Protocollen en de praktijk, het blijft passen en meten

Editorial bij de bijdrage van A.J.A. Verhoeven, Y.M.A. van Maaren, B. Voermans en C. Botermans, getiteld ‘Prospectieve analyse van de bloedtransfusieketen met behulp van de SAFER-methode’

WT1 proteins : functions in growth and differentiation

The Wilms' tumor 1 gene (WT1) has been identified as a tumor suppressor gene involved in the etiology of Wilms' tumor. Approximately 10% of all Wilms' tumors carry mutations in the WT1 gene. Alterations in the WT1 gene have also been observed in other tumor types, such as leukemia, mesothelioma and desmoplastic small round cell tumor. Dependent on the tumor type, WT1 proteins might either function as tumor suppressor proteins or as survival factors. Mutations in the WT1 gene can also result in congenital abnormalities as observed in Denys-Drash and Frasier syndrome patients. Mouse models have proven the critical importance of WT1 expression for the development of several organs, including the kidneys, the gonads and the spleen. The WT1 proteins seem to perform two main functions. They regulate the transcription of a variety of target genes and may be involved in post-transcriptional processing of RNA. The WT1 gene encodes at least 24 protein forms. These isoforms have partially distinct biological functions and effects, which in many cases are also specific for the model system in which WT1 is studied. This review discusses the molecular mechanisms by which the various WT1 isoforms exert their functions in normal development and how alterations in WT1 may lead to developmental abnormalities and tumor growth

Optimized (pre)analytical conditions and workflow for droplet digital PCR analysis of cell-Free DNA from patients with suspected lung carcinoma

\u3cp\u3eFor patients with suspected lung carcinoma, the analysis of circulating tumor DNA, obtained by liquid biopsy, has the potential to support cancer diagnosis and guide targeted therapy. To ensure sensitive and reproducible detection of circulating tumor DNA in routine clinical practice, a standardized (pre) analytical workflow is required. Plasma was obtained from patients and healthy volunteers. Using the QIAmp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), six different procedures for the isolation of cell-free DNA (cfDNA) were compared. cfDNA was analyzed by droplet digital PCR (ddPCR) for KRAS G12/13 mutations and for EGFR Ex19Del, L858R, and L861Q mutations using an in-house EGFR multiplex assay. A new isolation procedure that yields extracts with significantly higher cfDNA concentrations than described previously was selected (P < 0.001). EGFR and KRAS assay sensitivity of at least 0.2% fractional abundance was guaranteed for approximately 76% of patient samples in one run. A flowchart that includes validity criteria for a standardized analytical workflow of ddPCR analysis was designed. An improved protocol for cfDNA isolation enables a higher cfDNA input for ddPCR. The use of sensitive KRAS and EGFR multiplex assays and accompanying validity criteria allows for controlled and efficient testing of patient samples at lower costs. Using the suggested workflow, a guaranteed, reliable, and sensitive analysis of cfDNA can be performed using ddPCR in routine clinical practice.\u3c/p\u3

Allogene trombocytengel bij urologische patiënt met een verworven trombocytopathie

Applicatie van een trombocytengel kan de wondheling versnellen en het risico op complicaties (nabloeden, wondinfectie) verminderen. Deze casus betreft een patiënt met een verworven trombocytopathie, die operatief wordt behandeld voor een vergrote prostaat en blaasstenen, waarbij na de eerste operatie een gecompliceerd beloop optreedt. De patiënt heeft last van persisterende bloedingen, waarvoor enkele malen operatief stolsels uit de blaas worden geëvacueerd. In totaal ontvangt de patiënt 14 eenheden erytrocytenconcentraat en 7 eenheden vers bevroren plasma. Uiteindelijk wordt bij deze patiënt gebruik gemaakt van een trombocytengel op basis van een allogeen product (5 donoren trombocytenconcentraat), waarna geen stolselretentie meer optreedt en de patiënt snel herstelt. Autologe donatie bij patiënten met een verworven trombocytopathie, levert geen goede trombocytengel, het gebruik van allogene trombocyten is dan een goed alternatief

Dispersion of the HIV-1 epidemic in men who have sex with men in the Netherlands:a combined mathematical model and phylogenetic analysis

\u3cbr/\u3eBackground\u3cbr/\u3eThe HIV-1 subtype B epidemic amongst men who have sex with men (MSM) is resurgent in many countries despite the widespread use of effective combination antiretroviral therapy (cART). In this combined mathematical and phylogenetic study of observational data, we aimed to find out the extent to which the resurgent epidemic is the result of newly introduced strains or of growth of already circulating strains.\u3cbr/\u3eMethods and Findings\u3cbr/\u3eAs of November 2011, the ATHENA observational HIV cohort of all patients in care in the Netherlands since 1996 included HIV-1 subtype B polymerase sequences from 5,852 patients. Patients who were diagnosed between 1981 and 1995 were included in the cohort if they were still alive in 1996. The ten most similar sequences to each ATHENA sequence were selected from the Los Alamos HIV Sequence Database, and a phylogenetic tree was created of a total of 8,320 sequences. Large transmission clusters that included ≥10 ATHENA sequences were selected, with a local support value ≥ 0.9 and median pairwise patristic distance below the fifth percentile of distances in the whole tree. Time-varying reproduction numbers of the large MSM-majority clusters were estimated through mathematical modeling. We identified 106 large transmission clusters, including 3,061 (52%) ATHENA and 652 Los Alamos sequences. Half of the HIV sequences from MSM registered in the cohort in the Netherlands (2,128 of 4,288) were included in 91 large MSM-majority clusters. Strikingly, at least 54 (59%) of these 91 MSM-majority clusters were already circulating before 1996, when cART was introduced, and have persisted to the present. Overall, 1,226 (35%) of the 3,460 diagnoses among MSM since 1996 were found in these 54 long-standing clusters. The reproduction numbers of all large MSM-majority clusters were around the epidemic threshold value of one over the whole study period. A tendency towards higher numbers was visible in recent years, especially in the more recently introduced clusters. The mean age of MSM at diagnosis increased by 0.45 years/year within clusters, but new clusters appeared with lower mean age. Major strengths of this study are the high proportion of HIV-positive MSM with a sequence in this study and the combined application of phylogenetic and modeling approaches. Main limitations are the assumption that the sampled population is representative of the overall HIV-positive population and the assumption that the diagnosis interval distribution is similar between clusters. \u3cbr/\u3eConclusions\u3cbr/\u3eThe resurgent HIV epidemic amongst MSM in the Netherlands is driven by several large, persistent, self-sustaining, and, in many cases, growing sub-epidemics shifting towards new generations of MSM. Many of the sub-epidemics have been present since the early epidemic, to which new sub-epidemics are being added.\u3cbr/\u3

Hemoglobin in samples with leukocytosis can be measured on ABL 700 series blood gas analyzers

To compare lactate, bilirubin and Hemoglobin F concentrations obtained on ABL 700 series blood gas analyzers with those from laboratory methods. Pooled neonatal plasma, cord blood and adult plasma samples were used for comparison of bilirubin, hemoglobin F and lactate concentrations respectively. Results obtained on the ABL 700 series compared favorably (Deming regression slopes 0.97-1.13) with those from laboratory methods. For lactate ABL (y) = 1.13 Vitros (x) -0.43 with a CI (slope) of 1.10 to 1.16, CI (int) of -0.61 to -0.28. For hemoglobin F ABL(y) = 1.11 Variant (x) -8.0 with a CI (slope) of 0.88 to 1.33, CI (int) of -25.3 to 9.3. The three bilirubin comparisons are as follows: 1) Unistat (y) = 1.10 Vitros (x) -16.12 with CI (slope) of 1.06 to 1.14 and CI (int) of -25.3 to -6.9. 2), ABL (y) = 0.97 Vitros(x) -10.16 with CI (slope) of 0.94 to 1.00 and CI (int) of -17.6 to 2.73) Unistat (y) = 1.14 (x)-4.58 with CI (slope) of 1.09 to 1.18 and CI (int) of -13.6 to 4.5. The ABL 700 series gave comparable results for lactate, bilirubin and hemoglobin F with laboratory methods and may be used in patient care

Mutation of FLT3 is not a general phenomenon in CD117-positive T-ALL

CD117 is considered to be a marker of leukemic cells committed to the myeloid lineage, however up to 11% of T-ALLs have been found to express CD117 [1]. Activating mutations in the FLT3 gene are common in acute myeloid leukemia (AML) but are rarely found in acute lymphoblastic leukemia (ALL) [2]. Recently, a subset (3 out of 55) of adult T-ALLs characterized by expression of CD117 (in >90% of T-lymphoblasts) and FLT3 mutations (either internal tandem duplications (ITD) in the juxtamembrane region or mutations in the activation-loop coding region) was described [3]. These data suggested that CD117 expression in T-ALL lymphoblasts might identify a subset of T-ALLs in which activating FLT3 mutations are essential in oncogenesis. If FLT3 mutations would be present in all CD117-positive T-ALLs, up to 11% of all T-ALL patients could potentially benefit from therapy with FLT3 inhibitors, which are currently under investigation for AML treatment [2] and [4]. We report here on the FLT3 mutation status of a 75-year-old man diagnosed with CD117-positive T-ALL. The patient presented with pancytopenia and anemia. Bone marrow analysis revealed 70% blasts with an L1 ALL morphology according to the French–American–British classification. There was no cytochemical evidence of myeloid differentiation, i.e. Sudan black B, specific and non-specific esterase stains were negative. Flowcytometry demonstrated 85% blasts, 9% T-lymphocytes, 1% B-lymphocytes, 2% granulocytes, and 90% of the blast cells were positive for CD117, CD2, CD7, CD13, CD45, and CD56, whereas CD34, CD33, CD5, and CD19 were expressed on a subset of blast cells only (about 75, 30, 30 and 40% of blasts, respectively). Blast cells did not significantly express TdT, MPO, CD1a, CD4, CD8, CD10, CD14, CD15, CD22, CD65, CD133 and SmCD3 (al

A multicenter evaluation of a point of care CRP Test

\u3cp\u3eBackground: Point-of-care (POC) C-reactive protein (CRP) testing in the primary healthcare setting is a cost-effective approach for reducing antibiotic prescriptions, but has yet to be widely adopted. Methods: Analytical performance of the cobas CRP Test on the cobas b 101 system was evaluated at three POC sites and one reference laboratory. Within-run (repeatability), within-laboratory (intermediate precision), and between-laboratory precision (reproducibility) were assessed. Method comparison (reference test: CRPNX reagent [cobas c 501 module]) and matrix/lot-to-lot comparison experiments were conducted using prospectively collected blood samples from 217 adults (apparently healthy or with clinically relevant conditions). Usability and reliability were assessed by questionnaire and error reporting. Results: Coefficients of variation (CV) for repeatability and intermediate precision ranged from 1.7%–4.0% and 1.9%–4.5%, respectively, for human serum pools containing CRP 4.7–350.7 mg/L; repeatability in clinical samples ranged from 1.6%–5.9% (3.3–360.3 mg/L). CVs for reproducibility ranged from 2.5%–4.0% (4.7–344.3 mg/L). CRP concentrations were comparable for capillary whole blood, serum, Li-heparin whole blood/plasma, K2 and K3 EDTA whole blood/plasma (Pearson's r ≥ 0.996), and among three CRP Test lots (r ≥ 0.993). Clinically relevant CRP concentrations measured with the CRP Test showed good agreement with those measured by CRPNX reagent (serum, weighted Deming regression y = 0.97× + 0.11; Pearson's r ≥ 0.996). The overall mean usability score was 4.18/5 and the error rate across 9378 tests was 1.00%. Conclusions: The cobas CRP Test on the cobas b 101 system demonstrates robust analytic performance when used by healthcare professionals in the POC setting.\u3c/p\u3