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CLIN. CHEM. 27/3,466-468 (1981)
The Worthington “Ultrafree” Device Evaluated for Determination of
Ultrafiltrable Calcium in Serum
John Toffaletti,1 Dale Tompkins,1 and Gall Hoff2
We evaluated a commercially-available disposable device Materials and Methods
(“Ultrafree,” Worthington Diagnostics) for the anaerobic Subjects. The subjects, all volunteers, were employees of
preparation of protein-free ultrafiltrates from serum for the laboratory and hospital or blood donors. They were se-
measurement of ultrafiltrable calcium. Sufficient filtrate lected with an eye to a broad distribution by age and sex.
for the analysis is obtained within 10 mm from 0.2 to 0.4 Results for one subject who later was found to be hypertensive
mL of serum at room temperature. We assessed these were discarded. A group of six results for ionized calcium were
ultrafilters with regard to permeability of calcium citrate, not included, because they obviously were low (0.87-0.94
exclusion of proteins, frequency of leakage, and effect of mmol/L) for technical reasons during that particular day.
temperature on results. Within-run and day-to-day coeff i- Three abnormally high results for ultrafiltrable calcium ob-
cients of variation for human serum pools were 1.2 and tained early in the study were discarded on the basis that
1.5%, respectively. Reference intervals (in mmol/L) for leakage of protein through the filter probably accounted for
total (2.16-2.58), ultrafiltrable (1.44-1.67), dialyzable them.
(1.25-1.41), and ionized (1.04-1.25) calcium have been Analytical methods. Procedures for total calcium (5), di-
determined for a healthy population of 69 women and 81 alyzable calcium (6), and ionizedcalcium (7)were as described
men, ages 18 to 65 years. The device appears to be the c
previously.Ultrafiltrable alcium was determined with use
most practicable yet available for use in making this of Ultrafreefiltersttached to l-mL syringes.
a Positive pres-
measurement. sure was applied to the syringe plunger by an auto-injector and
ultrafiltrate was collected in the self-contained reservoir at
the top of the Ultrafree device. Both ultrafilter and auto-in-
AddItIonal Keyphrases: reference intervals low-cost
jector are available from Worthington Diagnostics. Between
methods for the laboratory . atomic absorption spectros- 200 and 400 L of serum was collected into the syringe for the
copy
ultrafiltration. After 100-150 zL of ultrafiltrate was collected
(at room temperature), it was analyzed for calcium content
Measurement of free ionized calcium in blood is generally
by atomic absorption spectroscopy (5).
believed to give the best clinical assessment of calcium me-
For measurement of ultrafiltrable, dialyzable, and ionized
tabolism, although many studies have reported few, if any,
calcium, serum was collected exposure to air in
with minimal
calcium-related cardiac effects during transfusions of citrated
1-mL tuberculin syringes, as described previously (8). These
blood in which ionized calcium concentrations became ex-
syringes were capped and kept refrigerated or on ice until the
tremely low (1-4). Measurement of ultraf’iltrable calcium
analysis. All analyses were done within 8 h of collection.
(protein-free ionized plus complexed calcium) might provide
Preparation of serum pool. A pooled specimen of human
a better index of the true physiological status, but because of
serum was prepared from patients’ samples left over from
technical limitations of older ultrafiltration procedures as well
The
routine analysis and stored for three or four days at 4#{176}C.
as a perceived lesser physiological importance of ultrafiltrable
pH was adjusted to about 7.4 by adding 10 L of 1 molfL HC1
calcium there are few reports related tothe measurement of
per milliliter of serum. After mixing, the serum was filtered,
ultrafiltrable calcium.
then injected into 3-mL evacuated blood-collection tubes. The
A disposable anaerobic ultrafiltration device (“Ultrafree,”
tubes were stored at -20 #{176}C individually thawed on the
and
Worthington Diagnostics, Div. of Millipore Corp., Freehold,
day of analysis.
NJ 07728) has recently become available with which a pro-
tein-free ultrafiltrate can be produced within 10 mm from
200-400 tL of anaerobically-processed serum. No additional Results
equipment is required other than a procedure for measuring Ultrafiltration of serum at room temperature and at 37#{176}C.
total calcium. The reliability of the technique therefore de- Sera from 20 of the healthy volunteers were ultrafiltered at
pends upon the integrity of ultrafilter and the reliability of both room temperature (23 ± 1 #{176}C) 37 #{176}C.
and at Capped sy-
the procedure for total calcium. From a practical standpoint, ringes containing serum were kept in a room at 37 #{176}C for 25
any laboratory can now measure ultrafiltrable calcium at a to 30 mm before ultrafiltration was begun. Ultrafiltrates were
nominal cost, whereas the measurement of ionized calcium transferred to plastic screw-top vials as soon as sufficient ul-
may cost more than many laboratories can justify based on trafiltrate was obtained. The results (Table 1) show that ul-
clinical use. trafiltrable calcium averaged 0.06 mmol/L less at 37 #{176}C than
We have evaluated this device with regard to temperature Individual differences due to temperature
at 23 #{176}C. ranged
effects, molecular permeability, precision, and other potential from 0.01 to 0.14 mmolfL, with a CV of 49%.
variables in the ultrafiltration process, and have determined Restriction of albumin and amytase. A human serum pool,
reference intervals for total, ultrafiltrable, dialyzable, and prepared as described above, was used for this study. The al-
ionized calcium from data on 150 healthy individuals, ages 18 busnin (Mr 66 000) and amylase (Mr 54000) in this pool
to 65 years. measured 39 g/L and 81 UfL. No detectable albumin or
amylase passed through four different filters we tested; the
limits of detection for these analyses were 1 g/L and 3 U/L.
1 Clinical Chemistry Laboratory, Duke University Medical Center,
Durham, NC 27710. Permeability of calcium citrate. Two solutions were pre-
2Wohjngtn Diagnostics, Freehold, NJ 07728. pared, each containing, per liter, 1.50 mmol of calcium, 150
D
Received Oct. 10, 1980; accepted ec. 1, 1980. mmol of NaCl, and 10 mmol of Tris, at pH 7.4. One of the so-
466 V
CLINICALCHEMISTRY, ol. 27,No.3,1981
Table 1. Ultrafiltrable Calcium at Room Table 3. Demographic and Analytical Data for
Temperature (RT) and at 37 #{176}C Reference Population
CaUF (RT) CaUF (37 #{176}C) Age and sex distribution
Mean, mmol/L 1.55 1.49 0.063
Age, yr 18-29 30-39 40-49 50-59 60-65
SD, mmol/L 0.055 0.055 0.03 1 No. men 27 24 12 14 4
CV, % 3.5 3.7 49
No. women 25 21 10 10 3
n = 20 different samples. Room temperature was 23 ± 1 #{176}C
Reference intervals, mmol/L
Mean 95th
Referenc.
lutions also contained 0.2 mmol of sodium citrate per liter, to ± 2 SD percentile Interval
chelate a portion of the calcium. Each solution was filtered 2. 16-2.58
Total calcium: 2.16-2.58 2.18-2.58
through four different filters; the ultrafiltrable calcium
measured the same, 1.50 mmolfL, in both solutions. Evidently Ultrafiltrabte
all the calcium citrate passed through the membrane. calcium: 1.44-1.67 1.45-1.66 1.44-1.67
Constancy of ultrafiltrable calcium during ultrafiltration. Dialyzable
We wondered if a change in protein concentration in the calcium: 1.25-1.40 1.26-1.41 1.25-1.41
sample during ultrafiltration caused a Donnan equilibrium Ionized
change to affect the ultrafiltrable calcium. To study this we calcium: 1.04-1.25 1.05-1.25 1.04-1.25
used four sera from different patients, collecting 600 zL of
each serum in syringes, and beginning the ultrafiltration.
When 300 jsL of concentrated serum remained in the syringe,
a new filter was attached and another ultrafiltrate was col- justify the cost of both instrumentation and reagents to
lected. Analysis of the first and second ultrafiltrates showed maintain typical semi-automated Ca-ion-specific electrodes,
no definite changes in ultrafiltrable calcium concentration the Ultrafree device may provide an economical alternative.
(Table 2).
Furthermore, virtually no additional space is required for the
Precision. A serum pool, prepared as described above, was ultrafilters and auto-injectors.
collected into 20 syringes and each was filtered through an The design of the device is especially well suited for deter-
Ultrafree device. Analysis of these ultrafiltrates gave a mean mination of free calcium. Wasted serum or “dead space” is
value for calcium of 1.52 (SD 0.018) mmol/L and a CV of only about 50 tL and the sample is maintained nearly an-
1.2%. aerobic during the ultrafiltration process. As a result, ultra-
Aliquots of another human serum pool were analyzed filtrable calcium can be measured in as little as 0.2 mL of
during a month, in which analyses were performed on 18 days. serum in about 10 min. The use of oil is not necessary, as it was
The mean ultrafiltrable calcium during this period was 1.58 in an adaptation of the Worthington antioonvulsant drug filter
(SD 0.024) mmol/L. The CV was 1.5%. to the measurement of ultrafiltrable calcium (9). As noted, we
Reference intervals. Samples from the 150 different ap- occasionally encountered problems with leakage of protein
parently healthy adults (Table 3) gave the reference intervals around the membranes, but by simply observing the color of
shown in Table 3, calculated from the mean ± 2SD and from the initial ultrafiltrate, errors due to this should be avoid-
values falling within the 2.5 and 97.5 percentiles. able.
Leakage through filters. The ultrafiltrates are ordinarily
In this study the ultrafiltrations were done at room tem-
clear and colorless, but about one filter in 50 of those used in perature. This is very convenient both in terms of time and
this evaluation gave a straw-colored ultrafiltrate and con- operation conditions. Theoretically, calcium fractionation
tained protein that apparently had leaked around the mem- should be done at 37 #{176}C, because the equilibrium can shift
brane. This problem can be eliminated in most of these sit- with temperature change. The importance of this in clinical
uations by observing the first droplet of ultrafiltrate to pass diagnosis has not been established, although there have been
through the membrane: if any color is seen, the syringe should several reports in which ionized calcium was measured at room
be removed from the auto-injector, a new filter installed, and temperature, apparently without obvious losses in diagnostic
ultrafiltration restarted. efficacy (10-12).
The discrepancy between ultrafiltrable and dialyzable
Discussion calcium is only partly explainable. The method for dialyzable
The Worthington Ultrafree filter provides a simple and calcium detects about 50% of calcium citrate, as compared to
rapid means to separate ultrafiltrable calcium. Additional 100% by Ultrafree. Considering the permeability of other
costs are small, because no new instrumentation is needed. In complexes, this amounts to a discrepancy of about 0.07
laboratories where the low volume would make it difficult to mmol/L. The difference between measurement of ultrafil-
trable calcium at room temperature and dialyzable calcium
at 37#{176}Caccounts for an additional 0.06 mmol/L. A small part
Table 2. Ultrafiltrable Calcium Content of Four of the remaining discrepancy, about 0.1 mmol/L, may pos-
Samples of Serum before and after Concentration sibly be due to the detection methods: spectrophotometry for
Original serum a Concd. serum b dialyzable calcium, atomic absorption spectroscopy for ul-
CaUF, mmOiIL trafiltrable calcium.
1.28 1.28 Possibly there are calcium complexes with relative molec-
1.43 1.40 ular masses between 2000 and 20000, that can permeate
through the ultrafilter but are mostly excluded by the mem-
1.73 1.70
brane used for dialyzable calcium. Peptides, phospholipids,
2.03 2.03 or amino acids such as ‘y-carboxyglutamate may also be in-
a Uitraflltrate collected as the serum was concentrated from 0.6 to 0.3 cluded in this group.
ml. The speed, precision, and simplicity of the Ultrafree fil-
Ultrafiltrate collected as the serum was further concentrated from 0.3 to tration device may encourage the clinical study of ultrafil-
0.15 ml.
trable calcium in situations where ionized calcium has been
CLINICAL CHEMISTRY, Vol. 27, No. 3, 1981 467
measured previously. Such measurements may provide insight the determination of total calcium in serum. Clin. Chem. 19, 1208-
into the lack of symptomatic hypocalcemia in the presence of 1213 (1973).
the very low concentrations of ionized calcium often observed 6. Toffaletti, J., and Kirvan, K., Spectrophotometric micromethod
for measurement of dialyzablecalcium by use of cresolphthalein
during transfusion with blood containing citrate and phos-
complexone and continuous-flow analysis. Clin. Chem. 26,1562-1565
phate (1-4). The low cost to initiate this procedure should also (1980).
be attractive for laboratories that regard high initial cost and
7. Husdan, H., Leung, M., Oreopoulos, D., and Rapaport, A., Mea-
cost per test of instrumentation for ionized calcium as exces- surement of serum and plasma ionic calcium with the “Space-Stat-20
sive. Ionized Calcium Analyzer.” Clin. Chem. 23, 1775-1777 (1977).
8. Toffaletti, J., and Bowers, G. N., Improvements in and clinical
utility of a continuous-flow method for routine measurement of di-
References alyzable (ultrafiltrable) calcium. Clin. Chem. 25, 1939-1943
1.Morse, E. E., Hohnadel, D.C., Genco, P., and Katz, A. J., Decreased (1979).
ionized p
calciumduringtherapeutic lasma exchangepheresis and 9. Eckfeldt, J., and Koehler, D. F., Measurement of ultrafiltrable
platelet pheresis. Johns Hopkins Med. J. 146, 260-263 (1980). calcium in serum with use of the Worthington Ultrafree Anticon-
2. Wieland,P.,Duc, G.,Binswanger,U., and Fischer, J. A., Pars- vulsant Drug Filter. Clin. Chem. 26, 1871-1873 (1980).
thyroid hormone response in newborn infants during exchange 10. Moore, E. W., Ionized calcium in normal serum, ultrafiltrates, and
a
transfusion ith blood supplemented with citratend phosphate:
w whole blood determined by ion-exchange electrodes. J. Clin. Invest.
of
Effect iv calcium. Pediatr. Res. 13,963-968 (1979). 49, 318-334 (1970).
3. Kahn, R. C.,Jascott,.,Carlon,G. C.,et al., Massive blood re-
D 11. Schwartz, H. D., McConville, B. C., and Christopherson, E. F.,
placement: Correlation of ionized calcium, citrate, and hydrogen ion Serum ionized calcium by specific ion electrode. Clin. Chim. Acta 31,
concentration. Anesth. Analg. 58, 274-278 (1979). 97-107 (1971).
4. Denlinger, J. K., Nahrwold, M. L., Gibbs, P. S., and Lecky, J. H., 12. Ladenson, J., and Bowers,G. N., Free calcium inserum.II. Rigor
Hypocalcemia during rapid blood transfusion inanaesthetized man. of homeostatic control, correlations with total serum calciumand
Br. J. Anaesth. 48,995-999 (1976). review of data on patients with disturbed calcium metabolism. Clin.
5. Cali, P. J., Bowers, G. N., and Young, D. S., A referee method for Chem. 19,575-582 (1973).
CLIN. CHEM. 27/3, 468-471 (1981)
An Optically Clear Hypercholesterolemic Hypeririglyceridemic Quality-
Control Material Prepared from Animal Lipid Sources
Gary J. Proksch and Dean P. Bonderman
A hyperlipidemic control serum can be simply prepared human or animal serum are commonly used to evaluate and
from animal lipid sources. Beta- and pre-beta-lipoproteins assure analytical performance. Hyperlipidemic human sera
containing cholesterol and triglyceride are removed from are expensive to prepare and sometimes difficult to obtain.
porcine serum by treatment with dextran sulfate and cal- Recently we examined 22 lots of commercial human-
cium ions. A triglyceride-rich fraction containing only trace serum-basedquality-control materials for several hepatitis-
amounts of cholesterol is isolated from chicken egg-yolks. related materials: HB5Ag, HBBAb, HBCAb, and HAVab (3).
The two fractions are then combined in 40 mmol/L sodium All were positive for one or more of these constituents.
bicarbonate to give the desired values for cholesterol and In view of the health hazard involved in handling quality-
triglyceride. The preparation is stabilized against surface control materials prepared from pooled human sera, we show
denaturation during long-term storage at 5 #{176}C,
perhaps for how to prepare hyperlipidemic serum from animal sources,
such serum is naturally free of hepatitis-associated constitu-
as long as two years, by adding 0.25 g of Triton X-100
ents.
surfactant per liter, and against an accidental exposure to
The concentration of lipids in the serum of herbivores such
short-term freezing by adding 10 g of sucrose per liter. We
as horses or cattle, from which large amounts of serum can be
used this solution as a diluent to reconstitute lyophilized readily obtained, is too low to be useful in monitoring lipid
bovine serum. The resulting product, having been prepared assays effectively (4). Because of the denaturation and inso-
from only animal sources, is free of hepatitis-associated lubiization of beta- and pre-beta-lipoproteins during ly-
constituents, and is remarkably clear, homogeneous, and ophilization, serum products with a high lipid content gen-
stable. Results obtained with it are precise. erally reconstitute slowly, and the reconstituted fluid is often
quite turbid (5). The lack of clarity and the inhomogeneity
The association of increased concentrations of certain related to the uneven dispersion of insoluble material may
serum lipid constituents and increased risk of heart disease decrease the usefulness of these products (6). A simple way
(1,2) dictates that the precision and accuracy of lipid assays is needed to increase the triglyceride and cholesterol con-
be carefully monitored. Pooled specimens of lyophilized centrations of animal sera without adversely affecting the
reconstitution rate or clarity of the product.
Department of Pathology, Indiana University Medical Center, 1100 Recently, we described a stable human lipoprotein diluent
West Michigan St., Indianapolis, IN 46223. for use in reconstituting lyophilized human serum for the
Received June 26,1980; acceptedNov. 24,1980. preparation of clear, hyperlipidemic quality-control materials
468 CLINICAL CHEMISTRY, Vol. 27, No. 3, 1981
