Ann M. Gronowski

Evidence-Based Approach for Interpretation of Epstein-Barr Virus Serological Patterns

ABSTRACT Diagnosis of Epstein-Barr virus (EBV) infection is based on clinical symptoms and serological markers, including the following: immunoglobulin G (IgG) and IgM antibodies to the viral capsid antigen (VCA), heterophile antibodies, and IgG antibodies to the EBV early antigen-diffuse (EA-D) and nuclear antigen (EBNA-1). The use of all five markers results in 32 possible serological patterns. As a result, interpretation of EBV serologies remains a challenge. The purpose of this study was to use a large population of patients to develop evidence-based tools for interpreting EBV results. This study utilized 1,846 serum specimens sent to the laboratory for physician-ordered EBV testing. Chart review was performed for more than 800 patients, and diagnoses were assigned based on physician-ordered testing, clinical presentation, and patient history. Testing for all five EBV antibodies was performed separately on all serum samples using the Bio-Rad BioPlex 2200 system. Presumed EBV diagnosis (based on previous publications) was compared to EBV diagnosis based on a medical record review for each serological pattern. Interestingly, of the 32 possible serological patterns, only 12 occurred in ≥10 patients. The remaining 20 patterns were uninterpretable because they occurred with such infrequency. Two easy-to-use tables were created to interpret EBV serological patterns based on whether three (EBV VCA IgG, IgM, and heterophile) or five markers are utilized. The use of these two tables allows for interpretation of >95% of BioPlex serological results. This is the first evidence-based study of its kind for EBV serology.

Circulating MicroRNA miR-323-3p as a Biomarker of Ectopic Pregnancy

BACKGROUND The use of serum human chorionic gonadotropin (hCG) and progesterone to identify patients with ectopic pregnancy (EP) has been shown to have poor clinical utility. Pregnancy-associated circulating microRNAs (miRNAs) have been proposed as potential biomarkers for the diagnosis of pregnancy-associated complications. This proof-of-concept study examined the diagnostic accuracy of various miRNAs to detect EP in an emergency department (ED) setting. METHODS This study was a retrospective case-control analysis of 89 women who presented to the ED with vaginal bleeding and/or abdominal pain/cramping and received a diagnosis of viable intrauterine pregnancy (VIP), spontaneous abortion (SA), or EP. Serum hCG and progesterone concentrations were measured by immunoassays. The serum concentrations of miRNAs miR-323-3p, miR-517a, miR-519d, and miR-525-3p were measured with TaqMan real-time PCR. Statistical analysis was performed to determine the clinical utility of these biomarkers, both as single markers and as multimarker panels for EP. RESULTS Concentrations of serum hCG, progesterone, miR-517a, miR-519d, and miR-525-3p were significantly lower in EP and SA cases than in VIP cases (P < 0.01). In contrast, the concentration of miR-323-3p was significantly increased in EP cases, compared with SA and VIP cases (P < 0.01). As a single marker, miR-323-3p had the highest sensitivity of 37.0% (at a fixed specificity of 90%). In comparison, the combined panel of hCG, progesterone, and miR-323-3p yielded the highest sensitivity (77.8%, at a fixed specificity of 90%). A stepwise analysis that used hCG first, added progesterone, and then added miR-323-3p yielded a 96.3% sensitivity and a 72.6% specificity. CONCLUSIONS Pregnancy-associated miRNAs, especially miR-323-3p, added substantial diagnostic accuracy to a panel including hCG and progesterone for the diagnosis of EP.

Diagnostic potential for miRNAs as biomarkers for pregnancy-specific diseases

Discovery of circulating miRNAs in maternal blood has not only facilitated the understanding of their role in normal pregnancy, but also paved new avenues for biomarker discovery to detect pregnancy-associated complications, such as preeclampsia, ectopic pregnancy, gestational diabetes mellitus, fetal growth restriction, recurrent pregnancy loss, and preterm delivery. In this review, we summarize the studies to date of miRNAs in maternal circulation and placental tissue in human. This brief review does not cover all aspects of this intriguing field but focuses on some new and interesting findings of diagnostic potential for miRNAs as biomarkers for pregnancy-specific diseases.

Evaluation of a Multiplexed Bead Assay for Assessment of Epstein-Barr Virus Immunologic Status

ABSTRACT Currently, serological assays using either indirect immunofluorescence assay or enzyme-linked immunosorbent assay (ELISA) are performed to evaluate the status of Epstein-Barr virus (EBV) infection in humans. Although these methods are reliable, they are limited to testing an antibody response to a single viral antigen per reaction, thus necessitating a panel of assays to complete the evaluation. In contrast, a new bead-based method (BioPlex 2200; Bio-Rad Laboratories, Hercules, Calif.) can analyze the humoral response to multiple antigens in a single tube. This approach potentially reduces overall cost, turnaround time, and sample volume. The aim of this study was to evaluate the multiplexed EBV serologic assays performed on the BioPlex 2200 platform compared to results of conventional heterophile and ELISA-based assays. A total of 167 nonconsecutive, stored serum samples from adult and pediatric patients submitted for EBV serologic studies were used in the evaluation. Concordance between results generated by the BioPlex 2200 system and conventional assays was calculated. The anti-EA-D assay had the lowest concordance at 91%. The BioPlex 2200 system showed 97% agreement with conventional heterophile and anti-nuclear antigen assays and 92% agreement with the anti-VCA IgG and immunoglobulin M assays. Agreement between the BioPlex 2200 system and conventional testing was 92% with respect to categorization of acute versus nonacute EBV disease. The correlation between these two systems with regard to assignment into one of four categories of EBV status was also good (82%). In summary, there is excellent correlation between contemporary EBV serologic testing and the BioPlex 2200 system.

False-Negative Results in Point-of-Care Qualitative Human Chorionic Gonadotropin (hCG) Devices Due to Excess hCGβ Core Fragment

BACKGROUND During pregnancy, human chorionic gonadotropin (hCG) immunoreactivity in urine consists of intact hCG as well as a number of hCG variants including the core fragment of hCGbeta (hCGbeta cf). We identified 3 urine specimens with apparent false-negative results using the OSOM(R) hCG Combo Test (Genzyme Diagnostics) qualitative hCG device and sought to determine whether an excess of 1 of the fragments or variants might be the cause of the interference. METHODS We measured concentrations of hCG variants in the urine from 3 patients with apparent false-negative hCG results. Purified hCG variants were added to urines positive for hCG and tested using the OSOM, ICON(R) 25 hCG (Beckman Coulter), and hCG Combo SP(R) Brand (Cardinal Health) devices. RESULTS Dilution of these 3 urine samples resulted in positive results on the OSOM device. Quantification of hCG variants in each of the 3 patient urine specimens demonstrated that hCGbeta cf occurred in molar excess of intact hCG. Addition of purified hCGbeta cf to hCG-positive urines caused false-negative hCG results using the OSOM and ICON qualitative urine hCG devices. CONCLUSIONS Increased concentrations of hCGbeta cf can cause false-negative results on the OSOM and ICON qualitative urine hCG devices.

Serum Human Chorionic Gonadotropin Concentrations Greater than 400,000 IU/L Are Invariably Associated with Suppressed Serum Thyrotropin Concentrations

BACKGROUND During pregnancy, when human chorionic gonadotropin (hCG) concentrations are highest, there is a transient suppression of serum thyrotropin (TSH). In normal pregnancy, TSH concentrations generally remain within nonpregnant reference intervals; however, in some patients TSH is suppressed. Here we sought to extend previous studies to examine the relationship between very high serum concentrations of hCG (>200,000 IU/L) and the thyroid hormones TSH and free thyroxine (FT(4)). The objective of this study was to determine: 1) if there is an hCG concentration above which TSH concentrations are suppressed (< or =0.2 microIU/mL); 2) how thyroid hormone concentrations change in response to changes in hCG concentrations; and 3) the clinical symptoms in patients with such extremely elevated hCG concentrations. METHODS Residual specimens sent to the laboratories for physician-ordered hCG testing were utilized. Over 26 months, 15,597 physician-ordered hCG tests were performed. Sixty-nine specimens from 63 women with hCG concentrations >200,000 IU/L were identified, and TSH and FT(4) concentrations were measured. Medical records were reviewed for clinical information. RESULTS Thirty-seven percent of subjects had hyperemesis gravidarum (HG) and 19% had gestational trophoblastic disease (GTD). TSH was suppressed (< or =0.2 microIU/mL) in 67% of the specimens with hCG concentrations >200,000 IU/L and 100% of specimens with hCG concentrations >400,000 IU/L. FT(4) concentrations were elevated above the reference interval (1.8 ng/dL) in 32% of specimens with hCG concentrations >200,000 IU/L and in 80% of specimens with hCG concentrations >400,000 IU/L. Only four subjects had documented signs of hyperthyroidism. Women with GTD had a median hCG concentration twofold higher than women with HG and a median TSH concentration one half that of women with HG. CONCLUSIONS 1) At hCG concentrations >400,000 IU/L, TSH is consistently suppressed; 2) serum FT(4) and TSH respond to changes in serum hCG concentrations; and 3) most patients with hCG concentrations >200,000 IU/L lack overt hyperthyroid symptoms.

Growth hormone rapidly activates rat serine protease inhibitor 2.1 gene transcription and induces a DNA-binding activity distinct from those of Stat1, -3, and -4.

Transcriptional regulation by growth hormone (GH) represents the culmination of signal transduction pathways that are initiated by the cell surface GH receptor and are targeted to the nucleus. Recent studies have demonstrated that the activated GH receptor can stimulate Stat1, a cytoplasmic transcription factor that becomes tyrosine phosphorylated and translocates to the nucleus, where it can interact with specific DNA sequences to modulate gene expression. GH also has been found to induce protein binding to a portion of the rat serine protease inhibitor (Spi) 2.1 gene promoter that is required for GH-induced transcription of Spi 2.1. Using GH-deficient hypophysectomized rats as a model, we show that GH treatment rapidly and potently induces both nuclear Spi 2.1 mRNA expression in the liver and specific nuclear protein binding to a 45-bp segment of the Spi 2.1 gene promoter. A GH-inducible gel-shifted complex appears within 15 min of systemic hormone administration and can be inhibited by an antiphosphotyrosine monoclonal antibody but is not blocked by a polyclonal antiserum to Stat1, Stat3, or Stat4, even though the nucleotide sequence contains two gamma interferon-activated sequence-like elements that could interact with STAT proteins. By Southwestern (DNA-protein) blot analysis, approximately 41- and 35-kDa GH-inducible proteins were detected in hepatic nuclear extracts with the Spi 2.1 DNA probe. Thus, a GH-activated signaling pathway stimulates Spi 2.1 gene expression through a unique mechanism that does not appear to involve known members of the STAT family of transcription factors.

Use of serum FSH to identify perimenopausal women with pituitary hCG

BACKGROUND Human chorionic gonadotropin (hCG) tests are performed on many female patients before performing medical procedures or administering medications that may harm a fetus. hCG of pituitary origin has been shown to increase with age. Therefore, mild increases in serum hCG in an older patient can be of pituitary origin and does not necessarily indicate pregnancy. The inability to rule out pregnancy in perimenopausal women can create clinical confusion and may delay needed therapies. Our objective was to determine the diagnostic utility of serum follicle-stimulating hormone (FSH) concentrations to rule out hCG of placental origin in perimenopausal women with a low concentration of serum hCG (5.0-14.0 IU/L). METHODS Seven testing centers performed 39 742 physician-ordered serum quantitative hCG tests over a 15-month period. From these, 100 samples from women 41-55 years of age with serum hCG concentrations 5-14 IU/L were identified. We performed FSH testing and patient chart review for each sample. RESULTS Twenty-three patients were found to have hCG of placental origin (pregnancy, resolving abortion, or gestational trophoblastic disease), and in those cases serum FSH was 0.4-43.8 IU/L. An FSH cutoff of 45.0 IU/L identified hCG of placental origin with 100% sensitivity and 75% specificity. FSH >45 IU/L was never observed when hCG was of placental origin (negative predictive value). CONCLUSIONS These data indicate that quantitative serum FSH can be used to rule out pregnancy and hCG of placental origin in women 41-55 years of age with mild increase in serum hCG concentrations.

Rapid Nuclear Actions of Growth Hormone

The mechanisms by which growth hormone (GH) initiates its effect on growth are largely unknown. In this report we examine the acute actions of GH with a focus on the intracellular signaling pathways leading from the cell-surface GH receptor into the nucleus, and culminating in the activation of specific target genes. We show that in vivo GH treatment leads to the rapid appearance of tyrosine-phosphorylated nuclear proteins and the equally rapid induction of c-fos and insulin-like growth factor I gene transcription. A model is proposed for a GH-activated intracellular signal transduction pathway.

The analytical specificity of human chorionic gonadotropin assays determined using WHO International Reference Reagents

BACKGROUND Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone with considerable molecular heterogeneity. There is uncertainty regarding which hCG variants are detected by different hCG assays. The analytical specificity of 8 hCG assays was investigated. METHODS WHO International Reference Reagents for hCG, nicked hCG (hCGn), beta subunit (hCGbeta), nicked beta subunit (hCGbetan), and beta core fragment (hCGbetacf) were individually added to hCG-free human serum. Specimens were analyzed with 8 commercially available hCG assays. Equimolar detection of hCG variants was defined as a recovery of 90-110%. RESULTS All assays detected hCG and hCGn with mean recoveries of 98.3 and 94.6%, respectively. Seven assays detected hCGbeta (mean recovery 103.8%) but with high variation, and equimolar detection was observed only in four. The mean recovery of hCGbetan was 85.5% but was highly variable with only two assays showing equimolar detection. With a mean recovery of 53.4%, two assays detected hCGbetacf and both underestimated it considerably. Information provided by the assay manufacturer regarding hCG variant analytical specificity was inadequate or unclear in 75% of the assays. CONCLUSIONS hCG assays vary considerably in their ability to detect different hCG variants. Manufacturers of hCG assays should clearly indicate the hCG variant specificity of their reagent systems.