CLIN. CHEM. 28/6, 1362-1363 (1982)
Effectsof Sample Freezingon Ion-SelectiveElectrodeDeterminationsof
Serum Calcium
Sandford B. Plant and David A. McCarron
We assessed in a prospective studythe effects of freezing,
thawing, and prolonged storage of serum samples for
ionized calcium determinations
with the “Clin :1: Ion.”
Forty-eight serum samples were collected before and
during infusions of EDTA and calcium into human subjects.
Values for ionized calcium in serum samples stored frozen
for one and six months were similar to those determined
for fresh specimens.
Samples stored for 12 months had
significantly
(p <0.001)
lower values. Thawing and refreezing the sample within the first six months did not alter
values. We conclude
that serum samples can be frozen
and stored for as long as six months without affecting re-
sults for ionized calcium determinations.
AddItIonal Keyphrases:
variation, source of
dling
ionized calcium
sample han-
Recent technical developments
in analytical chemistry have
resulted in the wider application
of direct measurements
of
ionized calcium in the serum of humans and laboratory animals (1-4). The reliability
of estimates of serum ionized calcium from total calcium measurements
recently has been
questioned (4-8). Apparently,
direct determination
of serum
ionized calcium concentrations is important in the precise
assessment of calcium homeostasis for clinical or research
purposes. Anticipating wider application of this analytical
technique, we sought to determine in a prospective study the
effects of freezing, thawing, and prolonged storage on directly
measured values for ionized calcium in serum collected from
the same subjects.
Materialsand Methods
Blood was sampled from hyperparathyroid
patients participating in a research protocol at The Oregon Health Sciences University. Forty-eight specimens were collected before
and during EDTA and calcium infusions. The blood was
centrifuged, the serum separated, and each serum specimen
was divided into two parts. Ionized calcium was determined
immediately for one group of specimens (Part 1), which were
then frozen; this determination was the “fresh” determination.
Part 2 specimens were frozen without any delay, at -20 #{176}C.
All samples were stored in plastic.
We determined
the stability
of the electrodes each day,
using lyophilized
calibration sera as the standard. These calibration standards were prepared freshly each day from ly-
ophilized human sera standards from the same lot, that were
individually sealed and stored in 5-mL vials at -20 #{176}C.
The
stability of the standards was previously established by concurrent measurement by atomic absorption spectroscopy.
Calibration curves of the electrodes were unchanged during
the course of the study.
At the end of one month, Part 1 sera were thawed and ionized calcium was again measured; these sera were then refrozen for an additional five months. At that time, sera from both
Parts 1 and 2 were thawed and ionized calcium was measured.
Values for ionized calcium were measured again on Part 2 sera
after an additional
six months
(a total of one year’s
storage).
To measure the ionized calcium concentrations in serum
samples, we used the ion-selective electrode method (“Clin
± Ion,” Model CI-3; Applied Medical Technology, Menlo
Park, CA 94043) (1,2). With two calcium-selective electrodes
we obtained duplicate measurements simultaneously. We used
paired and unpaired t-tests, analysis of variance, and regression analysis in the data analysis.
Results
Table 1 summarizes the values for ionized calcium in serum
stored frozen for various intervals. There was no statistically
significant
difference
between
values obtained
for fresh sera
and sera stored frozen for one month. The concentration
of
ionized calcium was slightly but insignificantly
lower in the
Part 1 samples assayed at six months. Values for the Part 2
samples (Figure 1) frozen for six months were significantly
(p
<0.005)lower than for the fresh determinations
on Part 1
samples. Linear regression analysis, however, demonstrated
a close correlation
(r = 0.87, p <0.001) between
values for
fresh determinations
and those done after six months on
previously unthawed samples. Part 2 samples, refrozen and
analyzed at 12 months (six additional
months of freezing),
showed a significantly
lower concentration
of ionized calcium,
and values correlated poorly with either the original values
for fresh samples or those determined
at six months. The
initial value for serum calcium was not consistently
related
to the variations observed for long-stored samples, although
calibration
curves at six months were similar for both fresh
and frozen standard solutions.
Discussion
Evidently,
serum samples for ionized
calcium determinations can be stored frozen as long as six months without significant changes in the values obtained.
There is a predictable
but modest decrease in values for ionized calcium in serum
over six months. Thawing and refreezing the samples at one
month did not influence the results obtained at six months,
but thawing and refreezing at six months appears to invalidate
results obtained at the end of one year.
Previous reports have documented that short-term freezing
does not alter free calcium determinations
(1,2, 4, 9, 10). Our
results, however, represent the first prospective evaluation
of the stability of specimens stored frozen for as long as 12
Table 1. Ionized Calcium Concentration after
Frozen Storage
Months frozen
0 (“fresh”)
1)
6 (Part 2)
6(Part
of Nephrology/Hypertension,
The Oregon Health Sciences
3181 S.W. Sam Jackson
Park Rd., Portland,
OR 97201.
Received Oct. 19, 1981; accepted Mar. 23, 1982.
Division
University,
1362 CLINICALCHEMISTRY,Vol. 28, No. 6,
1982
12
Ionized
calcium,
mean ± SD, mmoi/L
2.48
2.44
2.39
2.38
±
±
±
±
0.28
0.34
0.36
0.41
2.14±0.28
To validate our observations
for other currently
available
calcium electrodes
will require comparable investigation
with
those instruments.
1.6
1.5
This work was supported, in part, by a grant from the Oregon Heart
Association, and grant RR 00334 from the General Clinical Research
Branch of the Division of Research Resources. Jennifer Paquet prepared the manuscript.
1.4
-J
I
I.3
References
1.2
I
I.0
0.9
0.8
1’
#1
Fresh
Specimen
#2
6 months
Specimen
Fig. 1. Values for Ca2+ in fresh and frozen (six months) sera
months. Furthermore,
previous studies have not systematically assessed serum samples over as wide a range of ionized
calcium in the same population of subjects.
Our findings are specifically applicable to the Clin ± Ion
instrument, which restores the pH of the sample to pH 7.4. For
this method, serum samples for determination of ionized
calcium can be safely frozen and stored for at least six months.
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CLINICAL CHEMISTRY, Vol. 28, No. 6, 1982
1363