BACKGROUND: Nonhemolytic transfusion reactions (NHTRs) frequently occur after platelet transfusions. White cell (WBC)-derived inflammatory cytokines can cause these reactions, but they are rarely found in WBC-poor platelet preparations. Transfusion reactions were investigated with regard to the residual WBC content in the stored platelet concentrate in two consecutive study periods.STUDY DESIGN AND METHODS: In the first study period, platelet concentrates were WBC-reduced by bedside filtration. In the second period, all platelet concentrates were filtered before storage. Recipients who experienced transfusion reactions were examined with regard to their main clinical symptoms during and after transfusion. In the supernatant of the involved platelet concentrates, concentrations of interleukin (Il)-1 beta, IL-6, IL-8, tumor necrosis factor (TNF)alpha, macrophage inflammatory protein 1 alpha, and RANTES were analyzed.RESULTS: The incidence of transfusion reactions remained steady when the transfusion regimen was changed from bedside filtration to prestorage WBC filtration (1.63% and 1.56%; p = 0.84). In both periods, NHTRs were predominantly of allergic origin. Inflammatory mediators IL-IP, IL-6, IL-8, and TNF alpha were detectable in only a minority of platelet components involved in NHTRs. Platelet concentrates involved in allergic reactions contained high concentrations of RANTES (668 +/- 223 ng/mL).CONCLUSIONS: Prestorage WBC filtration did not reduce the incidence of these reactions, and inflammatory cytokines were of minor relevance. The proinflammatory platelet-derived chemokine RANTES, which accumulates even in WBC-reduced platelet concentrates, was associated with allergic transfusion reactions. Platelet-derived mediators may be a key to understanding NHTRs.

Recently, the leukocyte-derived cytokines interleukin (IL)-1 beta, IL-6, IL-8, and tumor necrosis factor(TNF)-alpha were found in considerable concentrations in platelet concentrates (PC) involved in nonhemolytic transfusion reactions (NHTR). We investigated transfusion reactions in two study periods over the last 3 years. In the 1st period, PC were leukocyte-depleted by bedside filtration. In the 2nd period we performed prestorage leukocyte filtration of PC. Recipients who experienced a transfusion reaction were tested for total and specific IgE, and reactions were analyzed with regard to the main symptoms. In the supernatant of platelets involved, we analyzed concentrations of IL-1 beta, IL-6, IL-8, TNF-alpha, macrophage inflammatory protein(MIP)-1 alpha, and RANTES. The incidence for NHTR did not change with the kind of PC transfused (n = 44, 1.63% and n = 46, 1.56%; p = 0.84), but allergic reactions were reduced in the 2nd observation period (0.92% versus 0.51%; p = 0.02). IL-1 beta, IL-6, IL-8, TNF-alpha, and MIP-1 alpha were not detectable in the majority of all products. PC involved in allergic reactions contained significantly higher concentrations of RANTES. We found evidence that the proinflammatory chemokine RANTES, stored in the platelet alpha-granules, is involved in allergic transfusion reactions. In contrast, leukocyte-derived inflammatory cytokines were of minor relevance in the onset of NHTR.

Primary and reactivated Epstein-Barr virus (EBV) infections after organ transplantation are associated with the development of posttransplant lymphoproliferative malignancies. Since viral reactivation frequently stays asymptomatic, early diagnosis and treatment are challenges during posttransplant patient monitoring. Both soluble-CD23 (sCD23) and intercellular adhesion molecule 1 (ICAM-1) cell surface expression as well as interleukin-10 (IL-10) production are closely associated with viral gene expression. Therefore, immunoglobulin M (IgM), IgG, IgA, sCD23, ICAM-1, and IL-10 concentrations were measured in serum samples from patients during EBV reactivation (n = 14) and were compared with those in samples from patients without EBV reactivation (n = 10) following renal transplantation. In addition, serum sCD23, ICAM-1, and IL-10 concentrations were measured longitudinally in weekly to biweekly samples from 10 patients with EBV reactivation for at least 20 weeks following transplantation. A significant elevation of sCD23 was found during viral reactivation (P < 0.05), whereas ICAM-1 levels showed a nonsignificant increase. The finding of a highly significant elevation of the serum IL-10 concentration during EBV reactivation (P < 0.001) may support speculations about its role in EBV-induced lymphoproliferation and in the development of opportunistic infections and secondary malignancies. Maximum serum IL-10 levels at the time of EBV reactivation were found in 7 of 10 patients. Well-defined ICAM-1 and sCD23 concentration peaks were found in 9 of 10 and 8 of 10 patients, respectively. Although both markers are not specific for EBV reactivation and therefore may not be useful for primary diagnosis, sCD23 and ICAM-1 might be potent tools for the clinical monitoring of EBV activity and virus-induced lymphoproliferation.

Platelets of atopic individuals differ in alpha-granular contents and in the amount of biologically active mediators released compared with platelets of nonatopic subjects. Because platelets carry the low-affinity IgE receptor (CD23), they may contribute to long-lasting IgE sensitivity by serving as a storage pool for IgE. We compared 45 atopic individuals with immediate-type allergies and 25 nonatopic control subjects with respect to storage and release of IgE by their platelets. Platelets of atopic individuals were characterized by a 10-fold higher median IgE content compared with those of nonatopic control subjects. The platelet IgE content correlated with the serum IgE level in the four atopic individuals with seasonal allergies who were followed up monthly over 1 year. Platelet stimulation with platelet activating factor, but not with thrombin or adenosine diphosphate, resulted in a release of 65% of the stored IgE. Conversely, platelet stimulation with monoclonal IgE/kappa resulted in the release of the chemokine RANTES. Platelet alpha-granules were identified as the main storage compartment for IgE by postembedding immunocytochemistry. Although more than half of the alpha-granules showed gold labeling for IgE, additional labeling was found on the external face of the plasma membrane and within the open canalicular system, indicating endocytosis and exocytosis of IgE. Moreover, the detection of CD23 not only on the plasma membrane but also on membranes of the alpha-granules further supports the existence of an exchange of IgE between the blood plasma and an internal storage compartment. Endocytosis could be confirmed by the uptake of an IgE myeloma protein coupled to colloidal gold. We conclude that platelets of atopic individuals may contribute to allergic inflammation by serving as a storage pool for IgE and by their increased capacity to liberate further mediators of allergy in response to IgE stimulation.

Background: Platelets contain several mediators, belonging to a family of proinflammatory cytokines named chemokines, that are stored in the organelles. Release and accumulation of these chemokines during storage of platelet concentrates (PCs) might be responsible for nonhemolytic transfusion reactions.Study Design and Methods: Analysis was done of pH and the levels of platelet factor 4, beta-thromboglobulin, interleukin 8, RANTES, macrophage-inflammatory protein-1 alpha, lactate dehydrogenase, and serotonin in the supernatant of stored PCs on Days 1, 3, 5, and 8. PCs were prepared by apheresis or from pools of four buffy coats. Puffy coat PCs were filtered before storage.Results: Nonfiltered apheresis PCs, which had a higher white cell contamination (p<0.01), contained significantly more platelets than did buffy coat PCs (p = 0.02). The pH decreased significantly in apheresis PCs (p = 0.01), whereas there was a significant increase in lactate dehydrogenase (p<0.001). In buffy coat PCs, pH remained stable and lactate dehydrogenase increased moderately. Concentrations of platelet factor 4 and beta-thromboglobulin increased steadily in both preparations over the storage period (p<0.001). Macrophage-inflammatory protein-1 alpha was hardly detectable in the supernatant of both PCs, while RANTES levels increased significantly with storage time (p<0.001). Interleukin 8 was not found in the supernatant of any PCs, with the exception of one apheresis PC with high white cell contamination (>10(9)/L). Serotonin levels were higher in apheresis PCs (p = 0.01), but the levels did not correlate with storage time.Conclusion: Platelet factor 4, beta-thromboglobulin, and RANTES were released from platelets during storage and accumulated over time in the PCs. These chemokines might play a causative role in nonhemolytic transfusion reactions because of their inflammatory potential, but the clinical effects of the transfusion of PCs with high chemokine contents remain to be investigated.

Die Pathophysiologie nichthämolytischer Transfusionsreaktionen (NHTR) nach Transfusion von Thrombozytenkonzentraten (TK) ist immer noch umstritten. Verschiedene Zytokine, der Gesamt-IgE-Spiegel und spezifische IgE-Antikörper wur-den mit NHTR in Zusammenhang gebracht. Wir untersuchten das Serum betroffe-ner PatIenten bezüglich IL-1β, IL-6, IL-8, TNF-α, der Gesamt-IgE-Werte und spezifischer IgE-Antikörper. Die gleichen Parameter wurden in den TK gemessen, nach deren Gabe NHTR auftraten. Unsere Ergebnisse zeigen, daβ die von uns untersuchten Zytokine im TK nur eine untergeordnete Rolle in der Pathophysiologie der NHTR in unserem Untersuchungskollektiv spielen. Der Nachweis spezifischer IgE-Antikörper im Serum der TK-Empfänger war hingegen mit der Art der NHTR dieser Patienten assoziiert. Atopische TK-Empfänger scheinen somit ein höheres Risiko als nichtatopische Patienten zu haben, an einer nichthämolytischen Überempfindlichkeitsreaktion zu leiden. Aus diesem Grund empfehlen wir eine All-ergiediagnostik für Patienten, die wiederholte TK-Transfusionen benötigen.

The cytokines RANTES and MIP-1 alpha are 8-kD peptides which belong to the C-C subfamily of chemokines. They are both potent chemotactic factors for basophils and eosinophils. Apart from lymphocytes, the RANTES peptides was recently shown to be released from human platelets stimulated with thrombin [Kameyoshi et al: J Exp Med 1992;176:587-592]. Employing postembedding immunocytochemistry, we could detect RANTES and for the first time also MIP-1 alpha within the alpha-granules of human platelets. To date, MIP-1 alpha was not reported to occur in platelets. In slightly activated platelets, as found in stored platelet concentrates (PC), label for RANTES and MIP-1 alpha could also be observed within cisterns of the open canalicular system and on the plasma membrane, indicating a release of both peptides. These findings were confirmed by in vitro studies in PC, by investigation of RANTES and MIP-1 alpha release into the suspending medium. Over a period of 8 days, RANTES was steadily released in relatively high amounts, whereas MIP-1 alpha was measured in rather small amounts in the suspending medium. As RANTES and MIP-1 alpha, besides their chemotactic activity on eosinophils and basophils, are able to mediate the release of histamine, it is tempting to speculate about a participation of platelets in inflammatory reactions in which eosinophils and basophils are involved.

We present a simple method for subclass typing of IgG paraproteins, with which we have demonstrated a large number of paraproteins that were undetected by conventional immunofixation techniques. The types and distribution of IgG subclass paraproteins were analyzed in 92 human sera in which IgG paraproteins had been demonstrated. The IgG subclass paraproteins were separated by agarose gel electrophoresis rapidly and simply and then typed with the use of sheep anti-human monospecific IgG1-IgG4 antibodies. In 24 of the sera analyzed, IgG subclass typing revealed 25 additional monoclonal bands that were not detected by conventional immunofixation electrophoresis with anti-IgG antisera. Most of these belonged to a different subclass type. The overall subclass frequencies were 68% IgG1, 13% IgG2, 16% IgG3, and 3% IgG4. The distribution of paraprotein subclasses, however, was different in monoclonal gammopathies of undetermined significance in which more IgG3 was shown, whereas in non-Hodgkin lymphomas the number of IgG2 paraproteins was greater than expected; this finding may have diagnostic and prognostic significance.

There has been some discussion whether the atopic disposition of a blood donor is associated with a potentially higher incidence of hypersensitivity nonhemolytic transfusion reactions (NHTRs). Serum samples from patients who had suffered from NHTRs and samples from the platelet concentrates (PCs) responsible for the reactions were examined for total and specific IgE as diagnostic markers for allergic events. In addition, the allergy prevalence among 1,088 blood donors was determined to analyze the allergy prevalence among our blood donors. Our results indicate that in 90% of cases, allergic NHTRs were associated with specific IgE antibodies in the recipient’s serum, indicating the allergic disposition of the patient. In contrast, specific IgE antibodies were detected in only 22% in the transfused PCs. However, among all investigated NHTRs, there was not a single case in which specific IgE antibodies were detected exclusively in the PC. The allergy prevalence among our blood donors was about 26%. In our opinion, the few cases in which the allergic disposition of blood donors in combination with the allergic disposition of the recipients was associated with NHTRs reflects the allergy prevalence among our blood donors in general (26%). On the basis of these findings, we conclude that allergy diagnosis for blood donors is only of minor value in the prevention and prediction of NHTRs, whereas allergy diagnosis for patients who require multiple PC transfusion might be helpful.

A novel family of structurally and functionally related polypeptides has recently been detected that are now referred to as chemokines. Within this family, a peptide with the acronym RANTES was shown to be chemotactic for memory T cells, monocytes, and eosinophilic and basophilic granulocytes, thus suggesting it plays an important role in chronic inflammatory and allergic diseases. Murine monoclonal antibodies as well as cDNA probes specific for human RANTES were raised and extensively characterized. With these antibodies, stimulated human dermal fibroblasts were shown to express intracellular RANTES peptide by immunocytochemistry. Furthermore, similar kinetics could be demonstrated in fibroblasts for both RANTES mRNA expression and secretion of RANTES peptide using Northern blot hybridization and sandwich-enzyme-linked immunosorbent assay, respectively. RANTES expression was induced upon stimulation with tumor necrosis factor-alpha as well as with interleukin-1 alpha and -beta in a concentration- and time-dependent manner. These results reinforce the role of both resident and circulating cells in the production and release of RANTES and their participation in inflammatory processes.

Platelet concentrates (PC) prepared from pooled buffy coat (BC-PC) contain a variable number of leukocytes from different donors. We questioned whether storage of BC-PC can lead to a lymphocyte activation in the sense of a mixed lymphocyte reaction. BC-PC were prepared from four ABO-identical buffy coats and we undertook leukocyte analyses and measurement of different cytokines on days 1, 3 and 5 of PC storage (n = 72). Cytokine content was also determined in freshly prepared plasma (n = 48) and PC prepared by thrombapheresis (SD-PC) (n = 12). As control, we studied lymphoproliferation of pooled peripheral blood mononuclear cells from four individuals in 10 mixed lymphocyte cultures (MLCs) under optimal conditions. In the BC-PC, whole blood count and lymphocyte analysis showed a mean leukocyte contamination of 64+/-28 x 10(6) per unit with a proportion of lymphocytes of 66.7+/-13%. In the MLC, levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were increased on day 3 and 5 of storage (p<0.001). In a proportion of BC-PC, tumor necrosis factor-alpha (72.2%) and IL-2 (43.1%) were detectable immediately after preparation, whereas IFN-gamma (4.2%), interleukin-1 beta (4.2%) and interleukin-8 (11.1%) were only found in some BC-PC. In all cases, initial values of cytokines did not increase during storage. Cytokine measurement in FFP and SD-PC showed similar results. The study demonstrates that cytokines are detectable in a variety of blood products immediately after preparation. Levels of cytokines did not increase in the preparations. BC-PC can be stored for up to 5 days without any signs of lymphocyte activation.

To test tetanus immunity, tetanus antitoxin titres were measured in the serum of 692 subjects (354 males, 338 females). aged one day (newborns) to 92 years (mean age 29 years). Those aged 18 to 65 years were first-time blood donours, the remainder were healthy newborns, while the children and those over 65 years were patients without immune-compromising disease. An inadequate protection (titre < 0.1 IU/ml) was found in 107 (15.5%), of whom 75 (70%) were females. Women aged 20 years and above also had significantly lower average antitoxin titres than men (1.7 vs. 3.5 IU/ml); P < 0.0001). The inadequate immunization protection of many young women is reflected in the lack of protective antibodies in 10 of the 49 examined newborns. In addition, 18% of children aged between 1 and 15 years had inadequate immunity against tetanus. In the whole group the titre level decreased with age, while the proportion of unprotected persons increased. - Apart from the obvious age and sex dependency of the demonstrated inadequacy of immunological protection against tetanus, attention should also be paid to the lack of protective antibodies in newborns and the marked gaps of immunity among children.

Non-febrile non-haemolytic transfusion reactions (NHTR) after platelet substitution vary from severe (anaphylactic shock) to mild (urticaria) forms. Although the symptoms of these immediate-type hypersensitivity reactions are well documented, little is known about their pathophysiology. We therefore examined sera from patients suffering from different forms of NHTR regarding total serum IgE, specific IgE antibodies and complement activity. Our results show that specific IgE antibodies in the recipients' sera were clearly associated with immediate-type hypersensitivity NHTR and conclude that atopic patients have a higher risk of suffering from these forms of transfusion reactions than non-atopic patients. Allergy diagnostic for patients who require platelet transfusions should be considered in case of multiple platelet substitution.

To test tetanus immunity, tetanus antitoxin titres were measured in the serum of 692 subjects (354 males, 338 females), aged one day (newborns) to 92 years (mean age 29 years). Those aged 18 to 65 years were first-time blood donors, the remainder were healthy newborns, while the children and those over 65 years were patients without immune-compromising disease. An inadequate protection (titre < 0.1 IU/ml) was found in 107 (15.5%), of whom 75 (70%) were females. Women aged 20 years and above also had significantly lower average antitoxin titres than men (1.7 vs. 3.5 IU/ml); P < 0.0001). The inadequate immunization protection of many young women is reflected in the lack of protective antibodies in 10 of the 49 examined newborns. In addition, 18% of children aged between 1 and 15 years had inadequate immunity against tetanus. In the whole group the titre level decreased with age, while the proportion of unprotected persons increased. Apart from the obvious age and sex dependency of the demonstrated inadequacy of immunological protection against tetanus, attention should also be paid to the lack of protective antibodies in newborns and the marked gaps of immunity among children.

Cytokines play an essential role in normal and malignant B-cell proliferation and isotype-switching. Therefore we determined serum levels of different B-cell activating cytokines including IFN gamma, IL-4 and IL-10, IgG subclasses, further immunoglobulins and the soluble B-cell activation marker sCD23 in 68 patients with recently diagnosed and previously untreated IgG myelomas in comparison with age- and sex-matched healthy controls. Our results demonstrated that 16% of the myeloma patients had elevated IL-10 levels up to 1000 pg/ml and 8% showed increased IFN gamma serum concentrations. By contrast, only 7% of the controls showed detectable IL-10 levels and 3% had measurable IFN gamma levels. While in men 67% of the paraproteins were of the kappa light chain type, we found an equal occurrence of kappa and lambda light chains in women. In addition, the distribution of IgG subclasses differed in men and women. In comparison with the controls, no alteration of sCD23 was detected in myeloma patients. We found an apparent correlation of elevated IL-10 levels and the IgG1 subclass.