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August 2001
Use of multicolor fluorescent in situ hybridization probes in congenital disorders
Multicolor fluorescent in situ hybridization probes use a number of fluorophores to label each chromosome so that each of the 24 human chromosomes can be visualized in a unique color. Work has been done to optimize the labeling of probes, filters, and image analysis in various tissue types, and the method has received a number of designations, among them multicolor FISH. The authors examined the utility of M-FISH in routine clinical cases for detecting a range of structural chromosomal anomalies and inherited disorders. They examined six cases selected to represent a range of congenital anomalies of chromosomes that reflect the strength of M-FISH analysis. The blood samples were processed by standard chromosome culture and banding procedures. The technical challenges involving M-FISH include sequential G bands by trypsin with use of Leishman-Giemsa stain/FISH, pretreatments for retention of good chromosome morphology, precautions for maintaining bright FISH signals, and comparison with the method used for whole-chromosome painting probes. The results of the study were that three familial and subtle derivative chromosomes were identified with M-FISH with relative ease. A small ring chromosome that was unidentifiable by banded-chromosome analysis was identified by M-FISH. A case of telomeric anomaly that was rather subtle could not be resolved without the use of telomeric-specific probes. The M-FISH results were confirmed by individual chromosome-specific painting probes. The authors concluded that M-FISH was helpful in identifying a range of congenital chromosomal anomalies, but for subtle chromosomal abnormalities, locus-specific probes may be necessary.
Jalal SM, Law ME, Lindor NM, Thompson KJ, et al. Application of multicolor fluorescent in situ hybridization for enhanced characterization of chromosomal abnormalities in congenital disorders. Mayo Clin Proc. 2001;76:16-21.
Reprints: Syed M. Jalal, PhD, Cytogenetics Laboratory, Mayo Clinic, 200 First St. SW, Rochester, MN 55905; sjalal@mayo.edu
Use of cardiac troponin T to risk stratify patients with idiopathic dilated cardiomyopathy
Idiopathic dilated cardiomyopathy (DCM) is a primary myocardial disease of unknown origin characterized by ventricular dilatation and depressed myocardial contractility. Although various functional and hemodynamic measurements are helpful in estimating adverse cardiac events in patients with this disease, it is difficult to assess prognosis. The diagnostic and prognostic value of cardiac troponin T has been established and reported in acute coronary syndromes, but its significance in DCM is not known. The authors reported that patients with idiopathic and secondary DCM and a poor prognosis have abnormally high serum concentrations of TnT in the absence of any increase in serum creatine kinase concentrations. Those patients with poor outcomes had persistently high TnT levels even when heart failure was compensated for by conventional treatment and when they were free of dyspnea or roentgenographic and auscultatory signs of pulmonary congestion. The authors studied consecutive patients with DCM who were examined at a university hospital in Japan. None of the patients had a history of myocardial infarction, infective myocarditis, metabolic disease, systemic illness, or heredofamilial disease. All patients underwent cardiac catheterization, coronary angiography, and endomyocardial biopsy. No significant coronary stenosis was found in any patient. At least three endomyocardial biopsy specimens were obtained from each patient and three to five specimens were stained with hemotoxylin-eosin. Microscopic examination revealed myocyte hypertrophy, nuclear hypertrophy, myocyte degeneration with myofibrillar attenuation, or interstitial and perivascular fibrosis in all patients. Serum TnT was measured at baseline with a commercially available second-generation immunoassay. Creatine kinase enzyme was measured by standard laboratory method, and left ventricular ejection fraction and left ventricular diastolic dimension were measured echocardiographically by two experts blinded to this study protocol. The authors identified
three evolutionary patterns of TnT concentrations. In 33 patients, concentrations of TnT were less than 0.02 ng/mL throughout the followup period (group one). In 27 patients, the initial serum concentrations of TnT were high, and in 10 of these 27 patients, the TnT decreased to less than 0.02 ng/mL during followup (group two). In 17 patients, the serum TnT concentrations were persistently high, despite the patients being treated for congestive heart failure via conventional means (group three). The echocardiographic measurements, left ventricular diastolic dimension, and left ventricular ejection fraction initially were not significantly different among the three groups. Followup echocardiography, however, showed significantly decreased left ventricular diastolic dimension and increased left ventricular ejection fraction in groups one and two compared with increased left ventricular diastolic dimension and decreased left ventricular ejection fraction in group three. The survival rate was lower in group three than in group one, and the cardiac event-free rate was significantly lower in group three than in groups one and two. The authors concluded that persistently increased TnT concentrations suggest ongoing subclinical myocyte degeneration associated with long-term deterioration of clinical status.
Sato Y, Yamada T, Taniguchi R, et al. Persistently increased serum concentrations of cardiac troponin T in patients with idiopathic dilated cardiomyopathy are predictive of adverse outcomes. Circulation. 2001;103:369-374.
Reprints: Yukihito Sato, MD, Div. of Cardiology, Dept. of Internal Medicine, Hyogo Prefectural Amagasaki Hospital, 1-1-1 Higashi-Diamotsu-Cho, Amagasaki, Hyogo 660-0828, Japan
Determining female sex hormone levels in saliva
A woman’s risk of developing breast and perhaps endometrial cancer is affected by long-term exposure to ovarian sex steroid hormones, primarily estradiol and progesterone. Studying the pathogenetic role of these compounds is difficult in premenstrual women, however, because of the great fluctuation of these hormone levels across the menstrual cycle. Standardized timing of blood samples can be improved by linking sampling to detection of ovulation or peaks in luteinizing hormone, but this testing peridine is cumbersome. The authors have developed ultra-sensitive radioimmunoassay procedures to directly measure estradiol and progesterone in saliva and have reported on the sensitivity, reliability, and serum-saliva correlations for these assays. Saliva potentially has several advantages over blood as a sampling medium. It can be easily collected by the subjects at repeated intervals; it requires no special collection or storage equipment; and the steroid concentrations measured exclude the fraction tightly bound to serum proteins and thus unavailable for biological action. Most importantly, consecutive daily samples can be grouped for analysis after the length of the menstrual cycle is known. The authors studied how salivary levels of estradiol and progesterone track within women from cycle-to-cycle to evaluate how cumulative levels of salivary steroid based on daily sampling can be used to discriminate interindividual from intraindividual differences. They also evaluated the effect of altering the number of consecutive daily samples considered and the method for locating a particular cycle day in relation to ovulation (day zero). They studied 12 healthy women who provided daily saliva samples for two consecutive ovulatory menstrual cycles. In each case, a single midluteal phase serum sample was collected seven to eight days after detection of a luteinizing hormone peak in urine. The authors plotted individual cycle profiles and computed correlation coefficients for various definitions of peak and cumulative daily
hormone level. For peak progesterone, which was determined as the maximal three day-mean running, the intraclass correlation coefficient was 0.68. For cumulative progesterone, based on eight consecutive cycle days, the correlation coefficients were 0.72 to 0.76. For estradiol, the correlation coefficients ranged from 0.74 to 0.79 for the five days preceding ovulation and from 0.85 to 0.92 for the 15 days around the center of the cycle. For estradiol and progesterone, selection of five or seven days for estimating the midluteal mean level provided separation of within- and between-subject variance that was comparable with a serum sample that was LH timed. The authors concluded that daily saliva samples could be used to combine and clarify the interindividual differences in estradiol and progesterone levels in premenopausal women and that these interindividual differences may be greater than was previously recognized.
Gann PH, Giovanazzi S, VanHorn L, et al. Saliva as a medium for investigating intra- and interindividual differences in sex hormone levels in premenopausal women. Cancer Epidemiol Biomarkers Prev. 2001;10:59-64.
Reprints: Peter H. Gann, Dept. of Preventive Medicine, Northwestern University Medical School, 680 N. Lake Shore Dr., Suite 1102, Chicago, IL 60611
Reproductive hormone ratios and hepatocellular carcinoma in male cirrhotic patients
Hepatocellular carcinoma is much more predominant in men than women but the reason for the large gender difference is unclear. In Japan, the prevalence of hepatitis C virus infection, which is the most important etiological factor of hepatocellular carcinoma in that country, has been shown to be similar or only slightly different between both genders, according to blood donor data. Japanese men have other potential risk factors that are more common than in women, including alcohol consumption and cigarette smoking, but heavy drinking has been estimated, epidemiologically, to account for only about 13 percent of the male occurrences of hepatocellular carcinoma in Japanese case control studies. The causal link, if any, between smoking and HCC is still uncertain. Animal experiments in carcinogenesis of liver cancer in mice have shown a promoting role of testosterone in this condition, although the role of estrogens remains in dispute. Various case control studies in different populations have yielded inconsistent results with respect to sex hormone level and incidence of hepatocellular carcinoma. The authors conducted a followup study of cirrhosis, mainly of hepatitis C virus origin, in males, taking into account the effect of other measurements that are possibly associated with serum hormone levels and HCC risk. They studied 46 Japanese male patients with liver cirrhosis predominantly of hepatitis C viral origin. They collected serum samples between December 1985 and December 1987 and continued complete longitudinal followup of the patients until the end of 1995, for an average of 5.1 years. Twenty patients developed hepatocellular carcinoma during followup. The authors measured serum levels of testosterone, free testosterone, estradiol, sex hormone binding globulin, and the ratio of testosterone to estradiol. The serum T:E2 ratio and testosterone were significant predictors of hepatocellular carcinoma on univariate analysis. The relative risks or hazard ratios of the middle and upper tertiles relative to the
lower tertile were 2.0 and 4.0 for the T:E2 ratio and 0.8 and 2.9 for testosterone. These hazard ratios were substantially increased by adjusting for age, serum albumin, hepatitis viral markers, and other biological variables. Serum free testosterone appeared to be associated with increased risk in multivariate analysis, but independent associations with estradiol and sex hormone binding globulin were not apparent. The authors concluded that elevated serum testosterone and decreased serum estrogens may promote the development of hepatocellular carcinoma in the context of hepatic cirrhosis.
Tanaka K, Sakai H, Hashizume M, et al. Serum testosterone: Estradiol ratio and the development of hepatocellular carcinoma among male cirrhotic patients. Cancer Res. 2000;60:5106-5110.
Reprints: Keitaro Tanaka, Dept. of Preventive Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Role of calcium metabolism in Graves disease
It has been hypothesized that concentrations of calcium and its regulating hormones may differ between the sexes in patients with Graves disease, leading to the varying incidences of postoperative tetany. Female gender has been reported to be the most important risk factor for postoperative tetany, and the predictive risk factors in women, after subtotal thyroidectomy, are preoperative decreased serum calcium concentration, younger age, increased serum alkaline phosphatase activity, large size of goiter, and increased thyroid-stimulating hormone binding inhibitory immunoglobulin (TBII). The authors conducted a study to examine gender differences in serum concentrations of parathyroid hormone, calcium, electrolytes, 25-hydroxyvitamin D, and 1.25 dihydroxyvitamin D. They studied 109 patients with Graves disease who were treated surgically at a hospital in Japan between June and August 1996, as well as an additional 114 patients treated between January and April 1998. The group consisted of 45 men and 178 women, none of whom had co-existing thyroid cancer or central node dissection. Women had significantly lower calcium concentrations than men (mean, 2.37 compared with 2.43). Serum calcium concentrations correlated significantly with concentrations of 25-hydroxyvitamin D. Of the women, 121 had vitamin D deficiency as defined as 25-hydroxyvitamin D of less than 25 nmol/L, compared with 13 of the men. Fifteen female patients and one male patient developed tetany postoperatively. The authors concluded that women with Graves disease are more susceptible to calcium and vitamin D deficiency than are men, which may account for the higher incidences of postoperative tetany among women with the disease.
Yamashita H, Noguchi S, Murakami T, et al. Calcium and its regulating hormones in patients with Graves disease: sex differences and relation to postoperative tetany. Eur J Surg. 2000;166:924-928.
Reprints: Hiroyuki Yamashita, MD, PhD, Noguchi Thyroid Clinic and Hospital Foundation, 6-33 Noguchi-Nakamachi, Beppu Oita 874, Japan
Using TFR and serum ferritin to diagnose iron deficiency
in infancy
Laboratory tests for iron deficiency have poor sensitivity and specificity. Using a battery of tests improves precision, but two problems continue to muddy the issue: the difficulty in accurately detecting mild iron deficiency and the identification of inflammation as a cause of changes in laboratory test results that are not due to iron deficiency. The serum transferrin receptor assay has shown some promise in clarifying these pitfalls because it is not influenced by acute or chronic inflammatory conditions and seems to be able to detect mild iron deficiency. Its usefulness in evaluating iron status in infancy, however, has not been fully evaluated. The authors evaluated the performance of serum transferrin receptor and the ratio of TFR to serum ferritin in assessing iron deficiency in infants and analyzed age-related changes in both variables. They studied 515 healthy infants, ages eight to 15 months, from whom a total of 716 blood samples were obtained. In 144 samples, in which all other laboratory indicators of iron status were within reference range, the median and 95th percentile for TFR and the ratio of TFR to serum ferritin were 8.5 mg/L and 497, respectively. These two measurements were significantly correlated with other laboratory indicators of iron status, and as the severity of the iron deficiency increased, the mean TFR concentration gradually increased. The sensitivity of a TFR greater than 13.5 mg/L and a TFR to serum ferritin ratio of greater than 975 for the diagnosis of iron deficiency was 23.6 percent and 68.4 percent, respectively. Specificities were 98.3 percent and 63.3 percent for TFR and the ratio TFR to serum ferritin, respectively. Receiver operator characteristic analysis showed that TFR and the ratio of TFR to serum ferritin were more accurate than serum ferritin alone.
Olivares M, Walter T, Cook JD, et al. Usefulness of serum transferrin receptor and serum ferritin in diagnosis of iron deficiency in infancy. Am J Clin Nutr. 2000;72:1191-1195.
Reprints: M. Olivares, Institute of Nutrition & Food Technology, Macul 5540 Casilla 138-11, Santiago 11, Chile
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