Current concepts for quality assured long-distance transport of temperature-sensitive red blood cell concentrates.

BACKGROUND AND OBJECTIVES: The German Armed Forces Blood Service in Koblenz supplies red blood cell concentrates (RBCs) to military and civilian institutions at home and to field hospitals during peacekeeping operations abroad. During long-distance transport, blood products can be exposed to extreme environmental conditions or inappropriate handling, which may compromise product quality. MATERIALS AND METHODS: Different active and passive cooling systems, cooling elements, packaging material and data loggers were examined in a climate chamber. A number of techniques for measuring temperature were investigated in order to preserve the blood products' quality during transport, including some field tests with multiparametric data recording. RESULTS: Any kind of active cooling systems, conventional cooling elements and customary packaging material, as well as temperature-sensitive labels, minimum-maximum thermometers and intra-product measurement were found to be unsuitable for military requirement. The best results were obtained when the passively cooling RCB 25 transport box (Dometic) was used together with latent heat/cold storage elements (deltaT) and Junior data loggers (Escort). CONCLUSION: The elaborated protocol allows temperatures to be maintained between 2 and 6 degrees C as required by European guidelines for at least 36 h each and between 1 and 10 degrees C as required by German guidelines for at least 48 or 64 h at ambient temperatures between -10 and 40 degrees C. Preliminary results indicate that care must be taken concerning additional factors such as air pressure variation or vibration.

Molecular epidemiology of West Nile Virus in humans.

After the introduction of West Nile Virus into the United States of America in 1999 followed by annual WNV epidemics during the mosquito seasons and spreading of the virus from the East (New York; 1999) to the West of the U.S. (California; 2003/2004) there appeared the question of whether a similar scenario could happen in Europe, too. To be able to answer this question the German Ministry of Health decided to investigate the prevalence and incidence of WNV infections in German blood donors. First a test algorithm was established taking into account the high level of cross-reactivity between different flavivirus infections in serological test systems. AntiWNV-suspicious specimens were further investigated for their neutralisation capacity and by an antiWNV confirmation assay developed in-house. As a preliminary result of our studies a very low prevalence of WNV infections in healthy German blood donors was measured. Development of highly specific test systems is necessary for accurate and reliable differential diagnosis of flavivirus infections.

Low mitogenic response to EGF and TGF-alpha: a characteristic feature of cultured Kaposi's sarcoma derived cells.

We have investigated the mitogenicity of Epidermal Growth Factor (EGF) and Transforming Growth Factor alpha (TGF-alpha) for cultured Kaposi's sarcoma derived cells (KS cells). In contrast to control fibroblasts from the same patients, KS cells revealed only a weak mitogenic response to these growth factors. Neither EGF nor TGF-alpha were able to substitute for Platelet-derived growth factor (PDGF) when KS cells were grown in PDGF-depleted Platelet-Poor-Plasma serum (PPPS)-supplemented medium. The low mitogenicity of EGF and TGF-alpha for KS cells is not based upon a reduced expression of EGF receptor mRNA and protein in KS cells. However, the binding of EGF to KS cells is about 50% lower than that to fibroblasts. This reduced binding is not due to an occupation of the receptors by TGF-alpha since the expression level of this mitogen in different KS cell lines does not correlate with their capacity to bind EGF. In contrast, the low EGF binding seems to be an intrinsic feature of the EGF receptors of KS cells. In spite of the low mitogenicity of EGF for the tumor cells, the expression of c-myc, PDGF-A and Fibroblast growth factor 5 (FGF-5) mRNA is equally induced by purified EGF in KS cells and fibroblasts. This shows that at least the signal transduction pathways which lead to the expression of these genes are functional in KS cells.

Depletion of PDGF from serum inhibits growth of AIDS-related and sporadic Kaposi's sarcoma cells in culture.

We have examined the mitogenic potential of platelet-derived growth factor (PDGF) on AIDS (acquired immune deficiency syndrome)-related and sporadic Kaposi's sarcoma cells in comparison to fibroblasts at physiological and subphysiological calcium concentrations of the culture medium. At low calcium concentrations in the presence of 3% human serum the growth rate of fibroblasts and Kaposi's sarcoma (KS) cells is similarly reduced to less than half of the growth rate at physiological Ca2+ concentrations. In the presence of 3% PDGF depleted, platelet poor plasma-derived serum (PPPS) growth of KS cells ceased completely, whereas fibroblasts made 1-2 cell divisions within 15 days. At physiological Ca2+ concentrations, the reduced PDGF content in 3% PPPS had no effect on human embryonal fibroblasts and little effect on adult skin fibroblasts. In contrast, KS cells became growth-arrested after one to two doublings. This is consistent with the observation that PDGF B-chain mRNA could not be detected in our KS cells whereas PDGF receptor mRNA was expressed.

Fibroblast growth factor 5 proto-oncogene is expressed in normal human fibroblasts and induced by serum growth factors.

Fibroblast growth factor 5 (FGF-5) is a member of the fibroblast growth factor family with transforming potential. It has been found to be expressed in several human tumor cell lines, but nothing is known about expression of this growth factor in normal cells and its biological functions. Here we show that the FGF-5 gene is expressed in exponentially growing normal human fibroblasts. In quiescent fibroblasts, expression of FGF-5 is strongly induced by serum and several growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). This induction can be mediated by at least two different pathways involving protein kinase C or cAMP-dependent kinases. Since the effect is independent of de novo protein synthesis, FGF-5 represents the product of a primary response gene. In addition our data suggest that FGF-5 is mitogenic for human fibroblasts, indicating the existence of an FGF-5-mediated positive feedback in these cells which could amplify and prolong the cellular response to the initial stimulus.

Infection of human fibroblasts and osteoblast-like cells with HIV-1.

Primary human skin- and lung-derived fibroblast cell cultures and continuous human osteoblast-like and fibroblast-like cell lines were infected with different strains of HIV-1. Infection was measured at the single-cell level using the immunoperoxidase staining method to detect viral proteins. No cytopathic effects were observed in HIV-1-infected cell cultures. One continuous cell line (LC5), derived from embryonic lung, was readily infectable with HIV-1 and showed continuous production of infectious virus. Infection of LC5 cells could be blocked with anti-CD4 monoclonal antibodies. These findings indicate that fibroblasts of skin and lung, and osteogenic cells may be considered as potential target cells for HIV-1, thereby possibly contributing to the establishment of local HIV reservoirs.

Clinical evaluation of autoantibodies to p53 protein in patients with chronic liver disease and hepatocellular carcinoma.

In hepatocellular carcinoma (HCC) of patients from the Western hemisphere, mutations in the p53 tumour suppressor gene are present in up to 37% of cases. Conformational change and cellular accumulation can initiate an immune response with generation of circulating autoantibodies to p53 protein. In the present study, we investigated 711 consecutive patients with chronic liver disease to evaluate the sensitivity and specificity of autoantibodies to p53 protein as a serological marker for HCC. Detection of p53 autoantibodies was performed using an enzyme-linked immunosorbent assay with immobilised recombinant p53 protein. Liver cirrhosis was present in 259 patients (36.4%) and a HCC was diagnosed in 75 patients (10.6%). Autoantibodies to p53 protein were detectable in 15 of 377 patients with chronic liver disease (4.0%) and in 10 of 259 patients presenting with liver cirrhosis (3.9%). All 25 p53 autoantibody-positive/HCC-negative patients were carefully investigated and no underlying malignancy was clinically detected, suggesting that elevated p53 antibody levels may not exclusively be detectable in patients with malignant disease. In patients with clinically manifest HCC, p53 autoantibodies were detected in 17 of 75 cases, thus resulting in a sensitivity of 22.7% and a specificity of 96.1%. In contrast, assessment of serum alpha-fetoprotein (AFP) resulted in a sensitivity and specificity of 69.3 and 91.8% (AFP > 20 ng/ml) and 53.3 and 99.1% (AFP > 100 ng/ml) for the detection of HCC, respectively. The data of the present study reveal that the presence of p53 autoantibodies in patients with chronic liver disease is not completely specific for HCC. Moreover, we obtained no direct evidence that p53 autoantibody formation precedes the clinical diagnosis of HCC. However, serological screening for HCC might be improved by combining AFP and p53 autoantibody assays.

Kaposi's sarcoma: a review of gene expression and ultrastructure of KS spindle cells in vivo.

The ultrastructural features and the gene expression pattern of Kaposi's sarcoma (KS) spindle cells in vivo suggest that KS is a tumor of the mixed cell type. The expression pattern of cytokines and cytokine receptors in the tumor lesion, together with the results obtained from in vitro characterization of KS-derived cells, provide evidence that paracrine mechanisms of growth factor action are important for the maintenance of KS. The reports on virus infection of KS cells suggest an indirect role of virus infection in the induction of KS, most likely mediated by immunostimulation and subsequent production of cytokines.

HIV-associated Kaposi's sarcoma: new developments in epidemiology and molecular pathology.

New epidemiological data give evidence for an unknown etiological agent of Kaposi's sarcoma (KS). Experimental support is provided by research on cultivated KS cells. These results contradict a direct involvement of HIV-1 in the pathogenesis of KS. Research on cultivated KS cells confirmed the hypothesis that KS spindle cells originate from endothelial cells and gave new insight into the pathogenesis of tumor cell growth. KS spindle cells secrete an autocrine acting growth promoting activity. Nevertheless, they seem to depend on several growth factors like PDGF and IL-6 provided by surrounding endothelial cells and macrophages, respectively. The results support the hypothesis of a tumor relying on paracrine acting factors more than on autocrine acting factors.

p53 autoantibodies in patients with pancreatitis and pancreatic carcinoma.

In human pancreatic carcinoma (PCa) mutations in the p53 tumor suppressor gene are present in up to 50% of cases. Conformational change and cellular accumulation, together with subsequent release of mutant and normal p53 protein from transformed cells, may initiate a B-cell response with generation of circulating autoantibodies to p53 protein (anti-p53). In the present study we analyzed the sera of 85 consecutive patients with acute pancreatitis (N = 19), chronic pancreatitis (N = 33), and PCa (N = 33) to evaluate the specificity of autoantibodies to p53 protein as a serological marker for PCa. Detection of anti-p53 was performed using an enzyme-linked immunosorbent assay system with immobilized recombinant wild-type p53 protein. Autoantibodies to p53 were detectable in 1 of 19 patients with acute (5.3%) and in 4 of 33 patients with chronic pancreatitis (12.1%). All anti-p53-positive patients with acute or chronic pancreatitis were carefully examined and no underlying malignant disease was found. During follow-up (range, 281-647 days; mean, 472 days) none of these patients showed any evidence for subsequent development of PCa or any other malignant disease. In patients with PCa, anti-p53 was detected in 6 of 33 cases, resulting in a sensitivity of 18.2% with a specificity of 90.4%. In contrast to anti-p53, detection of serum carbohydrate antigen (CA 19-9) resulted in a sensitivity and specificity of 69.7 and 71.2% (CA 19-9, > 37 U/ml) and 51.5 and 96.2% (CA 19.9, > 100 U/ml) for the detection of PCa, respectively. Taken together, the sensitivity of anti-p53 formation was low in patients with PCa (18.2%). Furthermore, the detection of anti-p53 was not specific for malignancy, indicating that severe inflammatory processes may also induce anti-p53 formation.

Anti-p53 autoantibodies in hepatitis C virus-infected patients.

Mutations in the p53 gene with generation of circulating autoantibodies to p53 protein (anti-p53) have been recently detected in a significant proportion of patients with different malignant diseases. Using ELISA methods we assessed alpha-fetoprotein (AFP) and anti-p53 as serological screening parameters for hepatocellular carcinoma (HCC) in 147 consecutive patients with hepatitis C virus-related chronic hepatitis. Liver cirrhosis was histologically diagnosed in 58 patients (39,5%) and a HCC confirmed in 7 patients (4.8%). Serum AFP levels were raised above 20 ng/ml in 26/147 patients (17.7%) and above 100 ng/ml in 5/147 patients (3.4%). In 6/7 patients with HCC serum AFP was raised above 20 ng/ml, but only in 3/7 cases above 100 ng/ml. Autoantibodies to p53 protein were detected in 3/7 patients with HCC, but in 0/140 patients without HCC (sensitivity 42.9%, specificity 100%). In conclusion, the presence of anti-p53 was specific for malignancy and independent of AFP status. Overall, the sensitivity of serological screening for HCC in patients with hepatitis C virus-related chronic hepatitis was improved by combining AFP measurements (level > 100 ng/ml) with the detection of anti-p53.

Heterosexual transmission of GB virus-C/hepatitis G virus infection.

GB virus-C (GBV-C) and hepatitis G virus (HGV) are recently identified non-A-E hepatitis-associated viruses belonging to the family Flaviviridae. The prevalence of GBV-C/HGV in the general population is high (1.2-3.0%), but data on possible transmission routes are sparse. In this report GBV-C/HGV RNA was detected in a couple by reverse transcription polymerase chain reaction (RT-PCR) using primers deduced from non-structural regions. Neither partner was coinfected with hepatitis B (HBV), hepatitis C (HCV) or human immunodeficiency virus (HIV). The child of the couple tested repeatedly negative for GBV-C/HGV by RT-PCR. The couple had lived in a stable monogamous relationship for 10 years and had never used barrier contraception. Other than sexual risk, factors for transmission were carefully excluded. GBV-C/HGV isolates obtained from the couple were sequenced and phylogenetically analysed together with control GBV-C/HGV isolates. The evolutionary distance between the sequences obtained from the couple (1%) was smaller than between any other GBV-C/HGV sequence. Taken together, the clinical and molecular data provide strong evidence for heterosexual but not vertical transmission of GBV-C/HGV in non-coinfected individuals.

Yield and future issues of nucleic acid testing.

Despite the much lower actual yield than that estimated for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) nucleic acid testing (NAT)-only positives in the USA and Germany, look-back procedures have revealed that no HCV transmission has occurred in Germany since the introduction of NAT. This indicates sufficient sensitivity of the pool-PCR approach. The slow ramp-up of hepatitis B virus (HBV) however, may require a different approach. It has been shown in Germany that the pooling of samples followed by virus enrichment results in a significant yield. Single donation testing for HBV would not increase the yield, because virus enrichment from mini-pool results in a similar sensitivity to that of single donation testing. Both strategies may be useful for extending future NAT to HBV screening. New candidate viruses for NAT are Parvo B19 and hepatitis A virus (HAV) because of their extreme resistance to inactivation procedures. Their low pathogenicity and epidemiologic characteristics, however, make them candidate viruses only for pooled source plasma. The main future issues of NAT will be related to the automation of pooling, extraction and amplification as a single homogeneous process. Depending on the throughput, automated single donation NAT as demonstrated by the 'Tigris' system may be an option, as far as all transfusion-relevant viruses will be included. In the near future high throughput systems will rely on pooled donor samples, most probably in conjunction with efficient enrichment procedures. For these systems, automation of the extraction and amplification process will be one of the first steps. These procedures will also limitthe costs of NAT and keep it available for use with future candidate viruses.

Balance module allows consistent monitoring and documentation of the pooling process for nucleic acid amplification technology testing.

BACKGROUND AND AIMS: When performing minipool testing of blood donations for transfusion-transmissible viruses, it is crucial to correctly dispense plasma from each donation into the pools. However, concerns regarding the monitoring and documentation of the pooling process exist. METHODS: A balance module with a tube holder has been developed, which can be easily integrated into liquid handling platforms. The existing software monitors and evaluates every single dispensing from primary samples to the minipool to confirm liquid arrival. The weighing accuracy of the balance module is approximately 2 mg per dispensing episode. RESULTS: Ten thousand one hundred and fifty-six blood donations were tested on a Tecan Genesis RSP pipetting system. Thirty-one donations were not pipetted because the pipetting workstation identified a clot or sample shortage in the primary tube. The balance module exclusively detected another 18 mispipettings, which were outside of the acceptance criteria of 100 +/- 10%. The mean pipetted volume for these samples was 34.2 microl (range 0-87 microl). Visual inspection of the corresponding primary tubes showed blood clots, short sample or no apparent cause. The average deceleration of the pooling process, using the balance, was determined to be about 22%. CONCLUSIONS: With the novel liquid arrival check system, complete and consistent process documentation of pooling for nucleic acid amplification technology (NAT) testing is feasible. It enables blood banks to monitor and compare every single dispense made with predefined, required volumes. Results can be transferred to the laboratory information management system for automated selective exclusion of inaccurately dispensed samples. ONE SENTENCE SUMMARY: Monitoring and documenting the pooling process for NAT testing using a balance-based liquid arrival check system.

Anti-HBc screening of blood donors: a comparison of nine anti-HBc tests.

BACKGROUND AND OBJECTIVES: Since voluntary introduction of hepatitis B virus (HBV) minipool nucleic acid amplification technology (NAT) at the German Red Cross, the expected residual risk of a transfusion-associated HBV infection has been estimated to be 1 : 500,000 - about 10 times higher than for human immunodeficiency virus (HIV) or hepatitis C virus (HCV) infection. Donors demonstrating chronic positivity for antibody to hepatitis B core antigen (anti-HBc), negativity for hepatitis B surface antigen (HBsAg) and polymerase chain reaction (PCR)-negative with a low virus load are a major cause of this increased risk. MATERIALS AND METHODS: Ten-thousand blood donors from our blood-donation centre were screened for anti-HBc using the current PRISM HBc and the new PRISM HBcore assay to evaluate the diagnostic sensitivity and specificity of these tests. PRISM HBc- or PRISM HBcore-reactive samples were further analysed using seven additional tests for anti-HBc, two tests for antibody to hepatitis B surface antigen (anti-HBs), one test for antibody to hepatitis B envelope antigen (anti-HBe) and three HBV NAT assays. RESULTS: From a total of 10,000 donors, nine and 14 samples were reactive only in the PRISM HBc and the PRISM HBcore, respectively, whereas 165 samples were reactive in both anti-HBc assays. Further analysis of these 188 anti-HBc-reactive specimens in a total of nine different anti-HBc assays revealed concordant results for 162 (86.2%) specimens. Sample cut-off values for anti-HBc were significantly (P < 0.01) lower for anti-HBc-only reactive samples compared with specimens that were also reactive for anti-HBs or anti-HBe. CONCLUSIONS: Both PRISM anti-HBc assays revealed that approximately 1.8% of non-prescreened blood donors from Germany were reactive for anti-HBc. Although sensitivity was comparable between both assays, specificity was increased significantly with the PRISM HBcore. High anti-HBc sample cut-off values were indicative for reactivity in other HBV parameters and for concordant results in the nine different anti-HBc assays. Look-back investigations are necessary to estimate the infection risk both of anti-HBc-only positive and of anti-HBc/anti-HBs-positive blood transfusions.