David G. Grenache

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.

α1-Antitrypsin Phenotypes and Associated Serum Protein Concentrations in a Large Clinical Population

BACKGROUND α1-Antitrypsin (AAT) deficiency variants reduce the concentration of serum AAT protease inhibitor and can lead to the development of pulmonary and hepatic disease. Relative frequencies of rare AAT variant phenotypes (non-M, Z, and S) and associated serum concentrations in the clinical population have not been thoroughly described. METHODS Protein phenotypes were determined by isoelectric focusing electrophoresis for 72,229 consecutive samples. Phenotype frequencies, median serum concentrations, and central 95% concentration intervals were determined for observed phenotypes. Concurrent AAT phenotype and concentration data were used to evaluate the efficacy of using serum AAT concentration alone to detect AAT deficiency. RESULTS Age, race, and sex had only slight effects on the median 95% serum protein concentration intervals of the 58,087 PiMM (wild type) phenotype specimens. Positive predictive values were calculated for the detection of potential deficiency phenotypes at different serum cutoff concentrations, aiding potential screening effort design. For example, the PiZZ deficiency phenotype (n = 814) could be detected at 99.5% sensitivity and 96.5% specificity using a cutoff of ≤ 85 mg/dL. However, at-risk specimens with two putative deleterious variants (Z, S, I, F, P, T, and Null variants) were detected with only 85.9% sensitivity at this cutoff (n = 1,661). Rare phenotype variants were observed in 2.5% of samples. CONCLUSIONS This analysis provides novel information on serum AAT concentrations associated with different AAT phenotypes and provides insight into the severity of depression of AAT concentration in the presence of rare deficiency variants. Additionally, it allows for evaluation of efficacy of testing algorithms incorporating AAT serum concentration determination.

Establishment of reference intervals for soluble ST2 from a United States population

BACKGROUND Soluble ST2 (sST2) is a protein in the interleukin-1 receptor family secreted by myocytes in response to mechanical strain. Elevated sST2 is strongly prognostic in patients with heart failure. METHODS sST2 was measured using the Presage ST2 ELISA. Evaluation included imprecision, linearity, recovery, analytical sensitivity, limit of quantification, stability, and sample type comparisons. Gender-specific reference intervals were established from 245 male and 245 female serum specimens. RESULTS At sST2 concentrations of 11.6, 26.9, and 88.0 ng/mL, the within-day CV was 7.6, 2.4, and 3.8%, respectively and the total CV was 11.5, 14.0, and 6.3%, respectively. The assay was linear over a concentration range of 2.8-161.1 ng/mL (y=0.95x+2.25; R(2)=0.997; Sy.x=3.03). The limit of quantification was 3.3 ng/mL. sST2 was stable for 2 days at room temperature, 10days at 4 °C, and 30 days at -20 °C. Concentrations of sST2 were significantly higher in males compared to females (24.9 vs. 16.9 ng/mL; p<0.0001) but were not correlated by age in either gender (r=-0.07; p=0.14). Reference intervals for sST2 were determined to be 8.6-49.3 and 7.2-33.5 ng/mL for males and females, respectively. CONCLUSION The Presage ST2 ELISA had acceptable performance characteristics for quantifying sST2 in serum or plasma. The assay is precise and linear over a wide sST2 concentration range and can measure low sST2 concentrations. Concentrations of sST2 are unaffected by age but are higher in males compared to females.

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.

Comparison of multiple methods for identification of hyperprolactinemia in the presence of macroprolactin

BACKGROUND Macroprolactin is a large, heterogeneous form of prolactin with limited bioavailability. Detection of macroprolactin by different immunoassays varies widely. The objectives of this study were to determine the immunoreactivity of macroprolactin by the Ortho Clinical Diagnostics Vitros((R)) ECi prolactin immunoassay, establish the most effective method for interpreting the prolactin concentration after PEG-precipitation, and correlate the clinical features of hyperprolactinemia with the presence of macroprolactin. METHODS PEG-precipitation was performed on 120 hyperprolactinemic specimens. Of these, 31 specimens with a recovery<80% were fractionated by GFC. Four different approaches for identifying true hyperprolactinemia were investigated. Clinical symptoms of hyperprolactinemia were determined by chart review. RESULTS Macroprolactin was detected by the Vitros ECi prolactin immunoassay. Use of a PEG modified prolactin reference interval was effective for identifying hyperprolactinemia in the presence of macroprolactin. There was no difference in the prevalence of abnormal menses, galactorrhea, or abnormal MRI between those with and without macroprolactin (p>0.05). Accounting for macroprolactin in patients with hyperprolactinemia reduced the number of idiopathic cases. CONCLUSIONS The Vitros ECi prolactin immunoassay detects macroprolactin. PEG-precipitation is an acceptable surrogate to detect hyperprolactinemia in the presence of macroprolactin when using a prolactin reference interval derived from PEG precipitated reference sera. Although testing for macroprolactin should not substitute for standard evaluation of hyperprolactinemia, identification of macroprolactin may clarify a diagnosis and direct appropriate therapy.

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.

Qualitative point-of-care and over-the-counter urine hCG devices differentially detect the hCG variants of early pregnancy

BACKGROUND Qualitative point-of-care (POC) tests for human chorionic gonadotropin (hCG) vary in their ability to detect purified hCG variants and there is data to suggest that over-the-counter (OTC) devices might also display similar variability. This could potentially influence the detection of urine hCG in early pregnancy. METHODS Six OTC devices were tested for their ability to detect 5 hCG variants. Ten early pregnancy urine specimens were selected for their diverse expression of hCG variants. The samples were tested with 6 brands of POC and 6 OTC devices. RESULTS OTC devices consistently recognized intact hCG, hCGn, and hCGbeta. hCGbetan was consistently recognized by 4 out of 6 brands. One brand inconsistently recognized hCGbetacf. OTC and POC devices varied greatly in their ability to detect hCG in early pregnancy urine, despite the fact that urine samples were adjusted to the same intact hCG concentration. Interestingly, we found that the OTC devices had better analytical sensitivity than the POC devices. Clinitest and First Response demonstrated the lowest hCG detection limits for POC and OTC devices, respectively. CONCLUSIONS Both OTC and POC devices are capable of detecting hCG concentrations in early pregnancy urine, and OTC devices demonstrated better analytical sensitivity relative to POC devices.

Performance Comparison of Capillary and Agarose Gel Electrophoresis for the Identification and Characterization of Monoclonal Immunoglobulins

The objective of this study was to compare gel- and capillary-based serum protein electrophoresis methods to identify and characterize monoclonal immunoglobulins (M proteins). Five reviewers interpreted 149 consecutively ordered serum specimens following agarose gel electrophoresis (AGE), capillary electrophoresis (CE), immunofixation electrophoresis (IFE), and subtraction immunotyping (IT). As a screening test for detecting M proteins, AGE and CE displayed similar sensitivity (91% and 92%, respectively). CE was less specific (74%) than AGE (81%). An analysis of interinterpreter agreement revealed that interpretations were more consistent using gel-based methods than capillary-based methods, with 80% of the gel interpretations being in complete (5/5) agreement compared with 67% of the capillary interpretations. After implementing the capillary-based methods, the number of tests per reportable result increased (from 1.58 to 1.73). CE is an analytically suitable alternative to AGE, but laboratories implementing it will need to continue IFE testing to characterize all M proteins detected by CE.

The relationship between PTH and 25‐hydroxy vitamin D early in pregnancy

Objective  Measure serum PTH and 25(OH)D in a cross‐sectional sample of pregnant women at 11th through 13th weeks’ gestation to examine vitamin D status and consider implications.

Immunoassay for Quantifying Squamous Cell Carcinoma Antigen in Serum

BACKGROUND Although the benefits of quantifying serum squamous cell carcinoma antigen (SCCa) have been reported, SCCa reagents were no longer available in the US by the late 1990s. Because SCCa quantification still has demonstrated clinical utility, we developed and validated a microtiter plate-based ELISA for measuring SCCa in serum. METHODS We coated microtiter strips overnight with capture anti-SCCa monoclonal antibody, washed the wells, added blocking buffer, and lyophilized the strips. For detection, we used a biotinylated anti-SCCa detection antibody, streptavidin/horseradish peroxidase conjugate, and tetramethylbenzidine/H(2)O(2) substrate. A novel blocking reagent against human antimouse antibodies (HAMA) was evaluated. A reference interval was established with sera from healthy individuals and was confirmed in smokers. RESULTS The assay was linear to 40 microg/L SCCa (slope, 1.00; y intercept, 0.695; R(2), 0.996) with a detection limit of 0.3 microg/L. The intraassay imprecision results [mean (CV)] were 2.5 microg/L (3.4%), 18.0 microg/L (3.0%), and 30.7 microg/L (2.4%); interassay imprecision results were 2.0 microg/L (9.9%), 20.0 microg/L (7.6%), and 36.3 microg/L (3.5%). A correlation analysis against an established automated assay generated a slope of 0.976 and a y intercept of -0.193 microg/L (r(2) = 0.916). An upper reference limit of 2.1 microg/L SCCa was established at 95% confidence level, with no difference observed in smokers. No correlation between SCCa concentration and age was observed (r(2) = 0.0003). At a blocking reagent concentration of 5 mg/L, HAMA interference was eliminated in 3 samples known to produce falsely increased SCCa results. CONCLUSIONS This SCCa ELISA demonstrates acceptable performance characteristics for quantifying serum SCCa and is effective in eliminating HAMA interference.