Characterization, isolation, and culture of spermatogonial stem cells in Macaca fascicularis.

Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-KitW/W (W) mutant mice. Collectively, GFRA1-enriched spermatogonia are monkey SSCs phenotypically both in vitro and in vivo. This study suggests that monkey might provide an alternative to human SSCs for basic research and application in human diseases.

The effect of maternal body mass index and gestational age on circulating trophoblast yield in cell-based noninvasive prenatal testing.

OBJECTIVE: To examine the effects of maternal body mass index (BMI) and gestational age (GA) on the number of single circulating trophoblasts (SCT). METHODS: Maternal blood was collected in 20 to 40 mL. All singleton pregnant women at any gestation were recruited. Trophoblasts were recovered by immunomagnetic enrichment and stained for cytokeratin and CD45. Candidate trophoblasts were identified by fluorescence microscopy. RESULTS: Blood samples were collected from 425 singleton pregnancies from April 2018 to December 2019. At least one candidate cell was identified in 88% (373/425). There was an inverse correlation between trophoblasts yield and increasing BMI (r = -0.19, P < .001). The mean ± SD number of trophoblasts/mL was 0.12 ± 0.22 in the underweight group (n = 5), 0.23 ± 0.25 in the normal weight (n = 169), 0.18 ± 0.19 in the overweight (n = 114), and 0.13 ± 0.15 in the obese (n = 109). Significantly more cells were identified in the normal weight than those in the obese (P = .001). In addition, the mean ± SD number of cells/mL was 0.21 ± 0.21 at GA of 10 to 14 weeks (n = 260), 0.14 ± 0.23 at GA ≥15 (n = 102) and 0.12 ± 0.12 at GA <10 (n = 63); P < .001. CONCLUSION: The lower number of SCT was identified from the samples of women with a high BMI. Cell recovery for SCT testing seems optimal at GA of 10 to 14 weeks, but earlier and later testing is still possible.

Heterogeneity of Human Pancreatic Islet Isolation Around Europe: Results of a Survey Study.

BACKGROUND: Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries. METHODS: A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan. RESULTS: Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities. CONCLUSIONS: The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria.

Nonspecific probe binding and automatic gating in flow cytometry and fluorescence activated cell sorting (FACS).

Flow cytometry is extensively used in cell biology to differentiate cells of interest (mutants) from control cells (wild-types). For mutant cells characterized by expression of a distinct membrane surface structure, fluorescent marker probes can be designed to bind specifically to these structures while the cells are in suspension, resulting in a sufficiently high fluorescence intensity measurement by the cytometer to identify a mutant cell. However, cell membranes may have relatively weak, nonspecific binding affinity to the probes, resulting in false positive results. Furthermore, the same effect would be present on mutant cells, allowing both specific and nonspecific binding to a single cell. We derive and analyze a kinetic model of fluorescent probe binding dynamics by tracking populations of mutant and wild-type cells with differing numbers of probes bound specifically and nonspecifically. By assuming the suspension is in chemical equilibrium prior to cytometry, we use a two-species Langmuir adsorption model to analyze the confounding effects of non-specific binding on the assay. Furthermore, we analytically derive an expectation maximization method to infer an appropriate estimate of the total number of mutant cells as an alternative to existing, heuristic methods. Lastly, using our model, we propose a new method to infer physical and experimental parameters from existing protocols. Our results provide improved ways to quantitatively analyze flow cytometry data.

Aislamiento y caracterización celular de exosomas de plasma para su uso como biomarcadores de diagnóstico / Isolation and cellular characterization of exosomes from plasma for their use as diagnostic biomarkers / Purificação e caracterização celular de exossomos para seu uso como biomarcadores de diagnóstico

Los exosomas son vesículas membranosas extracelulares esenciales en la comunicación intercelular a larga distancia, viajan en los fluidos corporales y entregan mensajes moleculares dirigidos a la mayoría de las células de todo el organismo. La liberación de mensajes vía exosomas ocurre en forma de ADN, ARN o proteínas; dicha liberación se ha asociado a diferentes condiciones fisiológicas normales y patológicas, como el cáncer. Por lo anterior, el aislamiento eficiente y caracterización celular de exosomas de plasma es clave para su uso como biomarcadores no invasivos de diversas enfermedades. En el presente estudio se purificaron exosomas a partir de muestras clínicas de plasmas de pacientes previamente diagnosticados con retinoblastoma y de individuos sanos como control. Los exosomas recuperados fueron caracterizados a nivel celular por microscopia electrónica de transmisión empleando una técnica de criogenia. Para demostrar la correcta purificación de exosomas se confirmó la presencia de las proteínas transmembranales CD63 y CD81 mediante immunoblot. Adicionalmente de los exosomas purificados, se identificaron ARNs pequeños no codificantes llamados microARNs. En general, se describe la purificación y caracterización celular de exosomas obtenidos de plasma humano para su potencial uso como biomarcadores.

Exosomes are small extracellular vesicles essential in intercellular communication; they act as vehicles of broad scope. They are travelling in body fluids and delivering molecular messages to cells in the organism. Messages released by exosomes like DNA, RNA and proteins are associated with different pathological conditions including cancer. Therefore, the efficient isolation and cellular characterization of exosomes from plasma is essential to use them as biomarkers in many diseases. Here, exosomes were purified from patients diagnosed with pediatric cancer and healthy individuals as control. The exosomes recovered were characterized using cryogenic transmission electron microscopy. Moreover, the presence of CD63 and CD81 transmembrane proteins was confirmed using Western blot. Besides, miRNAs presence was identified from exosomes. This work describes a complete technique to isolate and characterize exosomes from human plasma, recognizing their potential as biomarkers.

Os exossomos são vesículas membranosas extracelulares essenciais na comunicação intercelular de longa distância; eles viajam em fluidos corporais e entregam mensagens moleculares dirigidas à maioria das células de todo o organismo. A liberação de mensagens através dos exossomos ocorre em forma de DNA, RNA ou proteínas; essa liberação foi associada a diferentes condições fisiológicas normais e patológicas, tais como o câncer. Por tudo isso, o eficiente isolamento e caracterização celular de exossomos de plasma é chave para sua utilização como biomarcadores não invasivos de várias doenças. No presente estudo, exossomos foram purificados a partir de amostras clínicas de plasmas de pacientes que tinham sido diagnosticados previamente com retinoblastoma e de indivíduos saudáveis como controle. Os exossomos recuperados foram caracterizados a nível celular por microscopia eletrônica de transmissão usando uma técnica de criogenia. Para demonstrar a correta purificação dos exossomos, foi confirmada a presença de proteínas transmembranares CD63 e CD81 usando inmunoblot. Além dos exossomos purificados foram identificados ARNs não codificantes pequenos chamados microARNs. Em geral os métodos de purificação e caracterização celular de exossomos obtidos de plasma humano são descritos por seu potencial utilização como biomarcadores.

Actualización en el diagnóstico de la leptospirosis humana / Updating on the diagnosis of human leptospirosis

La leptospirosis humana es una enfermedad zoonótica de distribución mundial, con frecuencia subdiagnosticada por presentar un amplio espectro de manifestaciones clínicas. El objetivo es presentar una actualización sobre el diagnóstico de la leptospirosis humana. Se consultó la bibliografía disponible sobre el tema en las bases de datos de Scielo, HINARI, Pubmed-Medline y diferentes textos y artículos en los que se profundizaba en el diagnóstico. Las técnicas microbiológicas son la base de la confirmación de la enfermedad. La observación por microscopía de campo oscuro es poco sensible, ya que las leptospiras se pueden confundir con filamentos proteicos u otros artefactos. El aislamiento del agente etiológico constituye la prueba de oro, aunque ofrece un resultado retrospectivo. La reacción en cadena de la polimerasa es un método útil para el diagnóstico rápido de la infección. El diagnóstico serológico cobra vital importancia en esta entidad, pues supera en rapidez, sencillez y bajo costo al cultivo. La microaglutinación con antígenos vivos es la técnica de referencia. Las pruebas rápidas basadas en la inmunocromatografía de flujo lateral son una variante muy útil, ya que ofrecen el resultado entre 5 y 30 minutos.

The human leptospirosis is zoonotic disease with worldwide distribution, frequently present a difficult diagnosis because have a wide spectrum of clinical manifestations. The objective of this work is to present an upgrade on the diagnosis of the human leptospirosis; for it was consulted it the available bibliography on the topic in the databases of Scielo, HINARI, Pubmed-Medline and different texts and articles of the last five years to deepen in the diagnosis. The microbiologic diagnostic is the base of the confirmation of the illness; it depends on the taking of sample in the appropriate moment and the correct indication of the complementary one according to the clinical phase. The observation for microscopy of dark field is not very sensitive since the leptospiras can made a mistake with poetics filaments or other devices. The etiologic agent's isolation constitutes the gold standard test; although offers a retrospective result. The polymerase chain reaction is a method used for the quick diagnosis of the infection, the quantitative variant in real time shows substantial advantages, because it allows the immediate reading and avoids the electrophoresis step, these techniques are not available for its high cost. The serologic diagnostic charges vital importance in this entity, it overcomes in speed, simplicity and low cost to the cultivation; the microagglutinación with a live antigen is considered the reference technique. The detection of IgM for enzyme linked immunobsorbent assay has been broadly used. The rapid tests based on the lateral flow immunochromatography, are a very useful variant, since they offer the result between 5 and 30 minutes.

Rmax: A systematic approach to evaluate instrument sort performance using center stream catch.

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instrument's sort count. Yield and Recovery are often neglected, mostly due to difficulties in their measurement. Purity of the sort product is often cited alone but is not sufficient to evaluate sorting performance. All of these three performance metrics require re-sampling of the sorted fraction. But, unlike Purity, calculating Yield and/or Recovery calls for the absolute counting of particles in the sorted fraction, which may not be feasible, particularly when dealing with rare populations and precious samples. In addition, the counting process itself involves large errors. Here we describe a new metric for evaluating instrument sort Recovery, defined as the number of particles sorted relative to the number of original particles to be sorted. This calculation requires only measuring the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch (CSC), avoiding re-sampling the sorted fraction and absolute counting. We called this new metric Rmax, since it corresponds to the maximum expected Recovery for a particular set of instrument parameters. Rmax is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter, or any instrument related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument performance before single-cell sorting experiments. Because we do not perturb the sort fraction we can calculate Rmax during the sort process, being especially valuable to check instrument performance during rare population sorts.

HPC-A dose prediction on the optia® cell separator based on a benchmark CE2 collection efficiency: Promoting clinical efficiency, minimizing toxicity, and allowing quality control.

It has been shown that it is possible to predict the CD 34+ hematopoietic progenitor cell dose from collection procedures on TerumoBCT COBE Spectra® cell separator platform using simple variables available at the start of the procedure. In this article, we demonstrate that this can be done simply and reliably using TerumoBCT Spectra Optia® ("Optia") cell separator platform with a very close correlation between predicted and actual results (correlation coefficient 0.956). This knowledge can be used to optimize apheresis sessions and to minimize harmful effects and costs. In addition, we have shown differences in collection efficiency between healthy donors and cancer patients undergoing autologous donation. Finally, we have shown a small but significant improvement in collection efficiency for the Optia platform compared with the COBE Spectra platform.

Cushioned versus noncushioned centrifugation: sperm recovery rate and integrity.

It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10(6) sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), and 1800NC (96.7 ± 2.6) groups was significantly higher than the 900C (68.7 ± 4.6) and 1800C (79.6 ± 3.5) groups. The D0 TM and PM were not different between the CON, 900NC, 900C, and 1800C, but were lower for the 1800NC group. The D1 TM and PM of the 900NC (75.2 ± 3.8 and 71.1 ± 4.1) and 900C (76.2 ± 3.7 and 72.4 ± 4.0) groups were significantly higher than the 1800NC (71.7 ± 4.1 and 67.3 ± 4.4) and 1800C (71.6 ± 4.1 and 67.2 ± 4.4) groups, and the CON (66.2 ± 4.5 and 60.0 ± 4.8) group was significantly lower than the other groups. The D1 PLM of the CON, 900NC, 900C, 1800NC, and 1800C groups were not different. The ACR on D1 was significantly lower for the CON (93.0 ± 2.4) group compared with all other groups. Optimal RR preserving sperm integrity was obtained in the 900NC group.

Filtration parameters influencing circulating tumor cell enrichment from whole blood.

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·10(4)∶10(2)∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm(2) track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.

[Chimerism analysis after allogeneic haematopoietic stem cell transplantation. Interest of cell sorting: general review]. / Étude de chimérisme post-greffe de cellules souches hématopoïétiques. Intérêt du tri cellulaire : revue générale.

Haematopoietic stem cells transplantation, widely used these last decades, represent the ultimate treatment resource for patients with haematological malignancies. Long range success of this treatment is particularly affected by relapse of the initial disease, graft rejection or graft versus host disease. Chimerism analysis after transplantation had been used since several years to document engraftment, to determine the risk of relapse and to adapt therapy promptly when necessary. Usefulness of this analysis for the outcome of transplanted patients, as well as the impact of using high sensitive techniques coupled with specific cell populations sorted have been demonstrated by retrospective studies. Follow-up of chimerism would allow to operate efficiently before the onset of clinical signs in leukaemic patients with high risk of relapse and to control the expression of minimal residual disease when specific molecular markers could not be monitored.

Diffraction Phase Cytometry: blood on a CD-ROM.

We demonstrate Diffraction Phase Cytometry (DPC) as a single shot, full-field, high throughput quantitative phase imaging modality, dedicated to analyzing whole blood smears. Utilizing a commercial CD as a sample substrate, along with dynamic spatial filtering via a liquid crystal spatial light modulator, we have developed a compact instrument capable of making quantitative, physiologically relevant measurements. To illustrate the ability of the system to function as a highly sensitive cytometer we imaged a large number (N=1,537) of live human erythrocytes in whole blood without preparation. We retrieved a comprehensive set of geometrical parameters including cell volume and surface area, which are not directly available using existing cytometers. Furthermore, we retrieved the minimum cylindrical diameter, through which red blood cells can pass, and deliver oxygen. These initial results prove the concept for an inexpensive lab-on-a-chip blood screening device.

A questionnaire-based survey of perioperative blood conservation practice for revision hip arthroplasty in Scotland.

The aim of this study was to describe current blood conservation practice during revision hip surgery in Scotland and document practice variation. Revision hip surgery is associated with a high likelihood of blood transfusion. A decrease in the proportion of patients requiring blood transfusion has been documented, but the reasons for this are unclear. Various blood conservation practices are available to clinicians, but the extent to which these are used in Scottish hospitals is not known. A cross-sectional postal survey was sent to all consultant orthopaedic surgeons and consultant anaesthetists participating in revision hip surgery in Scottish hospitals. Responses were received from 92 of 120 (77%) surgeons, and 174 of 216 (81%) anaesthetists (62/92). A total of 62 of 92 (67%) surgeons and 78 of 174 (45%) anaesthetists surveyed participated in revision hip surgery. Blood conservation practice varied widely: 34 of 78 (44%) anaesthetists routinely assessed revision hip patients >or=1 week prior to surgery; 10 of 62 (16%) surgeons and 24 of 78 (31%) anaesthetists routinely used cell salvage; 7 of 78 (9%) anaesthetists and 2 of 62 (3%) surgeons routinely used tranexamic acid; and 45 of 62 (73%) surgeons use a transfusion protocol. A wide variation in the use of blood conservation strategies exists during revision hip surgery in Scotland.

Effects of magnetic-activated cell sorting on sperm motility and cryosurvival rates.

OBJECTIVE: To evaluate the effect of magnetic-activated cell sorting in cryopreservation-thawing protocols on sperm motility and cryosurvival rate. DESIGN: Prospective-controlled study. SETTING: Andrology department at a university-based medical institution. PATIENT(S): Ten healthy volunteer sperm donors. INTERVENTION(S): Sperm populations were separated using annexin-V magnetic-activated cell sorting before and after the cryopreservation-thawing process. MAIN OUTCOME MEASURE(S): Sperm motility and cryosurvival rate. RESULT(S): Annexin-negative sperm separated by magnetic-activated cell sorting had statistically significantly higher motility following cryopreservation-thawing than sperm that were not separated. Similarly, annexin-negative spermatozoa also had higher cryosurvival rate than sperm cryopreserved without magnetic-activated cell sorting and sperm that were annexin-positive. CONCLUSION(S): Superparamagnetic annexin V-conjugated microbeads can separate spermatozoa with externalized phosphatidylserine, which is considered one of the early features of late apoptosis. The separation of a distinctive population of nonapoptotic spermatozoa with intact membranes may optimize the cryopreservation-thawing outcome. Magnetic-activated cell sorting using annexin-V microbeads enhances sperm motility and cryosurvival rates following cryopreservation.

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

Quantification of immunosuppression by flow cytometry in stable renal transplant recipients.

The current standard of monitoring transplant patients by drug levels is not optimal because it does not take into account the different and individual effects of immunosuppressive drugs on each patient. In this study, the authors tested immune function assays for monitoring transplant patients. Blood was collected from stable renal transplant patients treated with cyclosporin, mycophenolate mofetil, and prednisone (n = 8), and from healthy volunteers (n = 12). Lymphocyte proliferation, expression of T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154), production of intracellular cytokines (IL-2, INFgamma, TNFalpha), and lymphocyte subsets (CD4, CD8, CD16, CD20) were assessed by flow cytometry. Lymphocyte proliferation, expression of T-cell surface activation antigens, and production of intracellular cytokines were significantly decreased in transplant recipients compared with healthy control volunteers. The combined effects of several immunosuppressive drugs in renal transplant recipients can be quantitated with immune function assays in whole blood. This new method may be helpful to achieve an optimal level of immunosuppression for each patient.

Limited efficacy of universal leucodepletion in reducing the incidence of febrile nonhaemolytic reactions in red cell transfusions.

This article demonstrates a 62% reduction in the number of febrile nonhaemolytic transfusion reactions (FNHTRs) and 50% reduction in febrile reaction rate associated with red cell transfusions following graded introduction of universal leucodepletion. Though this is a statistically significant reduction (P = 0.009), it shows limited efficacy in abrogating this complication. We also found a reduction in the proportion of cases of FNHTRs with lymphocytotoxic antibodies over the period studied from 54% in 1998, 28% in 1999 to 23% in 2000. This corresponds to a relative increase in the number of febrile reactions without human leucocyte antigen (HLA) antibodies following full implementation of universal leucodepletion, as the total number of reported reactions actually fell considerably during the period. The increase in the number of cases without HLA antibodies was directly proportional to the increase in the number of leucodepleted units used.

Improved methods for the isolation and purification of porcine islets.

Recent progress in human islet transplantation demonstrates the feasibility of using purified human islets for treatment of type 1 diabetes mellitus; however, a shortage of human pancreata remains a major obstacle. This report describes methods to isolate porcine islets using a modification of the automated chamber method. The pancreata from 2-year-old sows were trimmed and injected intraductally with Sevac, Sigma, or Liberase PI collagenase. The pancreata was placed in the chamber, shaken, and recirculated at 70 ml/min until an adequate number of islets were liberated. The digest was centrifuged and the pellets pooled with University of Wisconsin Solution + 10% horse serum and incubated at 4 degrees C for 1 h. The islets were purified using a continuous gradient of Hypaque Euroficoll on a refrigerated COBE 2991. The islets were collected in fractions, assessed for purity, sized, and then suspended in Medium 199. Collagenase preparations obtained from Sevac (2919 islet equivalents [IE]/g), Sigma (2543 IE/g), and Liberase PI (2901 IE/g) gave similar results with 94%-95% purity. In summary, we report a successful method for efficient isolation and purification of porcine islets, yielding nearly 3000 IE/gm, with different collagenase products.

Simultaneous differentiation and quantitation of erythroblasts and white blood cells on a high throughput clinical haematology analyser.

After the neonatal period, the presence of nucleated red blood cells (NRBC) in peripheral blood is indicative of pathology. Despite the clinical utility of such measurements, automated NRBC counting has hitherto not been available on routine automated blood cell counting analysers. To address this, an automated method for the analysis of NRBC was developed and incorporated into the Abbott Cell Dyn 4000 (CD4000) haematology analyser. The system white blood cell (WBC) reagent was specifically formulated to preserve concomitantly white blood cell (WBC) morphology, rapidly lyse red blood cell and NRBC membranes, and subsequently stain NRBC nuclei with a nucleotide specific fluorochrome dye (Kim et al. 1996a). The fluorochrome itself does not permeabilize the membrane of intact viable white blood cells. The sample is processed by flow cytometry and the signals generated from an argon-ion laser light source are analysed. Axial light loss (AxLL), intermediate angle light scatter (IAS) and red fluorescence (FL3) are used to discriminate between particles of various types. By using these discriminators in a three-dimensional approach, NRBC from a discrete cluster which can easily be separated from leucocytes and enumerated as a distinct cell population during the optical WBC differential analysis. Consequently, accurate absolute WBC counts and differentials can be obtained even in the presence of NRBC. Background 'noise' (both fluorescent and non-fluorescent) from platelets. Howell-Jolly bodies, basophilic stippling, RNA from lysed reticulocytes, and DNA from leucocyte and megakaryocytic fragments are essentially eliminated (Kim et al. 1996b). While the membranes of intact and viable leucocytes remain impermeable to the passage of the fluorochrome stain, leucocytes with damaged membranes are permeable to the dye and generate FL3+ signals. Such cells, which are commonly seen as a consequence of sample ageing as well as in some distinctive pathologies, are identified by the algorithm (using their AxLL signal size) and are labelled as non-viable. Moreover, because non-viable leucocytes are retained in the WBC count and differential analyses, the CD4000 is further able to provide both numerical and graphical data regarding the relative frequency of viable and non-viable components. This additional information can serve as valuable 'decision-drivers' in the laboratory data review process.