Prekallikrein deficiency in a 15-year-old boy with Ménière's disease: a case report.

Congenital prekallikrein deficiency is a rare disorder in which there is an in vitro clotting defect despite absence of bleeding or thrombotic tendency. In this report, a 15-year-old boy with an unexpected markedly prolonged activated partial thrombin time, a normal prothrombin time, and without personal nor familial history of bleeding or thrombosis is presented. Laboratory investigation revealed a severe prekallikrein deficiency. This case highlights the importance of following a diagnostic algorithm to establish the correct diagnosis. Moreover, by selecting appropriate laboratory tests, unnecessary and repeatedly testing can be avoided.

Evaluation of a real-time multiplex PCR for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./EIEC, and Yersinia enterocolitica in fecal samples.

Conventional diagnosis of infectious diarrhea caused by bacteria is time-consuming, labor-intensive, and has a suboptimal sensitivity. We have therefore developed a multiplex real-time polymerase chain reaction (PCR) for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), and Yersinia enterocolitica in fecal samples. No cross reactivity between the different pathogens was observed, and the multiplex setup of the assay did not have an impact on the sensitivity of the PCR. The analytical sensitivity was 87 CFU/mL for C. jejuni, 61 CFU/mL for Shigella spp./EIEC, 5,528 CFU/mL for Salmonella spp., and 1,306 CFU/mL for Y. enterocolitica. An extensive validation of the assay was performed by testing 1,687 patient samples by both PCR and with conventional techniques. The use of PCR increased the overall clinical sensitivity from 78 to 100 % (p < 0.0001), the specificity was 99.4 % for the PCR, compared with 99.9 % for conventional culture. The novel PCR assay allows for rapid, sensitive, inexpensive, and high-throughput testing of the most common bacterial causes of gastroenteritis.

Detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in clinical stool samples by using multiplex real-time PCR after automated DNA isolation.

Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated as an alternative approach for diagnosing Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in stool samples.Therefore, a total of 631 faecal samples were analysed both by microscopy as well as by real-time PCR following automated DNA extraction. Results showed that real-time PCR exhibited sensitivity and specificity of both 100%, whereas traditional microscopy exhibited sensitivity and specificity of 37.5% and 99.8% respectively. As real-time PCR provides simple, sensitive and specific detection of these three important pathogenic protozoan parasites, this technique, rather than microscopy, has become our diagnostic method of choice for the detection of enteric protozoan parasites for the majority of patients.

Validation of the minimal citrate tube fill volume for routine coagulation tests on ACL TOP 500 CTS®.

INTRODUCTION: CLSI recommends a minimal citrate tube fill volume of 90%. A validation protocol with clinical and analytical components was set up to determine the tube fill threshold for international normalized ratio of prothrombin time (PT-INR), activated partial thromboplastin time (aPTT) and fibrinogen. METHODS: Citrated coagulation samples from 16 healthy donors and eight patients receiving vitamin K antagonists (VKA) were evaluated. Eighty-nine tubes were filled to varying volumes of >50%. Coagulation tests were performed on ACL TOP 500 CTS(®) . Receiver Operating Characteristic (ROC) plot, with Total error (TE) and critical difference (CD) as possible acceptance criteria, was used to determine the fill threshold. RESULTS: Receiving Operating Characteristic was the most accurate with CD for PT-INR and TE for aPTT resulting in thresholds of 63% for PT and 80% for aPTT. By adapted ROC, based on threshold setting at a point of 100% sensitivity at a maximum specificity, CD was best for PT and TE for aPTT resulting in thresholds of 73% for PT and 90% for aPTT. For fibrinogen, the method was only valid with the TE criterion at a 63% fill volume. CONCLUSION: In our study, we validated the minimal citrate tube fill volumes of 73%, 90% and 63% for PT-INR, aPTT and fibrinogen, respectively.

Generation of T cells from adult human hematopoietic stem cells and progenitors in a fetal thymic organ culture system: stimulation by tumor necrosis factor-alpha.

To investigate the T-lymphopoietic capacity of human adult bone marrow (ABM) hematopoietic progenitor cells, CD34+Lin-, CD34+CD38+, and CD34++CD38- cells were cultured in a severe combined immunodeficient (SCID) mouse fetal thymic organ culture (FTOC). Direct seeding of these progenitors resulted in a moderate to severe cell loss, particularly for the CD34++CD38- cell fraction, and T cells could only be generated from the CD34+Lin- fraction. Preincubation for 36 hours with interleukin-3 (IL-3) and stem cell factor (SCF) led to an improved cell survival and proliferation, although T-cell development was seen only in the CD34+Lin- fraction. Addition of tumor necrosis factor (TNF)-alpha to IL-3 + SCF-supplemented preincubation medium resulted in optimal cell survival, cell proliferation. and T-cell generation of all 3 cell fractions. The TNF-alpha effect resulted in an up-regulation of CD127 (ie, the IL-7 receptor alpha-chain) in a small subset of the CD34+ cells. No evidence could be generated to support the possibility that TNF-alpha inhibits a cell population that suppresses T-cell differentiation. A quantitatively different T-cell generation potency was still seen between the 3 subpopulations: CD34+Lin- (100% success rate) > CD34+CD38+ (66%) > CD34++CD38- (25%). These data contrast with our previous findings using fetal liver and cord blood progenitors, which readily differentiate into T-lymphocytes in FTOC, even without prestimulation with cytokines. Our results demonstrate that adult CD34++CD38- cells, known to contain hematopoietic stem cells, can differentiate into T-lymphocytes and that a significant difference exists in T-lymphopoietic activity of stem cells derived from ontogenetically different sources. (Blood. 2000;95:2806-2812)

Developmentally regulated responsiveness to transforming growth factor-beta is correlated with functional differences between human adult and fetal primitive hematopoietic progenitor cells.

Important functional differences exist between primitive CD34++ CD38- hematopoietic progenitor cells derived from human fetal liver (FL) and adult bone marrow (ABM). FL progenitors are known to have higher proliferative capacities and lower cytokine requirements than their ABM counterparts. In this study, we isolated FL and ABM CD34++ CD38- cells and used a two-stage culture system to investigate the effects of transforming growth factor-beta (TGF-beta) and blocking anti-TGF-beta antibodies (anti-TGF-beta) on these cells. First, we demonstrate that FL progenitors are significantly less sensitive to the inhibitory effects of TGF-beta than ABM cells. Second, whereas ABM cells are significantly stimulated by anti-TGF-beta, only very limited effects are seen on FL cells. Third, we show that the effect of anti-TGF-beta is mainly situated at the level of the initial cell cycles of very primitive progenitor cells with a high proliferation potential. Fourth, we demonstrate that blocking the effects of endogenous TGF-beta reduces the growth factor requirements of ABM cells in order to proliferate and differentiate. Based on these data, we hypothesize that at least part of the functional differences that exist between adult and fetal stem cells can be accounted for by a developmental different responsiveness to TGF-beta.

CD34++ CD38- and CD34+ CD38+ human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source.

CD34++ CD38- and CD34+ CD38+ hematopoietic progenitor cells (HPCs) from human fetal liver (FL), cord blood (CB), and adult bone marrow (ABM) were isolated and investigated for their growth characteristics, cytokine requirements and response to two modulators of early hematopoiesis, interferon (IFN)-gamma and macrophage inflammatory protein (MIP)-1alpha. We observed first that a significantly lower percentage of CD34++ cells were CD38- in ABM than in FL and CB. Second, the functional differences between CD34++ CD38- and CD34+ CD38+ cells were less pronounced in FL and CB than in their ABM counterparts. Third, an inverse correlation was found between growth factor response and the ontogenic age of HPCs, and a direct correlation was noted between cytokine requirements and the ontogenic age of HPCs. Fourth, spontaneous colony formation in a classic semisolid culture system was reproducibly obtained only in the ontogenically earliest cells, that is, in FL but not in CB and ABM, in which no such spontaneous colony formation was observed. Fifth, the modulatory effects of IFN-gamma and MIP-1alpha were qualitatively different depending on the ontogenic age of the progenitor source: whereas IFN-gamma was only a selective inhibitor of primitive CD34++ CD38- ABM progenitor cells, it inhibited both CD34++ CD38- and CD34+ CD38+ FL and CB cells to the same extent. In contrast to the effects of MIP-1alpha on ABM, we could not find any consistently stimulatory or inhibitory effects on FL and CB progenitors. In conclusion, important functional and biologic differences exist between FL, CB, and ABM progenitor cells, and these differences could have major implications for the use of these cell populations in preparative protocols of ex vivo expansion, transplantation strategies, or gene transfer experiments.

The combined effects of all-trans retinoic acid and TGF-beta on the initial proliferation of normal human bone marrow progenitor cells.

We investigated the cell kinetic effects of retinoic acid (RA) and the functional interaction between RA and TGF-beta on normal human bone marrow progenitor cells (CD34+). Cell cycle progression throughout the first three consecutive cell cycles and alterations in cell kinetic responses were measured using the BrdU-Hoechst quenching technique. RA stimulates the IL-3-induced growth by additionally recruiting quiescent stem and progenitor cells out of the G0/G1-phase and by increasing the cell cycle traverse rate. In contrast, TGF-beta addition resulted in a significant decrease in the number of proliferating cells. Simultaneous addition of RA and TGF-beta resulted in a stronger inhibition compared to addition of TGF-beta alone. Preincubation experiments further showed that RA is capable of sensitizing the progenitors to the inhibitory action of TGF-beta: the inhibitory effect of TGF-beta was significantly increased when cells were pretreated with RA. These data show that, in combination with IL-3, RA additionally stimulates quiescent bone marrow progenitors in a simultaneous way, and that it increases sensitivity of the progenitors to the inhibitory action of TGF-beta. The combination of RA and TGF-beta on normal and leukemic hematopoiesis has to be further investigated, since this combination may possibly provide additional therapeutic benefit.

TGF-beta and MIP-1 alpha exert their main inhibitory activity on very primitive CD34+2CD38- cells but show opposite effects on more mature CD34+CD38+ human hematopoietic progenitors.

We investigated the effects of transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) on very primitive CD34++CD38- and on more mature CD34++CD38+ human hematopoietic progenitor cells by means of a two stage pre-colony-forming cell (pre-CFC) assay. The first (liquid) stage of this assay allows evaluation of the effects of TGF-beta and MIP-1 alpha on the "primary" proliferation of the progenitors under study and on the generation of "secondary" colony-forming cells (CFC, cells for which a second stage semisolid clonogenic assay was used as a read-out). TGF-beta inhibited the proliferation and CFC generation of CD34++CD38- and CD34+CD38+ cells, showing the strongest inhibitory activity on CD34++CD38- cells. MIP-1 alpha exerted a weaker inhibitory activity on CD34+2CD38- cells, whereas it enhanced the primary proliferation of CD34+CD38+ cells and generation of secondary CFC in this subpopulation. Thus, TGF-beta, and MIP-1 alpha both inhibit very primitive CD34+2)CD38- cells, but they are not equally potent. The effects of TGF-beta and MIP-1 alpha on more mature progenitor cells are more complex. Our results and data from the literature indicate that, as progenitor cells mature, they reach a "pivotal point" at a certain stage in their differentiation pathway, depending on the inhibitor, where they are no longer inhibited or where they may even be stimulated by the former inhibitor to proliferate.

Tumor necrosis factor alpha is a potent synergistic factor for the proliferation of primitive human hematopoietic progenitor cells and induces resistance to transforming growth factor beta but not to interferon gamma.

Since tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta have all been shown to be specific inhibitors of early human hematopoiesis, we wanted to investigate the interactions of these three cytokines on very primitive human adult bone marrow CD34++CD38- hematopoietic progenitor cells, using a pre-colony-forming cell (pre-CFC) assay, which detects the effects of these cytokines on the initial phases of the differentiation of these primitive progenitors, which are unresponsive to interleukin (IL) 3 alone. Surprisingly, TNF-alpha was a very potent stimulator of the proliferation of CD34++CD38- cells and was the most potent synergistic factor for the IL-3-induced proliferation of these cells of all cytokines tested (IL-1, IL-6, granulocyte colony-stimulating factor, kit ligand). TNF-alpha was the only cytokine that, as a single added factor, induced substantial proliferation in CD34++CD38- cells in the presence of IL-3, except for kit ligand, which induced very limited proliferation. TNF-alpha, moreover, induced a high degree of resistance to the inhibitory effects of TGF-beta in a dose-dependent way. The inhibitory effects of IFN-gamma, however, were not affected by the presence of TNF-alpha. We hypothesize that in situations of the hematopoietic stress, TNF-alpha may abrogate the inhibitory effect of ambient TGF-beta in the bone marrow microenvironment to allow primitive stem cells to proliferate and differentiate in response to an increased demand for mature blood cells.