Reference Intervals for Calcium, Phosphate, and ... - Clinical Chemistry

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the menopause on normal reference intervals for concentrations of calcium and inor- ganic phosphate, and alkaline phosphatase. (EC. 3.1.3.1) activity in plasma.
CLIN. CHEM. 32/1, 76-79

(1986)

Reference Intervals for Calcium, Phosphate, and Alkaline Phosphatase Derived on the Basis of Multichannel-Analyzer Profiles Terence

J. Slnton,1’2 David M. Cowley,’

and

Stewart

J. Bryant2

In an attempt to demonstrate a rapid and economical apto deriving reference intervals, we analyzed data on

proach

plasma

calcium and phosphate

from a multichannel analyzer

for more than 20 000 subjects and data on alkaline phosphatase from more than 10 000 subjects. Subjects were selected by the criterion that their results for constituents other than the one of interest were within current reference intervals. Thus we have been able to include older patients who have diseases that accompany old age, but that do not affect test results. The mean concentrations of calcium and phosphate decreased with increasing age in both sexes, except for an abrupt increase for women about the time of reaching menopause. Similarly, the mean alkaline phosphatase activity increased with age in both sexes, reflecting a skewed frequency distribution. Here also, there was an abrupt increase in the modal value for women near menopause. Additional pause

Keyphrases: osteoporosis

sex- and age-related effects frequency distributions of obserr’ed

val-

ues Interpretation of clinical chemical results depends in large part on the use of reference intervals. Therefore methods of deriving these values are of major interest and have generated an extensive literature. We have used a simple technique in which computer-archived patient information is searched quickly and relatively inexpensively to produce results from large numbers of subjects for statistical analysis. Using this technique, we have attempted to produos data that demonstrate the effect of inpatient and outpatient status, age, sex, and the menopause on normal reference intervals for concentrations of calcium and inorganic phosphate, and alkaline phosphatase (EC 3.1.3.1) activity in plasma samples from a hospitalized population. This approach was prompted by our subjective impression, in reviewing the daily results from our multichannel analyzer, that the reference interval for alkaline phosphatase activity in plasma might be higher for elderly women than for other groups. Bone is the source of much of the alkaline phosphatase in plasma, and the well-documented loss of bone mass with age is more rapid in post-menopausal women (1). Given that the changes in the activity of alkaline phosphatase in plasma of elderly women could be related to the increased rate of loss of bone mass, we considered also the possibility of parallel changes in the concentration in plasma of calcium and phosphate, the major bone constituents, age-related effects having been documented for inorganic phosphate and alkaline phosphatase activity in plasma in both normal children (2) and adults (3-8). Diseases such as arthritis are more common in the elderly and are sometimes considered a normal part of ‘Department of Pathology, Mater Miaericordiae Public Hospitals, Annerley Road, Brisbane, Australia 4101. 2Department of Pathology, Royal Brisbane Hospital, Herstan Road, Brisbane, Australia 4029 (address for correspondence). Received September 10, 1984; accepted September 17, 1985. 76

CLINICAL CHEMISTRY,

Vol. 32, No. 1, 1986

as

the aging process; such diseases often do not materially affect the commonly measured constituents of plasma. We wished, therefore, to include patients with such conditions in our study population-both to include “normal” elderly subjects, rather than including only “abnormally” diseasefree patients, and to avoid the computational expenses involved in excluding typical, but “unhealthy” subjects.

Subjects

and Methods

Analytical

Methods

Blood specimens were collected into tubes containing lithium heparin as anticoagulant and centrifuged. The separated plasma was then analyzed with a Technicon SMA 12/60 continuous-flow analyzer and reagents supplied by Technicon. The analytes assayed, and their respective reference intervals, previously established in our hospital, included sodium (135-145 mmol/L), potassium (3.5-4.5 mmolJL), chloride (92-109 mmol/L), bicarbonate (23-32 mmol/L), urea (3.5-7.5 mmolIL), urate (0.18-0.48 mmollL), creatinine (0.04-0.10 mmol/L), calcium (2.10-2.55 mmol/L), phosphate (0.8-1.5 mmolIL), aspartate aminotransferase (EC 2.6.1.1) (7-40 UIL), lactate dehydrogenase (EC 1.1.1.27) (125-220 UIL), and alkaline phosphat.ase (men 30-110 U/L; women 20-50 years old 20-110 U/L and women older than 50, 30-130 UIL). Enzyme activities were determined at 37#{176}C.

Subjects We selected those patients whose results for the analytes other than the one of interest were within these reference intervals. A computer program was written to search the archived ifies independently for calcium, phosphate, and alkaline phosphatase results for both inpatients (hospitalized at the time the specimens were collected) and outpatients. The computer collated calcium results from 8808 men and 11503 women over a four-year period, phosphate results from 8430 men and 10739 women over four years, and alkaline phosphatase results from 5599 men and 7299 women over 2.5 years. Table 1 shows the numbers of inpatients and outpatients, and the numbers of men and women in each age group for calcium, phosphate, and alkaline phosphatase.

Statistical Methods For each constituent a matrix was devised relating assay values and age groups, and the number of subjects fblfilling the criteria of age and assay value for each interval was entered. Separate matrices were constructed for each sex, and for inpatients and outpatients by sex. The assay values for calcium were 1.40-2.90 mmol/L (in 0.05 mmol/L increments), for phosphate 0.0-3.0 mmol/L (in 0.1 mmol/L increments), and for alkaline phosphatase 0-300 U/L (in 10 U/L increments). Graphical representations (Figure 1) indicate plausible gaussian distributions of these results (after a logarithmic transformation of the alkaline phosphatase activities). We calculated the intervals containing 95% of

Table

1. Num ber of Subjects

In Each Age Group Ag., years

Sex

Inpatlents

Outpatients

20-29

30-39

40-49

50-59

60-69

70-79

80+

267 123

Calcium 9

I

c

7194 5950

4046

2632

2034

1378

1759

1663 1339

803

2048

1997 1525

2107

2860

6862 5687

3876 2745

2284 1935

1975 1335

1931 1475

1992

1568

1279

750 601

239

1688

4564

2762 1968

1505 1302

1310

1359

1345

1003

1145

1082 749

521 421

177

894

636

Phosphate 9 d ‘A/k. phosphatase 9

3631

260

215

225 CALCIUM

235

245

255

215

O5

07

FIg.1. DistributIonsof concentrations

01

PHOSPHATE

(mmol/L)

of calcium (a), phosphate

Results The frequency distributions of alkaline phosphatase activities of inpatients and outpatients did not differ significantly; the comparable results for calcium and phosphate did (p 110 U/L

50-60

20-29 30-39

50-60 60-70

40-49 50-59 60-69 70-79 80+

% >110 U/I..

Modal value

2.6 3.0

40-50

4.6

15.6

13.1

1.1 1.2 1.9

40-50

50-60

5.3

50-60 50-60

8.1 9.0

40-50 60-70 60-70 60-70

70-80

9.4

70-80

7.1

8.5

Table 4 lists the normal reference intervals for calcium, phosphate, and alkaline phosphatase for each sex and age group. Again, parametric and non-parametric techniques both give similar results for calcium and phosphate but more divergent estimates of the upper limit for alkaline phosphatase activity, reflecting the positive skew in its distribution.

DIscussIon Interpretation of the result of a laboratory test depends on, among other things, knowledge of the distribution of results in a population. Reference subjects from this population should be volunteers known to be free of disease and should be available in sufficient numbers for statistical analysis (11). For elderly people, this approach is difficult and expensive if sufficient numbers of subjects are to be studied. One approach to overcoming such difficulties has

Table 4. Parametric

(and Nonparametrlc)

DECADES

of calcium

Values for Alkaline Phosphatase (U/L at 37#{176}C) Men

Age, years

concentrations

IN

Sex mmol/L

Phosphate,

mmol/L

(a), phosphate

Alk. phosphatase, U/L

Reference Intervals Phosphatase

20-29

30-39

2.14-2.56

2.12-2.55

d

(2.16-2.63) 2.15-2.61

(2.14-2.54) 2.15-2.57

2.12-2.55 (2.14-2.54) 2.13-2.56

(2.12-2.64) 0.67-1.35 (0.67-1.45) 0.62-1.36

(2.16-2.60) 0.66-1.30 (0.67-1.49) 0.61-1.31

(2.15-2.61) 0.64-1.31 (0.65-1.39) 0.60-1.30

(0.65-1.44) 25-98 (28-107) 33-110

(0.62-1.41)

(0.61-1.43)

9

(34-120)

78

CLINICAL

CHEMISTRY,

Vol. 32, No. 1, 1986

(b), and alkaline

phosphatase

SO’s IN

SO’s

70’.

SO’s

DECADES

activity

(C) with age

been proposed by Hoffman (12). However, this approach has been criticized (13). We used a computer to select subjects from our patient population, excluding those for whom any constituent other than the one of interest, measured on a multi-channel analyzer, fell outside the laboratory reference intervals. This has allowed us to accumulate a large number of subjects, apparently free of disorders affecting results, at low cost. Even in the elderly, many chronic diseases do not typically alter results of commonplace laboratory tests. We did not wish to exclude elderly patients with diseases such as osteoarthritis, benign chronic urinary disease, or ischemic heart disease, because such diseases commonly accompany old age, and may be considered part of the aging process itseli From our results the frequency distributions of calcium and phosphate for inpatients were found to differ statistically from those for outpatients (p 25% of the reference interval before a separate interval becomes justifiable. This is an arbitrary criterion but a reasonable one when com-

9

9

40’s AGE

for CalcIum,

Me.

Calcium,

30’.

40-49

Phosphate,

years

50-59

60-89

70.-19

(2.15-2.63) 2,12-2.56

(2.14-2.62)

(2.10-2.61)

0.72-1.34 (0.75-1.43)

0.71-1.33 (0.72-1.41)

2.06-2.53 (2.06-2.58) 0.70-1.30 (0.71-1.40)

0.60-1.28 (0.63-1.39)

0.59-1.22 (0.60-1.28) 37-154

0.58-1,16 (0.60-1.21) 36-152

(42-170) 35-135 (34-150)

(41-170) 37-144 (30-141)

25-94

27-104

33-131

(25-105) 32-111 (32-121)

(27-115) 33-116

(33-150)

(38-151)

34-123 (36-148)

34-130 (37-137)

2.07-2.58

80+

2.12-2.59 (2.14-2.65) 2.10-2.56

2.14-2.60

0.61-1.25 (0.62-1.35) 37-136

(34-138)

and AlkalIne

(2.09-2.65)

2.02-2.57 (2.05-2.64) 2.07-2.53

(2.10-2.60) 0.66-1.29 (0.68-1.39)

pared with inter- and intra-laboratory precision and accuracy on external quality-control surveys and when compared with the range of values seen in disease. The trends that we have identified on using this approach are similar to those found on using the more laborious IFCC-recomniended (11) approach. With increasing age of subjects, we have found a decrease in mean calcium and phosphate and an increased skew to higher values in the alkaline phosphatase distributions with little change in the modal value. In contrast, the changes in women at the menopause consist of an increase in values for calcium and phosphate, with a shift in the modal values of alkaline phosphatase towards the male distribution rather than an increase in skew. These differences suggest that the osteoporosis seen in the two circumstances is of different pathogenesis.

The age- and sex-related differences in calcium and phosphate (Table 4) are small when compared with interlaboratory variation and with changes typically seen in disease. For example, the upper limit for plasma phosphate declines with age in males, from 1.35 mmolfL to 1.16 mmolIL, which is clinically insignificant. Thus it seems valid to continue to use the one adult range for calcium and phosphate. The frequency distributions of plasma alkaline phosphatase activity in men and post-menopausal women are similar (Figure ic), whereas the frequency distributions of premenopausal women are different. In women, the mean activity of plasma alkaline phosphatase increases from 48.8 U/L to 70.0 U/L between the 4th and 6th decades, which is 29.5 and 26.3% of the premenopausal parametric and nonparametric reference ranges, respectively. Use of a separate reference interval for pre-menopausal women should therefore be considered. The positive skew of the alkaline phosphatase distributions gradually increases with age, so the parametric and percentile techniques give quite different estimates of the upper limit of normal. The use of separate reference intervals for the elderly is desirable when it avoids an excess of marginally abnormal results of dubious clinical significance. However, as the increasing skew may reflect diseases such as osteoarthritis rather than the effects of aging, we do not believe that either statistical approach is completely satisfactory for defining the upper limit of normal. Optimally, we believe that the effect of diseases such as osteoarthritis on plasma alkaline phosphatase activity should be

known and taken into account when the result is interpreted. In the present state of our knowledge, we should at least attempt to educate clinicians about the procedures we use to determine individual reference intervals. In summary: although this technique does not derive reference intervals in the way proposed by the IFCC, we believe it is an efficient, cost effective, and rapid way to collect appropriate information on large numbers of patients. It offers also the possibility of investigating other and more complex phenomena such as the effects of drugs and disease on results of common laboratory tests. Ref erences 1. Nordin BEC. Clinical significance sis. Br Med J 1971;i:571-6.

and pathogenesis

of osteoporo-

2. Round JM. Plasma calcium, magnesium, phosphorus, line phoephatase levels in normal British school children.

and alkaBr Med J

1973iii:137-40. 3. Roberts seventeen

LB. blood

The normal constituents.

for

ranges, with statistical analysis Clin Chim Acta 1967;16:69-78.

4. Eastman JR. Bixler D. Serum values for sex and age. Cliii Chem

nlkgline phosphatase: normal 1977;23:1769-70. 5. Sharland DE. Serum alkaline phosphatase: the levels and patterns of isoenzymes in the non-hospitalised elderly. Age Aging 1972;1:168-76.

6. Sharland DE. Alkaline phoephatase: the isoenzyme pattern in the elderly and changes in total serum levels with age. Clin Chim Acta 1974;56:187-98. 7. Hodkinson HM, McPherson CK. Alkaline phosphatase in a geriatric inpatient population. Age Aging 1973;2:28.-33. 8. Hobson W, Jordan A. A study of serum silks.Iine phosphatase levels in old people living at home. J Gerontol 1959;13:292-3. 9. Henry RJ, Cannon DC, Winkleman JW, eds. Clinical chemistzy principles and technics, 2nd ed. New York: Harper and Row, 1974:344-65. 10. Gardner MD, Scott R. Frequency distribution and “reference values” of plasma slkssline phosphatase (EC 3.1.3.1) activity in the adult

population

of

a

Scottish

new

town.

J

Clin

Pathol

1978;31:1202-6. 11. IFCC Expert Panel on the Theory of Reference Values. The concept of reference values. Clin Chem 1979;25:1506-8. 12. Hoffman RG. Statistics in the practice of medicine. J Am Med Assoc 1963;185:864-73. 13. Reed AH, Henry Ed, Mason WB. Influence of statistical methods used on the resulting estimate of normal range. Clin Chem 1971;17:275-84.

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