Comparative evaluation of supplemental hepatitis C virus antibody test systems

Implementation of routine blood donor screening using anti-hepatitis C virus (HCV) enzyme immunoassay (EIA) has resulted in an urgent need for well-characterized supplemental assays to confirm the presence of HCV antibodies. A comparative study of four commercially available supplemental assays is reported here: first- and second-generation versions of a strip recombinant immunoblot assay (RIBA-1 and RIBA-2), an HCV neutralization EIA, and HCV neutralization plus synthetic peptide EIA. Three hundred sixty-seven blood donor specimens that were repeatedly reactive on HCV EIA were studied. Most specimens (93%) were also evaluated by radioimmunoassay (RIA) with a six-antigen panel, and 60 selected specimens were tested for HCV RNA by the polymerase chain reaction (PCR). RIBA-1 and RIBA-2 gave concordant results with 86 percent of specimens, while an additional 13 percent were correctly classified by RIBA-2 but not RIBA-1. Neutralization EIA alone correctly identified 94 percent of the study group, while the remaining 6 percent required the peptide EIA or the combined neutralization-peptide assay system for correct classification. The RIBA-2 and neutralization-peptide assay system for correct classification. The RIBA-2 and neutralization-peptide assay systems yielded identical results for 86 percent of specimens, and these results were supported by RIA and selected PCR testing. Only 2 specimens (0.5%) were frankly discrepant, while 51 specimens were indeterminate on either (47) or both (4) assays. When either the RIBA-2 or neutralization-peptide assay yielded an indeterminate interpretation, the other system correctly classified the specimen (based on concordance with RIA and PCR data) in a high proportion (92%) of cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

丙型肝炎病毒感染临床检测程序的建立及结果报告与解释

HCV感染的临床检测指标主要有抗-HCV和HCVRNA,此外还有HCV核心抗原,但目前尚未在临床实验室推广应用.由于HCV RNA采用RT-PCR方法检测,对实验室有特定要求,因而临床实验室检测HCV感染的最常用的标志物是抗-HCV.抗-HCV是HCV感染的临床诊断、健康人群体检和血液筛查中的一个重要的血清学指标.抗-HCV初筛检测目前在临床实验室基本上均采用ELISA和化学发光免疫分析方法,确认方法则为RIBA和RT-PCR(针对RIBA不确定的标本).在临床实验室如何建立HCV感染的检测程序以及正确地报告和解释结果,对于及时正确地诊断HCV感染有决定性意义.     

Allelic dropout can cause false-positive results for Prader-Willi and Angelman syndrome testing

The diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing. Our laboratory uses a standard methylation-sensitive PCR (MSP) to target the differentially methylated SNRPN gene to test for Prader-Willi syndrome (PWS) and Angelman syndrome. One patient, a 27-month-old female, who lacked the classical clinical features of PWS, but had a molecular diagnosis of PWS by MSP by another laboratory, had repeat testing in our laboratory. Testing by MSP in our laboratory also identified an apparent loss of the unmethylated paternal allele, consistent with a diagnosis of PWS. Confirmatory testing using Southern blot analysis with a methylation-sensitive restriction enzyme showed a normal pattern of methylation, detecting both the methylated maternal and unmethylated paternal alleles. To investigate these discrepant results, we amplified and sequenced the SNRPN locus in this patient and identified a single nucleotide change within the binding site for the unmethylated DNA-specific primer. These results indicate this nucleotide change led to allelic dropout in the MSP analysis, yielding the false-positive result. Subsequently, MSP analysis using an alternate primer set that was developed by our laboratory detected both methylated and unmethylated alleles. These findings illustrate that allelic dropout due to the presence of rare polymorphisms can cause false-positive results in commonly used MSP assays and lead to molecular misdiagnosis.     

Integration of nucleic acid amplification test results into hepatitis C virus supplemental serologic testing algorithms: implications for donor counseling and revision of existing algorithms

BACKGROUND: The routine use of hepatitis C virus (HCV) nucleic acid amplification testing (NAT) donor screening assays has provided an opportunity for revision of the current HCV supplemental testing algorithm, which requires that recombinant immunoblot assay (RIBA) be performed on every HCV enzyme immunoassay (EIA)-repeat-reactive donation. The FDA has approved variance requests to use a new algorithm that eliminates the need to perform RIBA when HCV NAT results are reactive. Data are provided in support of this new algorithm. STUDY DESIGN AND METHODS: HCV EIA (including signal-to-cutoff optical density ratio), RIBA, and NAT data were compiled from 33.2 million donations screened over an approximately 4-year period by the American Red Cross and Blood Systems Laboratories. Further, donations having specific combinations of HCV EIA, RIBA, and minipool (MP) NAT results were evaluated, with more sensitive individual-donation (ID) NAT, to construct improved counseling messages for donors. RESULTS: Of 47,041 EIA-repeat-reactive donations, 49.3 percent were RIBA-positive, 17.1 percent RIBA-indeterminate, and 33.5 percent RIBA-negative. NAT-reactive rates were 79.2, 2.5, and 0.18 percent for RIBA-positive, -indeterminate, and -negative donations, respectively. The new algorithm classified an additional 1 percent of donations as HCV-infected while at the same time detecting all infections classified as HCV-infected under the current algorithm. An additional 2.4 percent of RIBA-positive, MP NAT-nonreactive donations were reactive when a frozen-thawed aliquot was retested by ID NAT. CONCLUSION: Integrating HCV NAT results with RIBA results for purposes of donor notification allows more appropriate counseling messages to be given to EIA-repeat-reactive donors. The new HCV supplemental algorithm is an acceptable alternative to the current algorithm because it provides equivalent or superior accuracy in formulating donor counseling messages and may also result in reduced costs and more timely notification of infected donors.     

两种方法检测低浓度乙型肝炎病毒表面抗体的探讨

目的 探讨对低浓度乙型肝炎(下称乙肝)病毒表面抗体(抗-HBs)定性与定量检测方法的差异.方法 采用酶免疫测定(EIA)一步法、时间分辨荧光法(TRF)、Axsym雅培试剂来测试抗-HBs.结果 采用EIA一步法对低浓度抗-HBs标本进行测定显示,阴性37例,阳性21例;再用TRF法进行复查,阴性标本37例中有3例呈弱阳性,阳性标本21例中有2例呈阴性;以上5例结果不一致标本,采用美国Abboutt公司生产的Axsym雅培试剂检测显示,与TRF法一致.结论 标志物提倡使用EIA二步法酶联试剂及定量方法,改善定性和定量检测结果差异性问题,最小限度避免结果上的差异,减少医患之间矛盾.     

3种酶联免疫吸附试验试剂检测丙型肝炎抗体结果分析

目的：通过对国产3种丙型肝炎病毒（HCV）抗体检测试剂盒间接法和夹心法检测的结果比较,分析评价双抗原夹心法试剂的性能。方法设计不同的试验方法,用3种试剂对收集的阳性和阴性标本进行丙型肝炎病毒抗体检测,对单试剂和双试剂呈阳性反应的标本同时进行第三组重组免疫印迹试验（RIBA）和核酸检测;另外,用已知浓度的质控物倍比稀释后,用3种试剂平行检测。对得到的所有检测结果进行比对,综合分析。结果与确证试剂相比,2种间接法试剂和1种夹心法试剂的假阳率分别为42.2％、57.8％和1.6％,阳性检出率均为100％,但夹心法的分析灵敏度比间接法高1～4倍。结论夹心法 ELISA 检测试剂在灵敏度、特异性方面优于间接法 ELISA 检测试剂。     

Mini-pool screening by nucleic acid testing for hepatitis B virus, hepatitis C virus, and HIV: preliminary results

BACKGROUND: The purpose of this study was to evaluate the feasibility of nucleic acid testing (NAT) of mini-pools as a blood donation screening test. STUDY DESIGN AND METHODS: The stepwise implementation of NAT of mini-pools began in January 1997. Since March 1997, all blood donations collected by the German Red Cross Blood Transfusion Service of Baden-Wurttemberg were tested for hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV nucleic acids. An extra barcoded serum sample is collected from each blood donor for NAT-based screening, which is performed only on hepatitis B surface antigen-, anti-HCV-, anti-HIV-, and anti-Treponema pallidum-seronegative donations. Samples are pooled to a maximum of 96. Positive results are resolved through intersecting subpools (a chessboard design). NAT-based screening does not include a virus concentration step before nucleic acid extraction. RESULTS: By the end of October 1997, 331, 783 donations in 3,779 pools had been screened. As yet, no viremic but seronegative blood donor has been found for the three markers. CONCLUSION: It is feasible to incorporate NAT-based screening of mini-pools into the routine virus diagnostics of a large blood transfusion service. It remains to be determined whether screening blood donations by NAT will indeed increase the safety of blood supply.     

The dangers of false-positive and false-negative test results: false-positive results as a function of pretest probability

This article focuses on the following key points of improving clinicians' use of laboratory tests: (1) laboratory tests are always imperfect, but some are much more imperfect than others; (2) limitations in human cognition compound the potential for misinterpretation of test results; (3) false-positive and false-negative laboratory results can lead to serious patient harm; and (4) judicious use of laboratory tests can help mitigate potential harm from spurious results.     

The effect on the safety of intravenous immunoglobulin of testing plasma for antibody to hepatitis C

BACKGROUND: The safety of intravenous immunoglobulin (IGIV), manufactured from units testing negative for antibody to hepatitis C virus (anti-HCV), was investigated. STUDY DESIGN AND METHODS: A study involving five chimpanzees was performed to determine whether the safety of IGIV would be compromised if units of plasma that reacted for anti-HCV were withheld from pools from which IGIV is manufactured. In the first phase of the experiment, two chimpanzees were infused with 25 mL per kg of unprocessed, pooled plasma from 2887 donors who did not react for anti-HCV in single-antigen (c100-3) enzyme-linked immunosorbent assays. In the second phase, each of three chimpanzees was infused with 1000 mg per kg of IGIV manufactured from the same plasma units. The immunoglobulin was made by seven United States- licensed manufacturers, each using its own approved method. Each chimpanzee received an equal dose of each manufacturer's IGIV. RESULTS: The two chimpanzees that received anti-c100-3-nonreactive, unprocessed pooled plasma became infected with HCV. The three chimpanzees infused with IGIV did not show any evidence of infection with HCV 15 months after inoculation. Two of these animals were challenged with human non- A,non-B hepatitis-infectious plasma, and both subsequently showed evidence of HCV infection. CONCLUSION: These studies demonstrate that, as determined by infectivity for chimpanzees, 1) the withholding of plasma units that react for anti-c100-3 from pools from which plasma products are manufactured does not render the source material noninfectious, and 2) the safety of IGIV manufactured from such plasma pools is not compromised by withholding the units that react for anti- c100-3.     

Performance of an algorithm for the reentry of volunteer blood donors deferred due to false-positive test results for antibody to hepatitis B core antigen

BACKGROUND: Blood donor testing for antibody to hepatitis B core antigen (anti‐HBc) has been used in the United States for more than 20 years as a surrogate to prevent transmission by transfusion of non‐A,non‐B hepatitis, as a human immunodeficiency virus surrogate, and to reduce transmission of hepatitis B virus (HBV). Nonspecific anti‐HBc assays have caused deferral of hundreds of thousands of otherwise qualified donors. A more specific anti‐HBc test and a sensitive HBV DNA test should permit donor reentry after false‐positive anti‐HBc. STUDY DESIGN AND METHODS: A total of 1324 otherwise eligible volunteer donors, deferred for anti‐HBc reactivity on more than one occasion, were recruited from four collection facilities. They were tested using a licensed, more specific anti‐HBc test, a licensed hepatitis B surface antigen (HBsAg) test, and a licensed HBV DNA assay with a 95 percent limit of detection of not more than 10 copies per mL. RESULTS: From 11 to 32 percent of donors contacted by participating sites entered the study. Overall, 488 (37%) of the donors were negative on the more specific anti‐HBc test. The proportion of putative false‐positive samples varied according to the test responsible for the original deferral. A single donor, negative for the presence of anti‐HBc and HBsAg, was positive for the presence of HBV DNA in one of three replicates. Repeat testing of this donor 10 months later was negative for the presence of all markers of HBV infection, and the donor had a history of HBV vaccination with documented postimmunization anti‐HBs seroconversion 10 years before her anti‐HBc deferral, and was considered HBV DNA false positive. CONCLUSION: These data support reentry of donors with false‐positive anti‐HBc results on the relatively nonspecific assays that have been in use in the United States for more than 20 years.     

自身免疫病患者血清中传染性非典型肺炎冠状病毒抗体阳性原因分析

目的 探讨传染性非典型肺炎又称严重急性呼吸综合征(SARS),冠状病毒(SARS-CoV)抗体在SARS病原学诊断中的特异性及其在系统性红斑狼疮(SLE)、类风湿性关节炎(RA)、干燥综合征(SS)和混合结缔组织病(MCTD)患者中的假阳性问题。方法 应用酶联免疫吸附试验(ELISA)和荧光定量RT-PCR技术检测了111名正常对照和58例SLE、20例RA、10例SS、16例MCTD患者做血清中SARS-CoV抗体的检测。结果 在111名正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3.6％;IgG抗体诊断SARS的特异性为96.4％,两种抗体同时阳性诊断SARS的特异性为100％。58例SLE患者中,IgM抗体和IgG抗体阳性率分别为8.6％和32.8％,IgG抗体和IgM抗体同时阳性为19％;在10例SS患者中只有1例两种抗体同时阳性(10％);在16例MCTD患者中,IgG抗体阳性6例(37.5％),两种抗体同时阳性1例(6.3％);在20例RA患者中只有1例IgG抗体阳性(5％)。经RT-PCR检测,上述自身免疫病患者中的阳性病例血清SARS-CoV抗体均为阴性。结论用非纯化抗原制备的ELISA试剂盒测定自身免疫病患者的SARS-CoV抗体,可能出现假阳性,两种抗体同时测定可降低诊断的假阳性率,提高诊断的特异性。在自身免疫病患者中出现假阳性的原因可能与包被的抗原有关。     

Low-positive anti-hepatitis C virus enzyme immunoassay results: an important predictor of low likelihood of hepatitis C infection

BACKGROUND: Tests for hepatitis C antibodies (anti-HCV enzyme immunoassays) are usually described as positive or negative. Several studies, mainly in blood donors, have found that specimens with low signal/cutoff (S/C) ratios are commonly negative when tested with a recombinant immunoblot assay (RIBA) or for HCV RNA. METHODS: We retrospectively reviewed 17 418 consecutive anti-HCV results from a screening program for high-risk veterans; 2986 (17.1%) samples were anti-HCV-positive, and 490 (16.4%) had S/C ratios 相似文献   

Patient attitudes toward testing for maternal serum alpha-fetoprotein values when results are false-positive or true-negative.

We investigated the attitudes toward the maternal serum alpha-fetoprotein (MSAFP) test of patients whose results from MSAFP testing were either false-positive or true-negative. Forty-six women who had previous false-positive results from MSAFP testing and 46 who had previous true-negative values participated. Patients were matched for age and socioeconomic status, and all were delivered of normal neonates. A significantly larger number of patients in the group with false-positive results believed they had not understood the MSAFP test than in the group with true-negative results (19.6% vs 0%). Significantly more women in the "false-positive" group felt that the test caused anxiety than in the "true-negative" group (65.2% vs 17.4%). Fewer women in the false-positive group than in the true-negative group said they would have MSAFP testing in a subsequent pregnancy (58.7% vs 91.3%). Further, the women in the false-positive group were less likely than those in the true-negative group to recommend MSAFP testing to a friend (52.1% vs 80.4%). In patients whose results were false-positive, there was a significant relationship between the patient's perception of understanding the MSAFP test and her attitude toward MSAFP testing.