Clinical Abstracts
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Clinical Abstracts |
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March 2005
Editors:
Homocysteine and fetal growth Intrauterine growth restriction describes a fetus whose weight is less than expected based on gestational age and gender, as determined by population standards; frequently chosen cutoffs are below the 10th percentile. The causes of intrauterine growth restriction (IUGR) are still unknown, although several determinants have been identified. On the basis that thrombophilic polymorphisms could affect placental circulation and thus fetal growth, the authors investigated the role of such maternal and newborn polymorphisms on IUGR. They also hypothesized that higher plasma homocysteine concentrations would be associated with a greater risk of IUGR or a reduction in birth weight through a thrombotic placental effect, regardless of the relative contribution of thrombophilic polymorphisms to maternal and newborn homocysteine concentrations. Mild hyperhomocysteinemia has been associated with pregnancy outcomes such as abruptio placentae, preeclampsia, and fetal loss. However, results of the association between plasma total homocysteine (tHcy) and atherothrombotic conditions in general are not consistent, and some authors have even suggested that the association is an effect rather than a cause—for example, the increase in plasma homocysteine could be caused by the decrease in renal function that is common in atherosclerosis. To address this issue, the authors conducted a case/control study that included all subjects born at their institution during a two-year period whose birth weight was below the 10th percentile for gestational age and gender according to Canadian norms. The infants used as controls were born during the same period at the same institution, were at or above the 10th percentile, and were matched on gestational age, race, and gender. Homocysteine was measured in cord and maternal blood. The analysis included 483 case and 468 control mothers and 409 case and 438 control newborns. The authors found that the homocysteine values largely were less than 15 µmol/L. Contrary to expectation, within that range of values, increased plasma homocysteine, particularly in the mother, was protective against IUGR. With case/control status as the outcome, the estimated odds ratio was 0.37 (95 percent confidence interval, 0.24-0.58) for a 5 µmol/L unit difference on the maternal homocysteine scale. With birth weight as the outcome, the estimated increase was 178.1 g (95 percent confidence interval, 92.5-263.7 g) for every 5 µmol/L unit increase in maternal homocysteine. Results were similar using newborn homocysteine concentrations. The authors concluded that, in contrast to the proposed hypothesis, mothers having smaller babies have lower homocysteine concentrations than those giving birth to larger babies. Infante-Rivard C, Rivard GE, Gauthier R, et al. Unexpected relationship between plasma homocysteine and intrauterine growth restriction. Clin Chem. 2003;49:1476-1482. Reprints: Claire Infante-Rivard, Dept. of Epidemiology, Biostatistics, and Occupational Health, Faculty of Medicine, McGill University, 1130 Pine Ave. West, Montreal, Quebec, H3A 1A3, Canada; claire.infante-rivard@mcgill.ca Cytogenetics of human blastocysts Key stages in understanding the origins and survival of different cytogenetic abnormalities during early development involve the fertilized oocyte and blastocyst. The fertilized oocyte defines the base level of abnormality due to meiotic and fertilization errors, and likely by the blastocyst stage, the majority of mosaic karyotypes will have been established. In addition, the first cell lineage differentiation processes will have occurred, and initial selection mechanisms against certain cytogenetic abnormalities already seem to be in place. This study concentrated on analysis of blastocyst stage embryos. Improved culture techniques permit the in vitro growth of surplus in vitro fertilization preimplantation embryos up to the blastocyst stage. In 1997, the authors published a novel and inexpensive, improved method for obtaining better quality G-banded metaphases from human blastocysts. Using this technique, thymidine-mediated synchronization of cell division, followed by gradual fixation, and then disaggregation of the blastomeres in acetic acid, allows the production of small numbers of good quality, analyzable metaphases. The method recently has been combined with fluorescence in situ hybridization (FISH) techniques. Abnormalities detected by karyotypic analysis are now dictating which FISH probes should be used to further investigate any mosaicism in interphase cells. The authors presented results from 438 blastocysts processed by this method, including FISH data where available. They compared these results with patterns of karyotypic abnormality seen at earlier and later gestations. Where analysis was possible, three percent appeared polyploid (mainly tetraploid), 29 percent were diploid:tetraploid mosaics, and 68 percent were uniformly diploid. Abnormalities observed included triploidy, trisomy 16, trisomy 2, trisomy for unidentifiable D-group chromosome, mosaic trisomy 3, and mosaic trisomy 3 and trisomy 7. The authors concluded that comparing results with existing data from first trimester pregnancies and cleavage stage embryos suggests significant loss of haploid and monosomic embryos, as well as loss of some trisomies, prior to the blastocyst stage. It appears that the general range and incidence of most main groups of constitutional abnormalities observed in the first trimester, including mosaic forms, are in place by the blastocyst stage. Clouston HJ, Herbert M, Fenwick J, et al. Cytogenetic analysis of human blastocysts. Prenat Diagn. 2002;22:1143-1152. Reprints: Dr. John Wolstenholme, Dept. of Cytogenetics, Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, United Kingdom; john.wolstenholme@ncl.ac.uk For epidemiological studies to be informative, they often need to be large (perhaps involving tens or hundreds of thousands of individuals), which in turn requires methods for blood collection that are practical and cost-effective. When blood samples are being collected for a study in a large number of separate sites, mailing of whole blood samples to a central laboratory for separation may be more convenient and cost-effective than making arrangements for local separation or for courier transport of chilled samples. Saliva samples have been successfully collected by mail for measurement of hepatitis B virus seropositivity, and the potential advantages of collecting blood samples by mail in epidemiological studies have been noted. There is limited evidence to suggest that some biochemical analytes, such as total cholesterol, may be stable in whole blood for several days at ambient temperature. If confirmed for a wider range of analytes of interest, use of mailed whole blood samples might allow blood collection to be included in studies where it would not otherwise be considered feasible. To assess the feasibility and reliability of transporting blood samples over several days at ambient temperature—for example, by mail—the authors evaluated the stability of various plasma analytes in samples stored at room temperature or chilled. Multiple vacutainers of blood containing EDTA and aprotinin as preservative were drawn from 12 volunteers and stored at 21°C or 4°C. Immediately after collection and one, two, three, four, and seven days later, vacutainers stored at each temperature were centrifuged, and the plasma was aliquoted and stored at -80°C. All aliquots from each individual subsequently were analyzed in one analytical run for a range of chemistries. In whole blood stored at room temperature for up to seven days, concentrations of albumin, apolipoproteins A1 and B, cholesterol, high-density lipoprotein, total protein, and triglycerides changed by less than four percent, and low-density lipoprotein by less than seven percent. While alanine transaminase, creatine kinase, creatinine, and γ-glutamyl transferase concentrations changed substantially at room temperature, there was less than four percent change when stored chilled up to seven days. In contrast, aspartate transaminase concentrations increased markedly under both conditions. The authors concluded that a wide range of important analytes, including lipids, change by only a few percent in whole blood during storage at room temperature for several days. Mailed transport of whole blood samples may, therefore, be a simple and cost-effective option for large-scale epidemiological studies. Clark S, Youngman LD, Palmer A, et al. Stability of plasma analytes after delayed separation of whole blood: implications for epidemiological studies. Int J Epidemiol. 2003;32:125-130. Reprint information not available. Homocysteine, hypertension, and blood pressure tracking Several epidemiologic studies have demonstrated that elevated plasma total homocysteine has a modest effect on risk of cardiovascular disease. This effect appears to be stronger in hypertensive people. It has been suggested that the adverse risk associated with hyperhomocysteinemia might be mediated in part by the positive association of homocysteine with hypertension. In the third National Health and Nutrition Examination Survey (NHANES III), subjects in the highest quintile of plasma homocysteine had a two- to three-fold increased prevalence of hypertension relative to those in the lowest quintile. Homocysteine-lowering treatment is associated with a reduction in systolic and diastolic blood pressure. No prior study has, according to the authors, prospectively examined the relation of plasma homocysteine to hypertension incidence. The authors attempted to test the hypothesis that hyperhomocysteinemia is related to the development of hypertension and blood pressure tracking in a community-based cohort of nonhypertensive subjects. They investigated the relations of baseline plasma total homocysteine levels to hypertension incidence and blood pressure tracking in 2,104 Framingham Heart Study participants (mean age, 57 years; 58 percent women) who were free of hypertension, myocardial infarction, heart failure, atrial fibrillation, or renal failure at baseline. Baseline mean ± SD plasma homocysteine was 10.1 ± 3.7 µmol/L. On followup four years from baseline, 360 people (17.1 percent) had developed hypertension, and 878 (41.7 percent) had progressed to a higher blood pressure stage. In unadjusted analyses, a 1-SD higher log homocysteine value was associated with increased odds of developing hypertension (odds ratio, 1.18; 95 percent confidence interval, 1.05-1.32) and increased odds of blood pressure progression (OR, 1.17; 95 percent CI, 1.07-1.27). The relationships of plasma homocysteine to the incidence of hypertension or blood pressure progression were statistically nonsignificant in age- and sex-adjusted logistic regression models (OR, 0.98; 95 percent CI, 0.87-1.11, and OR, 1.05; 95 percent CI, 0.96-1.16, respectively) and in multivariable models adjusted for age, gender, body mass index, diabetes, interim weight change, smoking, serum creatinine, baseline blood pressure, and blood pressure category (OR, 0.92; 95 percent CI, 0.81-1.06, and OR, 1.07; 95 percent CI, 0.97-1.18, respectively). In conclusion, the authors could find no major relation of baseline plasma homocysteine levels to hypertension incidence or longitudinal blood pressure progression in a large, community-based cohort of nonhypertensive subjects after adjusting for age, gender, and other important covariates. Sundström J, Sullivan L, D'Agostino RB, et al. Plasma homocysteine, hypertension incidence, and blood pressure tracking—The Framingham Heart Study. Hypertension. 2003;42:1100-1105. Reprints: Dr. Ramachandran S. Vasan, Framingham Heart Study, 73 Mt. Wayte Ave., Framingham, MA 01702-5803; vasan@fram.nhlbi.nih.gov A red cell genotype associated with atherothrombosis Moderately raised blood concentrations of total homocysteine are an independent risk factor for atherothrombotic vascular disease, including stroke. However, it is not known whether high homocysteine causes atherothrombosis or whether the disease is caused by a different component of the homocysteine metabolic cycle. In most tissues, homocysteine is re-methylated back to methionine by methionine synthase, with N5-methylenetetrahydrofolate reductase (MTHFR) acting on N5-methylenetetrahydrofolate (N5-MTHF), which becomes a methyl group donor, and cobalamin an essential cofactor. The formation of N5-MTHF depends on the presence of N5N10-methylenetetrahydrofolate and the enzyme MTHFR. The absence or reduced function of any of these components might be a cause of atherothrombosis. A substantial proportion of the population has a common point mutation-C to T substitution at nucleotide 677 [C677 T]—in the coding region of the gene for MTHFR, which is associated with a thermolabile variant that has reduced activity by about 50 percent. In association with poor folate status, this mutation has been linked with raised total homocysteine (tHcy) and an increased risk of atherothrombotic vascular disease. The intent of this study was to investigate the relation between total red cell folate, red cell N5-MTHF values, and the N5N10MTHFR genotypes found in patients with stroke. The authors hypothesized that patients with stroke who have the TT genotype would have lower red cell N5-MTHF than those with the CC and CT genotypes, particularly patients who are not taking folate supplementation. The authors' study comprised 120 consecutive patients presenting to a hospital with acute stroke. The subjects' use of multi-vitamin supplements was documented. Serum and red cell folate were measured by microbiological assays using Lactobacillus casei and Enterococcus faecalis and by the DPC-BioMediq Immulite TM 2000 analyzer. Total plasma homocysteine, serum cobalamin, and serum vitamin B56 were measured, and the C677T MTHFR genotype was determined. The authors found that there were no significant differences in blood tHcy or vitamin concentrations according to MTHFR genotype in the overall patient cohort. However, when patients taking vitamins were excluded, total red cell folate and red cell N5-MTHF were significantly lower in patients with the TT genotype compared with the CT or CC genotype. In the overall cohort, irrespective of genotype, red cell folate was significantly lower when assayed microbiologically than with the Immulite assay. This discrepancy remained after excluding patients taking vitamins. The authors concluded that total red cell folate and red cell N5-MTHF are significantly lower in stroke patients with the TT genotype than with the CT and TT MTHFR genotypes, particularly for those not taking vitamin supplements. Icke GC, Dennis M, Sjollema S, et al. Red cell N5-methyltetrahydrofolate concentrations and C677T methylenetetrahydrofolate reductase genotype in patients with stroke. J Clin Pathol. 2004;57:54-57. Correspondence: Dr. G. Icke, Division of Laboratory Medicine, Royal Perth Hospital, GPO Box X2213, Perth, WA, 6847, Australia; graham.icke@health.wa.gov.au Precision of point-of-care testing INR measurements Self-managed oral anticoagulant therapy is based on patients' international normalized ratio measurements with portable whole blood coagulometers. The authors previously estimated a precision (coefficient of variation) of 5.4 percent with the older version of CoaguChek (Roche Diagnostics, Mannheim, Germany), with measurements performed at home by patients on oral anticoagulant therapy. Other studies found that the precision of CoaguChek S (Roche Diagnostics) measurements based on liquid quality controls was 4.6 percent. The authors conducted a study to evaluate the precision of the CoaguChek S system in the hands of experienced self-managing patients. Fifteen patients on self-managed oral anticoagulant therapy performed measurements of international normalized ratio (INR) using the CoaguChek and CoaguChek S portable whole blood coagulometers at home for 10 weeks. The authors found that the coefficient of variation (CV) of INRs determined at home by CoaguChek S by patients on self-managed oral anticoagulant therapy was 5.5 percent (95 percent confidence limits: 4.9 percent, 6.1 percent). The biological CV of the INR within and between patients was 15 percent and 14.7 percent, respectively. The authors concluded that the precision of CoaguChek S is satisfactory. Attermann J, Andersen NT, Korsgaard H, et al. Precision of INR measured with a patient operated whole blood coagulometer. Thrombosis Res. 2003;110:65-68. Correspondence: J. Attermann at jorn.attermann@bisam.dk Assessing blood volume with fluorescein-labeled HES The circulating blood volume can be divided into the cellular component, red blood cell volume, and the noncellular fluid component, the plasma volume. Measurements of red blood cell volume (RCV) and plasma volume (PV) traditionally have been performed to confirm or refute erythrocytosis in patients with a raised hematocrit or features of polycythemia. Optimization of the circulating blood volume is important in managing critically ill patients to ensure an adequate supply of oxygen and nutrients to all cells. In high-dependency areas, most investigations performed routinely are intended to monitor circulatory status. On the basis of such investigations, clinical decisions are made regarding interventions necessary to adequately maintain blood flow and avoid regional ischemia. The gold standard method for measuring RCV, PV, and hence blood volume involves diluting a known amount of a measurable tracer substance in the appropriate compartment of the blood. The degree of dilution of the tracer can be used to calculate the volume of fluid it has been diluted to or distributed in. Unlabeled HES, a large carbohydrate molecule, has been used for a dilutional assay. By conjugation of the fluorescent molecule FITC to HES (FITC-HES), a fluorescent molecule considerably larger than albumin is produced. The degree of dilution of this fluorescence can be measured quickly, simply, and inexpensively to calculate PV. The authors investigated the clinical utility of FITC-HES as a tool to measure PV by comparing it with standard radioactive methods in a large number of relatively healthy patients undergoing routine investigations for polycythemia. They also conducted a pilot study to provide data about the potential usefulness of this method in critically ill patients undergoing blood volume assessment by comparison with the results obtained by standard methods. These two patient groups were chosen to assess the accuracy of the method and the effect of endothelial dysfunction on the results obtained with two molecules of differing size—albumin and FITC-HES. Seventy-nine ambulant outpatients and 18 intensive care unit patients were prospectively recruited. Measurements of RCV and PV were performed with radiochromium-labeled RBCs (51Cr), radioiodinated albumin (125I), and FITC-HES. Small molecules overestimate PV because of vascular endothelial dysfunction and increased capillary permeability; a reference value for PV was therefore derived with the RCV and hematocrit. The authors found that the mean PV with 125I dilution was 230 mL (standard deviation, 185 mL) greater than that with FITC-HES in outpatients. This difference was more exaggerated, at 345 mL (SD, 371 mL), in ICU patients likely to have endothelial dysfunction. Both the PV measured with FITC-HES and the 125I dilution correlated closely with the PV derived with RCV and hematocrit (r=0.950 and 0.925, respectively) in the ICU patients. The authors concluded that FITC-HES estimates PV more accurately than125I and that FITC-HES should replace radioactive tracers for assessing blood volume.7 Massey EJ, de Souza P, Findlay G, et al. Clinically practical blood volume assessment with fluorescein-labeled HES. Transfusion. 2004;44:151-15 Reprints: Edwin Massey, MRCP, MRCPath, Consultant Haematologist, National Blood Service, Southmead Rd., Bristol, BS10 5ND, United Kingdom; edwin.massey@nbs.nhs.uk Cystatin C in the elderly: relationship to health Serum creatinine level is used routinely in assessing glomerular filtration rate, although several widely recognized limitations compromise its usefulness in clinical work. The impact of muscle mass on serum creatinine values makes interpretation difficult, especially in the elderly. Serum cystatin C (Cys C) is believed to be a more sensitive indicator of glomerular filtration rate in the general population and specific patient categories. Determinants of elevated serum creatinine levels and microalbuminuria have been reported in population-based studies, including those involving elderly subjects, whereas Cys C has not been evaluated extensively in this respect in representative elderly populations. In this study, the authors assessed the associations of demographic and health status characteristics with levels of Cys C and serum creatinine and urinary albumin-creatinine ratio (ACR) by means of multivariate analyses in an elderly population to evaluate features of Cys C versus serum creatinine level in this context. The authors performed a cross-sectional, community-based survey in Lieto, Finland, measuring Cys C, serum creatinine, and ACR in 1,260 subjects aged 64 to 100 years. Associations of demographic characteristics and health status factors with levels of Cys C, serum creatinine, and ACR were assessed by linear models. The authors found that, in men, hypertension, coronary heart disease, urinary infection, rheumatoid arthritis, glucocorticoid treatment, older age, and lower functional status were significant predictors of higher Cys C values, whereas hypertension, coronary heart disease, urinary infection, older age, and increasing body mass index significantly predicted higher serum creatinine values. Among women, corresponding factors for Cys C were hypertension, glucocorticoid treatment, age, functional status, and body mass index, and corresponding factors for serum creatinine were hypertension, body mass index, and age. Diabetes was significantly associated with only ACR. These factors explained 35 percent of variation in Cys C values in men and 34.5 percent in women versus only 14.8 percent and 11.3 percent, respectively, for serum creatinine values. The authors concluded that glucocorticoid treatment should be recognized as an independent Cys C-increasing factor, presumably nonglomerular. In comparison with Cys C, a considerably greater proportion of total variation in serum creatinine values seemed to be explained by extrarenal factors. Wasén E, Isoaho R, Mattila K, et al. Serum cystatin C in the aged: relationships with health status. Am J Kidney Dis. 2003;42: 36-43. Reprints: Dr. E. Wasén, Institute of Clinical Medicine, General Practice, University of Turku, Lemminkaisenkatu 1, FIN-20014, Turku, Finland; elise.wasen@utu.fi The use of a potent combination of antiretroviral drugs has led to dramatic reductions in the morbidity and mortality associated with HIV-1 infection. However, approximately 20 percent of therapy-naive patients fail to achieve adequate virological response during their first year of triple therapy, with approximately 20 percent more patients experiencing virological failure during the second year. Variability in response to antiretroviral agents has been attributed, in part, to virological, immunological, pharmacokinetic, and adherence differences between patients. The first two issues have been partially addressed by drug-resistance testing and the monitoring of patients' CD4 cell counts as a standard of care. However, the latter two issues have proved to be difficult to objectively evaluate in a routine clinical setting. Interpatient variability in absorption, distribution, metabolism, elimination, and protein binding may put patients at risk for subtherapeutic or toxic exposures to antiretroviral drugs. Antiretroviral drug-to-drug interactions and interactions with other drugs or supplements, or both, can also contribute to less-than-optimal exposures. Therefore, it is reasonable to postulate that at least some unexplained cases of virological failure or adverse effects may result from pharmacokinetic problems. The authors investigated the predictive value of measuring protease inhibitor (PI) concentrations and non-nucleoside reverse-transcriptase inhibitor (NNRTI) concentrations in untimed plasma samples collected for virus-load testing in a population of antiretroviral-naive HIV-positive patients initiating highly active antiretroviral therapy (HAART). Given the pharmacokinetic characteristics of PIs and NNRTIs, they anticipated that patients treated effectively should maintain drug concentrations above predicted limits; therefore, they hypothesized that the detection of drug levels below predicted limits could reveal potential pharmacokinetic or adherence difficulties, or both. Plasma NNRTI and PI concentrations were retrospectively measured in all virus-load plasma samples collected during the first year of therapy for 122 patients in British Columbia, Canada, who initiated therapy between August 1996 and September 1999 and who had CD4 counts of less than 50 cells/µL. Drug levels were designated a priority as "low" if the concentrations were below the published C trough -SD. A single low drug level measured shortly after initiating therapy (median, six weeks) is common (30 percent) and is predictive of more rapid immunological failure (P=.06) and failure to achieve virologic success during the first year of therapy (P=.01). These results may reflect incomplete adherence, since a strong association (P<.001) was found between low drug levels and an imperfect prescription-refill record (less than 95 percent). Alexander CS, Asselin JJ, Ting LSL, et al. Antiretroviral concentrations in untimed plasma samples predict therapy outcome in a population with advanced disease. J Infect Dis. 2003;188:541-548. Reprints: Dr. Richard Harrigan, 613-1081 Burrard St., Vancouver, British Columbia, V6Z 1Y6, Canada; lab@hivnet.ubc.ca Significance of clinically established euthyroidism on the thyroid hormone reference range The classical problems in diagnosing and monitoring thyroid diseases are the difficulties in establishing a reference range for thyroid hormones and thyroid-stimulating hormone in blood without using measurements of both as a discriminator. A major problem is the cumbersome assessment of the clinical diagnosis of thyroid disease. Therefore, diagnosis and subsequent treatment are often based solely on thyroid function tests, S-T4 (thyroxine), s-T3 (triiodothyronine), s-TSH, and perhaps FT4 I (free thyroxine index) or FT4 (free thyroxine). Reference values are taken from the manufacturer's recommendations or determined from a previously established serum bank or among the staff in the local department of clinical chemistry, assuming that they are healthy and euthyroid. Parallel to the downgrading of the clinical aspects of thyroid disease, the number of thyroid tests has increased tremendously. It is now almost a rule that measurements of thyroid hormone concentrations and thyroid-stimulating hormone (TSH) define thyroid state. Furthermore, it recently has been demonstrated that, due to an individual set point for T4 and T3 , the distinction between subclinical and overt thyroid disease is arbitrary. The author conducted a study to examine whether persons selected for establishing a reference range of thyroid hormones and TSH, who are assumed to be euthyroid, are indeed euthyroid by clinical examination and by determination of basal oxygen consumption (VO2). The author also evaluated whether these supplementary examinations affected the reference range for thyroid hormones and TSH. Basal oxygen consumption at rest was chosen as an independent marker as it is generally accepted as the most satisfactory index of the overall metabolic effect of thyroid hormones. For the study, a clinical examination, including information on medication, was performed on 31 apparently healthy people. The following determinations were made for all of the subjects: basal oxygen consumption, serum TSH, serum total T3 , and serum total T4. T4 uptake for calculation of FT4 was also measured. The author found that the clinical examination and determination of VO2 reduced what initially appeared to be an euthyroid and healthy population by 32 percent. This also resulted in a narrower reference interval than that suggested by the manufacturer (range) (TSH=0.35-5.50 mU/L, T4 =58-140 nmol/L, T3 =0.9-2.8 nmol/L) or than that established in the initially selected population (TSH=0.76-3.90 mU/L, T4 =54-168 nmol/L, T3 =1.27-2.74 nmol/L, FT4 I=64-123 a.u.). In the final selected population, this was TSH=0.76-3.90 mU/L, T4 =59-127 nmol/L, T3 =1.45-2.74 nmol/L, and FT4 I=64-120 a.u. The author concluded that the method of selecting the population for establishing the reference interval for thyroid hormones and TSH influences the reference ranges. Neglecting to deal with this problem may invalidate screening procedures, especially for subclinical diseases. Kvetny J. The significance of clinical euthyroidism on reference range for thyroid hormones. Eur J Intern Med. 2003;14: 315-320. Correspondence: J. Kvetny at jkv@ribeamt.dk |
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