near 400 nm the spectral responses of both bilirubin sub- fractions ... base (support layer). ...... L. Evans for valuable technical support in the Jendrassik-Gr#{243}fdiazo assay. .... Powers, D., Lauff, J., Kasper, M. E., et al., Delta bilirubin in pe.
CLIN.
CHEM. 28/12, 2366-2372 (1982)
The KodakEktachem#{174} ClinicalChemistrySlidefor Measurementof Bilirubinin Newborns:Principlesand Performance Tal-Wing Wu,1 Glen M. Dappen,1 Donald M. Powers,2 Donald H. Lo,2 Royden N. Rand,2 and Richard W. Spayd1 In this slide, unconjugated bilirubin and its sugar conjugates interact with a cationic polymeric mordant to form spectrally enhanced complexes having similar absorptivities at “-‘400 nm. With reflection densitometry and appropriate mathematical transformation, readings at this wavelength are linearly related to bilirubin concentrations up to 260 mg/L. The slide requires 10 tL of serum, is precise (total CV 90% of the serum proteins of molecular size 60 000 daltons (as gauged by use of radioactively labeled proteins). Third, the combined action of surfactants, caffeine, and sodium benzoate coated therein facilitates the dissociation
of bilirubins from endogenous serum proteins. Finally, being largely opaque, it shields from optical detection most of the nonbilirubin serum pigments (including heme and lipochromes). The intermediate screen layer contains gelatin and titanium dioxide (Ti02). As its name implies, this layer further masks the potentially interfering spectral absorption of hemoglobin and provides an opaque background for reflection densitometry. The reaction layer contains a mordant (1-3), the structure of which is shown in Figure 2, and N,N-bis(2-hydroxyethyl)glycine(Bicine)buffer,pH 8.0.
Ti02, Cellulose Acetate Surfactants (Brij 98#{174}, TX-405#{174})
Spreading Layer
Caffeine Sodium benzoate
Screen Layer
Ti02 Gelatin
Reaction
Mordant
Layer
Gelatin
,
feasible, within 1-3 h of each other. Because serum bilirubins are labile to light and air, and because different assays for bilirubins may show different sensitivities to sample aging or handling, freshly sampled sera were kept in the dark and cold (e.g., on ice) and were tested, either on the film alone or concurrently by a comparative method, within 2-3 h after acquisition. Similar precautions were taken in testing sera supplemented with potential interferents or authentic bile pigments.
Results
Bicine
Film Base
Buffer,
Reflectance spectra of authentic (B,,) on experimental slides coated dant. Figure 3 ifiustrates, as reported
pH 8.0
Es/ar Bose
Fig. 1. Cross-section of the Kodak Ektachem clinical chemistry slide for neonatal serum bilirubin Diagrammatic, not drawn to scale Calibrating fluids. For all mechanistic studies indicated in the text, the serum-based standards of B,, and B were prepared by dissolving known weights of these pigments in a common serum pool with total biirubin (Br) less than about 3.0 mg/L. Each calibrating fluid was prepared in multiple aliquots and kept at -60 to -70 #{176}C under reduced pressure and desiccation until just before use. No vial was reused. For the performance evaluations, serum-based Kodak Ektachem calibrators were used with the Ektachem analyzers, and bilirubin standards prepared from SRM-916 bilirubin material (National Bureau of Standards, Washington, DC 20234) according to the specifications of Doumas et al. (6) were used to calibrate the Jendrassik-Gr#{243}fmethod (see below).
Reference
enhancement produced when a concentration series of serum-based B,, standards was spotted on a mordant-containing slide as compared with a control slide (without mordant). At every analyte concentration tested, the mordanted B,, shows a sharpened Ama,, at 460-470 nm, accompanied by at least a twofold increase in its reflection density (DR) at the new peak. Reflectance spectra of authentic diconjugated bilirubin (dB) on experimental slides coated with and without mordant. Figure 4A shows that authentic human dB has a broad reflectance spectrum (400-480 nm) on the control slide (without mordant). On the mordanted slide, however, dB
absorbs maximally at -420 nm, with increased peak (Figure 4B).
0,
C
a)
Sera
Serum samples from neonates (arbitrarily defined as infants up to “.14 days old) and older patients, where indicated, were tested on the film and with the diazo reference, wherever
C 0 U
a,
& Wavelength, nm
Wavelength, nm
Fig. 3. Reflectance spectra of unconjugated bilirubin (Be)on the slides with (right) and without (left) mordant (mg/L): (a) 237; (b) 167.2; (c) 97.3; ( 51.5. All scans were made through the transparent support ofthe slide fitted in a specially modified spectrophotometer (see text). Each spot developed wIth 10 zL of test fluid for 5 mm at 25#{176}C was compared with a spot developed with 10 tL of distilled water on an identical slide treated exactly like the test slide. Each spectral curve is the mean of at least five closely agreeing repeated scans
Concentrationsof bilirubin
0.8
0.8-
±CH2-CH--)-
DR at the new
Reflectance spectra of authentic monoconjugated biliru bin (mB) on experimental slides coated with and without mordant. For this demonstration, three representative con-
Method
The Jendrassik-Gr#{243}ftotal bilirubin method, as modified by Doumas et al. (6, see also 7) was used in the manual mode or, where indicated, in the automated mode. In the latter mode, a Rotochem IIa-36 centrifugal analyzer (American Instrument Co., Silver Spring, MD 20910) was used according to L. Evans and D. Grisley (private communication, 1981). The chief modification here of the usual mode was a decrease in the volume of the test fluid to 20 fLL (with no decrease in the final reagent concentrations).
Patients’
unconjugated bilirubin with and without morearlier (1-3), the spectral
-f-CH2-CH± I
Z
0 >.
0 0.6
0.6
-
(0
0)
C
C
C
a
C
0
0
U
a, a,
a, 360
400
440
480
520
Copoty[slyrene:
N-Vinylbenzyl-N.N-Dimethylbenzyl-
Ammonium Chloride; Divinylbenzene]
Fig. 2. Structure of the cationic polymeric mordant
360
400
440
480
520
Wavelength, nm
Wavelength, nm
Fig. 4. Reflectance spectra of diconjugated bilirubin (dBc) on the slides with (right) and without (left) mordant Concentrations of bilirubin (mgIL) of test fluids: (a) 92.3: (b) 46.2; (c) 22.8; ( 6.5. Test conditions as described for Fig. 3 CLINICAL
CHEMISTRY,
Vol. 28, No. 12, 1982
2367
0.8
400nm
0.8
0.5
0 0.6
,..
C
1.0
E..:::’
a)
0.4
C 0
0
C-)
o2yJ,b
0.2
0.5
U) C
C
0.4
460nm
>.
0.6
In
0,
420nm
a a
It 360
400
440
480
520
360
Wavelength, nm
400
440
480
520
200
0
00
200
0
200
(mg/L): (a) 45: (b) 22.3; (c) 11.2. Testconditions as
centrations of highly purified mB were used. This material is extremely unstable and is available only in small quantities.
Transformed density, 0.6 07, X l01
Reflection
density,
0.7
#{176}R
Nevertheless, repeated testing showed that its behavior was very similarto that of authentic dB on both the control and
0,5
mordant-containing slides4 (Figure 5). In the following sections, unless otherwise stated, the results obtained with dB will be assumed to be illustrative of mBa. To a first approximation, this assumption is valid. Detection wavelength.
100
Total bilirubin, mg/L, Manual Jendrassik-Grot (Doumas)
Fig. 5. Reflectance spectra of monoconjugated bilirubin (mBa) on the slides with (right) and without (left) mordant Analyte concentrations described for Fig. 3
100
0
Wavelength, nm
of total
bilirubin
(BT
= B,, + B)
0.4 0.3 0.2 0.I
at a single
Figure 6 presents typical response curves on the mordant-containing slide obtained with serum-based standards of B,, and authentic dB. The reflection density (DR) shown was not linear with analyte concentration at the three representative wavelengths (400, 420, and 460 nm). However, they clearly illustrate that: (a) at 460 nm, mordanted B,, absorbs more strongly than mordanted dB, as we had conjectured (see introductory paragraphs), and (b) at 420 nm, and especially at 400 nm, the response curves for B,, and dB match closely. The lower half of Figure 6 shows that the nonlinear response curve at 400 nm can be linearized by appropriate mathematical transformation of DR (4). Again, the responses
0
Total
00
bilirubin,
200
mg/L
Fig. 6. Typical response curves on the analytical slide for neonatal bilirubin at three representative wavelengths The top tfree frames depict the response curves obtained by using serum-based standards of B0and dB isolated from human bile. Reflection densities (D,) wore recorded at the ttee wavelengths Indicated (with approximate bandwidth of ±5 nm for each filter used) for spots developed with each test fluid and incubated at 37 #{176}C in a manually operated prototype Instrument built on the principles described earlier (4). The lower frame Illustrates the response curves at 400 nm, both as Op and as its transformed density (OT). vs analyte concentrations
of B,,and B match closely.
Similar trends have been duplicated on the film with natural or artifically reconstituted sera shown by “high-performance” liquid chromatography (8) to be enriched in one or more of the key bile pigments (B,,, mBa, or dB). These observations together suggest that the mordant-containing slide can estimate total bilirubin (Ri’ = B,, + mB + dB) based on (a) Be-only calibrators and (b) measurement of the absorbance at a singlewavelength (-‘400 nm). Accuracy. We used a Kodak Ektachem four-chemistry analyzer to compare the slides with the Jendrassik-Gr#{243}f method as used in a centrifugal analyzer as described under Materials and Methods. The National Committee for Clinical Laboratory Standards (NCCLS) comparison-of-methods experiment was carried out (9). All sera were sampled from patientslessthan 14 days old, transported in a dark container to our laboratory,and examined by both methods no more than a few hours apart. Over the nominal range of bilirubin covered (0-150 mgfL), results by the two assays correlate well (Figure 7), with a slope of 0.95, an intercept of 0.3, an S., of 3.4, and a correlation coefficient of 0.991. The mean values for the Ektachem and diazo methods were 86.9 and 89.8 mg/L, respectively. Precision. We evaluated the reproducibility of the slides Note,however,thatmB dissolved in human serum albumin solution (50 g/L, pH 7.0) absorbs with a broad spectrum over 400-460 nm and hasa nominalXma,, ‘-455 nm, whereas dB ina similar matrix absorbsover420-460nm witha nominalpeak -‘420nm. Apparently, the mB also interacts with thecoatedelementssuch thatitmimics
dB in its in-film spectrum. 2368
CLINICALCHEMISTRY,Vol. 28, No. 12, 1982
-J C)
E a, N >‘
Co
C 0 0
100
w 0 I-
w
N
102 = 0.95 Intercept = 0.3 Sy.x = 3.4 r = 0.991 =
Slope
I
50
0
50 100 150 200 Jendrassik-Grof Reference Method (mg/L)
250
Fig. 7. Comparison of results for total bilirubin obtained with the Kodak Ektachem clinical chemistry slide and with the .Jendrassik-Gr#{244}f method (Doumas et al. modification) automated on the Rotochem analyzer on the Kodak Ektachem 400 analyzer during 20 working days, using the NCCLS replication protocol (10). Table 1 summarizes the components of precision estimated by analysis of variance. Over the bilirubin range of particular relevance for
Table 1. Precision of the Slide Billrubin concn, mg/L
6.4 38.9
94.7 184.2
_________________ Within run ________________ Between runs SD, mg/L
CV, %
0.1 0.2 0.6 0.8
1.94 0.64 0.63 0.40
SO, mg/L
0.2 0.3 0.7 1.8
Between days SD, mgIL CV, % _________________
CV, %
2.80 0.87 0.76 0.97
0.3 0.5 0.8 0.4
5.16 1.26
0.84 0.21
SD, mg/L
0.4 0.6 1.2 2.0
Overall precision 95% confidence intervals, mg/L
0.3-0.5 0.5-0.8 1.0-1.5 1.6-2.5
CV, %
6.19 1.66 1.29
1.07
Precision of the slide was determined in the Kodak Ektachem 400 analyzer for lyophilized control samples during 20 days, according to NCCLS protocol EP3-P (10). Replication consisted of three aliquots of each sample per run (randomized order), two runs per day. Calibration was done once per week. Precision components were estimated by analysis of variance.
Table 2. Effect of Potential Interferents on Results with the Slide Substance
tested, concn
p-Acetylaminophenol, 50 mg/L Acetylsalicyclic acid, 300 mg/L Ammonium chloride, 0.25 mmol/L p.Aminosalicylic acid, 230 mg/L Ascorbic acid, 40 mg/L Calcium chloride, 4 mmol/L Cholic acid, 60 mg/L Ethanol, 3 g/L Palmitic acid, 3 mmol/L Gentisic acid, 5 mg/L fl.-o-Glucose, 6 gIL Heparin, 80 000 USP units/L Salicylic acid, 350 mg/L Triglycerides, 8 g/L Uric acid, 170 mg/L Hemoglobin, 1.0 g/L 1.5 g/L 2.Og/L 2.5 g/L pH 6.8 pH 8.8 Hyperalimentation fluid (Liposyn), 10-fold diluted Total protein, 100 g/L
(A/G =
Mean bias, a mg/L
95% confidence Interval
0.8 1.3 -0.6
-0.3-2.0 -0.2-2.8
-1.0 -0.4
-2.2-3.0 -1.0-1.8 -1.0-1.8 -1.9-1.4 -0.7-1.2 -1.9-2.2 -0.2-3.0 -1.6-1.2 -1.1-1.5 -0.1-3.1 -1.3-1.1 -0.3-1.5 -1.1-2.0 1.1-2.1 1.8-2.8 2.5-3.5 3.2-4.2 -2.6-1.0 -3.0-1.1 -0.8-0.0
-3.7
-9.7-2.3
-1.0
-3.4-1.4
0.0 0.4
-0.2 0.3 0.1
1.6 -0.2
0.2 1.5
-0.1 0.6 0.5
1.6 2.3
3.0 3.7
-0.8
043)b
Albumin, 50 g/L (A/G
=
2.5)
8interference tests were conducted at an unconjugatedbilirubinconcentration of 100 mgIL, except for hemoglobin, which was tested at different bilirubin concentrations (e.g. 10. 100, 200 mg/L) with essentially the same results. b Albumin/globulin ratio.
neonates (39-184 mg/L), the total coefficient of variation was 1.1-1.7%. Interferences. Table 2 lists some ofthe substances that been tested as potential interferents on the slide. A serum containing 100 mg of unconjugated bilirubin per liter
(CV)
have pool was divided, and portions were supplemented either with a stock solution of the test compound or with an equivalent volume of solvent. This diluted the portions by 1% or less. For estimating the effects of albumin and total protein, a 2 X 2 factorial experiment was used. Human serum albumin content was varied from 25 to 49 g/L, and ‘y-globulin content from 43 to 65 g/L. Their effects were estimated relative to a normal
serum containing
40 g of albumin and 70 g of total protein per
liter. For triglycerides, we used pools of lipemic human serum adjusted to 100 mg of B,, per liter and determined the bias by linear regression, using results by the Jendrassik-Gr#{243}f method as the reference. The results of comparisons on the Kodak Ektachem 400 analyzer (n = 6) showed that none of the compounds tested caused a bias (i.e., difference from expected bilirubin concentrations for the interferent-supplemented and control pools) exceeding 2.1 mg/L at 100 mg of bilirubin per liter (2.1%). The only exceptions were hemoglobin (see below) and total protein, which, at the abnormally high concentration tested (100 g/L), produced a mean negative bias of 3.7 mgfL at a bilirubin concentration of 100 mg/L. The case with hemoglobin deserves special mention for two reasons. First, hemoglobin is frequently increased in neonatal serum and it is well recognized as an interferent in many tests for bilirubin (11). Second, because the absorbance is measured at 400 nm, the assay is potentially sensitive to spectrometric interference from hemoglobin, which in solution has Xa,55at -‘418 nm. Indeed, early tests showed that hemoglobin was a potent spectral interferent in an experimental slide without the screen layer described in Materials and Methods. For instance, a 1 g/L hemoglobin solution produced a positive absolute bias of about 10 mg/L at either 10 or 200mg of bilirubin per liter. However, with a screen layer placed immediately beneath the spreading layer, this effect was greatly minimized. Thus, at hemoglobin concentrations up to 1.5 g/L, the apparent bilirubin concentration increased by only 2.3 mg/L, regardless of the analyte concentrations tested. This means that over the bilirubin range 60-100 mg/L, a range of particular relevance for neonates (11), 1.5 g of hemoglobin per liter will produce a positive bias of only 3.8-2.3%. Note that 1.5 g of hemoglobin per liter is at or near the highest hemoglobin concentration (1.47 g/L) found by Meites and Lin (12) in plasma of 417 children (including 176 < 14 days old) subjected to skin-puncture plasma sampling. Table 2 also shows that, even at 2-2.5 g of hemoglobin per liter, the positive biases produced at a bilirubin concentration of 100 mg/L were 3.0-3.7% (or 5-6% at 60 mg of bilirubin per liter). Linearity. A set of interrelated fluids (admixtures of highand low-bilirubin pools) was used to demonstrate the linear response of the system up to 260 mg/L. These results are shown in Figure 8. Stability of the slide. The slide is stable for about two years when stored at either -20 or 4 #{176}C and 50% relative humidity. Effect of light on bilirubin measurements. During development of the slide, we repeatedly observed that when neonatal samples or sera supplemented with B,, were left capped on the laboratory bench and exposed to ambient temperature and light for 3.5-7.5 h before testing, the slide typically registered less loss of bilirubin than did the Jendrassik-Grbf assay performed in parallel (Figure 9). This trend was intensified when the test fluids were similarly exposed for 2-3 days (Figure lOa). By contrast, if the fluids were left in the dark under otherwise identical conditions (Figure lOb), we saw no CLINICALCHEMISTRY,Vol. 28, No. 12, 1982 2369
300
(a)
270 -J
(b)
270
240
240
210
210
.
0
E 0 0
a) C 0
80
1811
50
ISO
20
20
90
90
60
60
.
.
30
Co ‘0
5
20
2.5
U
.0
No.of
.5
2.0
2.5
3.0
days
Fig. 10. Effect of long-term (a) illumination or (b) dark treatment of bilirubin on different assays Symbols same as In FIgure 9. Conditions of test are described In the text
change in bilirubin concentration by either method. The only exception to this trend was when the samples had been exposed to air. These observations demonstrate that the significant
0 2
04
0.6
0.8
I
Dilution of high pool with low pool
Discussion
Fig. 8. Linearity of response on the slide
200
J-G 160 -J
E C
40
EK
#{149} _IIa.
0 0 C
a) U
120
C 0 C.) C
100
.0 I.-
#{149} 80
60 . EK 40
F0
J-G
------------.
I
I
2
Exposure
3
4
I
I
5
6
to room light
I
7
I
8
(h)
Fig. 9. Effect on different assays of short-term exposure of billrubin to light EK, the neonatal bilirubin slide; J-G, the Jen&nsslk-#{244}fdiazo method. Except for the sample with starting bilirubin concentration at 86 mg/L. which was a pooled specimen of neonatal serum, the other test fluids were derived from a human-serum-based matrix supplemented with unconjugated biiirubln at the starting concentrations indicated. At each time point, Identical aliquots from each test fluid were assayed by both methods concurrently 2370
CLINICAL CHEMISTRY,
slide is much less sensitive to the effect of light on bilirubin than are its diazo counterparts.
Vol. 28, No. 12, 1982
We have thus described the Kodak Ektachem clinical chemistry slide for neonatal bilirubin (NBIL), which is based on the novel interaction between the analyte and a cationic polymer (mordant). Although the full mechanism of this reaction is still being explored, we surmise that it involves both hydrophobic and charge interactions. In particular, the mordant produces three effects on bilirubin that are pivotal to the assay. First, the mordant forms a stronger and more stable molecular complex with bilirubin than does human serum albumin. From fundamental studies we learned that in the presence of caffeine/benzoate, surfactants, or other iigands that challenge the albumin-B,, association, the mordant-B,, complex remains intact. Second, the mordant dramatically alters the spectrum of mB so that it is like that of mordanted dB. This greatly simplifies standardization and facilitates a future extension of the film assay (see the last section of this discussion). Third, responses of B,, and B on the mordant-containing film approximate each other over the region 400-420 nm. The multilayer embodiment of the mordant reaction further enhances some of the foregoing attributes. For example, the specificity inherent in the mordant complexation of bilirubin is reinforced in the slide (Figure 1) by (a) the strong buffering in the reaction layer, (b) the ability of the upper zones to dissociate bilirubin from albumin, and (c) the blocking of hemoglobin in the screen layer. Such specificity is also reflected by the resistance of the slide to many substances (Table 2) that reportedly interfere in various bilirubin assays. We have shown here that the slide isnot only precise, but also correlates well with the Jendrassik-Gr#{243}fmethod as modified by Doumas et al. Note, however, that whereas our slide was developed with research samples of authentic B,.1and B, similar tests have not, to our knowledge, been conducted on the diazo procedures. Thus, the issue of accuracy of either method should await the development of a definitive reference assay(s)for bilirubinand itsmajor subfractions in serum. We have also shown that the slide is distinctly less sensitive to the effect of light on bilirubin in vitro than are its diazo counterparts. This observation has two important consequences. First, it means that exposure of serum to ordinary room light can produce a false-positive bias in the slide relative to the diazo method, the magnitude of which will probably depend
on the intensity
and the duration
of light exposure.
Second, in monitoring the time course of a patient’s serum bilirubin concentrations, the clinician should ensure that a marked decline in the analyte concentration (based on diazo tests) is not clouded by in vitro light-induced effects that are unrelated to the subject’s health status. In fact, several workers (13-15) had reported similarly that diazo methods do detect greater loss of bilirubin after illumination of specimens than do direct spectrometric procedures used on solutions. In extending these observations, Ebbesen (15) demonstrated that such intermethod (direct spectrometry vs diazo) bias was insignificant for the sera of 125 jaundiced infants, including both pre-term and full-term neonates who had received “single light” (illumination from above), “double light” (illumination from both above and below), or no phototherapy. In explaining the apparent dichotomy of results between in vitro and in vivo studies, Ebbesen suggested (15) that the photoproducts of bilirubin generated in vitro are less diazo-reactive than is native bilirubin, but still absorb light in the wavelength region used in direct spectrometry. In vivo, however, the photoproducts could be excreted so rapidly from the body that their concentrations in plasma may be insignificant. Ebbesen’s hypothesis has since received strong experimental support from independent studies of Onishi et al. (16) and McDonagh et al. (17). These observations, taken together, suggest that phototherapy per se will not affect the determination of bilirubin by the diazo or direct spectrometric methods (of which the slide is a special case). However, the in vitro illumination of bilirubin will positively bias the slide relative to diazo tests. These facts also re-emphasize the importance of shielding serum from room light before bilirubin analysis. Finally, two other aspects of the slide assay deserve special mention. During the development of this slide, we confirmed by liquid chromatography (8, 18) the existence of a fourth bilirubin subspecies (in addition to B,,, mBa, and dB) in jaundiced adult sera. We recently isolated this serum entity, termed the “delta fraction” by Kuenzle et al. (19), and characterized it as a direct diazo-reacting bilirubin firmly attached to an albumin-like protein (18,20). When tested on the slide, this component was largely undetected, presumably by the same entrapment mechanism that prevents the detection of hemoglobin (18). Indeed, for several hundred jaundiced sera from adults that we examined on the slide relative to the Jendrassik-Gr#{243}fmethod for BT there was a good correlation (18) between the negative bias and the approximate content of (5bilirubin in the 7-8% of the adult serum population in which this component was increased (>25-30% of BT). Subsequent screening of more than 500 patients’ sera from different hospitals (21,22), however, has shown that 6bilirubin typically comprises
