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BACKGROUND: Autoimmune neutropenia of infancy (AIN) is a frequent cause of neutropenia in children. The disease is caused by antibodies against epitopes on the immunoglobulin G (IgG) Fc receptor type 3b (FcγIIIb). We investigated the possible association of human neutrophil antigens (HNA), human leukocyte antigen (HLA)-DR, and HLA-DQ alleles with AIN and the association of these genotypes with the presence of autoantibodies.METHODS: Eighty AIN cases with a median age of 13.5 months were included. Controls were healthy unrelated Danish blood donors. Anti-HNA-1a autoantibodies were detected using a flow cytometric granulocyte immunofluorescence test (Flow-GIFT) with phenotyped donor cells for detection of antibody specificity. Molecular determination of HNA genotypes was determined using real-time polymerase chain reaction (q-PCR). High-resolution HLA-DRB1 and HLA-DQB1 were determined by next-generation sequencing.RESULTS: Antibodies against HNA-1a were detected in 51% (n = 41) of AIN patients, and anti-HNA-1b was detected in 3% (n = 2) of cases. In 46% of cases, the antibodies were anti-FcγIIIb-reactive. FCGR3B*01+,*02-,*03- was more common (odds ratio, 6.70; P < .0001), and FCGR3B*01-,*02+,*03- was less common (odds ratio, 0.30; P < .0001) among AIN cases. HNA-1a antibodies were significantly more frequent among AIN cases with the FCGR3B*01+,*02-,*03- genotype (odds ratio, 3.86; P < .007). The HLA-DRB1*14 - HLA-DQB1*05:03 haplotype was significantly more common (odds ratio, 7.44; P < .0001) in AIN patients.CONCLUSION: The HLA haplotype HLA-DRB1*14 - DQB1*05:03 is associated with Danish AIN cases. Among Danish AIN patients, anti-HNA-1a is the most common autoantibody, and the antibody is more common in cases with the FCGR3B*01+,*02-,*03- genotype.

Clonal hematopoiesis of indeterminate potential (CHIP) is suspected of being a risk factor for patients with cancer. This study aimed to assess the clinical consequences of CHIP in patients with lymphoma intended for high-dose chemotherapy and autologous stem-cell transplantation (ASCT) in a population-based setting. We identified 892 lymphoma patients who had undergone stem cell harvest at all transplant centers in Denmark. A total of 565 patients had an available harvest sample, which was analysed for CHIP by next-generation sequencing, and the median follow-up was 9.1 years. Of the patients who were intended for immediate ASCT, 25.5% (112/440) carried at least one CHIP mutation. In contrast to previous single-center studies CHIP was not associated with inferior overall survival (OS) in multivariate analyses. However, patients with mutations in genes of the DNA repair pathway (PPM1D, TP53, RAD21, BRCC3) had a significant inferior OS (HR after 1 year of follow-up 2.79, 95% confidence interval 1.71–4.56; p < 0.0001), which also was evident in multivariate analysis (p = 0.00067). These patients had also increased rates of therapy-related leukemia and admission to intensive care. Furthermore, in patients who did not undergo immediate ASCT, a significant inferior OS of individuals with DNA repair mutations was also identified (p = 0.003).

The advent of novel immunotherapy (CAR-T cell therapy, bispecific CD20×CD3 antibodies) have highlighted the importance of T-cells in the treatment of lymphoma. However, overall T-cell characteristics have not been properly examined in patients receiving conventional chemotherapy. Next-generation sequencing (NGS) of the T-cell receptor (TCR) has enabled the possibility of identifying hundred thousands of unique T-cell clones in a single patient sample. Here we analyzed the impact of systemic TCR diversity and T-cell clonotypes in patients with Non-Hodgkin lymphoma (NHL) and Hodgkin-lymphoma (HL) receiving high-dose chemotherapy with stem cell support (HDT/ASCT). Autologous peripheral blood stem cell harvest samples from patients with lymphoma (predominantly B-cell NHL) were collected as part of a national population-based study (Husby et al. - Leukemia 2020). We performed high-throughput RNA-based sequencing of the V, D and J segment of the TCR β-chain to identify unique clonal rearrangements. To ensure supreme quality for TCR repertoire calculations, samples with less than 100.000 aligned reads to the TCR β chain were omitted from further analysis. By using the MiXCR bioinformatic pipeline we analyzed the number of unique clonotypes and TCR repertoire diversity, as calculated by the Simpson index. T-cell clonotype and diversity were for categorical analyses split in two groups by the median, respectively. A total of 96 patients with lymphoma who were intended for HDT/ASCT were included and analyzed for TCR characteristics. In brief, median age was 56 years, 64% were male and major subtypes were diffuse large B-cell lymphoma (37%), follicular lymphoma (24%), Hodgkin lymphoma (16%), and mantle cell lymphoma (14%). Median follow-up time was 6.7 years. Number of unique T-cell clonotypes was not associated with age (Fig. 1A), but low levels were highly associated with inferior survival (Fig. 1B, p=0.008), especially in the first year of follow-up. In contrast, elderly patients had a trend toward lower TCR diversity (Fig. 1C, p=0.08), but this did not impact overall survival (Fig. 1D). Low T-cell clonotype levels was also significantly associated with presence of clonal hematopoiesis (Fig. 1E, p=0.033). No association with clonal hematopoiesis was found with regard to TCR diversity (Fig. 1F). Furthermore, we investigated TCR repertoire in relation to subsequent severe infections (defined as sepsis, pneumonia, or invasive fungal infection). Number of unique T-cell clonotypes did not have an impact (Fig. 1F), but remarkably patients with a high T-cell diversity had significant increased incidence of severe infections in the first 500 days after sampling (Fig. 1G, p=0.029). This implies that patients who have a high T-cell diversity before high-dose chemotherapy, are more capable of mounting an immune response against infectious pathogens. These findings should be validated in larger homogenous cohorts. However, they imply the importance of inherent immune characteristics in patients with lymphoma. Although the immune response is exceedingly complex, we have identified systemic T-cell characteristics that associate with several important clinical variables. Assessment of systemic immunological parameters in patients with aggressive lymphoma may in the future inform on choice of optimal personalized therapy. Figure 1 Figure 1. Disclosures El-Galaly: ROCHE Ltd: Ended employment in the past 24 months; Abbvie: Other: Speakers fee. Larsen: Odense University Hospital, Denmark: Current Employment; Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Gilead: Consultancy.

Stringent complete remission (sCR) of acute myeloid leukemia is defined as normal hematopoiesis after therapy. Less sCR, including non-sCR, was introduced as insufficient blood platelet, neutrophil, or erythrocyte recovery. These latter characteristics were defined retrospectively as postremission transfusion dependency and were suggested to be of prognostic value. In the present report, we evaluated the prognostic impact of achieving sCR and non-sCR in the Danish National Acute Leukaemia Registry, including 769 patients registered with classical CR (ie

Background: Somatic driver mutations in hematopoietic cells may lead to clonal hematopoiesis of indeterminate potential (CHIP). In patients with lymphoma CHIP has been associated with increased risk of therapy-related myeloid neoplasms (tMN) and inferior survival after autologous stem cell transplantation as demonstrated in a large single center study and in a case-control study (Gibson CJ et al., JCO 2017 and Berger G et al., Blood 2018). Here, we investigated the clinical impact of clonal hematopoiesis in a nation-wide population-based cohort of Danish lymphoma patients undergoing autologous transplant with prospective data from four national patient registries. Methods: Patients with lymphoma who had undergone leukapheresis at all danish transplant centers from 2000 to 2012 were identified. DNA and RNA was extracted from mobilized peripheral blood products. Targeted sequencing of all samples was performed using an Illumina TruSeq Custom Amplicon panel (Illumina, San Diego, CA, USA) designed to cover >95% of mutations associated with CHIP (ASXL1, ASXL2, BCOR, BRCC3, CBL, CREBBP, DNMT3A, ETV6, GNB1, IDH1, IDH2, JAK2, KRAS, NRAS, PPM1D, RAD21, SF3B1, SRSF2, TET2, TP53). To allow detection of low-level mutations and secure variant calling, unique molecular identifiers (UMI's) were used. Filtering of variants was done by stringent criteria consistent with earlier studies. Assessment of mutations was performed blinded to the patients' clinical data. Prospective clinical patient data was obtained for all patients from four national registries, including the Danish Lymphoma Registry (diagnosis, involvement, lymphoma treatment, relapse and death), the Danish National Patient Registry (hospital admission diagnoses and treatments), the Danish Cancer Registry (primary and secondary cancer diagnoses) and the Danish Pathology Database (histopathological examinations and diagnoses), respectively. Results: Samples from 574 patients were included. The median age was 55.5 years (IQR: 45.3 - 62.2) and the median follow-up time for survivors was 9.2 years (IQR: 7.1 - 11.2). The lymphoma subtypes were typical of patients selected for autologous transplantation; diffuse large B-cell lymphoma (191 pts), follicular lymphoma (102 pts), mantle cell lymphoma (88 pts), Hodgkin's lymphoma (80 pts), peripheral T-cell lymphoma (77 pts) and other histologies (36 pts). Of the 574 patients analyzed, 191 (33.3%) of the patients had somatic mutations meeting CHIP criteria (total mutations called=210). The most commonly mutated genes were DNMT3A (n=59, 28%), TET2 (n=48, 23%), PPM1D (n=34, 16%), ASXL1 (n=21, 10%) and TP53 (n=18, 8%). As expected CHIP mutations were more frequent in patients above 60 years (p=0.002). Prevalence of CHIP was associated with an inferior overall survival (p=0.004) and event-free survival (p=0.03). It was also associated with increased risk of biopsy-confirmed tMN (p=0.03) and higher probability of receiving blood transfusions after autologous transplant (p=0.027). Especially patients with mutations in DNA damage response genes PPM1D and TP53 (found in 48 pts, 8.3%) had a significantly increased risk of adverse outcomes. Both overall survival and event-free survival were significantly poorer with the presence of DNA damage pathway mutations (p The impact of low-level mutations and statistical modelling of interactions between parallel outcomes will be presented at the meeting. Conclusion: To our knowledge this is the first population-based study of clonal hematopoiesis in patients with lymphoma. We find that CHIP and particularly mutations in DNA damage response genes (PPM1D/TP53) are associated with increased mortality, which confirms findings from single center studies. These data support the evaluation of CHIP for risk assessment in lymphoma patients before high-dose chemotherapy. Our study also identifies increased rates of several clinically relevant adverse outcomes (severe infections, blood transfusions and secondary cancers) in lymphoma patients with clonal hematopoiesis. Figure 1. Figure 1. Disclosures Grønbæk: Janssen Pharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees.

BACKGROUND: Allogeneic red blood cell (RBC) transfusion has been associated with new-onset postoperative atrial fibrillation (POAF) following cardiac surgery. Prolonged storage time of RBC may increase the risk. The primary aim of the study was to evaluate whether the storage time of RBC is associated with development of POAF.MATERIALS AND METHODS: Pre-, per- and postoperative data were retrieved from the Western Denmark Heart Registry and local blood banks regarding patients who underwent coronary artery bypass surgery, valve surgery or combined procedures in Aalborg or Aarhus University Hospital during 2010-2014. Multiple logistic regression was used to determine the risk of POAF according to transfusion of RBC on the day of surgery. Furthermore, we determined trend in storage time of RBC according to risk of POAF using restricted cubic splines. Patients with a history of preoperative atrial fibrillation, patients who received transfusions preoperative and patients who died at the day of surgery were among excluded patients.RESULTS: A total of 2,978 patients with a mean age of 66.4 years were included and 609 patients (21%) received RBC transfusion on the day of surgery. POAF developed in 752 patients (25%) and transfused patients were at an increased risk compared with non-transfused patients (adjusted Odds Ratios for patients receiving RBC: 1.37; 95% CI: 1.11-1.69, P-value = 0.004). However, RBC transfusion was not necessarily the cause of POAF and may only be a marker for development of POAF. There was no significant association between storage time of RBC and POAF.CONCLUSIONS: In contrast to intraoperative allogeneic RBC transfusion in general, increased storage time of RBC is not associated with development of POAF in cardiac surgery.

The origin of multiple myeloma depends on interactions with stromal cells in the course of normal B-cell differentiation and evolution of immunity. The concept of the present study is that genes involved in MM pathogenesis, such as immune response genes, can be identified by screening for single-nucleotide polymorphisms (SNPs) involved in the immune response and a subsequent statistical analysis that focusses on the association of SNPs, certain haplotypes or SNP-SNP interactions with MM risk and prognosis. We genotyped 348 Danish patients and 355 controls for 13 SNPs located in the TNFA, IL-4, IL-6, IL-10 and CHI3L1 gene promoters. The occurrence of single polymorphisms, haplotypes and SNP-SNP interactions were statistically analyzed for association with disease risk and outcome following high-dose therapy. Identified genes that carried SNPs or haplotypes that were identified as risk or prognostic factors were studied for expression in normal B-cell subsets and myeloma plasma cells. We observed a significantly reduced risk when harboring the TNFA-238A allele (OR = 0.51 (0.29-0.86)) and interactions between the TNFA-1031T/C * and IL-10 -3575T/A (p = .007) as well as the TNFA-308G/A * and IL-10-1082G/A (p = .008) allels. By statistical approaches, we observed association between prognosis and the TNFA-857CC genotype (HR = 2.80 (1.29-6.10)) and IL-10-1082GG + GA genotypes (HR = 1.93 (1.07-3.49)) and interactions between IL-6-174G/C and IL-10-3575T/A (p = .001) and between TNFA-308G/A and IL-4-1098T/G (p= .005). The 'risk genes' were analyzed for expression in normal B-cell subsets (N = 6) from seven healthy donors and we found TNFA and IL-6 expressed both in naïve and in memory B cells when compared to preBI, II, immature and plasma cells. The 'prognosis genes' CHI3L1, IL-6 and IL-10 were differential expressed in malignant plasma cells when comparing poor and good prognosis groups based on to the TC classification. In summary, these findings suggest that TNFA, IL-4, IL-6, IL-10 and CHI3L1 might be important players in MM pathogenesis during disease initiation and drug resistance in multiple myeloma.

Objective Prenatal and postnatal RhD prophylaxis reduces the risk of RhD immunization in pregnancies of RhDnegative women. Based on the result from prenatal screening for the fetal RHD gene, prenatal RhD prophylaxis in Denmark is targeted to RhD-negative women who carry an RhD-positive fetus. Here, we present a 2-year evaluation of a nationwide prenatal RHD screening. Methods Blood samples were drawn from RhD-negative women in gestational week 25. DNA was extracted from maternal plasma and analyzed for the RHD gene. The prenatal RHD results were compared with the serological typing of newborns in 12,668 pregnancies. Early compliance was assessed for 690 pregnancies. Results The sensitivity for the detection of fetal RHD was 99.9% (95% CI: 99.7–99.9%). Unnecessary recommendation of prenatal RhD prophylaxis was avoided in 97.3% of the women carrying an RhD-negative fetus. Fetuses that were seropositive for RhD were not detected in 11 pregnancies (0.087%). The sample uptake percentage was 84.2%, and the compliance for prenatal anti-D administration was 93.2%. Conclusion The high sensitivity, maintained over 2years, underlines the reliability of routine prenatal fetal RHD screening in RhD-negative pregnant women, specifically at 25weeks of gestation. The remaining challenges are logistical and are related to program compliance. © 2014 John Wiley & Sons, Ltd.

The rare memory B-cells in thymus are considered the cell of origin for primary mediastinal large B-cell lymphoma (PMBL). The goals for the present study were to characterize the normal memory B-cell compartment in thymus and support its association to primary mediastinal B-cell lymphoma. Seven paired human tissue samples from thymus and sternum bone marrow were harvested during cardiac surgery. B-cell subsets were phenotyped by Euroflow standard and FACS-sorted for microarray analysis on the Human Exon 1.0 ST Arrays platform. Differentially expressed genes between thymus and bone marrow memory B-cells were identified and correlated to the molecular subclasses of diffuse large B-cell lymphoma. Within thymus, 4% (median, range 2-14%) of the CD45(+) haematopoietic cells were CD19(+) B-cells with a major fraction being CD27(+)/CD38(-) memory B-cells (median 80%, range 76-93%). The bone marrow contained 14% (median, range 3-27%) of which only a minor fraction (median 5%, range 2-10%) was memory B-cells. Global gene expression analysis of the memory B-cell subsets from the two compartments identified 133 genes up-regulated in thymus, including AICDA, REL, STAT1, TNF family, SLAMF1, CD80 and CD86. In addition, Exon 4 and 5 in the 3`end of AICDA was significantly higher expressed in thymus compared to bone marrow. The thymus memory B-cell gene profile was over-expressed in primary mediastinal B-cell lymphoma when compared to other DLBCL subclasses. The present study describes a thymus memory B-cell subset and its gene profile correlated to primary mediastinal B-cell lymphomas, supporting that it may arise from thymus memory B-cells.

In this study, we report a novel real time polymerase chain reaction (Q-PCR) method using TaqMan probes for human neutrophil antigens (HNA)-1, -3, -4, and -5 genotyping. The method was validated in a Caucasian Danish population, a Zambian population, and in clinical samples using three different methods: an in-house polymerase chain reaction with sequence-specific primers (PCR-SSP) method, a commercial available PCR-SSP kit and a novel Q-PCR method. We observed no discrepancy in the genotype frequencies determined by the PCR-SSP methods and the TaqMan assay in the populations studied. In tests of a family of Nigerian origin and in samples carrying the rare SLC44A2*1:2 genotype, different results were produced by the commercial PCR-SSP kit and the real-time TaqMan assay. The TaqMan-based genotyping method was rapid and reproducible, allowing high-throughput HNA-1, -3, -4, and -5 genotyping.

Neutrophil antigens are implicated in a variety of clinical conditions, including neonatal immune neutropenia, transfusion-related acute lung injury, refractoriness to granulocyte transfusions, febrile transfusion reactions, and autoimmune neutropenia. In this report, we describe simultaneous genotyping of human neutrophil antigens (HNA)-1, -3, -4, and -5 using PCR with allele-specific TaqMan probes and end-point fluorescence detection, which is a robust, rapid, and reproducible method, allowing for high-throughput genotyping.

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) both depend on immune mediated survival and proliferation signals from the tumour microenvironment. Inherited genetic variation influences this complex interaction. Eighty-nine studies investigating immune-response genes in DLBCL and FL were critically reviewed. Relatively consistent association exists for variation in the tumour necrosis factor alpha (TNFA) and interleukin-10 loci and DLBCL risk; for DLBCL outcome association with the TNFA locus exists. Variations at chromosome 6p31-32 were associated with FL risk. Most important, individual risk alleles have been shown to interact with each other. We suggest that the pathogenetic impact of polymorphic genes should include gene-gene interaction analysis and should be validated in preclinical model systems of normal B lymphopoiesis and B-cell malignancies. In the future, large cohort studies of interactions and genome-wide association studies are needed to extend the present findings and explore new risk alleles to be studied in preclinical models.

Neutrophil antigens are implicated in a variety of clinical conditions, including neonatal immune neutropenia, transfusion-related acute lung injury, refractoriness to granulocyte transfusions, febrile transfusion reactions, and autoimmune neutropenia. In this report, we describe simultaneous genotyping of human neutrophil antigens (HNA)-1, -3, -4, and -5 using PCR with allele-specific TaqMan probes and end-point fluorescence detection, which is a robust, rapid, and reproducible method, allowing for high-throughput genotyping.

High-dose therapy (HDT) followed by autologous stem cell transplant (ASCT) has been adopted as the standard second-line approach for patients with relapsed chemosensitive aggressive non-Hodgkin lym...

Background Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. Methods We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B-cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that “overall, there are no phenotypic differences between cryopreserved and fresh B-cell subsets.” Subsequently, we performed an uncontrolled comparison of tonsil tissue samples. Results By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3, respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B-cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. Conclusions We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B-cell hierarchy; however, storage procedures need to be delineated by tissue-specific comparative analysis. © 2014 International Clinical Cytometry Society

Transfusion-related acute lung injury (TRALI) is recognized as the most frequent cause of transfusion-related severe morbidity and mortality. TRALI is characterized by post-transfusional respiratory distress, hypoxaemia and radiographic verified lung infiltration, in the absence of sign of circulatory overload. TRALI is predominantly triggered by human leukocyte antigen or human neutrophil antigen (HNA) antibodies from the transfused blood component. Particularly antibodies against the HNA-3a are involved in severe and fatal TRALI cases. The serological investigation is important to trace and exclude blood donors with TRALI antibodies.

Transfusion-related acute lung injury (TRALI) is recognized as the most frequent cause of transfusion-related severe morbidity and mortality. TRALI is characterized by post-transfusional respiratory distress, hypoxaemia and radiographic verified lung infiltration, in the absence of sign of circulatory overload. TRALI is predominantly triggered by human leukocyte antigen or human neutrophil antigen (HNA) antibodies from the transfused blood component. Particularly antibodies against the HNA-3a are involved in severe and fatal TRALI cases. The serological investigation is important to trace and exclude blood donors with TRALI antibodies.

Background Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. Methods We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B-cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that "overall, there are phenotypic differences between cryopreserved and fresh B-cell subsets". Subsequently, we performed a consecutive uncontrolled comparison of tonsil tissue samples. Results By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3, respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B-cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. Conclusions We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B-cell hierarchy; however, storage procedures need to be delineated by tissue specific comparative analysis. © 2014 Clinical Cytometry Society.

Following two workshops on standardization of enumeration of CD34+ cells in blood and leukapheresis products, the Nordic Stem Cell Laboratory Group (NSCL-G) evaluated the Milan/Mulhouse/Nordic standard in clinical practice during the third workshop (WS-III). This report documents an acceptable interlaboratory variation in the most clinically active laboratories, with a coefficient of variation (CV) below 0.19 in 7 of 8 analyses performed. The introduction of a pan-CD45 antibody in the analysis did not improve the CV. Comparison of two different CD34 class II antibodies on a total of 99 samples and procedures with and without washing on a total of 96 samples revealed a significant correlation (r2 > 0.99) for all analyses. Finally, subset analysis of uncommitted and lineage-specific progenitors revealed major gating difficulties, indicating that further improvements are necessary. In an analysis of more than 600 patients undergoing mobilization and harvest of blood progenitors, with about 500 patients autog...

SummaryThird generation oral contraceptives (OCs) are apparently stronger risk factors for venous thromboembolism (VTE) than other OCs, however, the increased risk may be due to confounding by indication related to differences in prescription behaviour.We estimated the risk of VTE associated with use of OCs with and without the presence of Factor V Leiden mutation, protein C-, protein S- or antithrombin deficiency.Sixty-seven cases with VTE were compared with 134 controls. The risk of VTE in the presence of thrombophilia was of the same magnitude for third generation OC users as for users of other OCs; OR: 52.5 (95% CI: 3.7-738.1) and OR: 63.3 (95% CI: 6.2-648.4), respectively.It is unlikely that confounding by indication entirely explains the risk of VTE associated with third generation OCs since the combined effect exceeds what could be explained if this source of error was the only determinant of the association.

Recent findings have suggested biological classification of B-cell malignancies as exemplified by the "activated B-cell-like" (ABC), the "germinal-center B-cell-like" (GCB) and primary mediastinal B-cell lymphoma (PMBL) subtypes of diffuse large B-cell lymphoma and "recurrent translocation and cyclin D" (TC) classification of multiple myeloma. Biological classification of B-cell derived cancers may be refined by a direct and systematic strategy where identification and characterization of normal B-cell differentiation subsets are used to define the cancer cell of origin phenotype. Here we propose a strategy combining multiparametric flow cytometry, global gene expression profiling and biostatistical modeling to generate B-cell subset specific gene signatures from sorted normal human immature, naive, germinal centrocytes and centroblasts, post-germinal memory B-cells, plasmablasts and plasma cells from available lymphoid tissues including lymph nodes, tonsils, thymus, peripheral blood and bone marrow. This strategy will provide an accurate image of the stage of differentiation, which prospectively can be used to classify any B-cell malignancy and eventually purify tumor cells. This report briefly describes the current models of the normal B-cell subset differentiation in multiple tissues and the pathogenesis of malignancies originating from the normal germinal B-cell hierarchy.

Primary autoimmune neutropenia (AIN) is characterised by severe neutropenia and the presence of granulocyte reactive autoantibodies. The pathogenesis of the disease remains unknown and the disease is believed to be underdiagnosed. AIN occurs predominantly at the age of 6-24 months and despite severe neutropenia the symptomatology covers mainly benign infections. Serious bacterial infections might occur and some patients thus may benefit from treatment with prophylactic antibiotics or granulocyte growth factor. Spontaneous remission usually occurs within 30 months from the time of diagnosis.

BACKGROUND: Malignant B-cell clones are affected by both acquired genetic alterations and by inherited genetic variations changing the inflammatory tumour microenvironment.METHODS: We investigated 50 inflammatory response gene polymorphisms in 355 B-cell non-Hodgkin's lymphoma (B-NHL) samples encompassing 216 diffuse large B cell lymphoma (DLBCL) and 139 follicular lymphoma (FL) and 307 controls. The effect of single genes and haplotypes were investigated and gene-expression analysis was applied for selected genes. Since interaction between risk genes can have a large impact on phenotype, two-way gene-gene interaction analysis was included.RESULTS: We found inherited SNPs in genes critical for inflammatory pathways; TLR9, IL4, TAP2, IL2RA, FCGR2A, TNFA, IL10RB, GALNT12, IL12A and IL1B were significantly associated with disease risk and SELE, IL1RN, TNFA, TAP2, MBL2, IL5, CX3CR1, CHI3L1 and IL12A were, associated with overall survival (OS) in specific diagnostic entities of B-NHL. We discovered noteworthy interactions between DLBCL risk alleles on IL10 and IL4RA and FL risk alleles on IL4RA and IL4. In relation to OS, a highly significant interaction was observed in DLBCL for IL4RA (rs1805010) * IL10 (rs1800890) (HR = 0.11 (0.02-0.50)). Finally, we explored the expression of risk genes from the gene-gene interaction analysis in normal B-cell subtypes showing a different expression of IL4RA, IL10, IL10RB genes supporting a pathogenetic effect of these interactions in the germinal center.CONCLUSIONS: The present findings support the importance of inflammatory genes in B-cell lymphomas. We found association between polymorphic sites in inflammatory response genes and risk as well as outcome in B-NHL and suggest an effect of gene-gene interactions during the stepwise oncogenesis.

As high-dose therapy for malignancies is now being applied to newly diagnosed patients as adjuvant therapy, it has become a requirement that quality and safety assessment of hematopoietic stem cell grafts be evidence-based. This process has developed a new institution in medicine, the stem cell laboratory. In most cases this speciality has evolved from or within hematological research laboratories. However, the increased routine technologies applied in quality evaluation, ex vivo manipulation and safety assessment in stem cell handling naturally places this activity in transfusion medicine. Multiparametric flow cytometry can identify progenitor subsets in normal human bone marrow and peripheral blood, and such subset quantification has been used retrospectively to predict three-lineage engraftment following high-dose therapy for malignancies. Published single center data have suggested an impact on clinical outcome, and a standardized technique for subset enumeration needs to be established before prospective multicenter trials can be initiated to document the prognostic value of such quality assessment in autografting. Based on experiences of CD34 enumeration, which we consider to be the first step in quality assessment of hematopoietic stem cell grafts, this review discusses flow cytometry subset identification by lineage-specific differentiation markers, stromal-dependent adherence molecules, and regulatory growth factor receptors from a technical point of view. The aim of this review is:To recommend a simple method based on the experiences of the Nordic workshop III on subset identification; To present new molecular genetic-based methods for future use in quality assessment; and To propose new endpoints necessary for validation of the likely clinical impact of subsets in prospective trials. As sample differences between blood and marrow result in technical difficulties, this review only focuses on the methodology of identifying subsets in blood and leukapheresis products. Methods for subset analysis in diagnostic bone marrow samples will be covered in a forthcoming review.

Introduction The present study investigates the association between single nucleotide polymorphisms (SNPs) in the chitinase 3-like 1 (CHI3L1) gene and serum concentrations of YKL-40 in Danish patients with rheumatoid arthritis (RA) and healthy controls as well as the association with RA in the Danish population. The CHI3L1 gene is located on chromosome 1q32.1 and encodes the YKL-40 glycoprotein. YKL-40 concentrations are elevated in the serum of patients with RA compared to healthy subjects, and YKL-40 has been suggested to be an auto-antigen and may play a role in development of RA and in inflammation. Methods Eight SNPs in the CHI3L1 gene and promotor were genotyped in 308 patients with RA and 605 controls (healthy blood donors) using TaqMan allele discrimination assays. Serum concentrations of YKL-40 were determined by an enzyme-linked immunosorbent assay (ELISA). Results We found significant association between the serum concentrations of YKL-40 and polymorphism in the CHI3L1 gene among both patients with RA and controls. The g.-131(C > G) polymorphism (rs4950928) was most strongly associated with age adjusted serum concentrations of YKL-40 in patients with RA (P < 2.4e-8) and controls (P < 2.2e-16). No significant allelic- or genotypic association with RA was found in this Danish cohort. Conclusions We suggest that the g.-131(C > G) promoter polymorphism has a substantial impact on serum concentrations of YKL-40 in patients with RA and healthy subjects. However, the polymorphism does not seem to confer risk to RA itself. The effect of CHI3L1 polymorphism on clinical outcome or the response to treatment in patients with RA remains to be investigated.

A prospective series of 47 total knee arthroplasties in 44 patients with gonarthrosis were followed for at least 1 year to detect patellar complications. In five knees the metal-backed patellar component failed, in one knee the cement fractured, and in one knee there was a spontaneous fracture of the patella. We regard this failure rate as unacceptable.

Perineal seeding of cancer of the prostate is a rare complication after transperineal prostatic biopsy. In the present case the biopsy responsible for the seeding was false-negative, but microscopy of the perineal lesion showed a differentiated adenocarcinoma.

Polymerase chain reaction in combination with sequence-specific oligonucleotide probes were used to analyze nine HLA-B27 subtypes among 51 healthy HLA-B27 positive Danish blood donors and 30 Danish HLA-B27 positive patients with ankylosing spondylitis (AS). In the group of healthy Danes we found two subtypes, B*2705 (90.2%) and B*2702 (9.8%), however, among the AS patients only the B*2705 subtype was detected. We did not find a significant evidence for associations between AS and a particular HLA-B27 subtype in a Danish population.