Klara Dalva

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

In multiple myeloma, the use of multiparametric flow cytometry in many laboratories is currently restricted to clinical research studies and the differential diagnosis of unusual cases. This article report the indications of the European Myeloma Network for flow cytometry in patients with monoclonal gammopathies, and the technical recommendations for the analysis of plasma cells. The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

The Shaping of Modern Human Immune Systems by Multiregional Admixture with Archaic Humans

Viral defense and embryo implantation mechanisms have been shaped by contributions from Neandertal and Denisovan genes. Whole genome comparisons identified introgression from archaic to modern humans. Our analysis of highly polymorphic human leukocyte antigen (HLA) class I, vital immune system components subject to strong balancing selection, shows how modern humans acquired the HLA-B*73 allele in west Asia through admixture with archaic humans called Denisovans, a likely sister group to the Neandertals. Virtual genotyping of Denisovan and Neandertal genomes identified archaic HLA haplotypes carrying functionally distinctive alleles that have introgressed into modern Eurasian and Oceanian populations. These alleles, of which several encode unique or strong ligands for natural killer cell receptors, now represent more than half the HLA alleles of modern Eurasians and also appear to have been later introduced into Africans. Thus, adaptive introgression of archaic alleles has significantly shaped modern human immune systems.

Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders

The European Myeloma Network has organized two workshops on fluorescence in situ hybridization in multiple myeloma. The first aimed to identify specific indications and consensus technical approaches of current practice. A second workshop followed a quality control exercise in which 21 laboratories analyzed diagnostic cases of purified plasma cells for recurrent abnormalities. The summary report was discussed at the EHA Myeloma Scientific Working Group Meeting 2010. During the quality control exercise, there was acceptable agreement on more than 1,000 tests. The conclusions from the exercise were that the primary clinical applications for FISH analysis were for newly diagnosed cases of MM or frank relapse cases. A range of technical recommendations included: 1) material should be part of the first draw of the aspirate; 2) samples should be sent at suitable times to allow for the lengthy processing procedure; 3) most importantly, PCs must be purified or specifically identified; 4) positive cut-off levels should be relatively conservative: 10% for fusion or break-apart probes, 20% for numerical abnormalities; 5) informative probes should be combined to best effect; 6) in specialist laboratories, a single experienced analyst is considered adequate; 7) at least 100 PC should be scored; 8) essential abnormalities to test for are t(4;14), t(14;16) and 17p13 deletions; 9) suitable commercial probes should be available for clinically relevant abnormalities; 10) the clinical report should be expressed clearly and must state the percentage of PC involved and the method used for identification; 11) a retrospective European based FISH data bank linked to clinical data should be generated; and 12) prospective analysis should be centralized for upcoming trials based on the recommendations made. The European Myeloma Network aims to build on these recommendations to establish standards for a common European data base to define subgroups with prognostic significance.

Thrombopoietic cytokines in patients with iron deficiency anemia with or without thrombocytosis.

Iron deficiency anemia is a cause of reactive thrombocytosis. A moderate increase in platelet numbers is common but sometimes counts may exceed 1,000 × 109/l. The mechanisms causing reactive thrombocytosis are unclear. In this study, we evaluated 15 women with iron deficiency anemia and thrombocytosis (platelets >450 × 109/l) and 16 women with iron deficiency anemia with normal platelet counts. Serum samples were taken before oral iron replacement therapy, after 1 and 3 months and at the end of replacement therapy. Thrombopoietin, erythropoietin (EPO), leukemia inhibitory factor, interleukin-6 and interleukin-11 levels were assayed. There was no change in the levels of thrombopoietic cytokines except for EPO. The correlation between high EPO levels and high platelet counts may suggest that EPO increases platelet counts, but the same EPO level changes can also be demonstrated in women with iron deficiency anemia but normal initial platelet counts. The fact that the levels of other cytokines remained unchanged during treatment suggests that either these cytokines have no effect on reactive thrombocytosis or the change in platelet counts in our patients is in a narrow range and is thus not affected by the cytokine levels.

Effects of Combined Conventional and Modified Ultrafiltration in Adult Patients

BACKGROUNDnModified ultrafiltration (MUF) improves hemodynamics and postoperative recovery in children. Ultrafiltration (UF) may have similar benefits in adults. The purpose of this study was to investigate the effects of UF in adult patients.nnnMETHODSnA total of 40 adult patients undergoing cardiac surgery were randomized into a study group of conventional UF during bypass + venovenous MUF after bypass and a control group with no UF. Perioperative clinical variables, cytokines, and endothelin-1 levels were compared between groups.nnnRESULTSnThere was no mortality in either group. The patients in the study group had a greater rise in hematocrit (5.7% +/- 2.4% vs 1.2% +/- 1.9%, p < 0.001), hemoglobin (1.7 +/- 0.8 mg/mL vs 0.5 +/- 0.6 mg/mL, p < 0.0005), and platelet levels (27,800 +/- 29,200 vs -9,000 +/- 30,970, p < 0.001). Mean arterial blood pressure and CI increased after MUF (from 64.2 +/- 16.9 mm Hg to 72.3 +/- 14.1 mm Hg, p = 0.05, and from 2.4 +/- 0.7 to 2.8 +/- 0.6, p < 0.03, respectively). Postoperative oxygenation was better in the study group (alveolo-arterial PO2 tension gradient 74.6 +/- 43.9 mm Hg vs 107.2 +/- 27.8 mm Hg, p = 0.03). Ultrafiltration reduced postoperative bleeding (522.2 +/- 233.4 mL vs 740 +/- 198.4 mL, p < 0.003).nnnCONCLUSIONSnA combination of conventional and modified UF is effective and safe in adult patients undergoing cardiac surgery. Ultrafiltration improved hemodynamics, hemostatic, and pulmonary functions. We recommend the use of combined UF in high-risk adult patients.

Effects of Preoperative Short Term Use of Atorvastatin on Endothelial Progenitor Cells after Coronary Surgery: A Randomized, Controlled Trial

ObjectivesWe investigated the effects of short-term use of atorvastatin on CD34+/VEGF-R2+/CD133+/CD45- endothelial progenitor cell (EPC) count after on-pump coronary artery bypass surgery (CABG).MethodsBetween Feb-2010 and May-2010, we randomly assigned, in a placebo-controlled, double-blind study, 60 consecutive patients who underwent isolated, first-time CABG to receive either 14-day atorvastatin (40xa0mg/day) or placebo preoperatively. Urgent CABG and recent myocardial infarction were excluded. EPCs were quantified (cells/μl) by flow cytometric phenotyping obtained from venous blood samples collected preoperatively (T1), 6-hours (T2), and on the 5th day postoperatively (T3). Levels of markers of inflammation and serum cardiac troponin I were also measured preoperatively and daily until day-5 after surgery.ResultsThere were no differences in baseline risk factors including cholesterol profiles, and EuroSCORES between the groups. The composite primary end-point, favored statin group with higher amount of circulating, early EPC count (cells/μl) at all time points compared with placebo (T1, 2.30u2009±u20090.02 versus 1.58u2009±u20090.03, pu2009<u20090.001; T2, 5.00u2009±u20090.06 versus 2.19u2009±u20090.06, pu2009<u20090.001; T3, 3.03u2009±u20090.08 versus 1.78u2009±u20090.02, pu2009<u20090.001). Postoperative hsCRP rise were inversely correlated with EPC count, and were significantly lower in the statin group (T1, 0.8u2009±u20090.1 versus 2.2u2009±u20091.5, pu2009<u20090.001; T2, 72.9u2009±u20093.2 versus 96.0u2009±u20093.6, pu2009<u20090.001; T3, 4.3u2009±u20091.2 versus 11.4u2009±u20094.1, pu2009<u20090.001). Furthermore, the incidence of postoperative atrial fibrillation was significantly lower in the statin group compared to placebo (3.3% versus 23%, pu2009=u20090.02).ConclusionsShort-term atorvastatin use increases circulating early EPCs both pre- and post-operatively and is associated with better preservation of sinus rhythm and reduced hsCRP levels. (ClinicalTrials.gov number, NCT01096875)

HLA typing with sequence-specific oligonucleotide primed PCR (PCR-SSO) and use of the Luminex™ technology.

The hybridization products obtained by PCR using sequence-specific oligonucleotides (PCR-SSO) can be traced either by colorimetric- (streptavidin- biotin), X-ray-(digoxigenin-CSPD), or fluorescence- (FITC, PE) based detection systems. To achieve a faster, reliable, automated typing, microbead and fluorescence detection technology have been combined and introduced to this field (XMAP technology). For each locus, a maximum of 100 microspheres, which are recognizable by their specific color originating from two internal fluorescent dyes, are used. Each microsphere is coupled with a single probe that is capable of hybridizing with the biotin labeled complementary amplicon. Once hybridization occurs, it can be quantified via the fluorescence signal originating from fluorescently (Streptavidin-PE) labeled amplicons captured by the beads. Currently, there are two commercially available systems that differ in the scale of probes and the method of amplification or denaturation. One of these will be described in detail in this chapter.

Stem cell therapy in spinal cord injury: in vivo and postmortem tracking of bone marrow mononuclear or mesenchymal stem cells.

ObjectiveThe aim of this study was to address the question of whether bone marrow-originated mononuclear cells (MNC) or mesenchymal stem cells (MSC) induce neural regeneration when implanted intraspinally.Materials and MethodsThe study design included 4 groups of mice: Group 1, non-traumatized control group; Groups 2, 3 and 4 spinal cord traumatized mice with 1xa0g force Tator clips, which received intralesionally either no cellular implants (Group 2), luciferase (Luc) (+) MNC (Group 3) or MSC (Group 4) obtained from CMV-Luc or beta-actin Luc donor transgenic mice. Following the surgery until decapitation, periodical radioluminescence imaging (RLI) and Basso Mouse Scale (BMS) evaluations was performed to monitor neural activity. Postmortem immunohistochemical techniques were used to analyze the fate of donor type implanted cells.ResultsAll mice of Groups 3 and 4 showed various degrees of improvement in the BMS scores, whereas there was no change in Groups 1 and 2. The functional improvement was significantly better in Group 4 compared to Group 3 (18 vs 8, pu2009=u20090.002). The immunohistochemical staining demonstrated GFP+Luc+ neuronal/glial cells that were also positive with one or more of these markers: nestin, myelin associated glycoprotein, microtubule associated protein or myelin oligodendrocyte specific protein, which is considered as indicator of donor type neuronal regeneration. Frequency of donor type neuronal cells; Luc + signals and median BMS scores were observed 48–64xa0% and 68–72xa0%; 44–80xa0%; 8 and 18 within Groups III and IV respectively.DiscussionMSCs were more effective than MNC in obtaining neuronal recovery. Substantial but incomplete functional improvement was associated with donor type in vivo imaging signals more frequently than the number of neuronal cells expressing donor markers in spinal cord sections in vitro. Our results are in favor of functional recovery arising from both donor MSC and MNCs, contributing to direct neuronal regeneration and additional indirect mechanisms.

Screening for hemochromatosis in Turkey.

In this study we screened 3060 consecutive blood donors for an unbound iron-binding capacity level of <28 μM and then performed HFE mutation analysis in these subjects. Sixty-five of the 75 subjects with a low initial unbound iron-binding capacity (all had normal ferritin levels) came back and only 5 (8%) had a low fasting unbound iron-binding capacity. Mutational analysis revealed H63D heterozygosity in two of five subjects. Four of five subjects had liver biopsy indication and none had increased liver iron. HFE genotyping of 60 subjects with a low initial but normal fasting unbound iron-binding capacity revealed heterozygote H63D in seven (11.6%). No allelic variant of position 282 or 63 was found in three previously diagnosed patients with hereditary hemochromatosis. In conclusion, full phenotypic expression of hereditary hemochromatosis is very rare in Turkey. The absence of HFE mutations in three patients with hereditary hemochromatosis suggests that hereditary hemochromatosis in Turkey occurs without common HFE mutations.

Role of Killer Immunoglobulin-Like Receptor and Ligand Matching in Donor Selection

Despite all efforts to improve HLA typing and immunosuppression, it is still impossible to prevent severe graft versus host disease (GVHD) which can be fatal. GVHD is not always associated with graft versus malignancy and can prevent stem cell transplantation from reaching its goals. Overall T-cell alloreactivity is not the sole mechanism modulating the immune defense. Innate immune system has its own antigens, ligands, and mediators. The bridge between HLA and natural killer (NK) cell-mediated reactions is becoming better understood in the context of stem cell transplantation. Killer immunoglobulin-like receptors (KIRs) constitute a wide range of alleles/antigens segregated independently from the HLA alleles and classified into two major haplotypes which imprints the persons ability to suppress or to amplify T-cell alloreactivity. This paper will summarize the impact of both activating and inhibitory KIRs and their ligands on stem cell transplantation outcome. The ultimate goal is to develop algorithms based on KIR profiles to select donors with maximum antileukemic and minimum antihost effects.