Ludo Muylle

Increased tumor necrosis factor alpha (TNF alpha), interleukin 1, and interleukin 6 (IL-6) levels in the plasma of stored platelet concentrates: relationship between TNF alpha and IL-6 levels and febrile transfusion reactions

Increased interleukin 6 (IL‐6) levels were found in 8 of 12 platelet concentrates (PCs) after 3 days of storage and in 10 of 12 PCs after 5 and 7 days of storage. Most of the PCs with an increased IL‐6 level also showed increased tumor necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL‐1 beta) levels. Levels of IL‐6 increased by 3 log10 over the base level during storage. Increased levels were found when the PC white cell count exceeded 3 × 10(9) per L. A linear correlation was found among the levels of TNF alpha, IL‐1 beta, IL‐1 alpha, and IL‐6 in the PCs (r > 0.885). Comparison of the TNF alpha, IL‐ 1 beta, and IL‐6 levels in samples taken at various storage times indicates that the increased levels are the result of an active synthesis and release of interleukins during storage. In a second part of the study, 45 transfusions of white cell‐reduced PCs were studied. Six transfusions were complicated by a febrile reaction. These reactions were related to high levels of IL‐6 and TNF alpha in the PCs (p < 0.0001). These cytokines are known as endogenous pyrogens. These findings indicate that transfusion reactions might be due to the intravenous administration of plasma with high cytokine levels and might not always result from an antigen‐antibody reaction.

Febrile reactions to platelet transfusion : the effect of increased interleukin 6 levels in concentrates prepared by the platelet-rich plasma method

Background: A relation between febrile reactions to platelet transfusion and high cytokine levels in platelet concentrates (PCs) was found previously. The levels of cytokines such as interleukin (IL)‐6 are related to the while cell content of the PC during storage. Therefore, early removal of white cells should prevent reactions.

Flow-cytometric analysis of erythrocytic blood group A antigen density profile.

Abstract. Blood group A antigen density on red blood cells (RBC) was studied using flow cytometry (FCM) and fluoresceinated polyclonal and monoclonal IgG anti‐A antisera. Agglutination was a problem, which could only be solved by prefixation of the RBC with glutaraldehyde or formaldehyde. However, this treatment resulted in a significant reduction of the number of antigen sites as compared to the native (i.e. nonfixed) RBC. Two major new findings came out of this study: (1) A antigen density on native RBC seems to be higher than previously recognized, and (2) A antigen density distribution is probably non‐Gaussian. The absolute number of A antigen sites was determined, using a human polyclonal IgG antiserum and commercially available absolute fluorescence standards. The site numbers on fixed RBC were comparable to those found by earlier radioimmunological studies (× 106/RBC): A1, 1.07 ± 0.28; A2,0.21 ± 0.09; A1B, 0.79 ± 0.26 sites (mean ± SD). The values found for native RBC were considerably higher (× 106/RBC): A1, 2.86 ± 0.95; A2,0.47 ± 0.29; A1B, 1.98 ± 0.58 sites (mean ± SD). With the 1 monoclonal and the 3 polyclonal antisera used in this study, and in contrast to Rh D, the erythrocytic A antigen density distribution of a given sample is highly asymmetrical. This non‐Gaussian distribution profile does not seem to be affected by such factors as antibody heterogeneity, variability in antibody fluoresceination range, RBC density and reticulocyte content. This suggests that the asymmetrical A antigen distribution may be an intrinsic property of the RBC population.

Flow cytometric analysis of erythrocytic D antigen density profile.

Abstract. Red blood cells (RBC) were studied with flow cytometry (FCM) after staining with a directly fluoresceinated anti‐D antiserum. The immunofluorescence distribution profiles were slightly asymmetrical, due to a significant, yet small skewness. This skewness was not associated specifically with D antigen positivity, making a homogeneous, normal D antigen distribution on the RBC very likely. With the exception of Du, D‐positive RBC could always be distinguished from D‐negative ones. FCM was successful in bringing the following phenomena to light: difference in mean fluorescence intensity between the different rhesus phenotypes, the D gene dosage effect, the depressing effect of C antigen on D antigenicity, the nonspecific attachment of IgG on (D‐negative) RBC and rhesus mosaicism. The absolute number of D antigen sites could be calculated, using an absolute fluorescence standard.

Linkage analysis of distal hereditary motor neuropathy type II (distal HMN II) in a single pedigree

We describe a six generation family affected with the autosomal dominant form of distal hereditary motor neuropathy type II (distal HMN II). The distal HMN shows similarities with the hereditary motor and sensory neuropathies type I and II (HMSN I and HMSN II) or Charcot-Marie-Tooth disease type 1 and 2 (CMT 1 and CMT 2) and with some proximal HMN or spinal muscular atrophies (SMA). Gene loci have been assigned to chromosomes 1q, 17p, and 19q for CMT 1 and to chromosome 5q for recessive SMA. In this study we excluded all four regions for the presence of distal HMN II, indicating that this neuropathy is genetically different from CMT 1 and recessive SMA.

Linkage and mutation analysis in an extended family with Charcot-Marie-Tooth disease type 1B.

Charcot-Marie-Tooth disease type 1 (CMT1) or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most families the disease segregates with a 1.5 Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with point mutations in the PMP-22 gene. In some families linkage has been found with markers located on chromosome 1q21-q25 (CMT1B) and more recently mutations have been identified in the P0 gene. We analysed an extended CMT1 pedigree (CMT-B) without the CMT1A duplication. Significant positive linkage with chromosome 1 indicated that this family is of the CMT1B subtype. Sequencing of the candidate gene P0 located in chromosome band 1q21-q23 showed a C to A point mutation at position 446 in exon 3 resulting in an Asp134Glu substitution. Since the P0 mutation cosegregated with CMT1 disease we suggest that this mutation is the primary genetic cause of CMT1B in family CMT-B.

The Duffy blood group is linked to the α-spectrin locus in a large pedigree with autosomal dominant inheritance of Charcot-marie-Tooth disease type 1

SummaryThe α-spectrin locus (SPTA) on chromsome 1 maps to 1q22–q25 and α-spectrin specific probes detect restriction fragment length polymorphisms (RFLPs) with the endonucleases MspI and PvuII. The Duffy blood group (FY) has been mapped to the 1p21–q23 region. We found positive linkage between the α-spectrin and the Duffy loci with a maximal Lod score of 3.81 at θ=0.0 using the computer program MLINK. This indicates that both loci are very closely linked and probably localized to 1q22–q23.

Nomenclature of Granulocyte Alloantigens

and the detection of new ones has made a new nomenclature for these antigens necessary. Neutrophil alloantibodies are known to cause neonatal alloimmune neutropenia, immune neutropenia after bone marrow transplantation, refractoriness to granulocyte transfusions, and severe transfusion-related acute lung injury. With respect to the increasing use of granulocyte transfusions this is of special importance, since alloimmunization to granulocyte antigens can decrease the recovery and half-life of transfused granulocytes, can prevent their migration to sites of infection and can cause severe pulmonary transfusion reactions. The Granulocyte Antigen Working Party of the ISBT agreed at their meeting in S’Agaró, Spain, in 1998, to establish a new nomenclature system for well-defined neutrophil alloantigens, which is based on the following rules: (1) Neutrophil antigens are to be called HNA for ‘human neutrophil antigen(s)’. (2) MemISBT Working Party

Exopeptidases in human platelets: an indication for proteolytic modulation of biologically active peptides

The determination in human platelets of four exopeptidases--aminopeptidase P, dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme--by means of fluorometric or liquid chromatography techniques was carried out. The results obtained show that the specific activities of dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme in intact and disrupted platelets are small compared to their specific activities in serum. However, for aminopeptidase P the specific activity of this enzyme is much higher in platelets than in serum. This suggests that circulating platelets may have a significant role as scavengers for circulating peptides containing bonds susceptible for aminopeptidase P.

The evolution of platelet procoagulant activity of remnant platelets in stored platelet concentrates prepared by the platelet-rich plasma method and the buffy coat method

Abstract Platelets stored as concentrates are gradually activated (storage lesion), a process associated with changes in the expression of platelet procoagulant activity (PPCA). The aim of the present study was to evaluate the evolution of PPCA and the mean platelet volume (MPV) of stored platelets prepared according to the platelet-rich method (PRM) and the buffy coat method (BCM). Using the platelet factor 3 availability clotting test (PF3AT) on appropriately diluted concentrate samples, we found a decrease in PPCA expression of remnant platelets as a function of storage time (0.025<p<0.01 between day 1 and 7) in PRM-derived but not in BCM-derived platelet concentrates. Using the PF3AT reduction test we found a more important clotting time reduction in samples obtained from BCM than in samples obtained from PRM platelet concentrates, suggesting a higher PPCA expression of BCM platelets, not significant after 1 day but highly significant after 3 days (p<0.0005) and after 7 days (p<0.0005) of storage, as compared with PRM platelets. For both PRM and BCM concentrates there were no significant MPV changes as a function of storage time, but at any storage day the MPV of BCM concentrates was significantly higher (p<0.0005) than the MPV of PRM concentrates. We conclude that the decrease of PPCA expression in PRM-derived concentrates as a function of storage time is in agreement with the gradual decrease of the platelet activation status in PRM concentrates during storage. There are probably several factors or variables causing platelets of BCM concentrates to express higher PPCA than those of PRM concentrates. Higher PPCA expression in BCM concentrates may be explained by an intrinsic platelet property, such as a difference in MPV between the two kinds of concentrates, or it may be related to an extrinsic factor such as different storage media, e.g., undiluted autologous plasma in PRM concentrates versus Plasmalyte A-diluted autologous plasma in BCM concentrates. Whether the difference in PPCA expression of remnant platelets in PRM and BCM concentrates is just an in vitro laboratory finding or may have consequences for the therapeutic efficiency of the concentrates is an interesting, still unresolved question.