Multinational Analysis of Children Transfused With Pathogen Inactivated Platelets.

BACKGROUND: Pathogen inactivated (PI) platelets are a technological advancement in blood safety; however, the pediatric experience is not well characterized. We studied pediatric patients who received transfusions of PI platelets across several centers and countries to determine if transfusion reaction rates differed when compared with conventional platelets. METHODS: This is a retrospective multisite study conducted during 2 time periods. The study period started at the time each site began using PI platelets on a widespread basis, and the control period was a similar timespan before PI introduction. Suspected acute transfusion reactions were compared. RESULTS: The study included 3839 pediatric patients who were 0 to 18 years of age who received >7930 platelet transfusions, in total, across 4 centers in 3 countries between 2013 and 2019. The age distribution of patients in the study and control period was not significantly different (P = .190). There was not a difference in the percentage of patients who had any type of transfusion reaction between the time periods (1.0% and 1.1%, P = .803). There were fewer patients with mild allergic reactions in the study period compared with the control period (0.2% and 0.7% of patients with reactions, respectively, P = .018). CONCLUSIONS: Pediatric patients have the same rate of acutely suspected transfusion reactions when receiving PI or conventional platelet transfusions. Subgroup analysis found fewer mild allergic reactions in the study period, which was contemporaneous to the addition of using platelet additive solution more broadly. Future studies of PI platelets should include children to better assess transfusion efficacy and hemostatic outcomes.

Severe antibody-mediated transfusion-related acute lung injury in an obstetric patient following transfusion of fresh frozen plasma from a non-transfused male blood donor.

Transfusion-Related Acute Lung Injury (TRALI) has been associated with neutrophil reacting antibodies in transfused blood products. We report a case of life-threatening TRALI in an obstetric patient triggered by transfusion from a non-transfused male blood donor. A residual risk of TRALI exist, even in a male-only plasma setting.

Association between human leukocyte antigens (HLAs) and human neutrophil antigens (HNAs) and autoimmune neutropenia of infancy in Danish patients.

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.

Clinical impact of clonal hematopoiesis in patients with lymphoma undergoing ASCT: a national population-based cohort study.

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).

Stringent or nonstringent complete remission and prognosis in acute myeloid leukemia: a Danish population-based study.

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, <5% blasts in the postinduction bone marrow analysis). Individual patients were classified as having sCR (n = 360; 46.8%) or non-sCR (n = 409; 53.2%) based on data from our national laboratory and transfusion databases. Survival analysis revealed that patients achieving sCR had superior overall survival (hazard ratio [HR], 1.34; 95% confidence interval [CI], 1.10-1.64) as well as relapse-free survival (HR, 1.25; 95% CI, 1.03-1.51) compared with those with non-sCR after adjusting for covariates. Cox regression analysis regarding the impact of the stringent criteria for blood cell recovery identified these as significant and independent variables. In conclusion, this real-life register study supports the international criteria for response evaluation on prognosis and, most importantly, documents each of the 3 lineage recovery criteria as contributing independently.

Interactions between SNPs affecting inflammatory response genes are associated with multiple myeloma disease risk and survival.

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.

Storage time of intraoperative transfused allogeneic red blood cells is not associated with new-onset postoperative atrial fibrillation in cardiac surgery.

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.

Characterization of memory B cells from thymus and its impact for DLBCL classification.

The rare memory B cells in thymus (Thy) are considered the cells of origin for primary mediastinal large B-cell lymphoma. The objectives of the present study were to characterize the normal memory B-cell compartment in Thy and to support its association with primary mediastinal B-cell lymphoma. Seven paired human tissue samples from Thy and sternum bone marrow (BM) were harvested during cardiac surgery. B-cell subsets were phenotyped by Euroflow standard and fluorescence-activated cell sorting for microarray analysis on the Human Exon 1.0 ST Arrays platform. Differentially expressed genes between Thy and BM memory B cells were identified and correlated with the molecular subclasses of diffuse large B-cell lymphoma. Within Thy, 4% (median; range 2%-14%) of the CD45(+) hematopoietic cells were CD19(+) B cells, with a major fraction being CD27(+)/CD38(-) memory B cells (median 80%, range 76%-93%). The BM contained 14% (median; range 3%-27%), of which only a minor fraction (median 5%, range 2%-10%) were memory B cells. Global gene expression analysis of the memory B-cell subsets from the two compartments identified 133 genes upregulated in Thy, including AICDA, REL, STAT1, TNF family, SLAMF1, CD80, and CD86. In addition, exons 4 and 5 in the 3' end of AICDA were more highly expressed in Thy than in BM. The Thy memory B-cell gene profile was overexpressed in primary mediastinal B-cell lymphoma compared with other diffuse large B-cell lymphoma subclasses. The present study describes a Thy memory B-cell subset and its gene profile correlated with primary mediastinal B-cell lymphomas, suggesting origin from Thy memory B cells.

Inherited Inflammatory Response Genes Are Associated with B-Cell Non-Hodgkin's Lymphoma Risk and Survival.

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.

Inherited variation in immune response genes in follicular lymphoma and diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) both depend on immune-mediated survival and proliferation signals from the tumor microenvironment. Inherited genetic variation influences this complex interaction. A total of 89 studies investigating immune-response genes in DLBCL and FL were critically reviewed. Relatively consistent association exists for variation in the tumor 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. Importantly, 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.

Allelic Discrimination by TaqMan-PCR for Genotyping of Human Neutrophil Antigens.

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.

Stable phenotype of B-cell subsets following cryopreservation and thawing of normal human lymphocytes stored in a tissue biobank.

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.

Stable Phenotype Of B-Cell Subsets Following Cryopreservation and Thawing of Normal Human Lymphocytes Stored in a Tissue Biobank.

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.

[TRALI is an overlooked severe complication related to blood transfusion].

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.

Validation and implementation of a method for microarray gene expression profiling of minor B-cell subpopulations in man.

BACKGROUND: This report describes a method for the generation of global gene expression profiles from low frequent B-cell subsets by using fluorescence-activated cell sorting and RNA amplification. However, some of the differentiating compartments involve a low number of cells and therefore it is important to optimize and validate each step in the procedure. METHODS: Normal lymphoid tissues from blood, tonsils, thymus and bone marrow were immunophenotyped by the 8-colour Euroflow panel using multiparametric flow cytometry. Subsets of B-cells containing cell numbers ranging from 800 to 33,000 and with frequencies varying between 0.1 and 10 percent were sorted, subjected to mRNA purification, amplified by the NuGEN protocol and finally analysed by the Affymetrix platform. RESULTS: Following a step by step strategy, each step in the workflow was validated and the sorting/storage conditions optimized as described in this report. First, an analysis of four cancer cell lines on Affymetrix arrays, using either 100 ng RNA labelled with the Ambion standard protocol or 1 ng RNA amplified and labelled by the NuGEN protocol, revealed a significant correlation of gene expressions (r ≥ 0.9 for all). Comparison of qPCR data in samples with or without amplification for 8 genes showed that a relative difference between six cell lines was preserved (r ≥ 0.9). Second, a comparison of cells sorted into PrepProtect, RNAlater or directly into lysis/binding buffer showed a higher yield of purified mRNA following storage in lysis/binding buffer (p < 0.001). Third, the identity of the B-cell subsets validated by the cluster of differentiation (CD) membrane profile was highly concordant with the transcriptional gene expression (p-values <0.001). Finally, in normal bone marrow and tonsil samples, eight evaluated genes were expressed in accordance with the biology of lymphopoiesis (p-values < 0.001), which enabled the generation of a gene-specific B-cell atlas. CONCLUSION: A description of the implementation and validation of commercially available kits in the laboratory has been examined. This included steps for cell sorting, cell lysis/stabilization, RNA isolation, RNA concentration and amplification for microarray analysis. The workflow described in this report will enable the generation of microarray data from minor sorted B-cell subsets.

Cell of origin associated classification of B-cell malignancies by gene signatures of the normal B-cell hierarchy.

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 in children]. / Primær autoimmun neutropeni hos børn.

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.

Promoter polymorphisms in the chitinase 3-like 1 gene influence the serum concentration of YKL-40 in Danish patients with rheumatoid arthritis and in healthy subjects.

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.