CLIN.CHEM.
30/4,553-556 (1984)
Effects of Storage Temperature and Time before Centrifugation on Ionized
Calcium in Blood Collected in Plain Vacutainer Tubes and Silicone-Separator
(SST) Tubes
John Toffaietti,”2 Nancy Biosser,2and Kathryn Klrvan3
We studied the stability of ionized calcium and pH in samples
stored at either room temperature or 4 #{176}C,
in centrifuged and
uncentrifuged blood-collection tubes and in centrifuged tubes
containing a silicone-separator gel (SST tubes).At room
temperature,inuncentrifuged blood from healthy individuals,
mean ionized calcium usually increased no more than 10
mol/L per hour; at 4 #{176}C
it did not change detectably for 70 h.
This stability was fortuitous, however: the concentrations of
both hydrogen and lactate ions in these samples increased,
apparently with offsetting effects on the concentration of
ionized calcium. Blood stored for 70 h at 4 #{176}C
in centrifuged
SST tubes, although showing a slightly greater change in
ionized calcium, had less change of pH and no change in the
ionized calcium corrected to pH 7.4. In 11 heparinized whole-
blood samples from eight patients in intensive care, the mean
change per hour in ionized calcium and pH after storage at
room temperature was + 10 mol/L and -0.04 units, respec-
tively.
AdditionalKeyphrases:pH lactate variation, source of
sample handling . ion-selective electrodes ionized calci-
um vs total calcium
One of the subcommittees of the AACC Ionized Calcium
Working Group was formed to gather information and
formulate guidelines for collection of samples for measuring
concentrations of ionized calcium; a preliminary report has
recently appeared (1). Here, we report our study of the
effects of temperature of storage, delay in cell separation,
and type of blood-collection tube on results for ionized
calcium, which may be useful in developing such guidelines.
We have observed little change in dialyzable calcium in
serum from blood left to clot for as long as 6 h at room
temperature (2), which is longer than usual for processing
samples for determinations of ionized calcium (3-5). Fogh-
Andersen et al. (6) have also reported that clotted blood may
be stored at 0-4 #{176}C for as long as 4 h without affecting
results for ionized calcium. The most rapid analyses require
the use of heparinized whole blood or plasma, but calcium
binding by heparin and problems with protein adherence to
electrodes are drawbacks to procedures involving heparin
(7).
By measuring the changes in ionized calcium, pH, and
related constituents, we wanted to determine whether less-
rapid collection and handling procedures would still give
satisfactory results for ionized calcium in serum. We mea-
sured ionized calcium, pH, and in some cases dialyzable
calcium, lactate, and phosphate in blood samples stored
without separation from the clot for as long as 24 h at room
temperature, and in samples stored at 4#{176}C
for as long as 70
‘Department of Pathology, 2 Clinical Chemistry Laboratory, and
S Blood Gas Laboratory, Duke University Medical Center, Durham,
NC 27710.
Received July 25, 1983; accepted January 18, 1984.
h with or without separation from the clot in both plain and
silicone-separator tubes. We then evaluated the potential
value of, and precautions for, using routinely collected
samples for measurement of ionized calcium.
Materials and Methods
Ionized calcium, pH, and ionized calcium corrected to pH
7.4 were measured simultaneously with a Radiometer ICA 1
ionized calcium analyzer (Radiometer America Inc., West-
lake, OH 44145). Dialyzable calcium was measured by
continuous-flow analysis (1), and lactate (8) and phosphate
(9) by centrifugal analysis. Measurements of ionized calci-
um, pH, and dialyzable calcium were done in duplicate.
Blood samples from apparently healthy volunteers, ages
23-57 years, were collected in large syringes, then injected
into evacuated blood-collection tubes containing either no
additive (plain Vacutainer Tubes) or a silicone gel for
maintaining separation of serum from erythrocytes after
centrifugation (SST), both from Becton Dickinson, Ruther-
ford, NJ 07070. For anticoagulants we used either sodium
heparin (Becton Dickinson) or a calcium-titrated heparin
solution (no. S 4500, Radiometer). Samples were stored at
room temperature or 4 #{176}C,
(22-25 #{176}C) as noted.
Eleven whole-blood samples, collected in heparinized sy-
ringes (no. MQ6O1 or MQ6O5LD; Marquest Medical Prod-
ucts, Inc., Englewood, CO 80112), containing 30 or 40 mt.
units of lithium heparin per milliliter, were selected without
conscious bias from eight patients who were either in
intensive care or undergoing surgery. These samples were
kept on ice before the initial analysis of the whole blood,
then stored at room temperature for 2 h before re-analysis.
Results
Table 1 shows the mean, SD, and range of changes in
ionized calcium, corrected ionized calcium, and pH in plas-
ma and serum from blood samples that were left uncentri-
fuged for as long as 3 h. In both plasma and serum the mean
change and most of the individual changes of ionized
calcium were within the imprecision of the method, about 20
molfL. One sample consistently had a greater change,
increasing by 40 to 55 mol/L in 1 to 3 h.
With longer storage before centrifugation, as shown in
Table 2, ionized calcium in blood from four of the five donors
changed by no more than 20 molJL after 4 h, although
blood from the other donor changed by 80 mo1!L at 4 h. By
6 h, the ionized calcium in all the samples increased by 50
mo1JL or more. The corrected ionized calcium, however, did
not change detectably in plasma or serum.
In general, the pH of the serum or plasma samples
changed by about 0.01 unit per hour of storage at room
temperature; lactate, in the study shown in Table 2, in-
creased by two- to threefold during 6 h.
Although not shown in Table 1, 28 mt. units of sodium
heparin per milliliter decreased both ionized and dialyzable
calcium by about 100 &moI/L from their concentrations in
serum. This indicates that the effect of heparin was not an
CLINICAL CHEMISTRY, Vol. 30, No. 4, 1984 553
 Table 1. Effect of Time before Centrifugation on Changes in Ca2, Ca27.4, and pH in Blood Samples
Stored at Room Temperature
aCa2, iimol,L 4tCa2 umol/L oH
h before
centrlf. Mean SD Range Mean SD Range Mean SD
Plasma containing sodium heparin (28 mt. units/mL)a
0.5 5 23 -15to45 2 22 -20 to40 -0.01 0
1.0 27 26 -5to55 23 18 0 to 40 -0.01 0.005
2.0 18 16 Oto4O 5 15 -15 to 25 -0.02 0.003
3.0 20 25 -lOtoSO -5 27 -40 to 20 -0.04 0.004
SerumL
1.0 33 18 10to45 8 11 -5 to20 -0.02 0.010
2.0 8 33 -35 to 45 -15 30 -50 to 25 -0.03 0.007
3.0 20 25 -45 to 40 -19 30 -60to15 -0.05 0.017
a Changesare relative
toconcentrations
inplasma from blood centrifugedwithin5 mm after collection.
bchangesare relativeto concentrations in serum from blood centrifuged within30 mm aftercollection.
n = 5 donors.

Table 2. Effect of Time before Centrifugation on Changes in Ca2, Ca27.4, pH, and Lactate in Blood
Samples Stored at Room Temperature
Mactate,
aCa2, pmol/L Ca2,4, imoI/L apH mmol/L
h before
centrif. Mean SD Range Mean SD Range Mean SD Mean SD
Plasma containing calcium-titrated heparin (22 mt. units/L)#{176}
2 20 19 5to45 3 5 -5tolO -0.04 0.024 1.5 0.1
4 35 30 20 to 80 3 6 -5 to 10 -0.06 0.049 2.5 0.5
6 65 20 50 to 95 3 8 -10 to10 -0.10 0.041 3.6 0.3
24 157 64 110to230 -130 45 -90 to -180 -0.37 0.150 10.5 2.2
Serumb
2 23 12 15 to40 9 9 0 to20 -0.02 0.011 1.5 0.6
4 19 13 5 to 35 -11 3 -15 to -10 -0.04 0.010 2.2 0.8
6 36 16 25 to 60 -1 17 -15 to 20 -0.06 0.015 2.7 0.7
24 72 11 65to85 -56 14 -4Oto-65 -0.14 0.039 5.4 1.1
Footnotes and n as in Table 1.

artifact of methodology. The use of calcium-titrated sodium Table 3. Changes in Serum Stored for 6 h at 4 #{176}C
heparin, 22 mt. units/mL, eliminated any detectable differ- after Centrifugatlon a
ence in ionized or dialyzable calcium between serum and Mean SD Range
plasma.
Plain tubes
We also measured dialyzable calcium in the samples ACa2, imol/L -2 7 -lOto5
represented in Tables 1 and 2. Up to 4 h, dialyzable calcium pH -0.01 0.003 -0.005 to -0.01
changed by no more than 30 JLmolJL in any sample; the lactate, mmol/L 0.0 0.1 -0.1 to 0.1
change after 6 h was no more than 50 mol/L, consistent iCa274, mol/L -8 10 -2OtoO
with an earlier report (2). SSTtubes
To determine the stability of ionized calcium and pH at Ca2, .mol/L 2 4 Oto7
ApH -0.01 0.003 -0.01 to -0.015
4#{176}C,
we collected blood samples from four volunteers into Alactate,mmol/L 0.0 0.2 -0.2 to 0.1
large syringes, injected the samples into both plain Vacu- Ca274, Lmol/L -3 4 -5toO
tamer Tubes and SST tubes, let them clot for 0.5 h, a Results are from four volunteers. Blood was allowed toclotfor30 mm at
centrifuged, then stored the unopened tubes at 4#{176}Cfor 6 h. room temperaturebeforecentrifugation.
Samples were analyzedimmediately
As Table 3 shows, serum separated from cells in either plain and 6 h aftercentrifugation.
tubes or SST tubes underwent no detectable changes in
ionized calcium, corrected ionized calcium, pH, or lactate for
6 h. However, there was consistently a bias between similar analysis, were divided into three groups: (a) uncentrifuged
samples from plain tubes and SST tubes. blood left in plain tubes for 24 or 70 h, (b) centrifuged blood
Of 12 blood samples simultaneously injected from a large left in plain tubes for 24 or 70 h, and (c) centrifuged blood
syringe into a plain tube and an SST tube, then allowed to left in SST tubes for 24 or 70 h before analysis. As Table 5
clot for 30 mm, ionized calcium was always slightly higher shows, ionized calcium was relatively stable at 4#{176}C under
in samples from SST tubes, as shown in Table 4. The higher all these conditions but most stable in the uncentrifuged
concentrations of lactate in samples from SST tubes appear tubes. The pH was altered most in the uncentrifuged tubes,
to account for these changes. The corrected ionized calcium and lactate, measured in one set of uncentrifuged samples,
was the same in samples from plain tubes or SST tubes. increased by almost threefold (data not shown). Values for
After the 6-h storage study, we wanted to determine how pH and ionized calcium corrected to pH 7.4 varied least for
long samples could be stored at 4#{176}C
before changes in the blood stored in the SST tubes.
ionized calcium or pH became significant. Sets of samples In addition to samples from healthy persons (Tables 1-5),
were collected from each of six healthy volunteers and left to we also studied 11 heparinized whole-blood samples from
clot for 20 mm at 4 #{176}C.
Some were then analyzed for baseline eight patients in intensive care or undergoing surgical
values of ionized calcium and pH; others, kept at 4#{176}Cuntil procedures. As shown in Table 6, the changes in ionized

554 CLINICALCHEMISTRY, Vol. 30, No. 4, 1984

 Table 4. Differences in Ca2, pH, and Lactate between Serum from Plain Tubes and from SST Tubes1
Plain tubes SST tubes DIfference

Mean SD Mean SD Mean SD Range

Ca2, hmol/L 1250 40 1270 40 23 9 10 to 40
Ca274, rnol/L 1270 40 1270 30 6 7 0 to 20
pH 7.44 0.04 7.41 0.03 -0.025 0.012 -0.01 to 0.04
Lactate,mmol/L 1.76 0.43 1.92 0.4.3 0.21 0.16 0 to 0.4
‘Blood allowed to clot for 30 mm at room temperature before centritugationand analysis.
n = 12 each.

on Values for Ca2, pH, and Ca2+74a

Table 5. Effect of Stora ge at 4#{176}C
PlaIn tubes, uncentrifuged Plain tubes, centrifuged SST tubes, centrIfuged
Storage,
h Mean SD Range Mean SD Range Mean SD Range
tCa, pmol’L
24 2 8 -10 to10 16 18 -20 to 30 25 8 20 to 40
70 0 16 -20 to20 21 12 10 to 40 30 12 10 to 40
ispH
24 -0.04 0.02 0 to -0.06 -0.03 0.02 0.01 to -0.06 -0.04 0.01 -0.02 to 0.06
70 -0.08 0.04 -0.01 to -0.13 -0.08 0.03 -0.03 to -0.12 -0.05 0.02 -0.04 to -0.08
iCa74, mo1/L
24 -21 15 Oto-40 2 8 -lOtolO 0 6 -lOtoO
70 -53 18 -20 to-70 -32 18 -10 to -60 -2 4 -10 toO
‘Results are from six different healthy individuals.

Table 6. Effect of Storage at Room Temperature on Ca2 and pH in Uncentrifuged Heparinized Blood
from Patients In intensive Care1
niolIL pH

O.2h 2.2h O.2h 2.2 h

Moan 820 840 24 7.32 7.28 -0.039
SD 340 330 19 0.17 0.16 0.021
Range 0.29-1.16 1.34-1.16 0 to 50 7.00-7.55 6.96-7.50 0 to -0.08
= 11 samples from eight patients.

calcium in any sample after 2 h of storage at room tempera-
ture ranged from 0 to 50 (mean 24) pmol/L, and the changes
in pH ranged from 0 to -0.08 (mean 0.04) pH unit, both
ranges slightly greater than those seen in the healthy
individuals.
Discussion
Among the clinical uses of calcium measurements are
monitoring of patients in surgery or intensive care, daily
monitoring of patients to maintain appropriate concentra-
tions of calcium, and determinations for purposes of diagno-
sis. Although special collection and handling procedures for
ionized calcium may be economically justified for the rela-
tively few patients in intensive care, they may be impracti-
cal for measurements on large numbers of other patients.
Given the large number of total calcium measurements
requested, more efficient collection and handling procedures
are necessary if ionized calcium becomes the predominant
clinical measurement for calcium.
The use of ordinary sodium heparin in plasma or whole
blood should be discouraged, because it decreases the values
for ionized calcium. Calcium-titrated sodium heparin would
be a better choice when fast analyses are needed; further-
more, results obtained on samples so treated should agree
with those from serum, the sample type used for most other
chemistry assays and which reportedly gives fewer prob-
lems with maintenance of the calcium electrodes (7) than
does plasma or whole blood.
Blood may remain uncentrifuged at room temperature for
as long as 2 h or at 4#{176}C
for 24 to 70 h without appreciable
change in the concentration of ionized calcium; use of SST
tubes to keep serum and cells separated after centrifugation
gives the least change in pH and corrected ionized calcium
after 70 h of storage at 4#{176}C. Nonetheless, we consistently
observed a 20 mo1/L bias in ionized calcium between serum
from plain tubes and that from SST tubes (Table 1), appar-
ently because of the lower initial pH in samples from the
SS’T tubes. As shown in Table 3, some heparinized whole-
blood samples from patients in intensive care had slightly
greater changes in calcium and pH after storage at room
temperature than did samples from healthy individuals,
although the maximum change in ionized calcium for any
sample was only 25 umo1/L per hour.
Our data from healthy volunteers and hospitalized pa-
tients indicate that ionized calcium in serum is stable,
especially if stored at 4#{176}C,
whether the blood is centrifuged
or not. However, we emphasize several cautions: First,
aerobic handling is not acceptable unless other means of pH
correction, perhaps such as that incorporated into the Radi-
ometer ICA 1 display, are used. Second, because most of our
studies involved healthy donors, we do not rule out the
possibility that samples from some patients may undergo
changes of pH that are not compatible with delays in
processing. Finally, this apparent stability of ionizedcalci-
um seems to be a dynamic process, such that the hydrogen
cation and the lactate anion from the lactic acid offset the
effect of the other, respectively releasing and chelating
ionized calcium. Although doubling or tripling the concen-
tration of lactate in samples stored at 4#{176}C did not apprecia-
bly affect our measurements of ionized calcium, this may
have been fortuitous for the samples we studied.
If total calcium remains the calcium measurement in
greatest use, then special handling of a few samples for
determinations of ionized calcium should present few prob-

CLINICAL CHEMISTRY, Vol. 30, No.4, 1984 555

lems. However, as instrumentation for ionized calcium
continues to improve, measurements of ionized calcium may
eventually displace total calcium, and more efficient proce-
dures for blood collection and handling will be needed.
Further work is needed to confirm conclusively that storage
of blood at room temperature for 2 h or at 4 #{176}C
for longer
periods does not alter ionized calcium values by more than
about 2%.
References
1. Graham G, Burritt M. Preliminary report: AACC Ionized Calci-
um Working Group on Reference Intervals. Clin Chem 29, 1187
(1980). Abstract.
2. Toffaletti J, Kirvan K. Spectrophotometric micro method for
measurement of dialyzable calcium by use of cresolphthalein com-
plexone and continuous-flow analysis. Clin Chem 26,1562-1565
(1980).
3. Ladenson J, Bowers GN. Free calciumin serum. I. Determina-
CLIN.CHEM. 30/4,556-559(1984)
Urea, Creatinine, and Glucose Determined in Plasma and Whole Blood by a
Differential pH Technique
M. Ripamonti, A. Mosca, E. Rovida, M. Luzzana, L Luzi, F. Ceriotti, F. Cottini, and L. Rossi-Bernardi
We report the conditions (buffer composition and enzyme
activity) required for estimating three frequently determined
analytes-urea, glucose, and creatinine-by use of an im-
proved version of the differential pH apparatus previously
described (C/in Chem 29: 80-85, 1983). For each analyte,
we used only one specific enzyme, thus avoiding a chain of
auxiliary and indicator reactions. The method requires about
a minute for each determination in undiluted plasma or whole
blood.
We previously described (1,2) a new instrument, based on
the differential measurement of pH, and illustrated its
application by the determination of glucose in plasma. We
believe that this pH technique can find interesting applica-
tions in clinical chemistry. Indeed, the two more interesting
advantages are that (a) the system automatically performs a
sample blank and (b) turbid solutions can be analyzed
directly. Moreover, many reactions lead to a change of pH in
the solution. The most relevant among them are: the
oxidoreductase-catalyzed reactions involving NAD/NADH
or NADP/NADPH interconversion; the reactions involving
transfer of phosphate residue; reactions producing CO2 and
NH3; and reactions involving ester bond synthesis or cleav-
age. Clearly, a potentially wide range of analytes and
enzyme activities can be detected by this technique.
We now describe our further refinement of the technique
and its extension to determination of urea, creatinine, and
glucose in plasma and whole blood.
Dipartimento di Scienze e Tecnologie Biomediche, Centro di
Fisiologia del Lavoro Muscolare del CNR, do Ospedale S. Raffaele,
University of Milan, Via Olgettina 60, 20132 Milan, Italy.
Received September 12, 1983; accepted January 5, 1984.
556 CLINICALCHEMISTRY, Vol.30, No. 4, 1984
tion with the ion-specific electrode, and factors affecting the results.
Clin Chem 19, 565-574 (1973).
4. Husdan H, L.eung M, Oreopoulos D, Rapaport A. Measurement
of serum and plasma ionic calcium with the “Space-Stat 20 Ionized
Calcium Analyzer.” Clin Chem 23, 1175-1777 (1977).
5. Fogh-Andersen N. Ionized calcium analyzer with built-in pH
correction. Clin Chem 27, 1264-1267 (1981).
6. Fogh-Andersen N, Christiansen TF, Komarmy L, Siggaard-
Andersen 0. Measurement of free calcium ion in capillary blood and
serum. Clin Chem 24, 1545-1552 (1978).
7. Larsson L, Finnstrom U, Nilsson B, Ohman S. Evaluation of
Radiometer ICA 1 as a routine instrument for serum ionized
calcium and its application for whole blood capillary samples from
newborn infants. Scand J Clin Lab Invest 43 (Suppl 165),21-26
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8. Pesce MA, Bodourian SH, Nicholson JF. Rapid kinetic measure-
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Materials and Methods
Instrumentation. pH was measured with a commercially
available differential pH analyzer (Delpas CL; Kontron AG,
Milan, Italy), an instrument similar in design to the home-
made instrument we previously described (2). Improved
features consist of a thermostated stainless-steel block to
ensure a better temperature control of the two electrodes, a
16-character alphanumeric display, a printer, and a key-
board for parameter entry and instrument control. All
measurements here reported were obtained by use of this
apparatus at 23 #{176}C except for creatinine, which was mea-
sured at 37 #{176}C. An IL919 analyzer (Instrumentation Labora-
tory, Milan, Italy) and IL-associated chemical procedures
were used as comparison methods for the determination of
urea and creatinine. Computations were performed by an
Altos ACS 8000/2 (Altos Computer Systems, Cuppertino,
CA). The software was written in PASCAL high-level lan-
guage.
Chemicals and solutions. Lyophilized jack-bean urease
(EC 3.5.1.5) was from Ames/Miles (Milan). A solution of it
was prepared by dissolving a suitable amount in 10 mL of
0.1 mol/L KC1 and adjusting the pH to 7.50 with 0.1 mol/L
K2C03; 15 mL of glycerol was added as a stabilizing agent.
The final activity was 1440 kUIL. For each urea determina-
tion, we routinely used 11.2 U of urease. Typically, the
measurement was taken 20 s after the enzyme was added.
No significant loss of urease activity in solution was detect-
ed after six months at 4 #{176}C, but a 12% loss of activity was
observed after four days at room temperature.
Creatinine (99% pure) and creatinine iminohydrolase (EC
3.5.4.21, microbial source) were from Farmitalia Carlo
Erba, Milan. The enzyme suspension was dialyzed against
0.1 molIL KC1 solution containing NaN3, 1 g/L. The pH was
then adjusted to 7.54 with 10 mmolIL NaOH, with continu-