of variation were estimated for 26 clinical chemistry analytes ... differences generated will be useful in routine clinical deci- ..... with a simple textbook (23).
CLIN. CHEM. 35/5, 783-786 (1989)
Biological Variability of 26 Clinical Chemistry Analytes in Elderly People 1. CummIngs,1StephenP. WIlkinson,2Ronald G. Nevllle,3 James D. E. Knox,3 Olga Ho,1 and RonaldS. MacWalter2 Callum G. Fraser,1 Steven
Analytical, within-subject, and between-subject components of variation were estimated for 26 clinical chemistry analytes from duplicate analyses of 10 specimens collected from 27 healthy elderly subjects over a period of 20 weeks. Withinsubject variations were similar to those generated previously by us in younger subjects. We conclude, therefore, that homeostasis is not compromised by age alone, and biological variability does not increase simply with age. All analytes except serum water had marked individuality, showing that conventional population-based reference values are of limited utility.The critical differences required for two results to be significantly (P 0.05) changed are not the same as those that prompt action by clinicians. Although heterogeneity of within-subject variation does exist, we believe that the critical differences generated will be useful in routine clinical decision making. AddItional Keyphrases: variation,sourceof A large
proportion
of hospitalized
geriatricchemistry patients
and those
seeking primary health care are elderly people, and the number of elderly persons in the population continues to increase (1). It has been suggested that: (a) homeostatic mechanisms may become diminished in the aging process (1), (b) there is an apparent increase in biological variability with age (2), and (c) biological variation is least between ages 30 and 50 years (3). Despite the fact that data on biological variation have been used for many purposes in clinical chemistry-including setting of analytical goals (4), deciding the significance of changes in serial results (5), and assessing the utility of conventional population-based reference values (6)-most information has been obtained on young subjects. Williams et al. (7), investigating biological variation in three groups, ages 18-35, 36-55, and >56 years, found little change with increasing age. However, elderly people have not been studied in detail. To examine hypotheses a-c, we assessed the analytical and biological components of variation for a range of serum analytes in a cohort of healthy elderly adults over a period of 20 weeks. To more clearly study the effect of age, we assessed a variety of analytes for which we had already generated data on within-subject and between-subject variation in young subjects, using exactly the same analytical methods and instrumentation, and sources of reagents, that we had used previously. The analytes we studied were those of value in assessing salt/water homeostasis, renal function, liver function, bone function, lipid metabolism, the immune system, carbohydrate metabolism, and pancreatic function: sodium, potassium, chloride, urea, creatimne, calcium, phosphate, proteins, albumin, alkaline phosphatase (EC 3.1.3.1),
Departments of 1 Biochemical Medicine, 2Medicine (Geriatrics), and 3General
Practice,
Ninewells
Hospital
and Medical School,
Dundee DD1 9SY, Scotland, U.K. Received January 10, 1989; accepted February
2, 1989.
and bilirubin (8); cholesterol, triglycerides, and apolipoproteins (apo) A-I and B (9); immunoglobulins G, A, and M, and and X light-chains (10); glucose (11); fructosamine (12); amylase (EC 3.2.1.1) and lipase (EC 3.1.1.3) (13); and osmolality and water (14).
Materials and Methods Subjects and Specimens The subjects were selected from the National Health (NHS) register of patients of a U.K. urban general practice. The study had ethical approval. The inclusion criteria were that the subjects should be ages 70 years or older, living at home independently, and Service
requiring hygiene.
no help with feeding, toileting, or maintaining The exclusion criteria removed patients with any
chronic medical condition, including dementia, a history of gastrointestinal surgery, or current drug therapy. After detailed review of the medical records of the practice, 32 subjects were invited by a letter from their own general practitioner to participate in the study. The research nurse then visited each subject to discuss the study, verified that the inclusion criteria were fulfilled, and confirmed the absence of dementia by the use of a structured questionnaire. At 14-day intervals, 12 mL of venous blood was collected from each subject, generally on 10 occasions. It was verified each day that the subjects were fasted, that no therapy had been instituted, and that lifestyle had not changed. The nurse collected the specimens between 0700 and 0900 h from seated subjects by conventional venepuncture with minimal stasis. The specimens were transported to the laboratory within 1 h; sera were separated by centrifugation (3000 x g, 15 mm) and stored at -30 #{176}C until analysis. Analytical Methods Instruments. The instruments used were the sc continuous-flow analyzer (Technicon Instruments Corp., Tarrytown, NY 10591) for sodium, potassium, chloride, urea, creatimne, calcium, phosphate, proteins, albumin, bilirubin, and alkaline phosphatase; a Cobas Fara centrifugal analyzer (Roche Products, Welwyn Garden City, Herts., U.K.) for cholesterol, triglycerides, ape A-I and ape B, IgG, IgA, 1gM, ic chains, A chains, glucose, amylase, and lipase; a Rotochem ila centrifugal analyzer (Aminco, Silver Spring, MD 20910) for fructosainine; and a Roebling automatic micro-osmometer (Herman Roebling, Berlin, F.R.G.) for osmolality. Reagents. Reagents for the SMAC were purchased from Technicon (Basingstoke, Hants., U.K.); for cholesterol and lipase from Boehringer Corp. London, Ltd., (Lewes, East Sussex, U.K.); for triglycerides from Roche Products; for ape A-I and ape B from Orion Diagnostica (Espoo, Finland); for IgG, IgA, IgM, K chains, and A chains from Kallestad
Diagnostics (Austin, TX 78701); for glucose from Beckman RIIC Ltd. (Risley, Warrington, U.K.); and for amylase from Behringwerke AG (Marburg, F.R.G.). Methods. Standard Technicon methodology was used with the SMAC. Cholesterol was assayed by the cholesterol esterCLINICALCHEMISTRY, Vol. 35, No. 5, 1989 783
ase/cholesterol oxidase/4-aminophenazone/phenol method; triglycerides by formazan production from iodonitrotetrazohum chloride; apolipoproteins, inununoglobulins, and lightchains by immunoturbidimetry; glucose by the hexokinase/ glucose-6-phosphate dehydrogenase method; fructosamine exactly as described previously (12); ainylase by hydrolysis of 4-nitrophenylmaltopentaoside/hexaoside substrate, lipase by turbidimetry; osmolality by freezing-point depression; and water by the osmometric method of Faye and Payne (15). Analytical Strategy
At the time of analysis of each analyte, all specimens were allowed to thaw at room temperature, and assayed in the smallest number of analytical batches possible. The specimens were assayed in no specific sequence in duplicate in separate batches. The same lots of reagents, standards, and quality-control materials were used throughout, and all analyses of a particular analyte were performed by the same analyst. Database and Statistical Analysis During the study, four subjects decided not to participate further, and one other subject was excluded because of initiation of antibiotic and diuretic therapy. The database therefore comprised results from 27 subjects, 14 men and 13 women, between 70 and 83 years of age. We examined the data rigorously at various strata before performing statistical analysis. Each pair of results was examined for differences; if; on the basis of the imprecision of the method, unexpected differences (> ± 2 SD) existed, the analyses were repeated, and the results not apparently aberrant were substituted. The results for each analyte for each subject were then examined against the reference values in use in the laboratory; if all results were outside such values, we excluded all of that subject’s results, because we wished to study healthy, elderly subjects. The mean and parametric standard deviation (SD) were calculated for each analyte, and if any result exceeded ± 2 SD from the mean, both results for that specimen were excluded. Before we determined immunoglobulins and lightchains, we assayed by serum protein electrophoresis (Multitrac agarose electrophoresis, Ciba Corning Diagnostics, Halstead, Essex, U.K.) the first specimen taken from each individual; further determinations were not performed if an expert reviewer considered that any abnormality was present. The remaining results were subjected to nested analysis of variance to dissect the total variance into analytical (SD3I),within-subject (SD?), and between-subject (SD) components.
Results and Discussion The number of results included in the nested analysis of variance; the mean values; and analytical, within-subject, and between-subject variations (as coefficients of variation) are shown in Table 1. Comparison of Elderly and Younger Subjects Table 2 shows the within-subject variation generated in this study, and the data previously obtained by us for younger subjects, ages 20-53 years. These sets of results can be compared directly because the same methods, instrumentation, and sources of reagents were used. The ratios of the CV1, called Qby H#{246}Lzel (16), are shown in 784
CLINICALCHEMISTRY, Vol. 35, No. 5, 1989
Table 1. Data Points, Mean Results, and Calculated Components of Variation5 Anelyte Sodium
Mean
No.b
Potassium Chloride Urea Creatinine Calcium Phosphate Proteins
518 518 516 516 516 500 516 518
141.8 4.45 107.2 5.94 93.8 2.36
67.5 gIL
Albumin
518
43.0
Bilirubin AIk.phos. Cholesterol
518
9.1 tmoVL 88.0 U/L
452
Triglycerides Apo A-I
486 522
6.30 mmol/L 1.47 mmol/L 1.17 gIL
Apo B
522
lgG
392
IgA
394
1gM
396
chains A chains Glucose Fructosamine Amylase Lipase Osmolality Water
370
516
390
512 276 530
498 476 438
mmol/L mmol/L mmol/L mmoIIL hmol/L mmoVL
CVA, %
CV,, %
CV0, %
0.75 2.0 0.95 2.5
0.86 4.8 1.2 10.3
0.88
6.7 1.6 13.4
2.8
4.3
0.95
1.6
18.3 2.8
1.7
4.8
13.3
1.2
2.6
3.4
1.3
2.6
3.5
6.0
16.5
27.8
1.9 1.9 3.4
6.5 5.8 15.3
24.4 11.0 27.8
1.05 gIL
2.3 1.9
6.6 8.3
13.8 25.7
9.47 gIL 2.24 gIL 0.92 gIL
3.5 3.6 3.7
6.2 8.6 9.3
16.5 36.8
2.2 2.1 2.4
6.1 5.4 4.7
17.7 20.6 5.4
1.19 mmol/L g/L
7.59 g/L 4.37 gIL 5.21 mmol/L 2.19 mmol/L 65.8 U/L 120.0 U/L 289.0 UIL 0.95
34.9
2.9
4.6
3.8
1.6 8.8 0.5 1.9
10.8 14.0 0.8 3.8
32.1 17.8 0.9 0.07
Analytical variation (V..), within-subject variation (CV,). and betweensubject variation (CV0). b No. of analytical resultssubjected to ANOVA.
Table 2. Q2,for Q >1, and 11Q2, for Q 1.4 are reference values of use. The indices of individuality for the analytes investigated are listed in Table 3. Except for serum water, they all have indices of individuality
