A Novel Marker for Screening Paroxysmal Nocturnal Hemoglobinuria Using Routine Complete Blood Count and Cell Population Data

BACKGROUND: Final diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) may take years demanding a quick diagnosis measure. We used the facts that PNH cells are damaged in acid, and reagents for measuring reticulocytes in Coulter DxH800 (Beckman Coulter, USA) are weakly acidic and hypotonic, to create a new PNH screening marker. METHODS: We analyzed 979 complete blood counts (CBC) data from 963 patients including 57 data from 44 PNH patients. Standard criteria for PNH assay for population selection were followed: flow cytometry for CD55 and CD59 on red blood cells (RBCs) to a detection level of 1%; and fluorescent aerolysin, CD24 and CD15 in granulocytes to 0.1%. Twenty-four PNH minor clone-positive samples (minor-PNH+) were taken, in which the clone population was <5% of RBCs and/or granulocytes. Excluding PNH and minor-PNH+ patients, the population was divided into anemia, malignancy, infection, and normal groups. Parameters exhibiting a distinct demarcation between PNH and non-PNH groups were identified, and each parameter cutoff value was sought that includes the maximum [minimum] number of PNH [non-PNH] patients. RESULTS: Cutoff values for 5 selected CBC parameters (MRV, RDWR, MSCV, MN-AL2-NRET, and IRF) were determined. Positive rates were: PNH (86.0%), minor-PNH+ (33.3%), others (5.0%), anemia (13.4%), malignancy (5.3%), infection (3.7%), normal (0.0%); within anemia group, aplastic anemia (40.0%), immune hemolytic anemia (11.1%), iron deficiency anemia (1.6%). Sensitivity (86.0%), specificity (95.0%), PPV (52.1%), and NPV (99.1%) were achieved in PNH screening. CONCLUSION: A new PNH screening marker is proposed with 95% specificity and 86% sensitivity. The flag identifies PNH patients, reducing time to final diagnosis by flow cytometry.

Diagnostic Significance of BAT in Anaphylaxis to Non-ionic Contrast Media / 法医学杂志

OBJECTIVE@#To investigate the diagnostic significance of basophil activation test (BAT) in anaphylaxis to non-ionic contrast media through testing the content of CD63, mast cell-carboxypeptidase A3 (MC-CPA3), and terminal complement complex SC5b-9 of the individuals by testing their levels in the normal immune group and the anaphylaxis groups to β-lactam drugs and non -ionic contrast media.@*METHODS@#The CD63 expression of basophilic granulocyte in blood was detected by flow cytometry. The levels of MC-CPA3 in blood serum and SC5b-9 in blood plasma were detected by ELISA.@*RESULTS@#The CD63 expression of basophilic granulocyte in blood, the levels of MC-CPA3 and SC5b-9 of anaphylaxis to non-ionic contrast media and β-lactam drugs were significantly higher than that in normal immune group (P < 0.05).@*CONCLUSION@#There is activation of basophilic granulocytes, mast cells and complement system in anaphylaxis to non-ionic contrast media. BAT can be used to diagnose the anaphylaxis to non-ionic contrast media.

Reliable, Accurate Determination of the Leukocyte Differential of Leukopenic Samples by Using Hematoflow Method / 대한진단검사의학회지

BACKGROUND: Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method. METHODS: We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500-2,000/microL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50-100 cells. RESULTS: The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count. CONCLUSIONS: The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.

Reliable, Accurate Determination of the Leukocyte Differential of Leukopenic Samples by Using Hematoflow Method / 대한진단검사의학회지

BACKGROUND: Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method. METHODS: We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500-2,000/microL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50-100 cells. RESULTS: The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count. CONCLUSIONS: The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.

Associations among white blood cells, CD34+ cells and GM-CFU in predicting the optimal timing of peripheral blood stem cell collections by apheresis.

AIMS: To analyze preapheresis blood CD34+ cells and corresponding apheresis products in order to investigate whether peripheral blood CD34+ cell counts correlate with peripheral blood progenitor cell (PBPC) yields and to determine the optimal timing for starting PBPC collections in our clinical setting. MATERIAL AND METHODS: Thirty-eight patients with hematological malignancies and undergoing varied mobilization regimens were enrolled. White blood cell counts (WBC), CD34+ cells and granulocyte-macrophage colony-forming units (GM-CFU) enumeration were performed on blood samples taken immediately prior to each apheresis procedure and in the corresponding PBPC collection. Results: A total of 61 apheresis procedures were performed, with a median of two collections per patient (range 1-3). The number of CD34+ cell/ml in the preapheresis blood correlated closely with CD34+ cells/kg and, to a lesser degree, with GM-CFU/kg in the apheresis products (r = 0.81 and r = 0.67, respectively, P < 0.0001). WBC showed significant but poor correlation with CD34+ cells/kg and GM-CFU/kg (r = 0.43 and r = 0.45, respectively, P = 0.004). A significant correlation was also found between CD34+ cells/kg and GM-CFU/kg in PBPC collections (r = 0.62, P < 0.0001). Linear regression analysis indicated that the minimum threshold of 2 x 10(6) CD34+ cells/kg might be attained with a single apheresis if the CD34+ cells/ml in the peripheral blood measured prior to apheresis on the day of collection is > or =26 x 10(3) CD34+ cells/ml. CONCLUSION: Collectively, these data demonstrate that circulating CD34+ cells is more useful than GM-CFU or WBC for predicting the optimal timing of PBPC harvests.

Use of flow cytometry to develop and characterize a set of monoclonal antibodies specific for rabbit leukocyte differentiation molecules

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.

CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34 + cells

A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34 + cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34 + cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34 + cells were compared with those in untreated CD34 + cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34 + cells. Here we describe an in vitro system in which CB CD34 + cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.

Immunophenotypic profile of adult acute myeloid leukemia (AML): analysis of 267 cases in Thailand.

Little data exists in Thailand and other Southeast Asian countries regarding the biological characteristics of adult acute myeloid leukemia (AML). In this study, we performed a flow cytometric analysis of 267 Thai adult AML cases to delineate the pattern of leukemic cell surface antigens. Forty-eight cases (18%) were identified as acute promyelocytic leukemia (M3) and 219 cases as non-M3. The most frequent subtype of AML in Thailand was M1/M2 and the least frequent was M7. M3 immunophenotypes were characterized by their unique lack of expression of CD34 and HLA-DR as contrast to the high mean expression of 50% and 70%, respectively, in non-M3. Overall, 60% of cases expressed CD34. Aberrant lymphoid antigens were uniquely seen in specific subtypes of Thai AML, including CD19 (33% of non-M3 vs 23% of M3) and CD2 (12% of M3 vs 2% of non-M3). CD56 was frequently expressed in both M3 and non-M3 while CD16 appeared to be associated with M4/M5 (24% of cases) and CD7 with M1/M2 (21% of cases). Eighty-one percent of non-M3 expressed CD38 while only 53% of M3 did. We found that most Thai adult AML patients were on average 15-20 years younger than those of the West or Japan with only 25% of Thai cases over 60 years of age, although the immunophenotypes were not markedly different. Biological studies of acute leukemia in various countries should help to provide epidemiological clues that play a role in the pathogenesis of leukemia in different geographic regions of the world. Our study represents the largest series of AML ever investigated in the Southeast Asian region.

Expression of hOGG1 protein during differentiation of HL-60 cells

Human 8-oxo-G-DNA glycosylase 1 (hOGG1) is a DNA glycosylase to cleave 8-oxo-7,8-dihydroguanine (8-oxo-G), a mutagenic DNA adduct formed by oxidant stresses. Here, we examined hOGG1 protein expression and repair activity to nick a DNA strand at the site of 8-oxo-G during differentiation of hematopoietic cells using HL-60 cells. Overall expression of hOGG1 protein was increased during granulocytic differentiation of HL-60 cells induced by DMSO and monocytic differentiation by vitamine D3. Greater level of hOGG1 protein was expressed in DMSO-treated cells. However, change in the DNA nicking activity was not in parallel with the change in hOGG1 protein expression, especially in PMA-treated cells. In PMA- treated cells, the level of hOGG1 protein was lowered, even though the DNA nicking activity was elevated, in a manner similar to the changes in serum- deprived HL-60 cells. These results indicate that hOGG1 expression change during differentiation of hematopoietic stem cells for adaptation to new environments. And the DNA cleaving activity may require additional factor(s) other than expressed hOGG1 protein, especially in apoptotic cell death.

Comportamento dos amebócitos circulantes de moluscos planorbídeos frente ao parasitismo por larvas de Schistosoma mansoni, inoculaçäo de tinta nanquim e fratura da concha / Behavior of hemolymph amebocytes from planorbidae in the presence of Schistosoma mansoni larvae parasitism, by inoculation of Indian ink or fracture of the shell

Foi estudado o comportamento dos amebócitos circulantes de Biomphalaria glabrata e Biomphalaria tenagophila mediante à infecçäo pelo Schistosoma mansoni, à inoculaçäo de tinta Nanquim e à fratura da concha. Foi realizada contagem diferencial de amebócitos na hemolinfa, dando-se ênfase aos tipos de células encontradas em cada amostra; avaliaçäo histopatológica dos moluscos submetidos à exposiçäo aos miracídios de S. mansoni; e análise da morfologia dos amebócitos através de microscopia de fase. Foi verificada correlaçäo entre a variaçäo do número de amebócitos circulantes e a reaçäo tecidual. Em Biomphalaria tenagophila somente houve aumento do número de amebócitos estrelados quando os moluscos eram infectados por S. mansoni, o que sugere a especificidade da reaçäo ao parasitismo. Comparando os resultados obtidos em B. glabrata e B. teganophila, conclui que esses moluscos apresentam comportamentos amebocitários diferentes frente aos diversos estímulos utilizados

Evidencia de que la célula blanco del factor estimulador de colonias de macrófagos y granulocitos es precursos de macrófagos / Evidence that the target cell of macrophage and granulocyte colony stimualting factor is the precursor of macrophages

En este trbajo se evaluó si el factor estimulador de colonias de macrófagos y granulocitos (GM-CSF) tiene al marcófago como única célula clanca. Para ello, se determinó la cinética de respuesta de precurores mieloides al GM-CSF en cultivos semisólidos. Los macrófagos son capaces de secretar el estimuladora de granulocitos (G-CSF), por tanto también se evaluó la posible secreción de G-CSF por los macrófagos inducidos con GM-CSF, Nuestros resultados indican que únicamente los precursores de macrófagos responden en forma temprana al GM_CSF, ya que al resembrar los grupos generados a los dos días se originan exclusivamente colonias de macrófago. Por otro lado el lisado de estas colonias promueve principalmente la formación de colinias granulocíticas. Finalmente se discute la posibilidad de que el GM-CSF sean en realidad un estimulador de macrófagos con capacidad de inducir la secreció de G-CSF, así como de la posible inexistencia de un precursor común demacrófagos y garnulocitos.

Conceptos en el diagnóstico de histoplasmosis humana / Concepts in diagnosis of human histoplasmosis

La histoplasmosis tiene una evolución muy similar a la de la tuberculosis. La diferencia principal entre ambas enfermedades consiste en la menor gravedad y más frecuente recuperación, en histoplasmosis. En ambas enfermedades hay que distinguir entre simple infección, formas activas y evolutivas y los casos recuperados y curados clínicamente; esta situación de cura; con secuelas, es mucho más frecuente en histoplasmosis, que en tuberculosis. El diagnóstico de actividad en histoplasmosis, es frecuentemente difícil y por esta razón el A. investiga el grado de parasitismo en los promonocitos como el más accesible medio de investigar el sistema reticuloendotelial.