Eight-color immunophenotyping of T-, B-, and NK-cell subpopulations for characterization of chronic immunodeficiencies

Background The heterogeneity of primary and secondary immunodeficiencies demands for the development of a comprehensive flow cytometric screening system, based on reference values that support a standardized immunophenotypic characterization of most lymphocyte subpopulations. Methods Peripheral blood samples from healthy adult volunteers (nâ= â25) were collected and split into eight panel fractions (100 l each). Subsequently, premixed eight-color antibody cocktails were incubated per specific panel of whole blood to detect and differentiate cell subsets of: (i) a general lymphocyte overviews, (ii) B-cell subpopulations, (iii) CD4+ subpopulations, (iv) CD8+ subpopulations, (v) regulatory T-cells, (vi) recent thymic emigrants (RTE), (vii) NK-cell subpopulations, and (viii) NK-cell activation markers. All samples were lysed, washed, and measured by flow cytometry. FACS DIVA software was used for data analysis and calculation of quadrant statistics (mean values, standard er ror of mean, and percentile ranges). Results Whole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naïve, nonswitched/switched, memory, (activated) CD21low, transitional B-cells, plasmablasts/plasmacells; (iii and iv) naïve, central memory, effector, effector memory, TH1/TH2/TH17-like, and CCR5+CD8-cells; (v) CD25+, regulatory T-cells (naïve/memory, HLA-DR+); (vi) /- and /-T-cells, RTE in CD4/CD8 cells; (vii) immature/mature CD56 bright, CD94/NKG2D+ NK-cells; and (viii) Nkp30, 44, 46, and CD57+NK-cells. Clinical examples and quadrant statistics are provided. Conclusion The present study represents a practical approach to standardize the immunophenotyping of most T-, B-, and NK-cell subpopulations. That allows differentiating whether abnormalities or developmental shifts observed in lymphocyte subpopulations originates either from primary or secondary immunological disturbance

Detection of Aspergillus DNA in Cerebrospinal Fluid from Patients with Cerebral Aspergillosis by a Nested PCR Assay

Invasive aspergillosis (IA), a complication with high mortality rates, especially in disseminated IA with cerebral involvement, is difficult to diagnose. Biopsy of cerebral lesions is often not feasible, and culture of Aspergillus spp. from cerebrospinal fluid (CSF) is frequently negative. New molecular methods have emerged for diagnosing IA. So far, there are only few reports of Aspergillus DNA detection in CSF. After modifying the DNA extraction protocol, we detected Aspergillus DNA in CSF samples by a previously described nested PCR assay. In six patients with hematologic malignancy and cerebral aspergillosis, CSF samples were investigated for Aspergillus DNA. IA was classified according to the EORTC/MSG 2002 criteria. Two patients each had proven, probable, and possible IA. Thirty-five CSF samples were investigated for Aspergillus DNA by nested PCR. Samples with positive results in the nested PCR assay were quantified by LightCycler PCR assay. Fourteen CSF samples showed positive results in the nested PCR assay. Of these, six samples gave positive results in real-time PCR. The range of CFU per ml was 2,154 to 63,100,000. The highest number of CFU per ml was found in a CSF sample of a patient with acute lymphocytic leukemia and probable cerebral aspergillosis. Detection of Aspergillus DNA in CSF samples is thus possible and has the potential to improve diagnosis of cerebral aspergillosis. Further prospective studies with larger numbers of patients must be performed to evaluate the clinical significance of Aspergillus PCR with CSF samples

Diagnostic criteria for hematopoietic stem cell transplant-associated microangiopathy: results of a consensus process by an International Working Group

BACKGROUND AND OBJECTIVES: There are no widely accepted criteria for the definition of hematopoietic stem cell transplant -associated microangiopathy (TAM). An International Working Group was formed to develop a consensus formulation of criteria for diagnosing clinically significant TAM. DESIGN AND METHODS: The participants proposed a list of candidate criteria, selected those considered necessary, and ranked those considered optional to identify a core set of criteria. Three obligatory criteria and four optional criteria that ranked highest formed a core set. In an appropriateness panel process, the participants scored the diagnosis of 16 patient profiles as appropriate or not appropriate for TAM. Using the experts' ratings on the patient profiles as a gold standard, the sensitivity and specificity of 24 candidate definitions of the disorder developed from the core set of criteria were evaluated. A nominal group technique was used to facilitate consensus formation. The definition of TAM with the highest score formed the final PROPOSAL. RESULTS: The Working Group proposes that the diagnosis of TAM requires fulfilment of all of the following criteria: (i) >4% schistocytes in blood; (ii) de novo, prolonged or progressive thrombocytopenia (platelet count <50 x 109/L or 50% or greater reduction from previous counts); (iii) sudden and persistent increase in lactate dehydrogenase concentration; (iv) decrease in hemoglobin concentration or increased transfusion requirement; and (v) decrease in serum haptoglobin. The sensitivity and specificity of this definition exceed 80%. INTERPRETATION AND CONCLUSIONS: The Working Group recommends that the presented criteria of TAM be adopted in clinical use, especially in scientific trials

Ultrashort echo time MRI of the lung in children and adolescents: comparison with non-enhanced computed tomography and standard post-contrast T1w MRI sequences

Objectives!#!To compare the diagnostic value of ultrashort echo time (UTE) magnetic resonance imaging (MRI) for the lung versus the gold standard computed tomography (CT) and two T1-weighted MRI sequences in children.!##!Methods!#!Twenty-three patients with proven oncologic disease (14 male, 9 female; mean age 9.0 + / - 5.4 years) received 35 low-dose CT and MRI examinations of the lung. The MRI protocol (1.5-T) included the following post-contrast sequences: two-dimensional (2D) incoherent gradient echo (GRE; acquisition with breath-hold), 3D volume interpolated GRE (breath-hold), and 3D high-resolution radial UTE sequences (performed during free-breathing). Images were evaluated by considering image quality as well as distinct diagnosis of pulmonary nodules and parenchymal areal opacities with consideration of sizes and characterisations.!##!Results!#!The UTE technique showed significantly higher overall image quality, better sharpness, and fewer artefacts than both other sequences. On CT, 110 pulmonary nodules with a mean diameter of 4.9 + / - 2.9 mm were detected. UTE imaging resulted in a significantly higher detection rate compared to both other sequences (p &amp;lt; 0.01): 76.4% (84 of 110 nodules) for UTE versus 60.9% (67 of 110) for incoherent GRE and 62.7% (69 of 110) for volume interpolated GRE sequences. The detection of parenchymal areal opacities by the UTE technique was also significantly higher with a rate of 93.3% (42 of 45 opacities) versus 77.8% (35 of 45) for 2D GRE and 80.0% (36 of 45) for 3D GRE sequences (p &amp;lt; 0.05).!##!Conclusion!#!The UTE technique for lung MRI is favourable in children with generally high diagnostic performance compared to standard T1-weighted sequences as well as CT. Key Points • Due to the possible acquisition during free-breathing of the patients, the UTE MRI sequence for the lung is favourable in children. • The UTE technique reaches higher overall image quality, better sharpness, and lower artefacts, but not higher contrast compared to standard post-contrast T1-weighted sequences. • In comparison to the gold standard chest CT, the detection rate of small pulmonary nodules small nodules ≤ 4 mm and subtle parenchymal areal opacities is higher with the UTE imaging than standard T1-weighted sequences

Targeted busulfan-based reduced-intensity conditioning and HLA-matched HSCT cure hemophagocytic lymphohistiocytosis

Reduced-intensity/reduced-toxicity conditioning and allogeneic T-cell replete hematopoietic stem cell transplantation are curative in patients with hemophagocytic lymphohistiocytosis (HLH). Unstable donor chimerism (DC) and relapses are clinical challenges. We examined the effect of a reduced-intensity conditioning regimen based on targeted busulfan to enhance myeloid DC in HLH. The European Society for Bone and Marrow Transplantation-approved reduced-intensity conditioning protocol comprised targeted submyeloablative IV busulfan, IV fludarabine, and serotherapy comprising IV alemtuzumab (0.5-0.8 mg/kg) for unrelated-donor and IV rabbit anti-T-cell globulin for related-donor transplants. We assessed toxicity, engraftment, graft-versus-host disease (GHVD), DC in blood cell subtypes, and overall survival/event-free survival. Twenty-five patients from 7 centers were treated (median age, 0.68 year). The median total dose and cumulative area under the curve of busulfan was 13.1 mg/kg (6.4-26.4) and 63.1 mg/L x h (48-77), respectively. Bone marrow, peripheral blood stem cell, or cord blood transplants from HLA-matched related (n = 7) or unrelated (n = 18) donors were administered. Donor cells engrafted in all patients (median: neutrophils d+20/platelets d128). At last follow-up (median, 36 months; range, 8-111 months), the median DC of CD151 neutrophils, CD3(+) T cells, and CD16(+)56(+) natural killer cells was 99.5% (10-100), 97% (30-100), and 97.5% (30-100), respectively. Eight patients (32%) developed sinusoidal obstruction syndrome, resolving after defibrotide treatment. The 3-year overall survival and event-free survival rates were both 100%. None of the patients developed acute grade III to IV GHVD. Limited chronic GVHD was encountered in 4%. This regimen achieves excellent results with stable DC in patients with HLH.Transplantation and immunomodulatio