Clinical Chemistry 44, No. 7, 1998
Fig. 1. (a) Cryoglobulin detection by simple diffusion in agarose gel at 4 °C.
Samples in which cryoglobulin was detected by the standard cryocrit tube
method were placed in wells 2, 4, and 6. Samples in which cryoglobulin
was not detected by the standard cryocrit tube method were placed in
wells 1, 3, and 5. In this study, the center well was not used. A clear
precipitate ring of cryoglobulin was observed in wells 2, 4, and 6. (b-d) For
identification of the precipitate ring of cryoglobulin, the sample was placed
in the center of the cooling agarose plate in this study. Cryoglobulin
precipitated in the center of the agarose plate was identified with the
Ouchterlony method by reacting it with antisera placed around the sample
well (P). Anti-gamma (G), anti-alpha (A), anti-mu (M), anti-kappa (K), and
anti-lambda (L) antisera were placed in each of the wells. (b) A sample
from a patient without cryoglobulinemia was placed in the center well (P).
No precipitate ring was observed by cooling diffusion, and a precipitin line
reacting with antisera was not observed by the Ouchterlony method. (c
and d) Samples of patients with cryoglobulinemia were placed in the
center wells; in (c) cryoglobulin reacted with all antisera; in (d) cryoglobulin
reacted with anti-mu and anti-lambda antisera to form the precipitin lines.
precipitate is found, the sample is centrifuged at 4 °C; the
supernatant is removed; cold physiological saline or phosphate buffer (pH 5.5–7.0) is added; and the mixture is heated
at 37 °C. If the precipitate redissolves after heating, the
precipitate is cryoglobulin. However, extremely small
amounts of precipitate are obtained by this procedure.
Cryoglobulin is apt to disappear in such small amounts, and
detection is not precise.
We have devised a very simple and reliable new
method of cryoglobulin detection, using a cooling agarose
plate, to replace this unreliable conventional method,
which requires a large quantity of sample. This method
can be performed with small samples of 10 mL. The
agarose plate was prepared as follows. Agarose (Sigma,
St. Louis, MO) was added to Dulbecco’s phosphate buffer
containing glycine (pH 7.0) so that the final concentration
was 6 g/L. This mixture was heated and dissolved until
clear. The gel was poured on a plate to a thickness of 2
mm. A well 5 mm in diameter was made in the plate.
1559
Blood was collected and centrifuged at 37 °C, and 20 mL of
the separated serum sample was placed in the well of the
agarose plate. The plate was allowed to stand at 4 °C, and
a precipitate ring was observed after 48 h. In cryoglobulin-positive samples, a clear precipitate ring was found
(Fig. 1a). After the agarose plate with the precipitate ring
was immersed in physiological saline at 4 °C for 48 h to
remove other proteins, the precipitate was redissolved at
37 °C, confirming that it was cryoglobulin. The detection
limit of our method was estimated by dilution with purified
cryoglobulin; the precipitate was seen at concentrations of
;50 mg/L and above. Identification of the components of
the cryoglobulin was possible, using the agarose plate with
the precipitate ring immersed in physiological saline at 4 °C
for 48 h. Twenty microliters each of anti-H chain gamma,
alpha, and mu, and anti-light chain kappa and lambda
antisera were placed in the wells for antisera located around
the sample well; the plates were allowed to react for 12 h at
37 °C, and the identification of the components was confirmed if there was a precipitin line (Fig. 1b-d). This cryoglobulin detection and identification can be performed on
one plate, and it is also effective for analysis of the physiological properties of cryoglobulin by changing the pH and
temperature of the gel (not shown).
References
1. Wintrobe MM, Buell MV. Hyperproteinemia associated with multiple myeloma. Bull Johns Hopkins Hosp 1933;52:156 – 65.
2. Agnello V, Chung RT, Kaplan LM. A role for hepatitis C virus infection in type
II cryoglobulinemia. N Eng J Med 1992;19:1490 –5.
3. Aiyama T, Yoshioka K, Okumura A, Takayanagi M, Iwata K. Hypervariable
region sequence in cryoglobulin-associated hepatitis C virus in sera of
patients with chronic hepatitis C: relationship to antibody response against
hypervariable region genome. Hepatology 1996;24:1346 –50.
4. Frangeul L, Musset L, Cresta P, Cacoub P, Huraux JM, Lunel F. Hepatitis C
virus genotypes and subtypes in patients with hepatitis C, with and without
cryoglobulinemia. J Hepatol 1996;25:427–32.
The Relation between the Ultrafiltrable Calcium Fraction and Blood pH and Concentrations of Total Plasma
Calcium, Albumin, and Globulin, Malcolm Cochran,* Brad
Rumbelow, and Glenn Allen (Department of Clinical Biochemistry, Flinders Medical Center, South Australia 5050,
Australia; * author for correspondence: fax 61-8-83740848, e-mail [email protected])
With the advent of various micropartition systems, typically as developed by Amicon, it became simple to
ultrafilter serum and to obtain an apparent measure of the
ultrafiltrable calcium fraction (UFCa). In studies of this
type, evidence of strict monitoring of serum pH, known to
affect calcium binding, was not generally reported (1– 4).
An earlier study (5), attempted to control temperature and
Pco2, but in our hands the study was difficult to reproduce (unpublished). We have determined the UFCa values of 54 samples of whole blood at 37 °C at or near
physiologic pH and sought an empirical relationship with
more readily measured biochemical variables, which
might enable us to predict UFCa from routine laboratory
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Technical Briefs
results. We were particularly interested in data in mild
renal failure.
Although any aqueous solution is subject to control by
the Henderson-Hasselbach relation, the relatively low pK
(6.1) means that the buffering capacity near pH 7.4 is poor.
Furthermore, modest changes in the Pco2 have a minor
effect on the HCO3 concentration, for a marked effect on
the pH. We found that the buffering capacity of whole
blood allowed confidence in our procedure because the
pH remained stable during handling. The Milliporet UFU
4 system allowed lateral filtration without the erythrocytes obstructing the membrane such that an ultrafiltrate
of whole blood could be obtained.
We used Millipore UFU 4 10KNML systems, shortened
to 20 mm by cutting off part of the upper reservoir.
Initially, we loaded 1 mL of plasma, but in our final work,
we used 1 mL of heparinized venous blood. In pilot
experiments, we took 1-mL aliquots of a pooled serum
sample (five specimens) and altered the pH by adding 0.1
mol/L HCl or 0.1 mol/L NaOH. The volumes added were
100, 80, 60, 40, 20, or 0 mL of HCl, respectively, and 20, 40,
60, or 80 mL of NaOH. When necessary, the added volume
was made up to 100 mL, in each case with 0.1 mol/L NaCl.
The starting pH was checked and ultrafiltrates were
obtained without gassing.
We then took 1-mL portions of four gassed heparinized
blood samples (see below) and observed the pH at 37 °C
over 3 min while the samples were exposed undisturbed
to air. We compared this with plasma from the same
samples, treated likewise.
Our final approach was to rotate the blood sample in
the collection tube, angled almost horizontally, in an
electrical heating block at 37 °C, passing over it a mixture
of 5.66% CO2 and 4.64% O2 in nitrogen for 3 min. The
blood was transferred to the Millipore filter, which sat in
a plastic serum tube shortened to 18 mm, and this in turn
was put in the steel centrifuge tube, similarly warmed. A
specially made nylon cap, with two small holes, plugged
the top of the steel centrifuge tube, and the same gas was
passed slowly through the system for 3 min, venting
through the cap. The holes were sealed, and the sample
was centrifuged at 1000g for 10 min in a fixed angle
Centra-3t centrifuge (IEC) placed in an incubator at 37 °C;
;100 mL was thus obtained for estimation of calcium
(UFCa). To test reproducibility, we used five blood samples, each divided into five portions. We obtained plasma
from the same blood samples and ultrafiltered the corresponding 25 portions in an identical way to allow comparison.
We analyzed, in this way, 54 samples from patients
chosen to include cases of mild renal failure. The pH,
Pco2, and ionized calcium (Ca21) of the gassed heparinized venous whole blood were measured before ultrafiltering. After filtering, the pH and Pco2 in the residual
blood (;90% of initial volume) were remeasured. The
calcium concentration of the ultrafiltrate was determined
using a Hitachi Boerhinger Mannheim 917 Automatic
Analyzer (Hitachi).
The pH, Pco2, and Ca21 were measured in a Radiom-
eter ABL 620 (Radiometer Medical A/S). A portion of
each blood sample had been separated previously to
allow estimation of plasma concentrations of total calcium
(TCa), magnesium, phosphate (P), bicarbonate, sodium,
potassium, chloride, albumin, total protein, and creatinine
in the Hitachi 917. The ion gap and globulin were calculated. The [H1] was calculated directly from the pH and
was assumed to be H1 activity.
Statistical analysis used SPSS.
In pilot experiments, we confirmed that the UFCa from
a sample of serum increased in a curvilinear fashion as the
pH decreased from 7.77 to 6.87. Near the physiologic
range, an ;1.2% change in UFCa resulted from each 0.02
pH unit change. When whole blood was exposed to air for
3 min, there was no measurable change in pH, but plasma
became steadily alkalotic, with a .10% fall in [H1] over
30 s and a .25% fall over 3 min. Measurement of calcium
in ultrafiltrates of blood and plasma gave almost identical
precision, the CVs being 1.28% and 1.31% respectively,
but blood yielded significantly higher UFCa values,
1.06 6 0.04 times greater (P ,0.05). After the centrifugation process, the pH of blood had increased slightly but
significantly, the initial mean pH 7.39 increasing to pH
7.42 afterward (P ,0.0001), and the Pco2 was comparably
lower (mean of 39.4, falling to 35.6 mmHg).
Among the 54 samples, creatinine ranged from 0.052 to
1.055 mmol/L (mean, 0.260; median, 0.153); TCa from 1.96
to 2.80 mmol/L (mean, 2.34; SD, 0.204); UFCa from 1.18 to
1.68 mmol/L (mean, 1.41; SD, 0.125); Ca21 from 1.0 to 1.36
mmol/L (mean, 1.20; SD 0.092); pH from 7.19 to 7.54
(median, 7.39) converted to [H1] from 29 to 64 nmol/L
(mean, 41; SD, 7.1); albumin from 17 to 44 g/L (mean,
34.3; SD, 7.8); and globulins from 17 to 53 g/L (mean, 35.1;
SD, 6.8).
We used UFCa as the observed variable and regressed
this on the other measured variables as predictors, except
for magnesium, which was considered separately. We
thought that the most useful predictors were likely to be
TCa, albumin (alb), globulin (glob), and [H1], but that ion
gap or phosphate might represent complexing radicals.
Using TCa, alb, glob, and H1, we obtained:
UFCa 5 0.6626~TCa! 2 0.0097~alb! 2 0.0040~ glob!
1 0.0068~H 1 ! 1 0.0540; df, 49; r2 5 0.743
The P value of each of the coefficients was ,0.0005, except
for glob, which was ,0.005.
Including ion gap or phosphate contributed nothing
useful. Excluding glob reduced the correlation, r25 0.647,
and increased the value of the constant. Using HCO3 as a
surrogate for H1 only slightly decreased the significance
of the 4-variable equation, r2 5 0.705; but the constant,
which also carried a large variance, became a major
determinant (0.647 mmol/L). We obtained the same
4-variable equation when we used SPSS to build the
optimal relationship by systematically rejecting variables
with nonsignificant partial correlation coefficients or by
Clinical Chemistry 44, No. 7, 1998
adding in predictors and accepting the simplest, most
significant correlation. The standardized coefficients
(beta) yielded values of 1.08 for TCa, 20.60 for alb, 0.38 for
H1, and 20.22 for glob.
When we used our equation to predict a UFCa value for
each case and obtained the difference between observed
and predicted, these residuals were scattered randomly
when plotted against the predicted UFCa.
Because we had measured the [Ca21] directly, we could
also test the ability of the data to predict this value. We
found the optimal equation was:
Ca 2 1 5 0.5155~TCa! 2 0.0052~alb!
2 0.0075~ion gap! 1 0.0080~H 1 !
1 0.0021; df, 49 r2 5 0.792
All P values of the coefficients were ,0.0005. Inclusion of
glob decreased the significance of the overall equation
and, furthermore, introduced a substantial value for the
constant. Similarly, we could see how [Ca21] was related
to UFCa. We found:
UFCa 5 1.065~Ca 2 1 ! 1 0.133~ 6 0.140!;
df, 52; r2 5 0.615
When Ca21was used to predict UFCa, taking the iongap into account, the equation was:
UFCa 5 1.022~Ca 2 1 ! 1 0.0071~ion gap!
1 0.03672; df, 51; r 5 0.683
2
The P values of the coefficients were ,0.0005 for Ca21 and
,0.01 for the ion gap. The constant was not significantly
different from zero. The standardized coefficient (beta) for
Ca21 was 0.752 and for ion gap was 0.240. The effect of pH
should have been implicit in the [Ca21] and including
[H1] was of negligible benefit to the equation.
The parathyroid responds, albeit weakly, to Mg21;
therefore, it was possible that this ion would influence the
setting of the Ca21 and, indirectly, the UFCa. However,
incorporation of Mg (mean, 0.85 mmol/L; SD, 0.15) into
our partial analyses had no helpful influence on any
regression coefficients that we tested.
Knowledge of the calcium filtered at the glomerulus
would be important in understanding renal calcium physiology, which is why we had set out to find an empirical
relationship between the UFCa and commonly measured
variables.
Near pH 7.4, the concentration of trivalent phosphate,
which binds calcium predominantly, is low (6), and
moreover, this concentration decreases sensitively as pH
falls. Thus, any potential effect of phosphate on the UFCa
might have been nullified in samples where there was
acidosis. Magnesium did not appear in the analysis as an
influence on either the UFCa or Ca21.
The use of whole blood ensured good control of pH
during the procedure, although a small decrease in Pco2
1561
was seen. We could not explain this by leakage of gas
from our system nor by oxidation of the metal holder. It
would be expected that the erythrocytes would continue
to take up the CO2 via carbonic anhydrase and also as
carbamate, given the low O2 atmosphere. We can only
speculate as to the cause of our finding that plasma
ultrafiltered by the same procedure always gave a slightly
lower UFCa value.
The UFCa was approximated as the Ca21 plus a constant, as would be expected. The variability of this constant obviously would have reflected the range of concentrations of dissociated, filterable moieties, such as calcium
citrate, bicarbonate, and phosphate, which are crudely
represented by the ion gap. More surprisingly, the Ca21
was not as good a predictor of the UFCa as TCa. The
standardized coefficients for [Ca21] and ion gap were
both inferior to those for TCa. However, given the merit of
its simplicity, Ca21 and ion gap might be considered more
practical predictors of UFCa than TCa, proteins, and
arterial pH. Although the standardized venous [Ca21] of
the ion-sensitive electrode would not exactly correspond
to the fraction in arterial blood pH that would determine
the glomerular UFCa, in practice it is likely to be a
satisfactory approximation.
Nordin et al. (7) devised an ingenious way of predicting
both UFCa and Ca21, using data obtained in a large series
of healthy middle-aged women (7). Our observed values
(as XUF or XCa) reasonably predicted that obtained from
his iterative method (as YNor), although with a rather high
degree of uncertainty (r2):
UFCa Nor 5 0.83XUF 1 0.10, r2 5 0.55;
Ca 2 1 Nor 5 0.72XCa 1 0.26, r2 5 0.53
The difference is easily explained. First, the hypothesis of
Nordin et al. (7) depends on Ca21, which is the major
component of UFCa, being held stable in a healthy
population, whereas in renal failure it is likely to be
variable. Second, at values ,24 mmol/L, HCO3 in our
series was a surrogate for [H1]. We could not assume an
unaffected pH and simply regard HCO3 as a possible
complexing agent.
In fact, it is impossible to determine the true glomerular
UFCa without micropuncture, because the average glomerular capillary protein concentration is higher than in
peripheral plasma as a result of the continuous loss of
filtered plasma water. Nevertheless, our equation does
allow the arterial pH to be taken into account, which
avoids approximations from empirical ultrafiltration procedures, whether they were carried out in a standardized
physiologic atmosphere or not. We found that the influence of increasing protein concentration was twice that of
lowering the [H1]. Given that the average increase (8) in
protein concentration in the glomerulus is ;10%, our
estimate might be systematically increased. However, the
Donnan effect counters much of this and probably obviates need for correction (9, 10).
From the results, we have therefore developed a relatively simple equation to predict the UFCa within 6 0.12
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Technical Briefs
mmol/L at 95% confidence from commonly estimated
biochemical variables. We plan to see whether our
method to calculate UFCa, which requires the concentrations of total Ca, alb, glob, and arterial blood pH, will
provide a tool to give insights into the altered tubular
handling of calcium in early renal failure.
References
1. Rose GA. A simple and rapid method for measurement of plasma ultrafiltrable and ionised calcium. Clin Chim Acta 1972;37:343–9.
2. Eckfeldt JH, Koehler DF. Measurement of ultrafiltrable calcium in serum with
use of the Worthington ultrafree anticonvulsant drug filter. Clin Chem
1980;26:1871–3.
3. Toffaletti J, Tompkins D, Hoff G. The Worthington ultrafree device evaluated
for determination of ultrafiltrable calcium in serum. Clin Chem 1981;27:
466 – 8.
4. D’Costa M, Cheng PT. Ultrafiltrable calcium and magnesium in ultrafiltrates
of serum prepared with the Amicon MPS system. Clin Chem 1983;29:519 –
22.
5. Robertson WG, Peacock M. New techniques for separation and measurement of the calcium fractions of normal human serum. Clin Chim Acta
1968;20:315–26.
6. Robertson WG. Factors affecting the precipitation of calcium phosphate in
vitro. Calcif Tissue Res 1973;11:311–22.
7. Nordin BEC, Need AG, Hartley TF, Philcox M, Wilcox M, Thomas DW.
Improved method for calculating calcium fractions in plasma: reference
values and effect of menopause. Clin Chem 1989;35:14 –7.
8. Pitts RF. Physiology of kidney and body fluids. Chicago: Year Book Medical
Publishers, Inc, 1963:202.
9. Thode J, Fogh-Andersen N, Sigaard-Andersen O. Ionized calcium and the
Donnan effect. Clin Chem 1983;29:1554 –5.
10. Payne RB. Clinically significant effect of protein concentration on ion
selective electrode measurements of ionized calcium. Ann Clin Biochem
1982;19:233–7.
tively rare conditions. Various hereditary Hb variants
have been reported to cause disproportionately high and
low HbA1c titers, as determined by chromatographic
methods such as HPLC (1– 6).
Recently, another simple and specific method for the
quantitation of HbA1c, a latex immunoagglutination (LA)
method that uses a monoclonal antibody against a glucose
moiety at the b-chain N-terminal, has been developed and
used in place of HPLC methods. However, the relevance
of the LA method for measurement of HbA1c concentrations in cases with Hb variants has not been tested.
We report a case of a Hb variant (Hb Niigata) (7) with
inappropriately increased and decreased HbA1c measured with HPLC and LA, respectively. Thus, LA may
have a limitation for the accurate quantitation of HbA1c in
some special cases.
A 58-year-old male (weight, 58.8 kg; height, 162.5 cm)
without any abnormal signs or symptoms was evaluated
with routine laboratory data at the time of an annual
health check provided by his employer. His HbA1c measured with LA was below the reference interval (2.9%),
but his fasting blood glucose (FBG) was 5.4 mmol/L (98
mg/dL). His medical records over the preceding two
years revealed HbA1c results by HPLC of 13.0% and
A Nondiabetic Case of Hemoglobin Variant (Hb Niigata) with Inappropriately High and Low HbA1c Titers
Detected by Different Methods, Tsuyoshi Watanabe,1*
Ken Kato,1 Daishiro Yamada,1 Sanae Midorikawa,1 Wakano
Sato,1 Masaru Shiga,1 Yoshihiko Otsuka,2 Masakazu
Miura,2 Keiko Harano,3 and Teruo Harano3 (1 Third Dept.
of Internal Medicine, Fukushima Medical Coll., Fukushima 960-12, Japan, 2 Res. & Dev. Dept., Mitsubishi
Kagaku Bio Clinical Lab., 3-30-1 Shimura Itabashi-ku,
Tokyo 174, Japan, and 3 Dept. of Biochem., Kawasaki
Medical School, Kurashiki 701-01, Japan; * address for
correspondence: Third Dept. of Internal Medicine,
Fukushima Medical Coll., 1 Hikari-ga-oka, Fukushima
960-12, Japan)
Human adult hemoglobin (Hb) consists of HbA (96% of
the total), HbA2 (3%), and HbF (1%). HbA contains a
number of subfractions, including HbA1a2, HbA1b, and
HbA1c (glycosylated Hb). HbA1c has been used as a
clinical marker for blood sugar control for the past one to
two months. Inappropriately low or high HbA1c concentrations in comparison with blood glucose concentrations
are caused by various conditions of Hb structure and
metabolism; under such conditions, the use of HbA1c as a
clinical marker cannot be warranted. Abnormally low
HbA1c concentrations are usually encountered in patients
with high turnover rates of Hb, whereas disproportionately high HbA1c concentrations are found under rela-
Fig. 1. Isoelectric focusing and sequencing gels showing presence of
abnormal b-chain.
(A) Isoelectric focusing patterns (pH range, 6 –9) of the hemolysates of the
proband (lower lane) and a control subject (upper lane). (B) Photograph of a
sequencing gel of the cloned cDNA encoding the b-globin gene of this case (left
lane) and a control subject (right lane).