Interference by Rheumatoid Factor in Immunoglobulin M-Class Herpes Simplex Virus Types 1 + 2 Immunoassays

Interference by Rheumatoid Factor in Immunoglobulin M-Class Herpes Simplex Virus Types 1 + 2... Abstract Herpes simplex virus (HSV) types 1 + 2 immunoglobulin (Ig)M antibodies assays are important tools for diagnosing pregnant women and their newborns. However, it is unclear whether high levels of rheumatoid factor (RF) interfere with serological methods used to test for HSV types 1 + 2 type-specific IgM antibodies. We report a case of a false-positive result for HSV 1 + 2 IgM antibodies due to gross overestimation of RF by enzyme-linked immunosorbent assay (ELISA). This result occurred in a 25-year-old Han Chinese woman who was 28 weeks pregnant with a fetus (sex of fetus unknown). Antinuclear antibody (ANA) screening was performed, and RF-absorbent material was used. Test results for 3 antibodies of ANA (SSA, Ro-52, and SSB) were positive; the rest of the results were negative. Also, RF levels were significantly higher than normal, as measured by ANA screening. We observed an obvious decrease in HSV 1 + 2 IgM antibodies after removal of RF. Based on our findings, the presence of high concentrations of RF may be responsible for false-positive HSV 1 + 2 ELISA results. rheumatoid factor, herpes simplex virus, immunoassay, IgM antibody, pregnant, ELISA It has been reported that various interferences can alter enzyme-linked immunosorbent assay (ELISA) outcomes, and that herpes simplex virus (HSV) assays are not immune to such problems. Rheumatoid factor (RF) is one of the most common sources of interference. The role of high-concentration RF as a cause of false decrease of surface antigen of the hepatits B virus (HBsAg) sample/cutoff (S/CO) values is well known.1 Also, the presence of RF in serum can cause falsely elevated results in thyroid-function tests and cytokine, troponin, and tumor-maker immunoassays.2-6 Herein, we report a case of evaluated RF interference in HSV 1 + 2 type-specific immunoglobulin (Ig)M ELISA (DIESSE Diagnostica Senese S.P.A). Case Report Slowed fetal heart rate (96 beats per minute) was diagnosed after a 25-year-old Han Chinese woman who was 28 weeks pregnant with a fetus (sex of fetus unknown) visited Yangxian Hospital, ShanXi Province, China. The treating physician recommended that fetal ultrasonography and chromosomal examination be performed, with reexamination performed after 4 to 6 weeks. Subsequently, the patient visited Hanzhong 3201 Hospital in Hanzhong City on the same day, where ultrasonography results showed that fetal heart rate was less than 89 beats per minute. That rate was 94 beats per minute when the patient visited the obstetrical department of the Northwest Women’s and Children’s Hospital in Xi’an 2 weeks later. During physical examination of the woman, her thyroid was impalpable, and no tubercule was observed under the lateral subcutaneous ankle. Previous medical examination had not revealed a family history of rheumatoid arthritis, systemic lupus erythematosus, or other special cases. The antistreptolysin O (ASO) and thyroid function test results for the patient (as determined by the ARCHITECT i2000, Abbott Laboratories Inc), including thyroid- stimulating hormone (TSH), total triiodothyronie (T3) and thyroxine (T4), free T3 and T4, thyroid peroxidase antibody (TPO Ab), and thyroglobulin antibody (TG Ab), were found to be lower than the reference values for the healthy population. Conversely, we observed a significantly increased RF concentration (Beckman Coulter AU5800, Beckman Coulter Inc). Toxoplasma gondii (TOX) IgM, rubella virus (RV) IgM, cytomegalovirus (CMV) IgM (DIESSE Diagnostica Senese S.P.A.), and parvovirus B19 IgM (Virion\Serion, Germany) results were determined to be negative. However, HSV 1 + 2 IgM results were to indicate elevated S/CO values, based on readings via an Absorbance Microplate Reader (Rayto Life and Analytical Sciences) (Table 1). Table 1. Laboratory Results for the Patient, a 25-Year-Old Han Chinese Woman Pregnant With a 28-Week-Old Fetus (Sex of Fetus Unknown) Parameter Result Reference Range TSH 1.24 mIU/L 0.35–4.94 mIU/L Total T3 1.55 nmol/L 0.88–2.44 nmol/L Total T4 124 nmol/L 62.9–151 nmol/L Free T3 3.03 pmol/L 2.63–5.70 pmol/L Free T4 12.0 pmol/L 9.01–19.1 pmol/L TPO Ab 0.1 IU/mL <5.61 IU/mL TG Ab 1.3 IU/mL <4.11 IU/mL RF 543 IU/mL 0–30 IU/mL ASO 13 IU/mL 0–170 IU/mL TOX IgM 0.311 S/CO <0.9 S/CO RV IgM 0.614 S/CO <0.9 S/CO CMV IgM 0.543 S/CO <0.9 S/CO HSV 1 + 2 IgM 3.09 S/CO <0.9 S/CO B19 IgM 0.328 S/CO <1.0 S/CO Parameter Result Reference Range TSH 1.24 mIU/L 0.35–4.94 mIU/L Total T3 1.55 nmol/L 0.88–2.44 nmol/L Total T4 124 nmol/L 62.9–151 nmol/L Free T3 3.03 pmol/L 2.63–5.70 pmol/L Free T4 12.0 pmol/L 9.01–19.1 pmol/L TPO Ab 0.1 IU/mL <5.61 IU/mL TG Ab 1.3 IU/mL <4.11 IU/mL RF 543 IU/mL 0–30 IU/mL ASO 13 IU/mL 0–170 IU/mL TOX IgM 0.311 S/CO <0.9 S/CO RV IgM 0.614 S/CO <0.9 S/CO CMV IgM 0.543 S/CO <0.9 S/CO HSV 1 + 2 IgM 3.09 S/CO <0.9 S/CO B19 IgM 0.328 S/CO <1.0 S/CO TSH, thyroid-stimulating hormone; TPO, thyroid peroxidase; Ab, antibodies; TG, thyroglobulin; RF, rheumatoid factor; ASO, antistreptolysin O; TOX, Toxoplasma gondii; IgM, immunoglobulin M; S/CO, sample/cutoff; RV, rubella virus; CMV, cytomegalovirus; HSV, herpes simplex virus; B19, parvovirus B19. View Large Table 1. Laboratory Results for the Patient, a 25-Year-Old Han Chinese Woman Pregnant With a 28-Week-Old Fetus (Sex of Fetus Unknown) Parameter Result Reference Range TSH 1.24 mIU/L 0.35–4.94 mIU/L Total T3 1.55 nmol/L 0.88–2.44 nmol/L Total T4 124 nmol/L 62.9–151 nmol/L Free T3 3.03 pmol/L 2.63–5.70 pmol/L Free T4 12.0 pmol/L 9.01–19.1 pmol/L TPO Ab 0.1 IU/mL <5.61 IU/mL TG Ab 1.3 IU/mL <4.11 IU/mL RF 543 IU/mL 0–30 IU/mL ASO 13 IU/mL 0–170 IU/mL TOX IgM 0.311 S/CO <0.9 S/CO RV IgM 0.614 S/CO <0.9 S/CO CMV IgM 0.543 S/CO <0.9 S/CO HSV 1 + 2 IgM 3.09 S/CO <0.9 S/CO B19 IgM 0.328 S/CO <1.0 S/CO Parameter Result Reference Range TSH 1.24 mIU/L 0.35–4.94 mIU/L Total T3 1.55 nmol/L 0.88–2.44 nmol/L Total T4 124 nmol/L 62.9–151 nmol/L Free T3 3.03 pmol/L 2.63–5.70 pmol/L Free T4 12.0 pmol/L 9.01–19.1 pmol/L TPO Ab 0.1 IU/mL <5.61 IU/mL TG Ab 1.3 IU/mL <4.11 IU/mL RF 543 IU/mL 0–30 IU/mL ASO 13 IU/mL 0–170 IU/mL TOX IgM 0.311 S/CO <0.9 S/CO RV IgM 0.614 S/CO <0.9 S/CO CMV IgM 0.543 S/CO <0.9 S/CO HSV 1 + 2 IgM 3.09 S/CO <0.9 S/CO B19 IgM 0.328 S/CO <1.0 S/CO TSH, thyroid-stimulating hormone; TPO, thyroid peroxidase; Ab, antibodies; TG, thyroglobulin; RF, rheumatoid factor; ASO, antistreptolysin O; TOX, Toxoplasma gondii; IgM, immunoglobulin M; S/CO, sample/cutoff; RV, rubella virus; CMV, cytomegalovirus; HSV, herpes simplex virus; B19, parvovirus B19. View Large Applying our combined clinical experience to this case, we suspected that the HSV 1 + 2 IgM result might be a false positive and so we investigated possible interference. Our investigation revealed that the results for antibodies against SSA, Ro-52, and SSB were positive, whereas the remaining envelope antigens on the antinuclear antibodies (ANA)–3 membrane (Omont Père et Fils) tested negative (Figure 1). However, we discovered that the patient had no related autoimmune disease history. We observed that RF concentration was 543 IU per mL, which is much higher than 30 IU per mL, the upper limit of the reference value for the healthy population. It has been reported7 that the minimal effective amounts of RF are 25 ng per mL for IgM measurement in RV solid-phase enzyme immunoassays. Therefore, after pretreating the serum specimen with RF adsorption materials (1:10 dilution according to instructions after 1:1 dilution; Virion\Serion, Germany), RF titers decreased from 20.8 IU per mL to 9.66 IU per mL (equal proportion of specimen diluents and RF adsorption materials) and 7.92 IU per mL (RF adsorption materials only), respectively. Using specimen material with negative RF and elevated HSV IgM as a control group, we discovered that the S/CO values of HSV1 + 2 IgM antibodies were decreased separately, from 2.95 to 0.487 (equal proportion of specimen diluents and RF adsorption materials) or 0.352 (RF adsorption materials only). We discovered similar results in the parallel double holes (Table 2). These results provide evidence for the determination of HSV1 + 2 IgM antibodies influenced by RF. Table 2. RF Concentrations and S/CO Values of HSV 1 + 2 IgM After RF Adsorption Pretreatment Method RF Titers Sample S/CO (Control Group) S/CO S/CO (Parallel Double Holes) Diluents from the kit 20.8 IU/mL 2.95 IU/mL 3.24 IU/mL 3.813 IU/mL Equal proportion of specimen diluents and RF adsorption 9.66 IU/mL 0.487 IU/mL 0.449 IU/mL 3.249 IU/mL RF adsorption 7.92 IU/mL 0.352 IU/mL 0.311 IU/mL 2.690 IU/mL Pretreatment Method RF Titers Sample S/CO (Control Group) S/CO S/CO (Parallel Double Holes) Diluents from the kit 20.8 IU/mL 2.95 IU/mL 3.24 IU/mL 3.813 IU/mL Equal proportion of specimen diluents and RF adsorption 9.66 IU/mL 0.487 IU/mL 0.449 IU/mL 3.249 IU/mL RF adsorption 7.92 IU/mL 0.352 IU/mL 0.311 IU/mL 2.690 IU/mL RF, rheumatoid factor; S/CO, sample/cutoff; HSV, herpes simplex virus; IgM, immunoglobulin M; RF, rheumatoid factor. View Large Table 2. RF Concentrations and S/CO Values of HSV 1 + 2 IgM After RF Adsorption Pretreatment Method RF Titers Sample S/CO (Control Group) S/CO S/CO (Parallel Double Holes) Diluents from the kit 20.8 IU/mL 2.95 IU/mL 3.24 IU/mL 3.813 IU/mL Equal proportion of specimen diluents and RF adsorption 9.66 IU/mL 0.487 IU/mL 0.449 IU/mL 3.249 IU/mL RF adsorption 7.92 IU/mL 0.352 IU/mL 0.311 IU/mL 2.690 IU/mL Pretreatment Method RF Titers Sample S/CO (Control Group) S/CO S/CO (Parallel Double Holes) Diluents from the kit 20.8 IU/mL 2.95 IU/mL 3.24 IU/mL 3.813 IU/mL Equal proportion of specimen diluents and RF adsorption 9.66 IU/mL 0.487 IU/mL 0.449 IU/mL 3.249 IU/mL RF adsorption 7.92 IU/mL 0.352 IU/mL 0.311 IU/mL 2.690 IU/mL RF, rheumatoid factor; S/CO, sample/cutoff; HSV, herpes simplex virus; IgM, immunoglobulin M; RF, rheumatoid factor. View Large Figure 1 View largeDownload slide Antinuclear antibody (ANA) results via scanning. CO indicates control; M2, antimitochondrial antibodies M2 subtype (or AMA-M2); RIB, ribosomal P protein; HI, histone; NUC, nucleosome; DNA, double-stranded DNA; PCNA, proliferating cell nuclear antigen; CB, centromere protein B (or CENP-B); Jo, Jo-1 antigen (or histidyl-transfer RNA synthetase); PM, polymyositis/scleroderma complex (or the exosome); Scl, topoisomerase I (or Scl-70); SSB, Sjögren-syndrome–related antigen B; 52, Ro-52/SS-A 52 kD; SSA, Sjögren-syndrome–related antigen A; Sm, Sm antigen; RNP/Sm, nonhistone nuclear protein/Sm complex; 0, negative; +, positive. Figure 1 View largeDownload slide Antinuclear antibody (ANA) results via scanning. CO indicates control; M2, antimitochondrial antibodies M2 subtype (or AMA-M2); RIB, ribosomal P protein; HI, histone; NUC, nucleosome; DNA, double-stranded DNA; PCNA, proliferating cell nuclear antigen; CB, centromere protein B (or CENP-B); Jo, Jo-1 antigen (or histidyl-transfer RNA synthetase); PM, polymyositis/scleroderma complex (or the exosome); Scl, topoisomerase I (or Scl-70); SSB, Sjögren-syndrome–related antigen B; 52, Ro-52/SS-A 52 kD; SSA, Sjögren-syndrome–related antigen A; Sm, Sm antigen; RNP/Sm, nonhistone nuclear protein/Sm complex; 0, negative; +, positive. Discussion Accurate laboratory assessment of HSV infections is critical for timely management and treatment in pregnancy. ELISA is a rapid, sensitive, commercialized, and automated serologic assay for detection of HSV-specific antibodies; its results can be affected by various types of interference. However, it is difficult to detect assay interferences in routine processing. It is colloquially understood that the identification of interferences requires clinical awareness. Laboratory personnel should consider that interference factors exist and therefore should communicate with clinical physicians in a timely manner when abnormal results emerge. We recommend that HSV-DNA be detected by fluorescence quantitative polymerase chain reaction (PCR) in patients with suspected HSV infection. RF is a type of autoantibody to the Fc fragment of IgG, representing a long-standing problem in terms of interference for immunometric assays. It has been reported8 that RF may lead to high levels of squamous-cell-carcinoma antigen (SCCA)–IgM in patients with cirrhosis. Salonen et al7 also suggested that RF can lead to false-positive results for IgM antibodies against rubella and influenza viruses by ELISA, but that RF had little to no effect on antibodies to cytomegalovirus in same-enzyme immunoassays. In the present study, cryopreservation of specimens for nearly 10 days may account for the slightly decreased RF titers. However, interference by RF was not excluded. We discovered that not only RF concentrations but also S/CO values of HSV 1 + 2 IgM antibodies were decreased after treatment with RF absorption. These findings support the theory that the presence of RF acts as an interferent. Our results are consistent with those of a previous report9 in which pretreatment with antihuman IgG can eliminate RF-interfering HSV IgM detection via ELISA. The mechanism whereby RF leads to the occurrence of false positives of HSV 1 + 2 IgM may involve RF bridging immobilized and enzyme- labeled antibodies, which form an immobilized complex consisting of enzyme-labeled antibodies and resulting in the appearance of false positives.10LM Abbreviations ELISA enzyme-linked immunosorbent assay HSV herpes simplex virus RF rheumatoid factor HBsAg surface antigen of the hepatits B virus S/CO sample/cutoff Ig immunoglobulin ASO antistreptolysin O TSH thyroid-stimulating hormone T3 triiodothyronine T4 thyroxine TPO thyroid peroxidase Ab antibody TG thyroglobulin TOX Toxoplasma gondii RV rubella virus CMV cytomegalovirus ANA antinuclear antibodies PCR polymerase chain reaction SCCA squamous-cell-carcinoma antigen IgM immunoglobulin M B19 parovirus B19 CO control M2 antimitochondrial antibodies M2 subtype (or AMA-M2) RIB ribosomal P protein HI histone NUC nucleosome DNA double-stranded DNA PCNA proliferating cell nuclear antigen CB centromere protein B (or CENP-B) Jo Jo-1 antigen (or histidyl-transfer RNA synthetase) PM polymyositis/scleroderma complex (or the exosome) Scl topoisomerase I (or Scl-70) SSB Sjögren-syndrome–related antigen B 52 Ro-52/SS-A 52 kD SSA Sjögren-syndrome–related antigen A Sm Sm antigen RNP/Sm nonhistone nuclear protein/Sm complex 0 negative + positive References 1. Xu L , Wang X , Ma R , et al. False decrease of HBsAg S/CO values in serum with high-concentration rheumatoid factors . Clin Biochem . 2013 ; 46 ( 9 ): 799 – 804 . Google Scholar CrossRef Search ADS PubMed 2. Berth M , Bosmans E , Everaert J , et al. Rheumatoid factor interference in the determination of carbohydrate antigen 19-9 (CA 19-9) . Clin Chem Lab Med . 2006 ; 44 ( 9 ): 1137 – 1139 . Google Scholar CrossRef Search ADS PubMed 3. Tsavaris N , Mavragani CP , Pikazis D , Dimitrios P . Rheumatoid arthritis: correlation between rheumatoid factor levels and CA-125 tumour marker elevation . Ann Rheum Dis . 2007 ; 66 ( 7 ): 980 . Google Scholar CrossRef Search ADS PubMed 4. Churchman SM , Geiler J , Parmar R , et al. Multiplexing immunoassays for cytokine detection in the serum of patients with rheumatoid arthritis: lack of sensitivity and interference by rheumatoid factor . Clin Exp Rheumatol . 2012 ; 30 ( 4 ): 534 – 542 . Google Scholar PubMed 5. Fleming SM , O’Byrne L , Finn J , Grimes H , Daly KM . False-positive cardiac troponin I in a routine clinical population . Am J Cardiol . 2002 ; 89 ( 10 ): 1212 – 1215 . Google Scholar CrossRef Search ADS PubMed 6. Norden AG , Jackson RA , Norden LE , Griffin AJ , Barnes MA , Little JA . Misleading results from immunoassays of serum free thyroxine in the presence of rheumatoid factor . Clin Chem . 1997 ; 43 ( 6 pt 1 ): 957 – 962 . Google Scholar PubMed 7. Salonen EM , Vaheri A , Suni J , Wager O . Rheumatoid factor in acute viral infections: interference with determination of IgM, IgG, and IgA antibodies in an enzyme immunoassay . J Infect Dis . 1980 ; 142 ( 2 ): 250 – 255 . Google Scholar CrossRef Search ADS PubMed 8. Biasiolo A , Tono N , Zaninotto M , et al. Specificity of squamous cell carcinoma antigen (SCCA)-IgM detection in patients with HCV infection and rheumatoid factor seropositivity . J Med Virol . 2013 ; 85 ( 6 ): 1005 – 1008 . Google Scholar CrossRef Search ADS PubMed 9. Juto P , Settergren B . Specific serum IgA, IgG and IgM antibody determination by a modified indirect ELISA-technique in primary and recurrent herpes simplex virus infection . J Virol Methods . 1988 ; 20 ( 1 ): 45 – 55 . Google Scholar CrossRef Search ADS PubMed 10. Kenny PR , Finger DR . Falsely elevated cardiac troponin-I in patients with seropositive rheumatoid arthritis . J Rheumatol . 2005 ; 32 ( 7 ): 1258 – 1261 . Google Scholar PubMed © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Laboratory Medicine Oxford University Press

Interference by Rheumatoid Factor in Immunoglobulin M-Class Herpes Simplex Virus Types 1 + 2 Immunoassays

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Abstract

Abstract Herpes simplex virus (HSV) types 1 + 2 immunoglobulin (Ig)M antibodies assays are important tools for diagnosing pregnant women and their newborns. However, it is unclear whether high levels of rheumatoid factor (RF) interfere with serological methods used to test for HSV types 1 + 2 type-specific IgM antibodies. We report a case of a false-positive result for HSV 1 + 2 IgM antibodies due to gross overestimation of RF by enzyme-linked immunosorbent assay (ELISA). This result occurred in a 25-year-old Han Chinese woman who was 28 weeks pregnant with a fetus (sex of fetus unknown). Antinuclear antibody (ANA) screening was performed, and RF-absorbent material was used. Test results for 3 antibodies of ANA (SSA, Ro-52, and SSB) were positive; the rest of the results were negative. Also, RF levels were significantly higher than normal, as measured by ANA screening. We observed an obvious decrease in HSV 1 + 2 IgM antibodies after removal of RF. Based on our findings, the presence of high concentrations of RF may be responsible for false-positive HSV 1 + 2 ELISA results. rheumatoid factor, herpes simplex virus, immunoassay, IgM antibody, pregnant, ELISA It has been reported that various interferences can alter enzyme-linked immunosorbent assay (ELISA) outcomes, and that herpes simplex virus (HSV) assays are not immune to such problems. Rheumatoid factor (RF) is one of the most common sources of interference. The role of high-concentration RF as a cause of false decrease of surface antigen of the hepatits B virus (HBsAg) sample/cutoff (S/CO) values is well known.1 Also, the presence of RF in serum can cause falsely elevated results in thyroid-function tests and cytokine, troponin, and tumor-maker immunoassays.2-6 Herein, we report a case of evaluated RF interference in HSV 1 + 2 type-specific immunoglobulin (Ig)M ELISA (DIESSE Diagnostica Senese S.P.A). Case Report Slowed fetal heart rate (96 beats per minute) was diagnosed after a 25-year-old Han Chinese woman who was 28 weeks pregnant with a fetus (sex of fetus unknown) visited Yangxian Hospital, ShanXi Province, China. The treating physician recommended that fetal ultrasonography and chromosomal examination be performed, with reexamination performed after 4 to 6 weeks. Subsequently, the patient visited Hanzhong 3201 Hospital in Hanzhong City on the same day, where ultrasonography results showed that fetal heart rate was less than 89 beats per minute. That rate was 94 beats per minute when the patient visited the obstetrical department of the Northwest Women’s and Children’s Hospital in Xi’an 2 weeks later. During physical examination of the woman, her thyroid was impalpable, and no tubercule was observed under the lateral subcutaneous ankle. Previous medical examination had not revealed a family history of rheumatoid arthritis, systemic lupus erythematosus, or other special cases. The antistreptolysin O (ASO) and thyroid function test results for the patient (as determined by the ARCHITECT i2000, Abbott Laboratories Inc), including thyroid- stimulating hormone (TSH), total triiodothyronie (T3) and thyroxine (T4), free T3 and T4, thyroid peroxidase antibody (TPO Ab), and thyroglobulin antibody (TG Ab), were found to be lower than the reference values for the healthy population. Conversely, we observed a significantly increased RF concentration (Beckman Coulter AU5800, Beckman Coulter Inc). Toxoplasma gondii (TOX) IgM, rubella virus (RV) IgM, cytomegalovirus (CMV) IgM (DIESSE Diagnostica Senese S.P.A.), and parvovirus B19 IgM (Virion\Serion, Germany) results were determined to be negative. However, HSV 1 + 2 IgM results were to indicate elevated S/CO values, based on readings via an Absorbance Microplate Reader (Rayto Life and Analytical Sciences) (Table 1). Table 1. Laboratory Results for the Patient, a 25-Year-Old Han Chinese Woman Pregnant With a 28-Week-Old Fetus (Sex of Fetus Unknown) Parameter Result Reference Range TSH 1.24 mIU/L 0.35–4.94 mIU/L Total T3 1.55 nmol/L 0.88–2.44 nmol/L Total T4 124 nmol/L 62.9–151 nmol/L Free T3 3.03 pmol/L 2.63–5.70 pmol/L Free T4 12.0 pmol/L 9.01–19.1 pmol/L TPO Ab 0.1 IU/mL <5.61 IU/mL TG Ab 1.3 IU/mL <4.11 IU/mL RF 543 IU/mL 0–30 IU/mL ASO 13 IU/mL 0–170 IU/mL TOX IgM 0.311 S/CO <0.9 S/CO RV IgM 0.614 S/CO <0.9 S/CO CMV IgM 0.543 S/CO <0.9 S/CO HSV 1 + 2 IgM 3.09 S/CO <0.9 S/CO B19 IgM 0.328 S/CO <1.0 S/CO Parameter Result Reference Range TSH 1.24 mIU/L 0.35–4.94 mIU/L Total T3 1.55 nmol/L 0.88–2.44 nmol/L Total T4 124 nmol/L 62.9–151 nmol/L Free T3 3.03 pmol/L 2.63–5.70 pmol/L Free T4 12.0 pmol/L 9.01–19.1 pmol/L TPO Ab 0.1 IU/mL <5.61 IU/mL TG Ab 1.3 IU/mL <4.11 IU/mL RF 543 IU/mL 0–30 IU/mL ASO 13 IU/mL 0–170 IU/mL TOX IgM 0.311 S/CO <0.9 S/CO RV IgM 0.614 S/CO <0.9 S/CO CMV IgM 0.543 S/CO <0.9 S/CO HSV 1 + 2 IgM 3.09 S/CO <0.9 S/CO B19 IgM 0.328 S/CO <1.0 S/CO TSH, thyroid-stimulating hormone; TPO, thyroid peroxidase; Ab, antibodies; TG, thyroglobulin; RF, rheumatoid factor; ASO, antistreptolysin O; TOX, Toxoplasma gondii; IgM, immunoglobulin M; S/CO, sample/cutoff; RV, rubella virus; CMV, cytomegalovirus; HSV, herpes simplex virus; B19, parvovirus B19. View Large Table 1. Laboratory Results for the Patient, a 25-Year-Old Han Chinese Woman Pregnant With a 28-Week-Old Fetus (Sex of Fetus Unknown) Parameter Result Reference Range TSH 1.24 mIU/L 0.35–4.94 mIU/L Total T3 1.55 nmol/L 0.88–2.44 nmol/L Total T4 124 nmol/L 62.9–151 nmol/L Free T3 3.03 pmol/L 2.63–5.70 pmol/L Free T4 12.0 pmol/L 9.01–19.1 pmol/L TPO Ab 0.1 IU/mL <5.61 IU/mL TG Ab 1.3 IU/mL <4.11 IU/mL RF 543 IU/mL 0–30 IU/mL ASO 13 IU/mL 0–170 IU/mL TOX IgM 0.311 S/CO <0.9 S/CO RV IgM 0.614 S/CO <0.9 S/CO CMV IgM 0.543 S/CO <0.9 S/CO HSV 1 + 2 IgM 3.09 S/CO <0.9 S/CO B19 IgM 0.328 S/CO <1.0 S/CO Parameter Result Reference Range TSH 1.24 mIU/L 0.35–4.94 mIU/L Total T3 1.55 nmol/L 0.88–2.44 nmol/L Total T4 124 nmol/L 62.9–151 nmol/L Free T3 3.03 pmol/L 2.63–5.70 pmol/L Free T4 12.0 pmol/L 9.01–19.1 pmol/L TPO Ab 0.1 IU/mL <5.61 IU/mL TG Ab 1.3 IU/mL <4.11 IU/mL RF 543 IU/mL 0–30 IU/mL ASO 13 IU/mL 0–170 IU/mL TOX IgM 0.311 S/CO <0.9 S/CO RV IgM 0.614 S/CO <0.9 S/CO CMV IgM 0.543 S/CO <0.9 S/CO HSV 1 + 2 IgM 3.09 S/CO <0.9 S/CO B19 IgM 0.328 S/CO <1.0 S/CO TSH, thyroid-stimulating hormone; TPO, thyroid peroxidase; Ab, antibodies; TG, thyroglobulin; RF, rheumatoid factor; ASO, antistreptolysin O; TOX, Toxoplasma gondii; IgM, immunoglobulin M; S/CO, sample/cutoff; RV, rubella virus; CMV, cytomegalovirus; HSV, herpes simplex virus; B19, parvovirus B19. View Large Applying our combined clinical experience to this case, we suspected that the HSV 1 + 2 IgM result might be a false positive and so we investigated possible interference. Our investigation revealed that the results for antibodies against SSA, Ro-52, and SSB were positive, whereas the remaining envelope antigens on the antinuclear antibodies (ANA)–3 membrane (Omont Père et Fils) tested negative (Figure 1). However, we discovered that the patient had no related autoimmune disease history. We observed that RF concentration was 543 IU per mL, which is much higher than 30 IU per mL, the upper limit of the reference value for the healthy population. It has been reported7 that the minimal effective amounts of RF are 25 ng per mL for IgM measurement in RV solid-phase enzyme immunoassays. Therefore, after pretreating the serum specimen with RF adsorption materials (1:10 dilution according to instructions after 1:1 dilution; Virion\Serion, Germany), RF titers decreased from 20.8 IU per mL to 9.66 IU per mL (equal proportion of specimen diluents and RF adsorption materials) and 7.92 IU per mL (RF adsorption materials only), respectively. Using specimen material with negative RF and elevated HSV IgM as a control group, we discovered that the S/CO values of HSV1 + 2 IgM antibodies were decreased separately, from 2.95 to 0.487 (equal proportion of specimen diluents and RF adsorption materials) or 0.352 (RF adsorption materials only). We discovered similar results in the parallel double holes (Table 2). These results provide evidence for the determination of HSV1 + 2 IgM antibodies influenced by RF. Table 2. RF Concentrations and S/CO Values of HSV 1 + 2 IgM After RF Adsorption Pretreatment Method RF Titers Sample S/CO (Control Group) S/CO S/CO (Parallel Double Holes) Diluents from the kit 20.8 IU/mL 2.95 IU/mL 3.24 IU/mL 3.813 IU/mL Equal proportion of specimen diluents and RF adsorption 9.66 IU/mL 0.487 IU/mL 0.449 IU/mL 3.249 IU/mL RF adsorption 7.92 IU/mL 0.352 IU/mL 0.311 IU/mL 2.690 IU/mL Pretreatment Method RF Titers Sample S/CO (Control Group) S/CO S/CO (Parallel Double Holes) Diluents from the kit 20.8 IU/mL 2.95 IU/mL 3.24 IU/mL 3.813 IU/mL Equal proportion of specimen diluents and RF adsorption 9.66 IU/mL 0.487 IU/mL 0.449 IU/mL 3.249 IU/mL RF adsorption 7.92 IU/mL 0.352 IU/mL 0.311 IU/mL 2.690 IU/mL RF, rheumatoid factor; S/CO, sample/cutoff; HSV, herpes simplex virus; IgM, immunoglobulin M; RF, rheumatoid factor. View Large Table 2. RF Concentrations and S/CO Values of HSV 1 + 2 IgM After RF Adsorption Pretreatment Method RF Titers Sample S/CO (Control Group) S/CO S/CO (Parallel Double Holes) Diluents from the kit 20.8 IU/mL 2.95 IU/mL 3.24 IU/mL 3.813 IU/mL Equal proportion of specimen diluents and RF adsorption 9.66 IU/mL 0.487 IU/mL 0.449 IU/mL 3.249 IU/mL RF adsorption 7.92 IU/mL 0.352 IU/mL 0.311 IU/mL 2.690 IU/mL Pretreatment Method RF Titers Sample S/CO (Control Group) S/CO S/CO (Parallel Double Holes) Diluents from the kit 20.8 IU/mL 2.95 IU/mL 3.24 IU/mL 3.813 IU/mL Equal proportion of specimen diluents and RF adsorption 9.66 IU/mL 0.487 IU/mL 0.449 IU/mL 3.249 IU/mL RF adsorption 7.92 IU/mL 0.352 IU/mL 0.311 IU/mL 2.690 IU/mL RF, rheumatoid factor; S/CO, sample/cutoff; HSV, herpes simplex virus; IgM, immunoglobulin M; RF, rheumatoid factor. View Large Figure 1 View largeDownload slide Antinuclear antibody (ANA) results via scanning. CO indicates control; M2, antimitochondrial antibodies M2 subtype (or AMA-M2); RIB, ribosomal P protein; HI, histone; NUC, nucleosome; DNA, double-stranded DNA; PCNA, proliferating cell nuclear antigen; CB, centromere protein B (or CENP-B); Jo, Jo-1 antigen (or histidyl-transfer RNA synthetase); PM, polymyositis/scleroderma complex (or the exosome); Scl, topoisomerase I (or Scl-70); SSB, Sjögren-syndrome–related antigen B; 52, Ro-52/SS-A 52 kD; SSA, Sjögren-syndrome–related antigen A; Sm, Sm antigen; RNP/Sm, nonhistone nuclear protein/Sm complex; 0, negative; +, positive. Figure 1 View largeDownload slide Antinuclear antibody (ANA) results via scanning. CO indicates control; M2, antimitochondrial antibodies M2 subtype (or AMA-M2); RIB, ribosomal P protein; HI, histone; NUC, nucleosome; DNA, double-stranded DNA; PCNA, proliferating cell nuclear antigen; CB, centromere protein B (or CENP-B); Jo, Jo-1 antigen (or histidyl-transfer RNA synthetase); PM, polymyositis/scleroderma complex (or the exosome); Scl, topoisomerase I (or Scl-70); SSB, Sjögren-syndrome–related antigen B; 52, Ro-52/SS-A 52 kD; SSA, Sjögren-syndrome–related antigen A; Sm, Sm antigen; RNP/Sm, nonhistone nuclear protein/Sm complex; 0, negative; +, positive. Discussion Accurate laboratory assessment of HSV infections is critical for timely management and treatment in pregnancy. ELISA is a rapid, sensitive, commercialized, and automated serologic assay for detection of HSV-specific antibodies; its results can be affected by various types of interference. However, it is difficult to detect assay interferences in routine processing. It is colloquially understood that the identification of interferences requires clinical awareness. Laboratory personnel should consider that interference factors exist and therefore should communicate with clinical physicians in a timely manner when abnormal results emerge. We recommend that HSV-DNA be detected by fluorescence quantitative polymerase chain reaction (PCR) in patients with suspected HSV infection. RF is a type of autoantibody to the Fc fragment of IgG, representing a long-standing problem in terms of interference for immunometric assays. It has been reported8 that RF may lead to high levels of squamous-cell-carcinoma antigen (SCCA)–IgM in patients with cirrhosis. Salonen et al7 also suggested that RF can lead to false-positive results for IgM antibodies against rubella and influenza viruses by ELISA, but that RF had little to no effect on antibodies to cytomegalovirus in same-enzyme immunoassays. In the present study, cryopreservation of specimens for nearly 10 days may account for the slightly decreased RF titers. However, interference by RF was not excluded. We discovered that not only RF concentrations but also S/CO values of HSV 1 + 2 IgM antibodies were decreased after treatment with RF absorption. These findings support the theory that the presence of RF acts as an interferent. Our results are consistent with those of a previous report9 in which pretreatment with antihuman IgG can eliminate RF-interfering HSV IgM detection via ELISA. The mechanism whereby RF leads to the occurrence of false positives of HSV 1 + 2 IgM may involve RF bridging immobilized and enzyme- labeled antibodies, which form an immobilized complex consisting of enzyme-labeled antibodies and resulting in the appearance of false positives.10LM Abbreviations ELISA enzyme-linked immunosorbent assay HSV herpes simplex virus RF rheumatoid factor HBsAg surface antigen of the hepatits B virus S/CO sample/cutoff Ig immunoglobulin ASO antistreptolysin O TSH thyroid-stimulating hormone T3 triiodothyronine T4 thyroxine TPO thyroid peroxidase Ab antibody TG thyroglobulin TOX Toxoplasma gondii RV rubella virus CMV cytomegalovirus ANA antinuclear antibodies PCR polymerase chain reaction SCCA squamous-cell-carcinoma antigen IgM immunoglobulin M B19 parovirus B19 CO control M2 antimitochondrial antibodies M2 subtype (or AMA-M2) RIB ribosomal P protein HI histone NUC nucleosome DNA double-stranded DNA PCNA proliferating cell nuclear antigen CB centromere protein B (or CENP-B) Jo Jo-1 antigen (or histidyl-transfer RNA synthetase) PM polymyositis/scleroderma complex (or the exosome) Scl topoisomerase I (or Scl-70) SSB Sjögren-syndrome–related antigen B 52 Ro-52/SS-A 52 kD SSA Sjögren-syndrome–related antigen A Sm Sm antigen RNP/Sm nonhistone nuclear protein/Sm complex 0 negative + positive References 1. Xu L , Wang X , Ma R , et al. False decrease of HBsAg S/CO values in serum with high-concentration rheumatoid factors . Clin Biochem . 2013 ; 46 ( 9 ): 799 – 804 . Google Scholar CrossRef Search ADS PubMed 2. Berth M , Bosmans E , Everaert J , et al. Rheumatoid factor interference in the determination of carbohydrate antigen 19-9 (CA 19-9) . Clin Chem Lab Med . 2006 ; 44 ( 9 ): 1137 – 1139 . Google Scholar CrossRef Search ADS PubMed 3. Tsavaris N , Mavragani CP , Pikazis D , Dimitrios P . Rheumatoid arthritis: correlation between rheumatoid factor levels and CA-125 tumour marker elevation . Ann Rheum Dis . 2007 ; 66 ( 7 ): 980 . Google Scholar CrossRef Search ADS PubMed 4. Churchman SM , Geiler J , Parmar R , et al. Multiplexing immunoassays for cytokine detection in the serum of patients with rheumatoid arthritis: lack of sensitivity and interference by rheumatoid factor . Clin Exp Rheumatol . 2012 ; 30 ( 4 ): 534 – 542 . Google Scholar PubMed 5. Fleming SM , O’Byrne L , Finn J , Grimes H , Daly KM . False-positive cardiac troponin I in a routine clinical population . Am J Cardiol . 2002 ; 89 ( 10 ): 1212 – 1215 . Google Scholar CrossRef Search ADS PubMed 6. Norden AG , Jackson RA , Norden LE , Griffin AJ , Barnes MA , Little JA . Misleading results from immunoassays of serum free thyroxine in the presence of rheumatoid factor . Clin Chem . 1997 ; 43 ( 6 pt 1 ): 957 – 962 . Google Scholar PubMed 7. Salonen EM , Vaheri A , Suni J , Wager O . Rheumatoid factor in acute viral infections: interference with determination of IgM, IgG, and IgA antibodies in an enzyme immunoassay . J Infect Dis . 1980 ; 142 ( 2 ): 250 – 255 . Google Scholar CrossRef Search ADS PubMed 8. Biasiolo A , Tono N , Zaninotto M , et al. Specificity of squamous cell carcinoma antigen (SCCA)-IgM detection in patients with HCV infection and rheumatoid factor seropositivity . J Med Virol . 2013 ; 85 ( 6 ): 1005 – 1008 . Google Scholar CrossRef Search ADS PubMed 9. Juto P , Settergren B . Specific serum IgA, IgG and IgM antibody determination by a modified indirect ELISA-technique in primary and recurrent herpes simplex virus infection . J Virol Methods . 1988 ; 20 ( 1 ): 45 – 55 . Google Scholar CrossRef Search ADS PubMed 10. Kenny PR , Finger DR . Falsely elevated cardiac troponin-I in patients with seropositive rheumatoid arthritis . J Rheumatol . 2005 ; 32 ( 7 ): 1258 – 1261 . Google Scholar PubMed © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Laboratory MedicineOxford University Press

Published: Apr 24, 2018

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