The Worthington “Ultrafree” Device Evaluated for Determination of

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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
                                                                                        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
                                                                                       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
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
  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
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
transfusion ith blood supplemented with citratend phosphate:
              w                                                         whole blood determined by ion-exchange electrodes. J. Clin. Invest.
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
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

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