Clin. Biochem.

15 (2) 113-115 (1982)

Laboratory Note

A "One-Minute ' Direct Bilirubin • 9

Determination Using the Abbott ABA-100

Bichromatic Analyser
S A M U E L Y. CHU, A G N E S K A C Z M A R E C K a n d V.E. T U R K I N G T O N

B i o c h e m i s t r y L a b o r a t o r y , D e p a r t m e n t of L a b o r a t o r y M e d i c i n e , O t t a w a G e n e r a l H o s p i t a l a n d
D e p a r t m e n t of P a t h o l o g y , U n i v e r s i t y of O t t a w a , O t t a w a , O n t a r i o , C a n a d a .
(Accepted November 23, 1981)

We describe a "one-minute" direct bilirubin procedure using added via an auxiliary dispenser to stop the reaction.
the ABA-100. The procedure correlates well with the manual The coloured complex is measured at 550 nm.
Jendrassik-Grof method and gives good precision at a relatively
low direct bilirubin level. In comparison with certain other Ab-
bott ABA-100 direct bilirubin methods, the proposed procedure MATERIALS AND METHODS
appears to be the least affected by the presence of high uncon- Instrument
j u g a t e d bilirubin levels.
ABA-100 Bichromatic Analyzer {Abbott Diagnostics,
Analytical Systems Division, South Pasadena, California
PROCEDURES FOR THE DETERMINATION OF DIRECT 91030}, was used for the assays. A small hole (1/32" size} was
BILIRUBIN using an ABA-100 analyzer have been pro- drilled in the cuvette cover to accept the auxiliary dispenser
posed (1, 2}. J a b e r g (1) used a 2-4 dichloroaniline coupl- probe (5).
ing r e a g e n t while Mort(2) employed a modified Jen- Reagents
drassik-Grof method with a 5-minute colour develop-
m e n t period. P r e l i m i n a r y i n v e s t i g a t i o n of t h e s e 1. Sulfanilic acid (5 g/L) - dissolve 5.0 g of sulfanilic acid in 15
ml of concentrated hydrochloric acid and dilute to 1 L with
methods showed a m a r k e d positive bias compared to distilled water.
the manual J e n d r a s s i k - G r o f "one-minute" procedure, as 2. Sodium nitrite i0.5 gO) - dissolve 500 mg of sodium nitrite
described by Routh (3). (NaNO 2) in distilled water and dilute to 100 ml. Prepare
Table 1 shows the results obtained by these methods weekly and store at 4°C.
3. Caffeine reagent - 50 g/L.
t o g e t h e r with our proposed ABA-100 "one minute"
4. Stabilizer - hydroxylamine sulfate 1.4 mol/L.
diazo procedure and the corresponding manual method.
The higher values obtained with the J a b e r g and Mori 120
p r o c e d u r e s may be due to the longer five-minute colour
d e v e l o p m e n t period. Lathe (4) has r e p o r t e d that the ap-
p a r e n t direct bilirubin reaction increases with time and
t h a t this may be due to the presence of indirect, noncon- E
j u g a t e d bilirubin. Considerable u n c e r t a i n t y exists in
the l i t e r a t u r e r e g a r d i n g the a p p r o p r i a t e conditions and ~ 80
reaction period for the m e a s u r e m e n t of direct bilirubin;
however, the "one minute reaction", although a r b i t r a r y ,
has been widely accepted. ~ 60
Recently, we introduced a simple modification of the T
ABA-100 to extend its use to two r e a g e n t t e s t
s y s t e m s (5). Using this device, we are t h e r e f o r e able to 40
obtain a one-minute reaction period for the measure- o
m e n t of direct bilirubin on the ABA-100. A modified
J e n d r a s s i k - G r o f method was used in which direct ~ 20
bilirubin reacts with diazotised sulfanilic acid in dilute
acid solution. The azobilirubin colour is allowed to
develop for 56.3 seconds and hydroxylamine stabilizer is
0 2'0 4'0 6'0 80 100 120
MANUAL J ENDRASSIK-GROF METHOD (mg/L)
Correspondence to: Dr. Samuel Y. Chu
Head, Biochemistry Laboratory, Fig. 1 -- Correlation b e t w e e n manual Jendrassik-Grof and
Dr. Everett Chalmers Hospital, automated A B A - I O 0 procedure for direct bilirubin measure-
Fredericton, N.B. E3B 5N5 ment.
114 CHU, KACZMARECK AND TURKINGTON

TABLE 1
COMPARISON OF VARIOUSABA-100 DIRECT BILIRUBINPROCEDURES WITH THE ONE-MINUTEMANUAL
J ENDRASSIK~.~ROF P ROCEDUREUSINGC ONTROLS ERA AND PATIENTS' S PECIMENS

Specimen Total Bilirubin Cone. Jaiberg (10) Mori (2) Proposed ABA-100 Manual (3l
mg/L mg/L mg~ mg/L mg/L
1. Am. Monitor Std. 1 66 26 21 10 5
2. Am. Monitor Std. 2 99 43 29 13 8
3. Am. Monitor Std. 3 198 72 32 20 14
4. Versatol Pediatric 200 24 15 10 10
5. Standard (in-house) 200 29 17 7 5
6. Patient A 35 21 16 11 10
7. Patient B 21 12 8 6 5
8. Patient C 67 47 36 24 25
9. Patient D 186 160 155 120 125
10. Patient E 19 10 9 8 7
11. Patient F 21 18 18 18 18
12. SMA Reference Serum 52 14 5 3 2
13. Baby A 103 21 11 7 7
14. Baby B 131 23 12 9 8

TABLE 2 the delay between primary and auxiliary dispensing, it is

INSTRUMENT SETTINGS FOR OUR PROPOSED A SBOTT A BA-100 necessary to place 6 empty cups immediately after the last
M ETHOD FOR DIRECT BILIRUBIN sample. A calibration factor is established using total bilirubin
standards (American Monitor Corp., Indianapolis, Indiana
Power ON 46268). Total bilirubin reagent is used as the working solution
Temperature 30°C for the determination of the calibration factor and the
Filter 550/650 stabilizer is added manually after completion of the analysis cy-
Mode End Point cle. Our calibration factor was found to be 17.8.
Direction Up Direct blank reagent is substituted for the direct bilirubin
Time 5 minutes reagent in a second run to determine the serum blank, which is
Revolution 2 then subtracted from the direct or total readings to derive the
Syringe Plate 1:26 results.
Decimal Setting 000.0 RESULTS AND DISCUSSION
Zero 000.0
Calibration Factor as determined T a b l e 1 s h o w s t h e r e s u l t s of v a r i o u s A B A - 1 0 0 pro-
Auxiliary Dispenser* 20o/0 STOP
c e d u r e s for d i r e c t b i l i r u b i n m e a s u r e m e n t s . R e s u l t s ob-
* Position dispenser probe at cup 26 tained by the proposed ABA-100 procedure were closer
to t h e m a n u a l m e t h o d t h a n t h o s e o b t a i n e d f r o m t h e
TABLE 3 other procedures.
"WITHIN.BATCH" AND "DAY-TO-DAY"PRECISION OF THE T h e w i t h i n - b a t c h a n d d a y - t o - d a y p r e c i s i o n s of t h e
PROPOSED METHOD d i r e c t b i l i r u b i n a n a l y s i s a r e s h o w n in T a b l e 3. T h e coef-
ficient of v a r i a t i o n (C.V.) for " d a y - t o - d a y " a n a l y s i s w a s
Within-Batch Precision 11.5o/o a t a l e v e l of 6.5 m g / L a n d less t h a n 4.0°/o a t t h e
Sample n Mean S.D. C.V. h i g h e r l e v e l of a p p r o x i m a t e l y 40 m g / L . S i m i l a r preci-
mg/L % sion v a l u e s w e r e o b t a i n e d for " w i t h i n - b a t c h " a n a l y s e s .
A 14 15.6 0.84 5.4 A c o m p a r i s o n w a s c a r r i e d o u t w i t h t h e m a n u a l one-
B 10 35.6 0.75 2.1 m i n u t e p r o c e d u r e (3) a n d t h e r e s u l t s a r e s h o w n in Fig. 1.
C 10 56.5 0.73 1.3 R e g r e s s i o n a n a l y s i s g a v e a n e q u a t i o n of Y -- 0.92 + 1.87,
Day-to-Day Precision a n d a c o r r e l a t i o n c o e f f i c i e n t ( r ) = 0.996. T h e S t u d e n t
C 24 6.5 0.75 11.5 t - t e s t s h o w e d t h a t t h e r e w a s no s i g n i f i c n t d i f f e r e n c e
D 17 44.3 1.7 3.8 b e t w e e n t h e m e a n s of t h e p r o p o s e d A B A - 1 0 0 a n d t h e
E 10 84.2 2.1 2.4
m a n u a l m e t h o d (t = 1.30, p>0.2}.
H e m o l y s i s h a s b e e n r e p o r t e d to d e c r e a s e d i r e c t
5. Diazo reagent -- mix 10 ml of sulfanilic acid solution with b i l i r u b i n v a l u e s (2, 6, 7). S i m i l a r e f f e c t s w e r e o b s e r v e d
0.25 ml of sodium nitrite solution. This reagent should be
prepared just before use. for t h e p r o p o s e d A B A - 1 0 0 p r o c e d u r e . T h e d e c r e a s e in
6. Direct bilirubin reagent -- 10 ml of 0.05 N HCI and 2.5 ml of d i r e c t b i l i r u b i n v a l u e s a p p e a r e d t o b e a r a l i n e a r rela-
diazo reagent. t i o n s h i p to t h e i n c r e a s e in f r e e h e m o g l o b i n c o n c e n t r a -
7. Direct blank reagent - 10 ml of 0.05 N HCI and 2.5 ml of tions. A n a p p a r e n t d e c r e a s e of 1.5 m g / L b i l i r u b i n p e r
sulfanilic acid solution. 100 m g / L h e m o g l o b i n w a s s e e n in o u r s t u d y . L i p e m i c
8. Total bilirubin reagent - 10 ml of caffeine solution and 2.5
ml diazo reagent. Prepare freshly and use within 30 s p e c i m e n s did n o t i n t e r f e r e w i t h t h e a n a l y s i s s i n c e a
minutes. blank correction was employed.
Our proposed ABA-100 method uses a small sample
Procedure v o l u m e (10 ~1) a n d is s u i t a b l e for p e d i a t r i c s p e c i m e n s .
The instrument settings are made as described in Table 2. Recently, neonatologists have shown increased interest
The syringe plate and reservoir are filled with the direct in s e r u m d i r e c t b i l i r u b i n l e v e l s in t h e m a n a g e m e n t of
bilirubin reagent and the auxiliary dispenser is filled with the
n e o n a t a l h y p e r b i l i r u b i n e m i a (8, 9). O u r " i n - h o u s e " non-
stabilizer reagent with the auxiliary dispenser probe placed in
cup 26 position over the cuvette. A carousel containing the con- c o n j u g a t e d 200 m g / L b i l i r u b i n s t a n d a r d a n d t h e q u a l i t y
trols and patients' sera is placed on the instrument. Because of c o n t r o l m a t e r i a l s ( S p e c i m e n s 1-5, 12, T a b l e I) g a v e
 ONE-MINUTE DIRECT BILIRUBIN DETERMINATION
minimal direct readings, suggesting that the procedure
does not significantly overestimate values in the
presence of elevated non-conjugated bilirubin. The
minimal reading obtained from new-born specimens
(Specimens 13, 14, Table I} supported this suggestion.
In view of the difficulty of measuring direct-reacting
bilirubin, the proposed "one-minute" procedure offers a
useful automated method using an Abbott ABA-100
Analyser.
Total bilirubin can also be assayed with this pro-
cedure by employing a five-minute reaction period with
the total bilirubin reagent. In this case, the auxiliary
dispenser containing hydroxylamine is not used.
REFERENCES
1. Jaberg, C., McConnel, K., Potter, J.L. and Platt, M.S.
Evaluation of the ABA-100 Procedure for totaland direct
bilirubin in serum or plasma. Clin. Chem. 21, 1511-1512
(1975).
115
2. Mori, L. Modified Jendrassik-Grof Method for bilirubin
adapted to the A b b o t t Bichromatic Analyzer. Cli~ Chem.
24, 1841-1845 (1978).
3. Routh, J.I. Liver Function. In: Fundamentals of Clinical
Chemistry. Tietz, N.W. (ed.}, W.B. Saunders Company
(publisher), 1976, pp. 1037-1040.
4. Lathe, G.H. and Ruthven, C.R.J. Factors affecting the rate
of coupling of bilirubin and conjugated bilirubin in the van
den Bergh reaction. J. Cli~ PathoL 11, 155 (1958).
5. Chu, S.Y. and Turkington, V.E. Modification of ABA-100 for
two-reagent test procedure. Cli~. Chem. 27, 1045 (1981).
6. Sims, F.J. and Horn, C. Some observations on Powelrs
method for the determination of serum bilirubin. Am. J.
Clin. Palh. 29, 412 (1958).
7. Portnoy, A.L., Harrigan, T. and Likeas, P. Evaluation of ef-
fect of hemoglobin on the determination of bilirubin. Cli~
Chem. 27, 1037 {1981).
8. Poland, R.L., and Ostrea, E.M., Jr. In: Neonatal Hyper-
bilirubinemia in Care of the High-Risk Neonate, 2nd Ed.
Klaus and Faranoff (eds.).W.B. Saunders Co., 1981, pp.
243-266.
9. Harper, R.G., Sra, C.G. and Kierney, C.M.P. In: Neonatal
Intensive Care, Clinics in Perinatology, W.B. Saunders Co.,
1980. Vol. 7, pp. 75-88.