Epstein-Barr virus antibodies in whole blood spots: a minimally invasive method for assessing an aspect of cell-mediated immunity.

OBJECTIVE: Study 1: Introduce and validate a method for measuring EBV p18-VCA antibodies in whole blood spots to provide a minimally invasive marker of cell-mediated immune function. Study 2: Apply this method to a large community-based study of psychopathology in children and adolescents. METHODS: The EBV antibody method was evaluated through analysis of precision, reliability, stability, and comparisons with plasma and indirect immunofluorescence methods. The effects of life events on p18-VCA antibody level were considered in a subsample of 9, 11, and 13 year-old children participating in the Great Smoky Mountains Study in North Carolina. The subsample was stratified by age, sex, and degree of overall life strain. RESULTS: Dried blood spots provided a convenient, sensitive, precise, and reliable method for measuring EBV p18-VCA antibody titer. Life events were positively associated with p18-VCA antibodies in girls but not in boys. CONCLUSIONS: The validity of the blood spot EBV p18-VCA antibody assay, as well as the ease of sample collection, storage, and transportation, may provide an opportunity for psychoneuroimmunology to explore a wider range of stress models in larger, community-based studies.

Hormone measures in finger-prick blood spot samples: new field methods for reproductive endocrinology.

Comparative endocrine studies have notably advanced understanding of ecological factors that contribute to variation in human reproductive function. Such research has relied on methodological advances that permit hormone determinations in samples that are easily and safely collected, stored, and transported, most recently on measurement of steroids in saliva. This report seeks to further expand the scope of endocrine research by demonstrating the value of blood spot samples collected by finger prick. As a sampling strategy, finger-prick blood spot collection offers the advantages of short collection time, low invasiveness, repeatability, absence of postcollection processing, low biohazard risk, and ease of sample storage and transport. We document good sample stability and present sensitive assay methods for a range of steroids and proteins (FSH, LH, PRL, T, E2, DHEAS, androstenedione, cortisol, SHGB) in blood spots that require sample volumes of 3-12 microliters and display good reliability, specificity, precision, accuracy, and convertibility of results to plasma/serum equivalent concentrations. Laboratory evaluation was augmented by a feasibility study at a remote site in Papua New Guinea that confirmed validity and stability of blood spot collections under field conditions. Research applications of blood spot sampling are illustrated with a series of studies, including cross-sectional surveys for developmental and life span endocrinology, a longitudinal, population-based developmental epidemiologic study of puberty, and serial sampling in a dynamic study of neuroendocrine response to suckling. We conclude that the sampling features and wide range of measurable biomolecules of blood spots do constitute a methodological advance for endocrine research.

Ratios of plasma and salivary testosterone throughout puberty: production versus bioavailability.

Because diffusion of testosterone (T) into the salivary gland is thought to be largely limited to the free, biologically active fraction, salivary testosterone is expected to provide a better measure of testosterone bioavailability in the body than is plasma testosterone. Matched saliva and blood spot samples were collected from 218 Zimbabwean males (age 11-23) who were at different stages of puberty, as assessed by self-reported Tanner genital stage ratings. Testosterone concentrations in these matched samples were highly correlated (r = 0.83). Both salivary and plasma testosterone (converted from blood spot value) showed expected significant increases across puberty. However, plasma testosterone distinguished among subjects at different stages of genital development more effectively than did salivary testosterone, suggesting the former to be a better marker of testosterone bioavailability. Sex hormone-binding globulin (SHBG) levels were also measured in a subgroup of 93 of these subjects. After controlling for plasma T concentrations, we found a small but significant inverse correlation between blood spot SHBG levels and the proportion of plasma testosterone recovered in salvia, supporting the hypothesis that SHBG-related changes in T bioavailability are detectable in saliva. We conclude that salivary testosterone accurately reflects testicular production of testosterone, but that neither salivary testosterone nor plasma testosterone is clearly superior to the other as a measure of testosterone bioavailability.

Prolactin response to suckling and maintenance of postpartum amenorrhea among intensively breastfeeding Nepali women.

The aim of the study was to determine the association between PRL responses to suckling and maintenance of postpartum amenorrhea among breastfeeding mothers. Three blood spot samples (5, 30, and 50 min following a timed nursing bout) were collected from 71 intensively breastfeeding Nepali women for PRL determination. Maternal age, BMI (weight/height2), menstrual status, caste, infant age, nursing bout length, and duration of supplementation were recorded at time of sample collection. Independent and paired t tests, linear regression analyses, and general linear models were used to evaluate differences between cycling (n = 36) and amenorrheic (n = 35) women and associations among variables. Logistic regression analyses were used to relate PRL measures to the odds of maintaining lactational amenorrhea. Amenorrheic breastfeeding mothers had higher (P < .001) PRL levels at all 3 collection times than cycling breastfeeding mothers, and PRL levels declined with time since birth (P < 0.05). The odds (OR) of having ceased lactational amenorrhea was significantly higher (OR = 5.0, 95% Cl = 1.3-19.9) among mothers with lower PRL levels (< or = 10 ng/mL) at 50 min post-sucking, and PRL at 50 min showed a significant dose response relationship with menstrual status. The association between 50 min PRL levels and lactational amenorrhea appears to be independent of time postpartum, maternal age, BMI, nursing bout length, and duration of supplementation. Among intensively nursing women, maintenance of elevated PRL levels across the interbout interval increases the odds of maintaining lactational amenorrhea.

PIP: In August 1991, in Nepal, all women aged 19-45 from the Tamang caste (agro-pastoralists of Tibetan origin) and from the Kami caste (low-caste blacksmiths of Aryan origin) who were intensively breast feeding and lived in remote villages in the foothills of the Himalayas were included in a study aiming to examine the association between post-suckling prolactin (PRL) and menstrual status. The study also aimed to determine what PRL response time after suckling best predicts the odds of having resumed menses. There were 36 women whose menses had returned and 35 women who remained amenorrheic. 50-minute post-suckling PRL levels were more linked to menstrual status than 5-minute levels (odds ratio [OR] = 5 vs. 2.1). They (but not 5-minute post-suckling PRL levels) also had a significant dose relationship with menstrual status. During the first year postpartum, PRL levels were significantly higher at all three collection times (5, 30, or 50 minutes) in amenorrheic women than cycling women (p 0.001). PRL levels fell as infant age increased (16 ng/ml for 11 months or less, 13.7 ng/ml for 11-15 months, and 7.4 ng/ml for 15 months; p 0.05). At 50 minutes post-suckling, mothers with PRL levels at or below 10 ng/ml were much more likely to have returned to menses than those with higher levels (OR = 5; p = 0.041). The association between 50-minute PRL levels and lactational amenorrhea were independent of time postpartum, maternal age, body mass index, nursing bout length, and duration of supplementation. The maintenance of high PRL levels across the interbout interval increased the odds of maintaining lactational amenorrhea (p 0.05). In conclusion, intensively nursing women whose PRL levels remain high across the interbout interval are most likely to maintain lactational amenorrhea.

Measurement of gonadotropins in dried blood spots.

We describe direct immunofluorometric assays for luteinizing hormone (hLH) and follicle-stimulating hormone (hFSH) in fingerstick blood spots dried on filter paper, based on modifications of commercially available kits. Determinations are made from 2.5-mm-diameter discs (3 microL of dried blood) punched out from blood spot standards and samples. Sample dose detection limits of the assays (IU/L) are 0.26 for LH and 0.13 for FSH, with mean interassay CVs of 11.6% (LH) and 7.8% (FSH) at low concentrations. Analytical recoveries of added hormone averaged 100% for LH and 95% for FSH. Clinical studies showed that values for blood spots (x) and directly assayed plasma (y) are highly correlated, so that results from blood spots can be converted directly to plasma equivalents, as follows: yLH = 0.07 + 1.90 xLH, and yFSH = 0.424 + 2.207 xFSH. These gonadotropins are stable in blood spots for at least a year under refrigeration; LH for at least 8 weeks and FSH 6 weeks at 22 degrees C; and both hormones for a week at 37 degrees C. These methods thus allow self-sampling, serial sampling, and mailing of specimens.

Attenuation of nursing-related ovarian suppression and high fertility in well-nourished, intensively breast-feeding Amele women of lowland Papua New Guinea.

Intense, sustained nursing lengthens inter-birth intervals and is causally linked with low natural fertility. However, in traditional settings, the effects of such nursing on fertility are difficult to disentangle from those of nutrition. Results from a prospective, direct observational study of reproductive function in well-nourished Amele women who nurse intensively and persistently but who also have high fertility are here presented. Endocrine measures show that ovarian activity resumes by median 11.0 months postpartum. Median duration of postpartum amenorrhoea is 11.3 months, time to next conception is 19.0 months, and the inter-birth interval is 28.0 months. Average life time fertility is 6.8. High fertility in Amele women is due both to refractoriness of reproductive function to suckling stimuli, and to maintenance of equivalent age-specific fertility rates across the reproductive life span.

PIP: Evidence is presented, from a study of 52 breast-feeding women with an infant under 5 years of age from 12 Amele villages in lowland Papua New Guinea, that women with good nutritional status experienced less variation in ovarian suppression than reported elsewhere among less well-nourished populations. Direct observations of nursing behavior were recorded during 660 hours. The average number of observations per child, which were conducted at different developmental stages, was 1.9. 1549 nursing events were recorded. 38 women had serum samples drawn to determine prolactin levels. Saliva samples were collected from 51 women for measurement of gonadal steroids. Women reported menstrual patterns in the preceding month. Measures were also taken of weight, skinfolds of the triceps and subscapular area, and mid-upper-arm circumference. Birth interval, completed fertility, and age at introduction of solid foods and denial of breast were obtained from ongoing demographic surveillance on nutrition in 1982-84 and 1982-83. Laboratory methods were described for serum prolactin and progesterone analysis. The results showed skewed results for the hormonal and some nursing variables, which were transformed by logarithmic functions. All Amele mothers practice indulgent demand feeding schedules. Supplementary foods are introduced at the median age of 7.4 methods. Early supplementation is liquids or semiliquids, followed by mashed starchy staples, and finally a standard diet. Cessation of breast feeding occurs at a subsequent pregnancy of the advanced age of the child (5 years). The median age of cessation was 36.3 months. The breast feeding pattern during the first 18 months was 1.5-3 times per hour for 1-3 minutes. Infant age was not linearly related to nursing frequency declines or feeding intervals. Nursing frequency was unrelated to morbidity. Prolactin declined strongly when bout frequency and interval were stable. Multiple regression findings were that duration of postpartum amenorrhea was explained by mother's parity and age, feeding interval, and time postpartum. Time of supplementary feeding was unrelated to duration of amenorrhea, nursing frequency, or feeding duration. 16% of interval variance was explained by length of postpartum amenorrhea, which was correlated with length of subsequent birth interval. 45% of the variance in prolactin was explained by triceps skinfolds and bout length.

Sensitive salivary estradiol assay for monitoring ovarian function.

Measurement of steroids in saliva has excited interest because of the numerous potential clinical applications; noninvasive, convenient sampling; and apparently accurate reflection of the concentrations of physiologically active unbound steroid in the circulation. Although assays of saliva for several steroid hormones are available and widely used, assays for salivary estradiol are not, primarily because of methodological limitations. By modifying a commercially available kit for serum estradiol, our laboratory has developed a procedure that is sensitive, highly specific, and reliable for measuring salivary estradiol. Assay sensitivity is 0.5 fmol (0.14 pg; sample concentration 1.3 pmol/L) with a mean interassay CV of 10.8% at low concentrations. Clinical studies showed that values for serum and saliva are highly correlated (P less than 0.001), and demonstrated reliable detection of estradiol peaks during normal ovulatory cycles in serial samples from 15 women. Salivary estradiol peaked at 5.4 (SD 1.9) pmol/L on cycle day 14.4 (SD 3.2), 1.2 (SD 0.8) days before ovulation detected by ultrasound. This assay may be particularly helpful in investigating ovarian function and free estradiol in women at various stages of the reproductive cycle.