in peripheral blood in 32 infants with protein-energy malnutrition [PEM] and compared with 14 healthy control infants. The study revealed thymus atrophy in patients with PEM, especially the oedematous type, accompanied by changes in the peripheral lymphocyte subsets. These changes were reversible after nutritional rehabilitation. However, they may affect the immune status of PEM patients and may require a longer duration of nutrition rehabilitation than required for recovery of anthropometric measures. We recommend proper assessment of the immune functions of PEM patients during nutritional rehabilitation until full recovery

Dificient expression of brouton's tyrosine kinase in monocytes from x linked agmmaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection

The B-cell defect in X-linked agammaglobulinemia [XLA] is caused by mutations in the gene for Bruton's tyrosine kinase [BTK]. BTK mutations result in deficient expression of BTK protein in peripheral blood monocytes. Using the anti-BTK monoclonal antibody [48-2H], a flow cytometric analysis of intra cytoplasmic BTK protein expression in monocytes was performed to identify Iranian patients with XLA phenotype. To examine the possible identification of XLA patients and female carriers by this assay, we studied 13 XLA families. The flow cytometric assay showed deficient expression of the BTK protein in 12 [92%] families. One patient exhibited a normal level of BTK expression. The cellular mosaicism of BTK expression in monocytes from obligate carriers was clearly shown in 9 of 12 [75%] families. The results suggested that most XLA patients have deficient expression of the BTK protein; therefore we conclude that deficient expression of BTK protein can be evaluated by a flow cytometric assay

assessment of NK cytotoxicity and CD56+/CD16+ phenotype by flow cytometry in PBL isolated from women with recurrent spontaneous abortion

Human peripheral blood NK cells constitutively express CD56 and CD16 antigens. Peripheral blood NK cells seem to be strongly related with decidual NK cells, and may reflect the decidual NK cell functional status. There are varied reports concerning the relationship between NK cell cytotoxicity in women with recurrent spontaneous abortion. To study NK activity in women with history of RSA by using a non-radioactive cytotoxicity assay. Peripheral blood lymphocytes from RSA and healthy multiparous women were obtained. Lymphocytes were isolated and mixed with K562 NK-sensitive cell line. A non-radioactive method for NK cell cytotoxicity assessment was utilized. Dead K562 cell populations were detected by FACS Calibur flow cytometry, and the data obtained was analysed on cell-Quest software. The proportion of CD16+/CD56+ cells was then calculated. The proportion of NK cells were 9.21% +/- 3.06 and 13.48% +/- 4.09 in healthy women and RSA, respectively. The percentage of cytotoxicity was determined to be 19.3% +/- 3.9 in healthy group and 27.1% +/- 6.5 in RSA group with an effector:target ratio of 50:1. The data shows an increase in PBLs potential for in vitro cytotoxicity assay in RSA individuals. The analyses indicate that there is a weak positive correlation between NK cytotoxicity potential and the percentage of NK cells in PBL population. The present study demonstrates that the percentage of CD56+/CD16+ cells increases in individuals with recurrent spontaneous abortion. We conclude that NK cytotoxicity as well as NK number is partially involved in RSA

survey on platelet surface glycoprotiens in patients with bernard-souliar syndrome by flowcytometry

The gold standard diagnosis of the Bernard-Souliar syndrome [BSS], a rare disease, is to prove the absence of Ib/IX surface complex on platelets with the use of aggregometric methods. Flowcytometry is an ideal method in analysis of surface markers on cells. The use of flowcytometric analysis in diagnosis of Bernard-Souliar syndrome. 15 suspected BSS, 20 healthy persons as control group and 3 ITP patints were selected to be analysed for the presence of GPIb alpha and GPIIIa on the surface of platelets with the application of FITC conjugated monoclonal antibodies using flowcytometry. All healthy persons in control group and 3 ITP patients showed normal expression of both glycoprotiens on platelets using flowcytometry. All 15 suspected BSS patients showed lack of GPIb? but a normal expression of GPIIIa on platelets. The application of flowcytometry for diagnosis of BSS is a quick, accurate, and precise method, which together with aggregometric method can be used for diagnosis of BSS

Primera experiencia en aislamiento de celulas endoteliales humans en Bolivia / First experience in the isolation of human endothelian cells in Bolivia

INTRODUCCIÓN: las células endoteliales pueden proveer información valiosa con respecto a muchos procesos patológicos como la aterosclerosis, inflamación, neoplasia y angiogénesis. Este trabajo describe la primera experiencia boliviana en el aislamiento y cultivo de células endoteliales humanas derivadas de la vena umbilical (HUVEC). MÉTODOS: Un segmento largo de cordón umbilical se utilizó para el aislamiento y fue tratado por digestión con colagenasa. Las células endoteliales fueron desprendidas de la íntima y posteriormente cultivadas en medio de cultivo M199 y suero fetal bovino. Técnicas morfológicas, inmunohistoquímicas y de citometría de flujo fueron utilizadas para identificar estas células. RESULTADOS: Se examinaron las células endoteliales obtenidas por análisis morfológico e inmunohistoquímico. El marcaje con CD34 fue positivo para más del 90% de las células obtenidas por digestión con colagenasa analizado por citometría de flujo. CONCLUSIÓN Se logró aislar y cultivar HUVEC de manera exitosa. Una gran variedad de experimentos y aplicaciones en ciencias biomédicas pueden ser potencialmente factibles.

INTRODUCTION: endothelial cell study yields valuable information concerning many pathologic processes such as atherosclerosis, inflammation, neoplasia and angiogenesis. This paper describes the first Bolivian experience in isolation and culture of human umbilical vein endothelial cells (HUVEC). METHODS: a long segment of the umbilical cord was processed by collagenase digestion. Endothelial cells were detached from intima and further cultured using M199 culture media. Morphologic, immunochemistry and flow cytometry approaches were used to identify these cells. RESULTS: morphologic and immunochemistry analysis of the pool of obtained cells were positive for HUVEC. CD34 staining was positive in more than 90% of the cells obtained by collagenase digestion as assessed by flow cytometry. CONCLUSION: HUVEC were successfully isolated for the first time in Bolivia. A great deal of further experiments and applications in biomedical sciences is possible

Flow cytometric quantitation of phagocytosis in heparinized complete blood with latex particles and Candida albicans

Se da cuenta de un método rápido para la cuantización del flujo citométrico de la fagocitosis en sangre periférica completamente heparinizada (HCPB), mediante la utilización de partículas de látex phycoerythrin-conjugadas de 1µm de diámetro disponibles comercialmente. El método es más rápido y presenta mayor reproducibilidad que la técnica estandar de Bjerknes' (1984) utilizando propidium iodide-teñida Candida albicans, aplicada convencionalmente a la capa leucocitica de sangre periférica pero modificada por HCPB. Tambien damos cuenta de una modificación de Bjerknes' Intracellular Killing Test para permitir su aplicación a HCPB.

We report a rapid method for the flow cytometric quantitation of phagocytosis in heparinized complete peripheral blood (HCPB), using commercially available phycoerythrin-conjugated latex particles of 1 micron diameter. The method is faster and shows greater reproducibility than Bjerknes' (1984) standard technique using propidium iodide-stained Candida albicans, conventionally applied to the leukocytic layer of peripheral blood but here modified for HCPB. We also report a modification of Bjerknes' Intracellular Killing Test to allow its application to HCPB.