and preterm
Table 1. Some Mean Clinical and LaboratoryData
Full-term
Pre-term
Gender, M/F
8/6
9/5
Delivery: vaginal/cesarean
11/3
10/4
Apgar score 1-mm >6
Gestational age,weeks
Birth weight, g
Cord-blood pH
Cord-blood base deficit, mmol/L
Plasma albumin, gIL
14
39.9 (SD 1.2)
3401 (SD 371)
7.31 (SD 0.04)
8.1 (SD 1.2)
3.55 (SD 2.2)
14
30.0 (SD 1.9)
1230 (SD 258)
7.32 (SD 0.05)
7.6 (SD 1.4)
2.86 (SD 4.6)
C’,
0
‘U
I
J.Pediatr. 95, 521-527
6. Perlman, M., Kapitulnik, J., Blondheim,
S. H., et al., Bilirubin binding and neonatal
acidosis. Clin. Chem. 27, 1872-1874 (1981).
7. Snedecor, G. W., and Cochran, G., Statistical Methods. Iowa State Univ. Press,
Ames, IA,
1973, p 115.
M. Perlman’
Dept. of Pediatr.
Hadassah
Univ. Hosp.
Hebrew Univ. Hadassah
Med. Sch.
Jerusalem,
Israel
Present address and address for correspondence: Perinatal Div., Hospital for Sick
Children, 555 University
Ave., Toronto, Ontario, M5G 1X8 Canada.
1.oo
z
infants.
(1980).
o.eoo
.
.
Albuminby BromcresoiGreen-A
Case of LaboratoryConservatism
0
0
F00
3 0.400
To the Editor:
In 1967 Northam
and Widdowson
(1)
published
their much-quoted
paper
concerning automation
of the bromcresol green (BCG) dye-binding method for
human albumin originally introduced by
Rodkey
(2). This simple procedure
‘U
S
z
0
0
0.200
-l
0.00
0.5
1.0
tS
2fl
BILIRUBIN/ALBUMIN MOLAR RATIO
Fig.1. Regressioncurvesrelating
plasma bilirubin/albumin
molar ratios(x-axis)
to
“loosely bound” bilirubin in the “late” Sephadex G-25 column eluate (y-axis)
Values for pre-term infants are indicated by closed circles, for full-term infants by open circles. The curve
on the right is for pre-term infants
tion, and the bilirubin adsorbed onto the
gel was eluted and measured as previously described (6). Plasma samples
from full-term and pre-term infants
were run in parallel, to minimize interassay variation. Regression curves were
plotted for each set of data (Figure 1),
and we calculated (7) the statistical
significance of the difference between
the curves for the whole range of molar
ratios studied (0.3-2.2), and for the
clinically relevant range (0.5-1.0).
Plasma from pre-term infants proved
to have less “loosely bound” bilirubin
than full-term infant plasma (Figure 1);
the p value of the difference between the
regression curves was <0.001. In the
clinically relevant range of bilirubin/
albumin molar ratios (0.5-1.0), there
were no significant differences in binding between pre-term and full-term infants’ plasma. Plasma albumin concentrations were lower in pre-term infants
(Table 1) by a mean factor of 0.81.
Evidently, in the clinically relevant
range, plasma albumin concentration
alone may be considered to be the major
determinant of bilirubin binding dif-
ferences
full-term
between well pre-term
and well
infants.
“Kernlute”
was graciously supplied by
Ames-Yissum, Ltd., Jerusalem, Israel. I
thank Drs. S. H. Blondheim and J. Kapitulnik for their advice and assistance.
References
I. Harris, R. C., Lucey, J. F.,and MacLean,
J. R., Kernicterus in premature infants associated with low concentrations of bilirubin
in plasma. Pediatrics 21,875-884 (1958).
2. Gartner, L. M., Snyder, R. N., Chabon, R.
S., and Bernstein, J., Kernicterus: High incidence in premature infants with low serum
bilirubin
906-917
concentrations.
(1970).
Pediatrics
45,
3. Hugh-Jones,
K.,Slack,
J.,Simpson,K.,et
al.,
Clinical
courseofhyperbilirubinemia
in
premature
infants. N. Engi.J. Med. 263,
1223-1229
(1960).
4. Shiller, J. G., and Silverman, W. A., “Uncomplicated” hyperbilirubinemia
of prematurity. Am. J. Dis. Child. 101, 587-592
(1961).
5. Cashore,W. J.,Free bilirubin
concentrationand bilirubin
bindingaffinity
in term
quickly replaced the tedious and inaccurate method whereby globulins were
precipitated with sodium sulfate and
albumin was measured in the supernate
with the biuret reagent (3).
With the passage of time, deficiencies
in the BCG methodology became apparent. BCG reacts rapidly with albumin but also, more slowly, with ceruloplasmin, C3 component of complement,
and haptoglobin (4), and with transferrin (5). The reaction of BCG with globulins becomes more apparent with decreasing albumin concentrations, such
that in those conditions (nephrotic
syndrome, liver disease) where albumin
measurements are most critical there
may be clinically serious overestimates
of this protein. Some modifications of
the original procedure have been published, based on either measuring the
color developed in the first few seconds
(6-8) or incorporating high concentrations of NaCl to reduce nonspecific
binding (9, 10).
It is a curious
feature
that
in 1968
Louderback et al. (11) showed that
bromcresol purple (BCP) almost instantly and specifically binds to human
albumin, with no increase in color on
standing. Other plasma proteins, either
separately or in combination, did not so
react. In 1978 Northam, this time in association with Pinnell (5), automated
the BCP procedure and confirmed the
specificity
of this dye for human albumin. They found the correlation with an
electroimmunoassay
(EIA) method to
CLINICAL CHEMISTRY, Vol. 28, No. 6, 1982
1407
#{149}
ability ofreferencematerialthatisnot
S
S
0
U
U
I’
I.
a
I.
4.
20
___
2
#{149}_
4
4
I
lid
10
20
60
(NUN)
Fig.1. Comparative ratesof colordevelopment with BCG and BCP
Plasma pool, open symbols; nephrotlc syn&ome
plasma, closed symbols. Cl, #{149},
BCG; 0, #{149},
BCP
be (in g/L) BCP = 0.95E1A + 1.72 for
serum samples. (Immunological methods for measuring albumin are accepted
as being more desirable than dye-binding assays but lack the low cost and ease
ofreagentpreparationand handlingof
the latter.) Subsequently,
Northam (12)
stated: “I believe that thereare now very
strong reasons for not using the BCG
methods.”
Figure 1 shows the relative rates of
colordevelopment with BCG and BCP
of a plasma pool and of plasma from a
patient with nephrotic syndrome. For
both samples there is a rapid color development with BCP, which does not
increase further, quite unlike the situation with BCG.
Despite the obvious superiority of
BCP over BCG as a reagent suitable for
use in busy hospital laboratories, the
great majority nevertheless continue to
use the latter material. An indication of
this resistance to change may be seen
from an examination
of the relative
usage of both dyes in three international
External Quality Assurance Programs
(Table 1).
Two objections
have been raisedto
use of BCP for measuring plasma albumin. First, many calibration materials
contain bovine serum albumin and the
color developed with BCP is only 26% of
that of an equivalent amount of human
albumin
(our measurement).
This,
however, only emphasizes the unsuit-
Table 1. Relative Use of Dye
Methodologies
in Measuring
AlbumInin ExternalQualityAssurancePrograms
Dy.
BCG
BCP
Other
American
Dade
U.K.
Internal.
W.iIcorne
N.O.C.S.
Q.A.P.
0.C.P.
(Oct. 1981) (Oct. 1981) (sept. 1981)
277
16
24
61
2
9
947
18
153
‘Our laboratory, even though no results are returned for albirnin, because the material used in this
proam isbovinebased.
1408
CLINICAL CHEMISTRY,
of human origin. Second isthereported
(13)increase in color of the BCP-albumm complex at the heparin concentrations used in obtaining plasma samples.
Bonvicini et a!. (14) reported that interference from heparmn could be overcome by incorporating hexadimethine
bromide (Polybrene;AldrichChemical
Co.), 450 mg/L of reagent. We found
that this concentration
of Polybrene
prevented the increase in absorbance
from heparin but also greatly diminished the sensitivity of the method.
However, Polybrene at a concentration
of 50 mg/L in the BCP reagent completely abolished the interference from
heparin without affecting the absorbance readings. Thus only a slight modification of the Pinnell and Northam
procedure (5)is required.
It is encouraging that a solid-phase
strip for serum albumin has been recently developed in which BCP rather
than BCG is used (15). Also, in 1979,
Fredrichsen and Kierulf (16)in a Letter
also recommended BCP over BCG. And
yet BCG is still preferred over BCP in
hospital laboratories by a factor of 67 to
1 (Table 1).
References
1. Northam, B.E., and Widdowson, G. M.,
Determination of serum albumin
by AutoAnalyzer
Clin.
using
Biochem.,
bromcresol
Tech.
green. Assoc.
Bull.
No.
11, 1
11. Louderback, A., Mealy, E. H., and Taylor, N. A., A new dye-binding technic using
bromcresol purple for determination of albumin in serum. Clin. Chem. 14, 793-794
(1968). Abstract.
12. Northam,
B. E., Whither
automation?
Ann. Clin. Biochem. 18, 189-199 (1981).
13. Perry, B. W., and Doumas, B. T., Effect
of heparin on albumin determination
by use
of bromcresol green and bromcresol purple.
Clin. Chem. 25, 1520-1522. Letter.
14. Bonvicini, P., Ceriotti, G., Plebani, M.,
and Volpe, G., Heparin interferes with albumin determination by dye-binding methods.
Clin. Chem. 25, 1459-1460 (1979).
15. McCleary, A. R.,A solid-phase strip test
for the determination of serum albumin. Clin.
Chem. 26, 1058 (1980). Abstract.
16. Fredrichsen, P., and Kierulf, P., A more
accuratedye-binding method for the routine
determination of serum
25, 1180 (1979). Letter.
albumin. Clin. Chem.
Jennifer
P. Finbarr
Duggan
Duggan
Biochem.
Lab.
Regional Hosp.
Cork, Ireland
ImprovedContinuous-Flow
DeterminatIon
ofAlbuminwith
BromcresolGreen
(1967).
2. Rodkey,
F. L., Binding
of bromcresol
green
by human serum albumin. Arch. Biochem.
Biophys. 108, 510-516 (1964).
3. Varley, H., Determination
of plasma
proteins. In Practical Clinical Biochemistry,
William Heinemann Medical Books Ltd.,
1963,pp 184-186.
J. E. C., Improved specificity
of serum albumin determination and estimation of “acute phase reactants” by use of
the bromcresol green reaction. Clin. Chem.
London,
4. Gustafsson,
22,616-622
5. Pinnell,
automated
(1976).
A. E., and Northam, B. E., New
dye-binding
method for serum
albumin determination with bromcresol
purple. Clin. Chem. 24,80-86 (1978).
6. Webster, D., The immediate reaction between bromcresol green and serum as a
measure of albumin content. Clin. Chem. 23,
663-665 (1977).
7. Gustafsson, J. E. C., Automated serum
albumin determination by use of the immediate reaction with bromcresol green reagent.
Clin. Chem. 24, 369-373 (1978).
S., and Raabo, E., Improved
and more specific bromcresol green methods
8. Ingwersen,
for the manual and automatic determination
of serum albumin. Clin. Chim. Acta 88,
545-550 (1978).
9. O’Donnell,
N., and Lott, J. A., Reducing
the interference from globulins in the bromcresol green (BCG)
determination
of serum
albumin. Clin. Chem. 24, 1004 (1978). Abstract.
10. Sandford, K. J., and Eikenberry, J. N.,
Albumin assay in a multilayered analytical
reagent using bromcresol green. Clin. Chem.
26,1059(1980). Abstract.
Vol.28,No.6,1982
To the Editor:
Dye-binding methods for serum a!bumin assessment are simple, easily
automated, and economical (1).In our
SMAC (Technicon Instruments Corp.,
Tarrytown, NY 10591) we use bromcresol green (BCG) as the dye (2),but
the agreement with immunochemical
methods is poor, especially in the lower
concentration range (3). To improve the
accuracy in the lower range, we tried
another BCG method (4), prediluting
the sample in water. Even though this
modification resulted in obviously more
nearly accurate values in the lower
range, as compared with the original
method (2), still better accuracy was
desirable. Our study shows that, when
the sample predilution (4) is modified
by using succinate buffer instead of
water,a furtherimprovement can be
achieved, and results then correlate well
with those by the BCG method recently
introduced by Technicon (5).
We obtained serum samples from 57
patients attending the regional hospital
of Linkoping. Each sample was divided
intofourportions and keptfrozen(-20
#{176}C)
until assay. Each aliquot was first
assayed immunochemically foralbumin,
by nephelometry (6). These resultshomogeneously representing the range
15-54 g of albumin per liter-were used
as reference and compared with the results from three variations of the BGC
method (A, B, and C), all performed on