C. Micha Nübling

Roadmap for Harmonization of Clinical Laboratory Measurement Procedures

Results between different clinical laboratory measurement procedures (CLMP) should be equivalent, within clinically meaningful limits, to enable optimal use of clinical guidelines for disease diagnosis and patient management. When laboratory test results are neither standardized nor harmonized, a different numeric result may be obtained for the same clinical sample. Unfortunately, some guidelines are based on test results from a specific laboratory measurement procedure without consideration of the possibility or likelihood of differences between various procedures. When this happens, aggregation of data from different clinical research investigations and development of appropriate clinical practice guidelines will be flawed. A lack of recognition that results are neither standardized nor harmonized may lead to erroneous clinical, financial, regulatory, or technical decisions. Standardization of CLMPs has been accomplished for several measurands for which primary (pure substance) reference materials exist and/or reference measurement procedures (RMPs) have been developed. However, the harmonization of clinical laboratory procedures for measurands that do not have RMPs has been problematic owing to inadequate definition of the measurand, inadequate analytical specificity for the measurand, inadequate attention to the commutability of reference materials, and lack of a systematic approach for harmonization. To address these problems, an infrastructure must be developed to enable a systematic approach for identification and prioritization of measurands to be harmonized on the basis of clinical importance and technical feasibility, and for management of the technical implementation of a harmonization process for a specific measurand.

Standardization of Hepatitis E Virus (HEV) Nucleic Acid Amplification Technique-Based Assays: an Initial Study To Evaluate a Panel of HEV Strains and Investigate Laboratory Performance

ABSTRACT The performance of hepatitis E virus (HEV) RNA nucleic acid amplification (NAT)-based assays has been investigated using a panel of HEV-containing plasma samples. The panel comprised 22 HEV-positive plasma samples representing 10-fold serial dilutions of HEV genotypes 3a, 3b, 3f, and 4c obtained from blood donors. Two negative-control plasma samples were included. All samples were blinded. The plasma samples were prepared as liquid/frozen materials and distributed to participants on dry ice. Laboratories were requested to test the panel using their routine HEV assays and to score samples as either positive or negative and could optionally return data in copies/ml for HEV RNA. Twenty laboratories from 10 different countries participated in the study. Data were returned by all participating laboratories; 10 laboratories returned quantitative data. All assays except one were developed in-house using conventional or real-time reverse transcriptase PCR (RT-PCR) methodologies. There was a 100- to 1,000-fold difference in sensitivity between the majority of assays, independent of the virus strain. Although the quantitative data were limited, for the samples in the range of ∼6 to 4 log10 copies/ml, the standard deviations of the geometric means of the samples ranged between 0.38 and 1.09. Except for one equivocal result, HEV RNA was not detected in the negative samples. The variability of assay sensitivity highlights the need for the standardization of HEV RNA NAT assays.

Sensitivity of HCV core antigen and HCV RNA detection in the early infection phase

BACKGROUND : Various countries have introduced HCV NAT to exclude infectious donations collected during the preseroconversion window phase (PWP). For the same purpose, an ELISA has also been developed to detect HCV core antigen (cAg).

World Health Organization International Standard to Harmonize Assays for Detection of Hepatitis E Virus RNA

Nucleic acid amplification technique–based assays are a primary method for the detection of acute hepatitis E virus (HEV) infection, but assay sensitivity can vary widely. To improve interlaboratory results for the detection and quantification of HEV RNA, a candidate World Health Organization (WHO) International Standard (IS) strain was evaluated in a collaborative study involving 23 laboratories from 10 countries. The IS, code number 6329/10, was formulated by using a genotype 3a HEV strain from a blood donation, diluted in pooled human plasma and lyophilized. A Japanese national standard, representing a genotype 3b HEV strain, was prepared and evaluated in parallel. The potencies of the standards were determined by qualitative and quantitative assays. Assay variability was substantially reduced when HEV RNA concentrations were expressed relative to the IS. Thus, WHO has established 6329/10 as the IS for HEV RNA, with a unitage of 250,000 International Units per milliliter.

Hepatitis B virus genotype G monoinfection and its transmission by blood components

An acute hepatitis B virus (HBV) infection was diagnosed in a regular apheresis (plasma/platelet) donor by the hepatitis B surface antigen (HBsAg) assay and minipool nucleic acid amplification technology (NAT). The acute infection was confirmed by detection of anti‐HBc (IgM) and anti‐HBs 2 weeks later. The donor showed no clinical symptoms and had normal alanine aminotransferase levels. He had a history of weekly apheresis plasma or platelet donations. Archived material from the donor and the respective recipients was investigated by sensitive HBV NATs as part of a look‐back procedure. HBV DNA was detectable in previous donations as well as in two recipients transfused with platelet concentrates. The rare HBV genotype G was identified in all HBV‐DNA‐positive samples. Strong evidence of genotype G monoinfection was obtained by clonal sequencing, HBV genotype line probe assay, genotype‐specific NATs, and restriction pattern analysis. In contrast to previously described genotype G infections, which all appeared as coinfections with genotype A, neither the hepatitis B e antigen (HBeAg) nor anti‐HBe was detectable in any of the samples. This shows that HBeAg is dispensable for viral replication. The delay in detecting HBsAg in both the donor and recipient samples may be explained by either decreased genotype G–specific synthesis of incomplete viral forms in early HBV infection or the lower sensitivity to genotype G of the current HBsAg assays. In conclusion, this reported case of an HBV infection was caused exclusively by genotype G. (HEPATOLOGY 2006;44:99–107.)

Experience of mandatory nucleic acid test (NAT) screening across all blood organizations in Germany: NAT yield versus breakthrough transmissions.

BACKGROUND: Mandatory nucleic acid test (NAT) blood screening was introduced in Germany in 1999 for hepatitis C virus (HCV) RNA and in 2004 for human immunodeficiency virus Type 1 (HIV‐1) RNA. Minimal sensitivity limits of 5000 IU HCV RNA/mL and 10,000 IU HIV‐1 RNA/mL were defined for the individual donation facilitating testing of minipools (MPs). The NAT yield obtained from all blood organizations is summarized. Transfusion‐associated virus transmissions despite NAT screening (“breakthrough transmissions”) are analyzed.

Frequencies of GB virus C/hepatitis G virus genomes and of specific antibodies in German risk and non‐risk populations

The prevalence of the new flavivirus GB virus C/hepatitis virus G (GBV‐C/HGV) in different German populations was investigated by detection of viral genomes and anti‐E2 antibodies. While blood donors had an overall prevalence of 10.4% there were increased rates for hemophiliacs (54.7%), hemodialysis patients (30.2%), male homosexuals (30.2%) and intravenous drug users (74.4%). Most GBV‐C/HGV positive samples were either viral genome positive or antibody positive, exclusively. Samples with the rare constellation “positive for both GBV‐C/HGV genome and specific antibody” originated in almost all cases from patients who were additionally infected with HIV or HCV. Probable transmission of GBV‐C/HGV by PCR‐positive blood transfusions was observed in 5 of 6 cases approximately six months after transfusion. J. Med. Virol. 53:218–224, 1997.

Blood screening nucleic acid amplification tests for human immunodeficiency virus Type 1 may require two different amplification targets.

BACKGROUND: Five cases of human immunodeficiency virus Type 1 (HIV‐1) RNA–positive blood donations are described that escaped detection by three different CE‐marked nucleic acid amplification technique (NAT) screening assays. These events were associated with two HIV‐1 transmissions to recipients of blood components. The implicated NAT assays are monotarget assays and amplify in different viral genome regions (group‐specific antigen or long terminal repeat). Investigations into the cause of the false‐negative test results were initiated.

Viral nucleic acids in human plasma pools

The identification of viruses in human blood is required for epidemiologic surveillance and to detect potentially emerging threats to blood transfusion safety.

West Nile virus and blood product safety in Germany

West Nile Virus (WNV) is a mosquito‐transmitted flavivirus, widely distributed throughout Africa, Asia and the Middle East. WNV may cause epidemics of human meningoencephalitis. The unexpected emergence of WNV (New York, 1999) and its rapid spread throughout North America during the following years caused a number of blood transfusion‐ and organ transplant‐associated transmissions of WNV. In order to estimate the potential WNV threat for Central Europe, we analyzed the anti‐WNV prevalence and WNV‐RNA incidence among 14,437 and 9,976 blood donors from Germany. There was a high rate of initially anti‐WNV reactives (5.9%), but only a few cases (0.03%) were confirmed as anti‐WNV positive by neutralization assay. No WNV‐RNA positive blood donor was identified in this study. Whereas WNV‐RNA was frequently detected in manufacturing plasma pools from the US, none was detected in pools of European or Asian origin. Virus inactivation steps integrated into the manufacturing process of plasma derivatives were shown to be sufficient to assure the WNV safety of plasma derivatives. A well‐characterized WNV reference material was prepared, showing 340 WNV‐RNA copies per infectious dose. J. Med. Virol. 80:557–563, 2008.