JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Supplement 2, 1991 Mary Ann Liebert, Inc., Publishers

Calcium Determination in Primary Hyperparathyroidism JACK H . LADENSON

ABSTRACT Calcium is the most closely controlled substance in the blood. The biologic variation of total calcium is ~ 2 and % of the biologically active free (ionized, ionic) calcium only l070.('-~) Thus, the monitoring of calcium in blood requires analytic procedures of high precision and accuracy. For patients with asymptomatic primary hyperparathyroidism, calcium monitoring involves the measurement of total calcium and free calcium. This review first considers the measurement of total calcium and then free calcium.

MEASUREMENT OF TOTAL CALCIUM

Analytic approaches NUMBER O F ANALYTIC PROCEDURES have been utilized to measure total calcium over the last 50 years. Because of the small biologic variation, the need for methods of high precision and accuracy has long been apparent, and this analyte was one of the first to be addressed by professional organizations in laboratory medicine and clinical chemistry with interest in improving the performance of clinical laboratories. These efforts are now coordinated by the National Reference System for the Clinical Laboratory (NRSCL), formerly called the National Reference System in Clinical Chemistry. The NRSCL is a component of the National Committee for Clinical Laboratory Standards (NCCLS), which is a nonprofit organization that involves government, professional organizations, and industry and is the leading standards-setting organization for clinical laboratories in the United States. The components involved in the development and application of a standardization system are shown in Fig. 1. The key to the system are steps I1 through V. For calcium, a definitive method based on isotope dilution mass spectroscopy was developed by workers at the National Institute of Standards and Technology (NIST, formerly the National Bureau of Standards).(41A primary reference material of calcium carbonate is available (standard reference material, SRM 915, available from NIST) and a reference method based on atomic absorption spectroscopy has been developed and validated.") In addition, a secondary reference material consisting of lyophilized sera with an assigned calcium value is available (SRM 909, NIST).

A

These activities, along with the evolution of more automated procedures, have had a dramatic effect on the agreement among clinical laboratories for the measurement of total calcium. This improvement is readily apparent when comparing the interlaboratory precision 01' total calcium measurements with the survey samples of the College of American Pathologists quality control survey program (Table 1). These data show that the interlaboratory precision of total calcium measurements decreased from =6Vo in the early 1970s to 3.3% in 1990. The precision in an individual laboratory can be even better ihan 3.3%. In the clinical chemistry laboratory at Barnes Hospital, the precision of total calcium measurements over the last year has been I .8-2.1 OJo for one type of analyzer and 2.3-3.1 Yo for another analyzer. It is my opinion that the current state of the art of total calcium measurement is not likely to further improve over the next few years. The precision is still too high for ready detection of the biologic variation of = 2070, but surveys of clinicians have indicated a variation of 4.8-7.0% to be acceptable depending on the initial calcium value.('' Furthermore, monitoring of total calcium in 698 individuals for 7.5-9 years showed that a change of = 10% was required to be significant at 95% confidence limits.(*' The discrepancy between 2% biologic variation and 10% observed variation is likely due to nonanalytic sources of error, discussed later. The analysis of total calcium has been performed by a variety of analytic techniques over the years. Today, dyebinding techniques are utilized by 98% of laboratories. The dye-cresolphthalein complexone is used by 73.7% of laboratories and Arsenazo 111 by 24.3%. Some of the

Department? of Pathology and Medicine, Washington Univeryity School of Medicine,

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CALCIUM DETERMINATION IN HYPERPARATHYROIDISM the sample. These interferences should rarely present a problem when monitoring patients with asymptomatic primary hyperparathyroidism. With some of the current instruments, 1 have heard anecdotal reports of low calcium values. Use of the national reference system for calcium should allow mathematical correction factors to be defined and incorporated into the instrument output if a laboratory suspects its calcium values are systematically low.

Nonanalytic sources of error

s35

tration of protein and protein-associated substances. The effect is more pronounced if forearm exercise, for examplc clenching the fist, is also used to facilitate placing the needle in the vein and can easily lead to increases of 1007o in total calcium values.(16' A number of other factors have been studied that influence calcium results. Blood should not be obtained from an arm being infused by an intravenous line. Exercise produces few or only short-term effects. There is little influence of food consumption. Seasonal changes in total calcium have not been found in North America,'" '"I and changes with the menstrual cycle are not observed.'"' The use of thiazide diuretics has been linked to hypercalcemia in population studies,('*) but thiazide administration produces inconsistent responses in total calcium.("' The histamine H,-receptor blocker cimetidine has been implicated in altering parathyroid hormone concentration, but it does not appear to affect blood calcium.12z1In =8O% of patients treated with lithium carbonate an increase in calcium (and parathyroid hormone) is noted within 1 and clear hypercalcemia is found in 12Vn after several months of treatment. It is now clear that a small diurnal variation in free calcium concentration exists but that the changes in total calcium are predominantly related to changes in protein concentration through the day.(z4'The nadir for total calcium is found in the very early morning, and the difference between the highest and lowest values is of the order of 0.1 mmol/liter. Results change little for patients with primary hyperparathyroidism sampled between 0800 and 1 6 0 0 . ~ 2 ' ~

The observed variation in total calcium values in an individual patient has been reported to be about 10% (Ref. 8; see earlier), even though the biologic variation is < 2070 and the intralaboratory precision can approach 2%. The most likely cause of this discrepancy between observed and actual biologic variation is nonanalytic sources of variation that can occur when monitoring total calcium. Such sources of variation have been previously reviewed(14)and are likely to be very important when monitoring patients with asymptomatic primary hyperparathyroidism over time. Heparinized plasma and serum give similar results for total calcium, but anticoagulants that form calcium complexes, such as EDTA or citrate, cannot be used with dyebinding procedures. Calcium in separated sera is quite stable for at least 10 days even at room temperature. Sera are also stable up to 2 days in contact with the clot at room temperature, so no special precautions are necessary for the transport of samples to the laboratory. Calcium contamination of the sample from the collection tube or laboReference range ratory glassware has been reported on occasion but is not generally a problem. Cork stoppers should never be utiThe reported reference intervals for total calcium in lized because they have sufficient calcium to totally invali- adults have varied widely, as reviewed previously,") with date calcium analysis. A negative error due to the apparent upper limits of 2.52-2.85 mmol/liter (10.1-1 1.4 mg/dl) beabsorption of calcium into polystyrene sampling cups has ing reported. In a survey of laboratories in Canada bebeen described, and samples should not be stored in such tween 1974 and 1980, the lower limits of the reference cups. range fell between 1.98 and 2.42 mmol/liter and upper A major source of variability in total calcium measure- limits between 2.50 and 2.90 mmol/liter even though overments relates to the posture of the patient before obtaining all interlaboratory precision was only 4.9%. Furthermore the blood sample. Variation in posture, for example from there was no significant difference in total calcium for the upright to sitting or supine, causes redistribution of fluid survey samples between laboratories using a reference between the intravascular and interstitial compartments. In range of 1.21-2.50 or 2.25-2.75 mmol/liter.~z6~ Thus it is the standing position, fluid leaves the intravascular com- likely that the lack of an accepted reference range leads to partment, presumably as a result of an increase in hydro- considerable variation in the definition of hypercalcemia static pressure. Cells, larger proteins, and protein-associ- from laboratory to laboratory. ated substances, such as calcium, do not redistribute and At the time of my last review of reported ranges for total so their concentrations are highest when standing and low- calcium,''' it was concluded that the reference values for est when supine. We have found the average changes in females varied little with age, but for males total calcium total protein are 11.5% and total calcium are 4.6%, 30 values clearly decreased with age concomitantly with albuminutes after going from the upright to supine position, or min values. In the 20-50 year age group total calcium valvice versa.('" Other data indicate that redistribution takes ues are generally 0.05 mmol/liter higher in males than feabout 20 minutes to completion. Thus, for outpatients, the males. The additional studies reported since 1980 have not time the patient is in the sitting position before drawing clarified the situation with regard to the influence of age blood may significantly affect the total calcium values. on the reference range. When obtaining a blood sample, a tourniquet is generIn our laboratory we utilize a reference range of 2.22ally utilized. This creates a pressure gradient and causes 2.58 mmol/liter for both males and females 20-60 years. fluid to leave the intravascular space below the tourniquet. This range fits the distribution of total calcium values from The blood subsequently obtained has an increased concen- the = 1500 relatively healthy outpatient samples we analyze

S36 each month and is also consistent with the median calcium values of 2.32 mmol/liter for inpatients and 2.38 mmol/ liter for outpatients we observed over the last 4 months. However, we again emphasize the need for either further studies or a consensus agreement on the reference range or action limits for total calcium to be utilized when monitoring patients with asymptomatic primary hyperparathyroidism.

MEASUREMENT OF FREE CALCIUM Analytic approaches In recent years, several manufacturers have introduced second-generation semiautomated analyzers based on ionselective electrodes (ISE) that rapidly and reliably measure free calcium and pH. Previously utilized procedures based on bioassay or metal indicator dyes are now of only historical interest.") In 1%7, ROSS'") described a liquid ion exchanger suitable for use in a calcium-sensitive electrode. The problems with the first commercial adaptation of this method, the Orion model 99-20, have been well documented.(lI They include a long start-up and analysis time, a variation in electrode performance from electrode to electrode and during the lifetime of a single electrode, and a negative magnesium error. These problems created a poor reputation for the analysis of free calcium, but improvements in technology have overcome most of these problems and very reliable instruments are not available. In our laboratory, the long-term precision of free calcium analysis is now better than total calcium, with a coefficient of variation (CV) of 1.2-2.3% for two instruments from the same manufacturer. Similar precision has been found by others.(2B) Although the intralaboratory precision with a given instrument is very good, there are problems with interlaboratory precision and accuracy. The 1990 CAP quality control survey indicates an interlaboratory precision of 6.6-7.7%. A study that included five different models of free calcium analyzers and 24 laboratories showed good precision for a particular model, 2.4-3.1 Yo, but a considerable difference in the mean free calcium values obtained between the instruments.12P)A mean difference of 0.03 mmol/liter between the two most widely used free calcium analyzers has been reported,1281and a study of the free calcium analyzers available in France in 1988 confirmed the excellent precision of the analyzers and emphasized discrepancies between different types of analyzers.(3o' The discrepancies among instrument measurements are likely a result of the differences in calcium electrodes, composition and type of reference electrodes, and composition of the calibrating s o l u t i o n ~ . ~The ~ ~ )difference in calibrating solutions is mainly due to other inorganic materials depending o n whether the instrument was designed for the analysis of free calcium alone or for a battery of tests including other electrolytes. In essence, free calcium analysis today is in a similar state as was total calcium in the 1970s, with different methods giving different values. As noted in the discussion of total calcium procedures, this

LADENSON was resolved by the evolution of definitive and reference methods and primary and secondary reference materials (Fig. 1). The development of methods and materials for the standardization of free calcium measurements is proceeding as an international involving the National Reference System for the Clinical Laboratory, the European working group on ion-selective electrodes of the International Federation of Clinical Chemistry, and the electrolytes and blood gas division of the American Association for Clinical Chemistry. These efforts should be encouraged and supported so that free calcium analysis can be more effectively utilized in the monitoring of patients with primary hyperparathyroidism.

Differences between free and total calcium Calcium exists in three major forms in plasma.'35) The free (unbound, ionized, ionic) calcium is a little over 50% of the total calcium; calcium associated with protein (mainly albumin) is ==@To,and the remainder is soluble complexes with such anions as bicarbonate, phosphate, and lactate. These distributions have been known for some time, and correction of total calcium for protein-associated calcium has long been advocated since only the free calcium is physiologically important. The advent of direct measurement of free calcium allowed the accuracy of corrected total calcium determinations to be tested. A number of studies have concluded that measured free calcium is a better indicator of calcium status than corrected total calcium, 136-421 although some disagree.'".") In work from our laboratory we found measured free calcium to be more effective than corrected total calcium calculated using any of 13 different published algorithms.'36' The reasons for the lack of efficacy of the corrected calcium compared to the measured free calcium is not clear since knowledge of total protein, albumin, and pH was expected to be sufficient to calculate protein-bound calcium. A possible explanation is modulation of calcium binding to albumin by other substances. Modulation by b i l i r ~ b i n ~ ' ~ ) and fatty acid('6) has been reported, and the effect of fatty acids shown to be due to a conformational change in albumin.(461 A number of studies now define more precisely differences between free and total calcium in a variety of conditions. Artefactual results for total calcium are found in conditions with altered albumin concentration; altered binding of calcium to albumin, such as the excess fatty acids that occur in acute pancreatitis; altered pH; excess soluble calcium chelators, such as citrate from blood products; and calcium binding to some myeloma proteins. These d o not significantly affect data in patients with primary hyperparathyroidism, however. The available data from studies on free and total calcium in hyperparathyroidism are summarized in Table 2. The larger studies, including one from our laborato~y,'~') find that a higher percentage of preoperative samples in confirmed primary hyperparathyroidism show elevated free calcium as opposed to total calcium. This is also observed in patients following renal tran~plantation,'~'.~') who generally have elevations

CALCIUM DETERMINATION IN HYPERPARATHYROIDISM TABLE2. REPORTSOF FREECALCIUM

Reference 47 48 49 50 51 3 52 53 54 55 56 57*

V A L U E S IN

PRIMARY HYPER PARATHYROID ISM^

Year

Free calcium methods

No. of palienha

1958 1962 1973 1973 1973 1973 1976 1977 1979 1983 1984 1973

Chemical Chemical Orion 99-20 Orion 99-20 Orion 99-20 Orion 99-20 Orion 99-20 Chemical Orion 99-20 (modified) Orion SS-20 Orion SS-20 Orion 99-20

17 (17) 14 (13) 15 (14) 1(1) 27 (?)

12 (12) 48 (43) 69 (60) 93 (93) 10 (10) 19 (l9)c 17 (17)

s37

No. of samples

% 1 Cu,

19 17 68 8 86 19 149 ? 142 10 151 109

94.7 52.9 98.5 100

9 1.9" 94.7 99.3 99- 100 89.5 80 88.7 79

To t

('(I,

94.7 94.1 75.0 62.5 91.9 84.2 92.6 86 83.1 60 30.5 35

aNumber confirmed histologically is shown in parentheses. The percentage elevation for free calcium (Ca, ) and total calcium (Ca,) is based on the number of samples and the reference range used in the particular study. bThis study used a calculated pH-corrected Cab. CThis study included only patients who had at least 40% of the total calcium values within reference limits before obtaining samples for simultaneous measurement of Ca, and Cap dpostrenal transplantation.

in parathyroid hormone and relatively normal renal function. The rationale for the higher diagnostic efficacy of free calcium in this setting is not totally clear. There appears to be a small increase in the percentage of free calcium in patients with primary hyperparathyroidism that is not explained by changes in protein, albumin, or pH.IS4) The ratio of Cab- to C a r in patients with primary hyperparathyroidism correlates weakly but significantly with the parathyroid hormone concentration in preoperative samp l e ~ , ( ~ and ~ . " the ~ ratio of CaF/CaT was found to decrease after parathyroidectomy by one group(471but not another. We administered parathyroid hormone to rats to determine whether a similar increase in the percentage of Cak exists. We found a small but significant increase in the percentage of Caf, not accompanied by changes in protein or pH (CaF/Car = 52.5 0.8% for PTH treated, 51.3 + 1.3% for controls; 10 animals in each group). Because of the small degree of change in the percentage of Cap we doubt it will be productive to further investigate its cause at this time. However, since patients with asymptomatic primary hyperparathyroidism are likely to have only mild abnormalities in blood calcium, the measurement of free calcium in addition to total calcium will likely be helpful to monitor such patients.

*

Nonanalytic sources of error Calcium-chelating anticoagulants, such as EDTA and citrate, must obviously be excluded for CaF- measurement in blood. Heparin can be utilized, but the ratio of heparin to blood should be kept constant since heparin, a polyanion, binds some calcium ( ~ 2 0 7 0in a full heparinized vacuum collection tube). This error is greater with incompletely filled tubes and could be a significant problem

when samples are obtained via syringes containing variable amounts of liquid heparin.ls8] Calcium-titrated heparin solutions have been developed to minimize this influence of heparin binding to "') It is recommended that samples be collected in tubes or syringes with consistent amounts of solid rather than liquid heparin if plasma or whole blood is needed. Whole blood and plasma give similar values with currently available (second-generation) free calcium instruments.(60.611 Vacuum collection tubes containing a gel cell separator aid may give slightly higher (0.02 mmol/liter) Ca, values.(s91 Once collected, the Cat. is reasonably stable if precautions to avoid introducing air into the sample are taken. Introduction of air leads to a loss of CO, and therefore an increase in pH and lower Cab. Without air admixture, thc stability of the sample is a function of in vitro metabolism that is minimized by storage at 4°C. The time at which the Capvalues are significantly altered is a function of the balance of the increase in acid (decrease in pH) and the increase in calcium binding species, such as lactate. U n opened, full collection tubes appear stable for up to a day at 4°C and at least a few hours at room temperature.'sR-hzl Once serum is anaerobically removed to a syringe, it is stable for days at 4°C.(s"1 Because of the difficulties in ensuring that a sample has remained anaerobic from the collection of the sample until its analysis, a number of workers have advocated the use of a pH-corrected Ca,. value (Gal.- 7.40). Such a value is calculated by virtually all second-generation analyzers using an in vitro slope for A log Ca,../ApH of -0.22 to -0.24.163' Sachs et al.'641recently reported an in vitro slope of -0.17 and raised questions about the accuracy of the algorithm used in commercial analyzers. The CaF7.4 appears to correct adequately for the loss of CO, during sample handling and storage,(59-hz) at least

LADENSON

S38 TABLE3. REPORTED REFERENCE RANGES FOR FREECALCIUM Reference

Instrument

N

33 28 62

ICAl ICAl ICAl

105 ? 95

61

ICA I

20

69 74

ICA 1 ICA 1

76

ICAl

371 Female 5 12 Female 465 Male 53

77 72 75 70

ICAl NOVA 2 NOVA 2 NOVA 7

49 152 120 402

until increases in lactate cause the relationship of Ca and pH to vary.(s86z1The relationship of Ca, and pH also varies with the concentration of albumin and bicarbonate.(63) There is some question about the accuracy of the algorithm to be used for calculated CaF(pH 7.4), but the greater question is for which patients will it be valid. For instance, if the patient has an actual pH value that deviates significantly from pH 7.40, then Cap 7.40 gives an erroneous impression of in vivo free calcium status,(61 as has been demonstrated for uremic patients with acidosis.(616s) Thode et compared Cab and Ca,7.40 in 183 patients with chronic calcium metabolic disorders, including 37 patients with primary hyperparathyroidism, and found the two values equivalent for clinical use. At this time there are not sufficient data to justify using Ca, 7.4 for the monitoring of patients with asymptomatic primary hyperparathyroidism who may be acidotic. The potential benefits of such a measure involve correction for aerobic sample handing; the potential problem would be inappropriate correction of Caf. when there is acidosis. Only a much larger study could answer the question of whether the benefit is greater than the problem. The influence of patient posture on free calcium values is much smaller than for total calcium.('5)However, there may be significant increases as a result of venous stasis when a tourniquet is employed in drawing blood.('61The major cause of the increase in Ca, is the decrease in pH due to the lactic acid buildup when the forearm is exercised with a tourniquet blocking venous return. Prolonged bed rest (immobilization) has been reported to lead to increases in CaF within 4 The administration of radiographic contrast media may lower Ca, as a result of calcium binding.(68) Seasonal variation in Ca, is not observed(I8) nor are changes with the menstrual cycle.('91 There is a diurnal variation in CaF, with the nadir at = 1630 and a range from lowest to highest value of = 0.05 mmol/liter. ( I 4 )

Range

Sample

1.09- 1 .39 1.17-1.33 1.20-1.36 1.20- 1.34 1.15-1.35 1.19- 1.39 1.17- 1.37 1.12-1.28 1 .14- 1.26 1.16- 1.30 1.18- 1.32 1.18- 1.32 1.19- 1.30 1.14- 1.38 1.11-1.27 1.15- 1.3 1 I . 19- 1.35

Frozen sera Serum, pH corrected Serum pH corrected Capillary blood Venous blood Either, pH corrected Serum, pH corrected Frozen serum, pH corrected Frozen serum, pH corrected Serum, pH corrected Venous blood Capillary blood Venous plasma Venous blood Venous blood Serum

Reference range The influence of age, if any, on CaFappears to be small, with some studies reporting no i n f l u e n ~ e , ( ~one ~ . ~a ~de.~~) crease with and two report slight increases with age.(72.73) Likewise, any influence of sex is quite small, with most studies finding no d i f f e r e n ~ e ( ~ or ~ ~men ~ ~ .hav'~) ing slightly higher (0.02-0.03 mmol/liter) values than w~men.'~~.'~) Thus, it does not appear necessary to use reference ranges distinct for age and sex. Some of the reported reference ranges with more modern instruments are noted in Table 3 . There appears to be more variation of reported reference ranges than would be accounted for by the random variation of a given type of instrument. Thus, better definition of the reference range for free calcium needs to be established.

CONCLUSION The homeostatic control of calcium in blood is the tightest of any substance known, with a biologic variation of less than 2%. Because this variation is small, methods of high accuracy and precision are required, particularly for patients needing monitoring over a long time, as in asymptomatic primary hyperparathyroidism. The precision and accuracy of total calcium procedures are now quite adequate for monitoring patients with primary hyperparathyroidism. The variation in the reported reference ranges is beyond that expected for the variation of the analysis, and a consensus on the reference range and action limits for total calcium should be established. The biologically active form of calcium, free calcium (ionized or ionic), can also be reliably measured. Clinically, measurement of free calcium has been shown to be more sensitive for the detection of hypercalcemia in patients with primary hyperparathy-

CALCIUM DETERMINATION IN HYPERPARATHYROIDISM roidism. Variations between instruments lead to unacceptably large interlaboratory variation for free calcium, although these measurements are highly precise in a given laboratory. Current international efforts to standardize the measurement of free calcium should be actively supported with a view toward establishing a consensus reference range.

19.

20.

21.

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