Axel Pruss

Activity of clotting factors in fresh-frozen plasma during storage at 4°C over 6 days

BACKGROUND: Fresh‐frozen plasma (FFP) requires thawing, which delays availability. We investigated clotting factor activity and bacterial contamination of FFP when stored at 4°Cu2003±u20032°C for 6u2003days.

Detection of Oncofetal H19 RNA in Rheumatoid Arthritis Synovial Tissue

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

Immune hemolysis-serological and clinical aspects

Abstract.The differential diagnosis of anemia must consider immunenhemolytic anemias as a frequent cause. Whereas detection ofnanti-red blood cell (RBC) alloantibodies frequently induced bynimmunogenic stimuli (transfusion, pregnancy) is performed bynroutine serology, diagnosing autoimmune hemolytic anemias orndrug-induced hemolytic anemias remains a challenge, usuallynrequiring close collaboration of a number of disciplines.nPositive direct antiglobulin test (Coombs’ test) represents ancentral criterion in diagnosing immune hemolytic anemias,nleading to further detailed analyses. The most-severe type ofnimmune-mediated hemolysis is acute intravascular hemolysis afternABO incompatible RBC transfusion. This review highlightsnunderlying biochemical aspects, immunohematological diagnostics,nand the clinical relevance of RBC allo- and autoantibodies,nincluding paroxysmal nocturnal hemoglobinemia and drug-inducednhemolysis. Finally, current and partly experimental therapeuticnstrategies of immune hemolytic anemias are summarized.

Clinical efficacy and compatibility of allogeneic avital tissue transplants sterilized with a peracetic acid/ethanol mixture.

In the course of the past 20 years a quantity of approximately 60,000 allogeneic avital tissue grafts sterilized with the peracetic acid–ethanol method (PES) were transplanted successfully. Based on a retrospective report of clinical experience of the years 1997–2001 on the overall scope of tissue grafts manufactured by the Tissue Banks of the University Hospital Charité and the German Institute for Cell and Tissue Replacement, the clinical efficacy and side effects of 18.3% (3.087/16.823) of all transplants were studied. Cancellous (1.601/3.087) and cortical (291/3.087) bone transplants as well as amnion (1.027/3.087) constituted the greatest part. In 91% of the examined patients (2.369/2.592) tissue integration ratios ranging from good up to very well could be observed. The transplant function of defect replacement or of a spacer respectively could be obtained for all types of tissue. The clinical effect caused by the transplant resulted in more than 99% of the transplants in primary integration or in the desired aim of the therapy (defect replacement, stabilization in case of palliative operations, etc.). In less than 1% (9/2.592) of cases a secondary healing occurred for cancellous bone transplantations or, revisional operations became necessary. In all cases severe side effects, in particular transmission of infectious diseases or transplant rejections, were not observed.

Agonistic AT1 receptor autoantibodies and monocyte stimulation in hypertensive patients

BACKGROUNDnAgonistic AT(1) receptor autoantibodies (AT(1)-AA) have been described in hypertensive and preeclamptic patients. Furthermore, monocytes are activated in hypertensive patients. We investigated and compared the ability of angiotensin II (Ang II) and AT(1)-AA to stimulate monocytes from hypertensive and normotensive persons. The adhesiveness of the monocytes to endothelial cell layers, tissue factor expression, and chemiluminescence were determined.nnnMETHODSnBlood samples were obtained from 17 patients with essential hypertension and from 20 normotensive subjects. Peripheral blood monocytes were isolated by Dynabeads and used in adhesion experiments. Adherence assays, Western blotting, and reactive oxygen species release by chemiluminescence were done.nnnRESULTSnMonocyte adhesion to human aortic or umbilical vein endothelial cell layers was significantly higher after stimulation with AT(1)-AA, compared to Ang II or no stimulation. The effect was blocked with tissue factor antibody or epitope peptide preincubation. Eposartan was partially effective in blocking the effects. Western blotting after AT(1)-AA or Ang II stimulation showed that the monocytes expressed tissue factor. The AT(1)-AA and Ang II induced significantly higher chemiluminescence in monocytes from hypertensive than control subjects. Endothelial cells, on the other hand, showed much less chemiluminescence.nnnCONCLUSIONSnThese data show that monocytes can be stimulated by AT(1)-AA and Ang II to adhere, produce tissue factor, and probably reactive oxygen species. They underscore the importance of monocyte activation in hypertensive patients. The relevance of AT(1)-AA in hypertension will require further studies.

Significance of Autoantibodies in Neonatal Lupus Erythematosus

Autoantibodies produced by the mother and transported into the fetal circulation are of significant importance in the diagnosis of neonatal lupus syndromes. These humoral autoimmune findings provide an unique opportunity to assess the pathogenic role of autoantibodies against the Ro(SS-A)/La(SS-B) complex, most notably for congenital heart block. Current knowledge about the involved autoantibody-autoantigen systems, including recent therapeutic concepts of these autoimmune syndromes, is summarized.

Acute intravascular hemolysis after transfusion of a chimeric RBC unit

BACKGROUND:u2002 Natural blood cell chimerism rarely occurs in humans. The case of a patient who developed transfusion reaction due to the transfusion of chimeric RBCs is reported.

Increased RBC autoantibody production in pregnancy.

BACKGROUND: Autoimmunization against RBCs is generally believed to occur very rarely during pregnancy and to represent a high risk for those affected. The occurrence of benign RBC autoantibodies in pregnancy is reported.

Thawing procedures and the time course of clotting factor activity in fresh-frozen plasma: a controlled laboratory investigation.

BACKGROUND:In this study, we evaluated the effects of the thawing process of 2 commercially available devices on the activity of clotting factors, inhibitors and activation markers of the hemostatic system in fresh-frozen plasma (FFP). In an experimental procedure, FFP was thawed under running warm water at 42°C. METHODS:Plasma of 20 healthy donors was sampled, separated, and distributed in 3 plasma bags. Within 2 h after sampling plasma bags was frozen at a temperature of −30°C to −40°C and stored for at least 8 wk. After sampling (baseline) as well as immediately and 1, 2, 4, and 6 h after thawing, the activity of FV, FVII, FVIII, fibrinogen, fibrin monomers (FM), d-dimers (DD), α2-antiplasmin (α2-AP), and protein S (PS) was determined from each plasma bag. RESULTS:From 1 h to 6 h after thawing, no significant differences in the activity of the investigated coagulation markers dependent on the thawing procedure were found. However, immediately after thawing and independent of the thawing procedure, the activity of FVII was significantly decreased (P < 0.01), whereas FM were significantly increased (P = 0.001). CONCLUSION:The thawing procedures studied exhibited no significant influence on activity and stability of the investigated markers of coagulation over the study period. The decreased activity of FVII and the clinical significance of the increase in FM require further research.

Validation of the Microbiological Testing of Tissue Preparations Using the BACTEC™ Blood Culture System

Background: Since blood culture bottles are validated by the manufacturer for blood only, an additional validation for the use with fluids of tissue preparations is necessary. Methods: Two 10-ml samples of cornea culture medium, histidine-tryptophan-ketoglutarate (HTK) solution, or Ringer solution at the end of femur head thermo-disinfection were given into blood culture bottles (BD BACTECTM Plus Aerobic/F, Anaerobic/F for cornea culture medium and BD BACTECTM Standard Aerobic/ Anaerobic for HTK and Ringer solution) and subsequently spiked with 10–100 colony forming units (CFU) of bacteria or fungi (aerobic bacteria: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa; anaerobic bacteria: Clostridium sporogenes; fungi: Candida albicans, Aspergillus brasiliensis) according to the European Pharmacopoeia Chapter 2.6.1. Results: All tested bacteria and fungi could be detected in all solutions. All positive and negative controls were tested correctly. Compared to the positive controls, the microbial growth was delayed in the antibiotic-containing cornea culture medium, and negative in two cases of B. subtilis spiking. Conclusion: The use of BACTECTM blood culture bottles seems to be a suitable method for microbiological testing of HTK solution, Ringer solution, and, with limitations, also for testing of the antibiotic-containing cornea culture medium.