Clinical Abstracts

November 2002

• Fresh frozen plasma and platelet utilization
• DHEA-S levels and depressive symptoms in perimenopausal women
• Apoptotic cells in bronchoalveolar lavage
• Detecting epitestosterone in sports medicine
• Determining the etiologic agents in sarcoidosis
• Biomarkers for endometriosis
• Umbilical cord plasma leptin in preeclampsia
• NMP22 false positives

Novis DA, Renner S, Friedberg RC, et al. Quality indicators of fresh frozen plasma and platelet utilization. Arch Pathol Lab Med. 2002;126:527-532.

Reprints: Dr. David A. Novis, Dept. of Pathology, Wentworth-Douglass Hospital, Dover, NH 03820; ynpa@nh.ultranet.com

DHEA-S levels and depressive symptoms in perimenopausal women
Relatively little is known about the endocrine changes that occur in women in the late reproductive and premenopausal years and how these are affected by racial factors. Young adult women have high levels of the adrenal androgen dehydroepiandrosterone sulfate (DHEA-S). These levels decrease with age to about 25 percent of peak levels in the aged, and it has been thought that DHEA-S plays an important role in the aging process. In studies of predominately Caucasian women or those in whom race was not specified, low DHEA-S levels in the elderly and declining levels in midlife have been associated with increased risk of depressive symptoms. The authors undertook a cross-sectional study using a population-based urban sample recruited through random-digit telephone dialing. They developed a sample composed of 338 women aged 35 to 47 with regular menses. Half of these women were African-American and the other half were Caucasian. Women in the younger half of this cohort were found to have higher DHEA-S levels, which were associated with depressive symptoms, whereas in the older half of this cohort, lower DHEA-S levels were associated with depressive symptoms. The direction of the relationship of DHEA-S and depressive symptoms changes with age, being a positive relationship in younger women and an inverse relationship in the older women in this cohort. The relationships of DHEA-S levels, depressive symptoms, and other hormones in the hypothalamic-pituitary-adrenal axis need to be better understood in the premenopausal age group to draw firm conclusions about these results.

Morrison MF, Have TT, Freeman EW, et al. DHEA-S levels and depressive symptoms in a cohort of African American and Caucasian women in the late reproductive years. Biol Psychiatry. 2001;50:705-711.

Reprints: Dr. Mary F. Morrison, Merck Research Laboratories, 10 Sentry Park, Mail Drop BL2-5, Blue Bell, PA 19422

Apoptotic cells in bronchoalveolar lavage
Apoptosis is a mechanism of cell death in healthy tissues and a key regulator of balance between pro- and anti-inflammatory processes. Apoptosis is down-regulated by pro-inflammatory mediators, increasing the life span of neutrophils, macrophages, and other cells. This prolonged survival may cause an unbalanced tissue load of neutrophils and macrophages and uncontrolled release of toxic metabolites. Apoptosis has also been shown to be responsible for remodeling various organ cells during inflammatory response in animals. The mode of lung cell death in particular patients with severe sepsis is unknown. The authors conducted a study to determine whether apoptosis is present in bronchoalveolar lavage cells, particularly alveolar macrophages of septic patients. The authors carried out a prospective study in the ICU and surgical intensive care and trauma unit of a large university hospital in Athens, Greece. They obtained BAL specimens from 20 consecutive patients admitted to two ICUs who met the criteria for sepsis. They also obtained BAL specimens from nine volunteers without lung disease who served as controls. Apoptosis was detected using annexin V binding, terminal deoxynucleotidyl transfer-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL), DNA laddering, light microscopy, and immunohistochemistry. The spontaneous apoptosis of BAL cells, particularly alveolar macrophages, was significantly decreased in septic patients compared with nonseptic controls. This finding was confirmed using morphologic criteria and the TUNEL method. Gel electrophoresis of DNA obtained from BAL cells revealed that DNA fragmentation was not necessarily associated with apoptotic cell death. An inverse correlation was found between the percentage of apoptotic alveolar macrophages and the severity of sepsis. The authors concluded that prolonged survival of lung cells in septic patients, especially alveolar macrophages, can be attributed to the inhibition of apoptosis. The host in this situation may be attempting to increase the defense capacity to kill the invading microorganism, resulting in an unbalanced tissue load of cells and an uncontrolled release of toxic metabolites downstream. The inhibition of apoptosis in septic patients also may explain why lung function is impaired, leading to sepsis-induced acute respiratory distress syndrome and death.

Liacos C, Katsaragakis S, Konstadoulakis MM, et al. Apoptosis in cells of bronchoalveolar lavage: a cellular reaction in patients who die with sepsis and respiratory failure. Crit Care Med. 2001;29:2310-2317.

Reprints: Dr. Manousos M. Konstadoulakis, Kalvou 24, P. Psychico, 154 52, Athens, Greece; labsures@med.uoa.gr

Detecting epitestosterone in sports medicine
Epitestosterone is a naturally occurring steroid found in urine in concentrations similar to those of testosterone. It is produced in the testes and probably by the ovaries and adrenals, but it has minimal or no androgenic activity. It is not available as a pharmaceutical agent, but it can be purchased in bulk from chemical companies. The compound is of great interest in the field of doping control because it is the denominator in the testosterone/epitestosterone (T/E) ratio, an indirect marker of testosterone administration. When testosterone is administered, the excretion rate of urinary testosterone increases, the excretion rate of epitestosterone declines, and the T/E ratio tends to increase. The increased T/E ratio is also an indirect marker of androstenedione and dehydroepiandrosterone administration. If the T/E ratio exceeds six, doping control labs report the case to the sport authorities, who then investigate. One technique for circumventing the T/E test is to self-administer epitestosterone. Consequently, the International Olympic Committee has classified epitestosterone as a urine-manipulating agent and labs are required to report epitestosterone concentrations of more than 200 µg/L. The authors investigated the carbon isotope ratio of chemical epitestosterones and determined the δ¹³C value of urinary epitestosterone in urine samples obtained from healthy control subjects and athletes. They developed a gas chromatography-combustion-isotope ratio mass spectrometry method for measuring the δ¹³C values for urinary epitestosterone. Sample preparation involved deconjugation with β-glucuronidase followed by a solid-phase extraction and semipreparative high-pressure liquid chromatography. Gas chromatography-mass spectrometry was used to determine epitestosterone concentrations in urine obtained from a control group of 456 healthy males. The epitestosterone δ¹³C values were determined for 43 control urines with epitestosterone concentrations of 40 µg/L or more and 10 athletes' urines with epitestosterone concentrations of more than 180 µg/L. The authors found that the log epitestosterone concentration distribution was gaussian (mean, 3.30; standard deviation, 0.706). The δ¹³C values for four synthetic epitestosterones were low and differed significantly. The standard deviations of between-assay precision studies were low (< 0.73 percent). The mean δ¹³C value for urine samples from the 43 healthy males was -23.8 percent. Nine of 10 athletes' urine samples with epitestosterone concentrations of more than 180 µg/L had δ¹³C values within ± 3 standard deviations of the control group. The δ¹³C value of epitestosterone in one sample was -32.6 percent, suggesting that epitestosterone was administered. The authors concluded that determining δ¹³C values for urinary epitestosterone may be useful for detecting cases of epitestosterone administration because the mean δ¹³C values for a control group are high compared with the δ¹³C values for synthetic epitestosterones.

Aguilera R, Hatton CK, Catlin DH. Detection of epitestosterone doping by isotope ratio mass spectrometry. Clin Chem. 2002; 48:629-636.

Reprints: Don H. Catlin, UCLA Olympic Analytical Laboratory, 2122 Granville Ave., Los Angeles, CA 90024; dcatlin@ucla.edu

Determining the etiologic agents in sarcoidosis
Medical researchers have known about sarcoidosis for more than 100 years, but the cause of sarcoid lesions is still unknown. The condition most likely results from exposure of a genetically susceptible subject to some specific environmental agent or agents. No specific infectious agent, however, has been identified. The clinical, histologic, and immunologic similarities between sarcoidosis and tuberculosis have led some researchers to think that the cause may be an atypical mycobacterium, but Mycobacterium tuberculosis has never been isolated in culture from sarcoid lesions. Mycobacterium avium subspecies paratuberculosis was identified by polymerase chain reaction in cultured isolates in one study. In Japan, however, Propionibacterium acnes have been isolated in culture from biopsy samples in 31 of 40 lymph nodes from 40 patients with sarcoidosis. The authors collected biopsy samples of lymph nodes from patients with sarcoidosis and tuberculosis and from control samples in two Japanese institutes and three European institutes. The specimens were obtained from each of the 108 patients with sarcoidosis and 65 patients with tuberculosis, as well as from 86 control samples. Quantitative real-time PCR was used to detect genomes of P. acnes, P. granulosum, M. tuberculosis, M. avium subspecies, paratuberculosis, and Escherichia coli (as the control). P. acnes or P. granulosum was found in all but two of the sarcoid samples from all locations. M. avium subspecies paratuberculosis was not found in the sarcoid samples. M. tuberculosis was found in zero to nine percent of the sarcoid samples and 65 to 100 percent of the tuberculosis samples. There were far more genomes of P. acnes or P. granulosum in the sarcoid lymph nodes than in the tuberculosis lymph nodes. The authors concluded that Propionibacterium species are more likely than Mycobacteria species to be etiologic agents in sarcoidosis, not only in Japanese patients with sarcoidosis but also in European patients with the condition.

Eishi Y, Suga M, Ishige I, et al. Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis. J Clin Microbiol. 2002;40:198-204.

Reprints: Yoshinobu Eishi, Dept. of Human Pathology, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; eishi.path@tmd.ac.jp

Biomarkers for endometriosis
Endometriosis is an extremely common, benign gynecological condition. There are, however, no easy ways to diagnose the condition short of laparoscopy. A considerable amount of evidence implicates disorders of the immune system in the pathogenesis of endometriosis. Several studies have found that immunological components of the peritoneal fluid play an essential role in the pathogenesis and progression of the condition. The peritoneal fluid macrophages and their cytokine secretory products specifically appear to play an active role. The authors studied this phenomenon prospectively by analyzing peritoneal fluid samples from 130 women undergoing laparoscopy for pain, infertility, tubal ligation, or sterilization reversal. Concentrations of the cytokines (IL)-1β, IL-6, IL-8, IL-12, IL-13, and TNF-α (tumor necrosis factor alpha) were measured in serum and peritoneal fluid, and reactive oxygen species were measured in peritoneal fluid. Fifty-six of the women were diagnosed with endometriosis, eight with idiopathic infertility; 27 underwent tubal ligation or reanastomosis (control group); and 39 were excluded because of bloody taps of peritoneal fluid. Only serum IL-6 and PF TNF-a could be used to discriminate between patients with and without endometriosis with a high degree of sensitivity and specificity (P<0.001). Using a threshold cutoff value of 15 pg/mL for PF TNF provided 100 percent sensitivity and 89 percent specificity. A threshold cutoff value of 2 pg/mL for serum IL-6 provided 90 percent sensitivity and 67 percent specificity. The authors concluded that these markers may be useful as a nonsurgical diagnostic tool, but the findings should be verified in a larger study.

Bedaiwy MA, Falcone T, Sharma RK, et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod. 2001;17:426-431.

Reprints: T. Falcone, Dept. of Obstetrics-Gynecology, The Cleveland Clinic Foundation, 9500 Euclid Ave., A81, Cleveland, OH 44195; falcont@ccf.org

Umbilical cord plasma leptin in preeclampsia
Leptin is the product of the obesity gene. It is expressed primarily in adipocytes where, through negative feedback loop inhibition between adipose tissue and the hypothalamus, it may contribute to the regulation of obesity by affecting satiety mechanisms. Leptin is highly expressed in the placenta in pregnancy. It has been detected in umbilical cord blood from fetuses at week 18 of gestation, with increasing levels from the middle of the third trimester toward term. This increase appears to coincide with the development of fetal adipose tissue. Some studies indicate that cord blood levels of leptin are positively correlated with fetal adiposity at birth. Preeclampsia increases fetal risk of being born small for gestational age, particularly in cases of early and recurrent disease. Wasting of subcutaneous fat is seen in these growth-retarded infants, thus one would expect lower levels of leptin in umbilical cord blood from preeclamptic than from normotensive pregnancies. At least one previous small study, however, found no difference in cord leptin levels between cases of preeclampsia and controls with delivery at term. The authors of this study compared umbilical cord plasma levels of leptin between pregnancies with preeclampsia and normotensive control pregnancies to assess the relation between fetal adiposity at birth and leptin levels. On the basis of a population of approximately 13,000 deliveries, the investigators compared cord plasma leptin from 256 preeclamptic and 607 control pregnancies after taking into account differences in gestational age and ponderal index. The investigators found that cord plasma leptin levels increased markedly with gestational age in the preeclamptic group and control subjects. At each gestational age, however, the preeclamptic group had higher leptin levels than the control subjects, and this was highly significant (P<.01). The adjustment for higher ponderal index among the controls did not alter the difference in leptin levels between the groups. The authors concluded that higher levels of umbilical cord plasma leptin are found in mothers with preeclampsia than in controls.

Ødegård R, et al. Umbilical cord plasma leptin is increased in preeclampsia. Am J Obstet Gynecol. 2002;186:427-432.

Reprint information not available.

NMP22 false positives
Urinary tumor markers have not yet proved to be reliable enough to replace cytology and cystoscopy as the major modalities for diagnosis and followup of bladder cancer. The NMP22 nuclear matrix protein is a candidate tumor marker in voided urine. These markers, however, have low specificity and low positive predictive value due to false-positive results. The false positives tend to occur in the same clinical categories typically associated with hematuria and pyuria. This is a serious problem in bladder cancer because 85 percent of patients present with hematuria. The authors investigated the effect of hematuria and pyuria on NMP22 results. They examined 202 urine samples from 30 healthy individuals, 20 with symptomatic urinary tract infections, and 32 with known bladder carcinoma. In the first group, the median urinary NMP22 was 4 U/mL, with a range of 1.6 to 9.5. When blood was added to these urine samples, NMP22 level increased in parallel to the increase in the amount of red cells in the sediment. When more than 2 µL/mL of blood was added to the urine of a healthy individual, the NMP22 level was comparable to levels seen in patients with bladder cancer. The leukocyte count in urine sediment also had a significant impact on the NMP22 levels in the group with pyuria. Hematuria and pyuria did not significantly affect NMP22 measurements in the group with bladder cancer. The NMP22 clinical parameters were sensitivity of 78.1 percent, specificity of 66 percent, positive predictive value of 59.5 percent, and negative predictive value of 82.5 percent. Test sensitivity increased as grade and stage progressed. The authors concluded that in an experimental model, pyuria and hematuria significantly affected urinary NMP22 and that the effect of white blood cells was more pronounced than that of red blood cells. The source of NMP22 in isolated hematuria remains to be elucidated.

Atsü N, Ekici S, Öge Ö, et al. False-positive results of the NMP22 test due to hematuria. J Urol. 2002;167:555-558.

Reprint information not available.