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  Clinical Abstracts





October 2010

Michael Bissell, MD, PhD, MPH

A new total activin B ELISA: development and validation A new total activin B ELISA: development and validation

Activins and inhibins are members of the transforming growth factor-β superfamily and are dimeric in structure. Activins are hetero- or homodimeric molecules, whereas inhibins are heterodimeric and comprise an alpha and beta subunit. Activins have been found in most tissues, including placenta, bone marrow, brain, and reproductive organs. They also have been co-localized with their receptors, suggesting autocrine/paracrine mechanisms of action. Activins can be neutralized by being bound by the high-affinity binding protein follistatin, such that most of the activin measured in the circulation is bound to follistatin. Consistent with their widespread localization, activins have been reported to be involved in numerous processes and conditions, including stimulation of follicle-stimulating hormone release from the pituitary, folliculogenesis in the ovary, erythroid differentiation, wound healing, osteoporosis, inflammation, nerve cell survival, and tumor and embryonic development. Descriptions of most of these functions have used measurements or effects of activin A, mainly because of the lack of tools for measuring activin B. Reports of activin B have mostly considered expression of βB subunit mRNA or the effects of recombinant/purified protein in cell systems or other bioassays. The best known and most widely used method for measuring these proteins is a highly specific enzyme-linked immunosorbent assay for activin A. ELISAs developed for measuring activin B in biological fluids are few and are not used widely. Therefore, the authors conducted a study to develop and validate a total activin B ELISA for use with human biological fluids and to establish concentrations of activin B in the circulation and fluids from reproductive organs. The new ELISA was validated and then used to measure activin B levels in the circulation of healthy study participants, in vitro fertilization patients, and pregnant women, as well as in ovarian follicular fluid and seminal plasma. By sampling the healthy adult subjects (n=143), subjects from an in vitro fertilization clinic (n=27), and pregnancy groups (n=29), the authors found that the sensitivity of the assay was 0.019 ng/mL. Validation of the activin B ELISA showed good recovery (90.7±9.8 percent) and linearity in biological fluid and cell culture media and low crossreactivity with related analytes (inhibin B, 0.077 percent; activin A, 0.0034 percent). A negative correlation was noted between activin B concentration (r=–0.281; P<0.011) and females with increasing age. Patients visiting in vitro fertilization clinics had significantly lower levels of activin B compared with gender-matched control subjects. Ovarian follicular fluid and seminal plasma had 50- to 80-fold higher levels of activin B (mean, 5.35 and 3.66 ng/mL, respectively) than sera (mean, 0.071 ng/mL). The authors concluded that this fully validated ELISA for activin B offers tremendous utility for measuring the protein in a variety of normal physiological processes and in various clinical pathologies.

Ludlow H, Phillips DJ, Myers M, et al. A new ‘total’ activin B enzyme-linked immunosorbent assay (ELISA): development and validation for human samples. Clin Endocrinol. 2009;71:867–873.

Correspondence: Helen Ludlow at

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Cell membrane fatty acid and cardiovascular disease Cell membrane fatty acid and cardiovascular disease

Erythrocyte (RBC) membrane composition varies with such factors as diet, oxidative stress, and genetic variation. This is important because the RBC traverses the microvasculature and is subject to turbulence and deformation when the vasculature and microvasculature are altered by atherosclerotic disease. Therefore, factors that change RBC membrane fluidity, such as the RBC lipid composition, affect the progression of atherosclerotic disease. A number of drugs also modify membrane composition, including antiepileptic drugs and drugs that modify cholesterol synthesis. Well-characterized dietary effects include the protective effects of fatty acids found in fish oils that have unsaturation at the third carbon from the terminal (omega-3) carbon of the fatty acid. Mammalian polyunsaturated fatty acids, including arachidonic acid (cis5, cis8, cis11, cis14-eicosatetranoic acid), are omega-6 fatty acids. Erythrocyte fatty acid measurements have been shown to better correlate with dietary intake than serum fatty acids. The authors conducted a study to determine if cell membrane fatty acid composition is a useful indicator of vascular disease. They obtained blood samples from healthy volunteers, unselected hospitalized patients, and patients with newly diagnosed clinical myocardial damage who were selected by cardiac troponin I screening. The authors separated fatty acids extracted from RBCs by gas chromatography and identified them using mass spectrometry. Fatty acids with abundance greater than one percent of total—hexadecanoic (C16:0), octadecadienoic (C18:2), cis- and trans-octadecenoic (C18:1), and eicosatetrae-noic (C20:4) acids—were quantified and compared. Deuterated standards established the proportionality of fatty acid recovery. The cis- and trans-C18:1 identification was verified by comparison with standards. The authors found that in troponin-positive samples, C18:2 to C18:1 ratios were increased by 30 percent compared with those in healthy controls or random patient samples. Erythrocyte trans-C18:1 had a wide variation of approximately 10-fold in all groups but no differences between groups. Replicates showed that the wide range of RBC trans-fatty acid load is not due to analytic variation. In healthy subjects, the RBC content of lower molecular weight fatty acids (C16–C18) correlated with serum low-density lipoprotein cholesterol. But despite the established relationship between dietary trans-fatty acid and increased low-density lipoprotein cholesterol, lipid profiles did not correlate with RBC trans-fatty acid content. The authors concluded that erythrocyte accumulation of unsaturated fatty acid may be a useful indicator of vascular disease, whereas the wide range in trans-fatty acids suggests that diet and genetic variation affect RBC trans-fatty acid accumulation. Unsaturated fatty acids increase membrane fluidity and may reflect a natural response to subclinical vascular changes, which may in turn reflect increased risk of clinical disease.

Sepulveda JL, Tanhehco YC, Frey M, et al. Variation in human erythrocyte membrane unsaturated fatty acids: correlation with cardiovascular disease. Arch Pathol Lab Med. 2010;134:73–80.

Correspondence: Harry C. Blair at

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Value of serum CA125 levels in early stage ovarian cancer Value of serum CA125 levels in early stage ovarian cancer

Although survival is significantly higher for those with early stage epithelial ovarian cancer than for those with advanced cancers, approximately 20 to 30 percent of such patients will die of the disease. Observation is recommended for patients with stage IA or IB disease and favorable histology because survival is more than 90 percent with surgical treatment alone. However, other patients with early stage epithelial ovarian cancer—stage IA or IB and unfavorable histology, including grade 3 or clear cell, stage IC, or stage II—are candidates for adjuvant treatment based on significant five-year recurrence rates of approximately 25 to 45 percent. Carboplatin plus paclitaxel has become the standard adjuvant chemotherapy for epithelial ovarian cancer based on data demonstrating that the combination of cisplatin plus paclitaxel is superior to that of cisplatin plus cyclophosphamide and that carboplatin has a better therapeutic index than cisplatin. The serum CA125 level has been used as a tumor marker for diagnosing epithelial ovarian cancer and detecting recurrence after primary treatment. Several recent studies have reported on the significance of the serum CA125 level after primary adjuvant chemotherapy in the survival of patients with epithelial ovarian cancer. The studies have also recommended optimal cutoff values for evaluation. Nonetheless, it is still difficult to determine the prognostic significance of the serum CA125 level in predicting recurrence of high-risk, early stage epithelial ovarian cancer. The authors conducted a study to evaluate the prognostic significance of the serum CA125 level in patients with high-risk, early stage epithelial ovarian cancer who have achieved a complete response to adjuvant chemotherapy. Between January 1998 and April 2004, the authors reviewed the records of 95 patients with high-risk, early stage epithelial ovarian cancer who had elevated CA125 levels at the time of diagnosis and who were complete responders after six cycles of adjuvant paclitaxel/carboplatin chemotherapy. A receiver operating characteristic curve was used to determine the most useful CA125 level for predicting disease progression. Cox proportional hazards models adjusted for covariates were used for analyses. The authors found that the five-year progression-free survival rate was 70.5 percent. The optimal cutoff point for CA125 after completing adjuvant chemotherapy to predict disease progression was 12 U/mL (sensitivity, 71.4 percent; specificity, 82.1 percent). On multivariate analysis, a CA125 level greater than 12 U/mL after completing adjuvant chemotherapy was an independent prognostic factor predictive for disease progression. The risk of recurrence was higher for a CA125 level greater than 12 U/mL (hazards ratio, 10.567; P<0.001). The five-year progression-free survival rate for patients with a CA125 level of 12 U/mL or less was 83.3 percent, which was higher than the progression-free survival rate of 37.5 percent for CA125 greater than 12 U/mL (P<0.001). The authors concluded that a CA125 level after six cycles of adjuvant chemotherapy is a strong independent prognostic factor for high-risk, early stage epithelial ovarian cancer after achieving a complete response.

Kang WD, Choi HS, Kim SM. Value of serum CA125 levels in patients with high-risk, early stage epithelial ovarian cancer. Gyn Oncol. 2010;116:57–60.

Correspondence: Seok Mo Kim at

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<sup>210</sup>Pb as a biomarker in cigarette smoke exposure 210Pb as a biomarker in cigarette smoke exposure

The World Health Organization estimates that nearly 1 billion men and 250 million women worldwide smoke a combined total of more than 15 billion cigarettes daily. In many situations, an accurate accounting of a person’s current and former smoking habits is essential. Such information is needed to predict a person’s risk of developing one or more smoking-related diseases, as well as for epidemiologic studies in which smoking may act as a confounder and for clinical trials in which smoking can alter drug metabolism. Cotinine is the most commonly used biomarker of exposure to cigarette smoke. However, with an average half-life in plasma of only 16 hours, cotinine is indicative of a person’s exposure to nicotine for just a few days. In addition, single-nucleotide polymorphisms within the gene encoding CYP2A6 have been noted and can affect a person’s ability to metabolize nicotine to cotinine. Lack of a biomarker that identifies long-term exposure to mainstream cigarette smoke has presented a challenge to the scientific community. Given the limitations of self-reporting, additional biomarkers of smoking are needed. Because the tobacco of cigarettes contains trace quantities of radioactive lead-210 (210Pb) and polonium-210 (210Po), which are volatilized and inhaled when a cigarette is smoked, the authors hypothesized that urinary 210Pb and 210Po activity concentrations could serve as biomarkers of exposure to mainstream cigarette smoke. For their study, the authors recruited people (n=250) from Beijing, China, and reported their smoking habits. Each subject provided a 24-hour urine sample, which was assayed for its 210Pb and 210Po activity concentrations. Although the urinary 210Po activity from smoking was very low compared with background levels, the urinary 210Pb activity correlated with the number of cigarettes smoked per day (CPD; p=0.38; P<0.001) and the urinary cotinine concentration (p=0.52; P<0.001). In a linear regression model, a one-unit increase in CPD was associated with an increase of 0.13 mBq in urinary 210Pb activity. In a logistic regression model, a one-unit increase in urinary 210Pb activity was associated with an estimated 25 percent increase in the odds of being a smoker. These data were modeled using the respiratory, gastrointestinal, and biokinetic models of the International Commission on Radiological Protection. When the final model was applied to a long-term smoker (20 CPD) that suddenly quit, the predicted urinary activity decreased to 50 percent of the steady-state activity in about 90 days. The authors concluded that based on this estimate and the regression results, urinary 210Pb can be used to assess the probability of having smoked in past months.

Schayer SR, Qu Q, Wang Y, et al. 210Pb: A predictive biomarker of retrospective cigarette smoke exposure. Cancer Epidemiol Biomarkers Prev. 2010;19:338–350.

Correspondence: Beverly S. Cohen at

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Use of D-dimer to diagnose VTE in pregnant women Use of D-dimer to diagnose VTE in pregnant women

Pregnant women are at increased risk for thrombosis, and thromboembolic complications are the leading causes of maternal and fetal morbidity and mortality in developed countries. The overall incidence of venous thromboembolism (VTE) is one per 1,000 pregnancies. Risk of thrombosis is increased throughout pregnancy and is particularly high after delivery. The clinical diagnosis of VTE in pregnancy is unreliable because such problems as leg swelling and discomfort are also common in normal pregnancy. The pri-mary diagnostic tool for deep venous thrombosis is compression ultrasono-graphy. This test does not reliably detect isolated iliac vein thrombosis that occurs relatively frequently during pregnancy nor calf vein thrombosis. Failure to identify VTE places the mother’s life at risk, while unnecessary treatment exposes her to anticoagulants and labels her as having had a VTE, which can impact her health care in the future. Therefore, tests that are objective, quick, non-invasive, and safe to the fetus are required. Although D-dimer testing plays an important role in ruling out VTE in pregnancy, the inclusion of D-dimer in algorithms of VTE diagnosis in pregnancy has not been adequately studied. It is difficult to establish the role of D-dimer in the diagnosis of VTE in pregnancy because D-dimer increases substantially throughout gestational age. The authors conducted a study to investigate the level of D-dimer during each trimester of pregnancy and to establish the reference range for each trimester. They followed 89 healthy pregnant women to establish the reference ranges. They also performed D-dimer testing on 12 women with clinical suspicion of VTE and then compared the results with the reference range of the reference group and with recorded ultrasound findings to define sensitivity and correlation with the ultrasonography findings. The authors found that in the first trimester, 84 percent of women from the reference group had normal D-dimer. This level declined to 33 percent in the second trimester and to only one percent in the third trimester, suggesting that D-dimer has no practical diagnostic use in ruling out VTE if using the threshold of 230 ng/mL for abnormal. All pregnant women with thrombosis who had positive ultrasound findings also had a statistically significant elevation of their D-dimer level when considering the established reference range of the corresponding trimester. The sensitivity for D-dimer testing was 100 percent. Women who developed thrombosis in the first trimester had a 6.7 to 7.6 times higher level of D-dimer than the mean value in the reference group. In the third trimester, thrombotic women had a 2.0 to 3.8 times higher level of D-dimer (P<0.0001). The authors concluded that D-dimer testing with the new thresholds of 286 ng/mL for the first trimester, 457 ng/mL for the second, and 644 ng/mL for the third can be useful in diagnosing pregnancy-related VTE.

Kovac M, Mikovic Z, Rakicevic L, et al. The use of D-dimer with new cutoff can be useful in diagnosis of venous thromboembolism in pregnancy. Eur J Ob/Gyn Reprod Biol. 2010;148:27–30.

Correspondence: Valentina Djordjevic at

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Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.