Neonatal blood lymphocyte subpopulations: a different perspective when using absolute counts.

We compared the absolute counts of lymphocyte subpopulations in 15 neonates, and 9 adults using the whole lysed blood technique with 15 different triple immunostainings. To obtain accurate absolute lymphocyte counts in neonatal cord blood samples, the flow cytometric 'lympho-gate' was corrected for the erythroid cell contamination by normoblasts and unlysed erythrocytes. In contrast to earlier studies where relative frequencies were reported, we found that the major difference between neonatal and adult lymphocyte subpopulations concerned the much larger pool of naive 'untriggered' cells in neonates, standby for participation in primary immune responses.

Longitudinal survey of lymphocyte subpopulations in the first year of life.

Age-matched reference values for lymphocyte subpopulations are generally obtained via cross-sectional studies, whereas patients are followed longitudinally. We performed a detailed longitudinal analysis of the changes in lymphocyte subpopulations in a group of 11 healthy infants followed from birth up to 1 y of age, with special attention for early developmental markers, markers of maturation, and markers of activation. We found that T and B lymphocytes increased at 1 and 6 wk of age, respectively. In contrast, NK cells showed a sharp decline directly after birth, suggesting that they are more important during pregnancy than thereafter. CD45RA+--mainly CD4+--naive T lymphocytes were high at birth, and increased further during the first year of life; they form a large expanding pool of cells, ready for participation in primary immune responses. The absolute counts of CD45RO+ memory T lymphocytes were similar in infants and adults, albeit with a lower level of expression of CD45RO on infant T lymphocytes. Almost all infant T lymphocytes expressed CD38 throughout the first year of life. The abundant expression of CD38 on an infant's T lymphocytes might be related to a greater metabolic need of the large population of naive untriggered cells that are continually involved in primary immune responses during the first year of life. The high B lymphocyte counts in infants mainly concerned CD38+ B lymphocytes throughout the first year of life. Also, the relative frequencies of CD1c+ and CD5+ B lymphocytes were higher throughout the first year of life than in adults. Therefore, CD1c, CD5, and CD38 could be markers of untriggered B lymphocytes. In conclusion, our longitudinal survey of T and B lymphocytes, NK cells, and their subpopulations during the first year of life helps to complete the picture of lymphocyte development in infants. This information contributes to the correct interpretation of data from infants with possible immune disorders.

Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.

Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies.

Thermolabile methylenetetrahydrofolate reductase in coronary artery disease.

BACKGROUND: Hyperhomocysteinemia, an independent and graded risk factor for coronary artery disease (CAD), may result from both environmental and hereditary factors. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the methyl donor in the remethylation of homocysteine to methionine. A 677C-->T mutation in the MTHFR gene has been associated with elevated homocysteine concentrations in homozygous (+/+) individuals. METHODS AND RESULTS: We assessed the frequency of this common mutation in 735 CAD patients from the Regression Growth Evaluation Statin Study (REGRESS), a lipid-lowering coronary-regression trial, and in 1250 population-based control subjects. Furthermore, the association between the mutation and serum homocysteine concentrations was studied. The frequency of the homozygous (+/+) mutation was 9.5% among patients versus 8.5% among control subjects, resulting in an odds ratio of 1.21 (95% confidence interval [CI], 0.87 to 1.68), relative to the (-/-) genotype. Homocysteine concentrations were significantly elevated in both (+/+) and (+/-) individuals compared with (-/-) individuals (median homocysteine levels, 15.4, 13.4, and 12.6 micromol/L, for (+/+), (+/-), and (-/-) individuals, respectively). For a summary estimation of the risk of the (+/+) genotype for CAD, we performed a meta-analysis on 8 different case-control studies on thermolabile MTHFR in CAD. In the meta-analysis, the homozygous (+/+) genotype was present in 299 of 2476 patients (12.1%) and in 257 (10.4%) of 2481 control subjects, resulting in a significant odds ratio of 1.22 (95% CI, 1.01 to 1.47) relative to the (-/-) genotype. CONCLUSIONS: Both the homozygous (+/+) and heterozygous (+/-) genotype result in elevated homocysteine concentrations. From our meta-analysis, we conclude that the homozygous (+/+) genotype is a modest but significant risk factor for CAD.

Changes in bone mineral density, body composition, and lipid metabolism during growth hormone (GH) treatment in children with GH deficiency.

Adults with childhood onset GH deficiency (GHD) have reduced bone mass, increased fat mass, and disorders of lipid metabolism. The aim of the present study was to evaluate bone mineral density (BMD), bone metabolism, body composition, and lipid metabolism in GHD children before and during 2-3 yr of GH treatment (GHRx). Forty children with GHD, mean age 7.9 yr, participated in the study of bone metabolism and body composition; and an additional group of 17 GHD children, in the study of lipid metabolism. Lumbar spine BMD, total body BMD, and body composition were measured with dual-energy x-ray absorptiometry. Volumetric BMD (bone mineral apparent density, BMAD) was calculated to correct for bone size. BMD, BMAD, lean tissue mass, bone mineral content, fat mass, and percentage body fat were expressed as SD scores (SDS), in comparison with normative data of the same population. Lumbar spine BMD and BMAD and total body BMD were all decreased at baseline. All BMD variables increased significantly during GHRx, lumbar spine BMD SDS, already after 6 months of treatment. Lean tissue mass SDS increased continuously. Bone mineral content SDS started to increase after 6 months GHRx. Fat mass SDS decreased during the first 6 months of GHRx and remained stable thereafter. Biochemical parameters of bone formation and bone resorption did not differ from normal at baseline and increased during the first 6 months of GHRx. Serum 1,25 dihydroxyvitamin D increased continuously during GHRx, whereas PTH and serum calcium remained stable. Lipid profile was normal at baseline: Atherogenic index had decreased and apolipoprotein A1(Apo-A1) had increased after 3 yr of treatment. In conclusion, children with GHD have decreased bone mass. BMD, together with height and lean tissue mass, increased during GHRx. GHRx had a beneficial effect on lipid metabolism.

Creatinine and surveys: an assessment.

We analysed the results of surveys on creatinine held in The Netherlands during the years 1992, 1993 and 1994. Assay results of 113 samples were reviewed: 88 human sera and 25 samples of animal origin. The results of 5 creatinine assays, 4 based on the Jaffé reaction and 1 enzymatic procedure, are discussed. The enzymatic assay showed by far the best performance, while some of the Jaffé methods differed considerably. All results were evaluated by reference to a HPLC-based selected method for creatinine. Our study shows the need for caution when applying survey performance criteria for creatinine.

Effect of dietary triacylglycerol fatty acid positional distribution on plasma lipid classes and their fatty acid composition in preterm infants.

Seven premature infants were each fed, for 1 wk in a crossover design. The beta formula contained triacylglycerols resembling the stereoisomeric structure of human milk fat (25.4% by wt 16:0, 76.1% of which is at the sn-2 position), whereas in the alpha formula 87.3% of total 16:0 (25.7% by wt was at the sn-1,3 positions. Plasma lipids and their fatty acid compositions were determined at the end of each 1 wk study period. Infants fed with the beta formula had higher percentages of palmitic acid in plasma sterol esters, triacylglycerols, and free fatty acids, and lower linoleic acid in triacylglycerols than with the alpha formula. Premature infants fed formulas with triacylglycerols 16:0 predominantly in the sn-2 rather than the sn-1,3 positions had alterations in their plasma fatty acids consistent with enhanced absorption of 16:0 from the sn-2 compared with the sn-1,3 positions.

Testing the accuracy of total cholesterol assays in an external quality-control program. Effect of adding sucrose to lyophilized control sera compared with use of fresh or frozen sera.

We studied the suitability of various types of human serum preparations to test the accuracy of total cholesterol measurements in the External Quality Assessment scheme in The Netherlands, in which approximately 180 laboratories participate. Checked against the certified Abell/Kendall Reference Method, large reagent-dependent negative biases were observed with lyophilized serum that was insufficiently cryoprotected. The biases for the reagents of Du Pont, Roche, and Beckman averaged -16.7%, -9.2%, and -7.6% respectively; the least bias, -0.4%, was obtained with reagent from Boehringer Mannheim. The beneficial effect of cryoprotection with sucrose was demonstrated by the decrease in interreagent variation from 5.4% to 1.9%, the latter value being comparable with the values for fresh and once-frozen pooled serum (1.3% and 1.7%, respectively). We conclude that the detrimental effect of lyophilization on serum matrix can be minimized by suitable cryoprotection with 200 g/L sucrose.

Effects of lipid lowering by pravastatin on progression and regression of coronary artery disease in symptomatic men with normal to moderately elevated serum cholesterol levels. The Regression Growth Evaluation Statin Study (REGRESS).

BACKGROUND: Intensive lowering of serum cholesterol may retard progression of coronary atherosclerosis in selected groups of patients. However, few data are available on the potential benefit of serum cholesterol reduction in the broad range of patients with coronary atherosclerosis and normal to moderately elevated serum cholesterol levels who undergo various forms of treatment. The Regression Growth Evaluation Statin Study (REGRESS) addresses this group of patients. METHODS AND RESULTS: REGRESS is a double-blind, placebo-controlled multicenter study to assess the effects of 2 years of treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor pravastatin on progression and regression of coronary atherosclerosis in 885 male patients with a serum cholesterol level between 4 and 8 mmol/L (155 and 310 mg/dL) by quantitative coronary arteriography. Primary end points were (1) change in average mean segment diameter per patient and (2) change in average minimum obstruction diameter per patient. Clinical events were also analyzed. Of the 885 patients, 778 (88%) had an evaluable final angiogram. Mean segment diameter decreased 0.10 mm in the placebo group versus 0.06 mm in the pravastatin group (P = .019): The mean difference between treatment groups was 0.04 mm, with a 95% CI of 0.01 to 0.07 mm. The median minimum obstruction diameter decreased 0.09 mm in the placebo group versus 0.03 mm in the pravastatin group (P = .001): The difference of the medians between the treatment groups was 0.06 mm, with a CI of 0.02 to 0.08 mm. At the end of the follow-up period, 89% (CI, 86% to 92%) of the pravastatin patients and 81% (CI, 77% to 85%) of the placebo patients were without new cardiovascular events (P = .002). CONCLUSIONS: In symptomatic men with significant coronary atherosclerosis and normal to moderately elevated serum cholesterol, less progression of coronary atherosclerosis and fewer new cardiovascular events were observed in the group of patients treated with pravastatin than in the placebo group.

Reference values for apolipoprotein A-I and apolipoprotein B in serum still depend on choice of assay techniques.

In a study with young children receiving growth hormone treatment, it was necessary to re-establish reference values for various blood components, e.g. apolipoproteins A-I and B. We find that considerable method-to-method variation still exists. We used the DuPont Dimension analyzer (D) and the Beckman Array analyzer (B) systems and a third procedure with Orion reagents and a Kone Analyzer (K). We investigated if assay results may be pooled or exchanged within our study. In 59 serum samples we measured apolipoprotein A-I and apolipoprotein B (n = 58) and calculated the orthogonal regression equations y = a(Sa) x + b(Sb). For apolipoprotein A-I the results are: (I) B = 1.165 (0.065) D - 0.193 (0.077), Syx = 0.055; with r = 0.922 and (II) K = 0.831 (0.056) D - 0.190 (0.066), Syx = 0.055; with r = 0.898. For apolipoprotein B the equations are: (III) B = 1.586 (0.137) D - 0.246 (0.100), Syx = 0.061; with r = 0.840 and (IV) K = 0.869 (0.065) D + 0.251 (0.048), Syx = 0.044; with r = 0.875. According to Passing & Bablok, the slopes and intercept values are 1.093 and -0.126; 0.848 and 0.167; 1.500 and -0.185; 0.880 and 0.249. The overall impression is the same for both regression methods: comparability had not yet been achieved by early 1993, particularly not for apolipoprotein B, and reference values differ significantly depending on the selected methodology.

Total and HDL-cholesterol in The Netherlands: 1987-1992. Levels and changes over time in relation to age, gender and educational level.

BACKGROUND: To gain insight into the prevalence of and trends in plasma cholesterol levels in the general population of the Netherlands, a monitoring project was carried out from 1987 to 1992. METHODS: Each year a random sample of men and women aged 20-59 years in three towns in the Netherlands was invited to participate in the study. The overall response rate was 50% for men and 57% for women and a total of almost 42,000 men and women participated. Total (TC) and high density lipoprotein cholesterol (HDL-C) was measured and the non-HDL-C/HDL-C ratio was computed. Data were age-standardized to the Dutch population distribution aged 20-59 years. RESULTS: The prevalence of hypercholesterolaemia (TC > or = 6.5 mmol/l) in men ranged from 5% in the youngest (20-29 years) to 29% in the oldest age group (50-59 years), and from 4% to 38% in women. Low HDL-C levels (< or = 0.9 mmol/l) in men ranged from 15% in the youngest to 26% in the oldest age group, and in women from 4% in the youngest to 7% in the oldest age group. The lipid profile of those with a higher educational level was more favourable than that of the less educated. From 1987 to 1992, in men, TC decreased by 0.12 mmol/l, HDL-C decreased by 0.07 mmol/l and the non-HDL-C/HDL-C ratio increased by 0.22. In women no statistically significant changes were observed. Changes over time did not differ according to age and educational level. CONCLUSION: The prevalence of hypercholesterolaemia is still high in the Netherlands. During the period 1987-1992 the lipid profile worsened in men and remained stable in women.

Evaluation of the Cholestech L.D.X. desktop analyser for cholesterol, HDL-cholesterol, and triacylglycerols in heparinized venous blood.

The analytical performance of the Cholestech L.D.X. lipid analyzer for total and HDL-cholesterol and triacylglycerols in heparinized venous blood was evaluated, using two Cholestech L.D.X. analysers and two reagent cassette lots. Within-day and day-to-day precision were checked with commercial quality control sera. Within-day coefficients of variation (CVs) were 2.0 to 4.7% for total cholesterol, 3.4 to 5.5% for HDL-cholesterol, and 2.1 to 4.8% for triacylglycerols. Between-day CVs were < or = 6.0% for triacylglycerols, and < or = 7.0% for HDL-cholesterol. A method comparison study, according to the NCCLS EP9-P guidelines, was performed for all three analytes. Cholestech whole blood values (y) were compared with serum values (x) generated by a standardized enzymatic method for cholesterol, and by current state-of-the-art methodology for HDL-cholesterol and triacylglycerols. Correlation coefficients were all > 0.98. Mean slopes and intercepts for the Passing & Bablok regression equations of total and HDL-cholesterol were not significantly different from one and zero, respectively. Overall, the Cholestech means differed by < 1.8% for total cholesterol, and < 5% for HDL-cholesterol versus the comparison method means. For total cholesterol the National Cholesterol Education Program (NCEP) requirements for accuracy and precision of < or = 3% were met. For triacylglycerols, significant negative intercepts ranging from -0.26 to -0.33 mmol/l were observed, with slopes significantly greater than one.

Multicenter evaluation of Reflotron direct dry-chemistry assay of high-density lipoprotein cholesterol in venous and fingerstick specimens.

The Reflotron HDL Cholesterol test (Boehringer Mannheim GmbH) directly separates and analyzes high-density lipoprotein (HDL) cholesterol in plasma collected with EDTA in an integrated dry-reagent system suitable for alternative site testing of lipoproteins. We describe a multicenter evaluation of this test by two US and six European laboratories experienced in lipid analysis. Each laboratory compared the Reflotron with the same conventional wet-chemistry method, Boehringer phosphotungstate-Mg2+ precipitation with enzymatic cholesterol assay. Imprecision was within accepted guidelines, with CVs of < or = 8% for fresh and frozen plasmas (median CV 1.7-3.9%) and for lyophilized sera (median CV 3.8-4.7%), similar to those of the conventional method. Results of linear-regression analysis were as follows: Reflotron HDL Cholesterol = 1.03 conventional - 3.9 mg/L, r = 0.987. The Reflotron results were somewhat low in the two US laboratories, demonstrating the need for general standardization of methods for measuring HDL cholesterol. Results from capillary fingerstick plasma agreed well with those from venous-derived plasma; capillary = 1.04 venous + 4.5 mg/L, r = 0.967. The system is relatively insensitive to interference from hemoglobin (< or = 0.75 g/L), ascorbic acid (< or = 0.3 g/L), bilirubin (< or = 50 mg/L), cholesterol (< or = 3.5 g/L), and triglycerides (< or = 4 g/L). The relative ease of operation and the rapid availability of results (within 90 s for plasma collected in EDTA) make the method appropriate for use by well-trained, but not necessarily technical, operators in the physician's office or other alternative sites.

[Variation in serum cholesterol level in the same person: what is the true value and when is it called a significant change?]. / Schommeling van het serumcholesterolgehalte bij één persoon: wat is de werkelijke waarde en wanneer is er sprake van een significante verandering?

The blood cholesterol level is not constant over time. Changing values may be found when rechecking high values, especially in patients on a diet or receiving medication. Repeated measurements are necessary. It will then be possible to estimate the 'true mean value'. This is the theoretical average of a large number of measurements taken from one person. The study concentrated on the intra-individual variation of the serum cholesterol and the consequences for screening and follow-up. For this purpose, during a period of four weeks, cholesterol levels were measured 12 times in 33 men aged 25-40. The mean coefficient of variation was 5.7%, with wide differences between participants, ranging from 2.9% to 9.8%. The position of the 'true mean value' was estimated (with 90% confidence), after I resp. 3 determinations. These findings have consequences for the classification of subjects in the different risk categories as defined in the Dutch Cholesterol Consensus. It is also possible to determine if, after a period of intervention, there is a significant decline in the cholesterol level. Roughly, a decline of 10-12% indicates a significant difference.

[The importance of HDL-cholesterol level determination in the classification of persons at increased risk of coronary heart disease]. / Het belang van de bepaling van het HDL-cholesterolgehalte voor het classificeren van personen met een vergroot risico van coronaire hartziekten.

The evidence is growing that not only total cholesterol, but also HDL cholesterol is an important predictor of coronary heart disease. In the Framingham Study, the total cholesterol/HDL cholesterol ratio gave the best prediction for the coronary heart disease risk. With data of the Netherlands Monitoring Risk Factor Project it was investigated to what extent persons with a high ratio (greater than or equal to 7) were identified when the criteria of the Netherlands Cholesterol Consensus were applied. Between 1987 and 1989 total and HDL cholesterol were determined in about 22,000 men and women aged 20-59. Twenty per cent of the men had hypercholesterolaemia (total cholesterol greater than or equal to 6.5 mmol/l). Of the hypercholesterolaemic men, 60 per cent did not have a high total/HDL cholesterol ratio. Eighteen per cent of the women were hypercholesterolaemic. Of all hypercholesterolaemic women, 80 per cent did not have a high total/HDL cholesterol ratio. Therefore, it is important that after a first screening on total cholesterol, HDL cholesterol is measured at the second cholesterol determination. Subsequently, a decision about treatment should be made, based on the total/HDL cholesterol ratio and the presence of other risk factors (hypertension, smoking, obesity, diabetes and a family history of cardiovascular disease.

Protein restriction affects fat intake and serum lipids in children with chronic renal failure.

Cardiovascular complications in patients with chronic renal failure have been associated with the hyperlipidemia present in many of these patients. Since a protein-restricted diet is often prescribed in an attempt to preserve renal function, we performed a randomized controlled study in children with chronic renal failure on the effect of a protein-restricted diet on fat intake and serum lipid profiles. Although total fat intake did not change, we found a lower cholesterol intake and a higher polyunsaturated/saturated fat ratio in the patients with the protein-restricted diet. This is probably caused by the restriction of animal protein which results in the replacement of animal fat by vegetable fat in the protein-restricted group. Moreover, we observed an increase of plasma cholesterol and low-density lipoprotein cholesterol in patients with a normal protein intake which was absent in the protein-restricted group. This suggests a favourable effect of the institution of a protein-restricted diet on lipid intake and plasma profile.

Trend in serum total cholesterol level in 110,000 young adults in The Netherlands, 1974 to 1986.

Data from two screening projects on cardiovascular risk factors were used to analyze the trend in serum total cholesterol level in the Netherlands between 1974 and 1986. Cholesterol levels were measured in a single reference laboratory of the World Health Organization throughout the entire study period. Between 1974 and 1980, about 30,000 men and women aged 37 to 43 years (mean age, approximately 40 years) were screened. A decrease in mean serum total cholesterol level was observed until the end of 1977, when it was followed by an increase. This resulted in a net change over the entire study period of -0.07 mmol/liter (3 mg/dl) in men and -0.03 mmol/liter (1 mg/dl) in women. Between 1981 and 1986, about 80,000 men aged 33 to 37 years (mean age, 35 years) were screened. During this period, a decrease of 0.20 mmol/liter (8 mg/dl) in the mean total cholesterol level was observed. In spite of the decline in the mean total cholesterol level, the prevalence of cholesterol values of greater than or equal to 6.5 mmol/liter (greater than or equal to 251 mg/dl) in young adult men was still high in 1986 (16 percent). A further reduction is therefore desirable. The decline in the mean total cholesterol level in young adults might indicate that a further decline in mortality from coronary heart disease can be expected.