WO1995000843A1

WO1995000843A1 - Assay for total bilirubin

Info

Publication number: WO1995000843A1
Application number: PCT/US1994/007157
Authority: WO
Inventors: Jerry W. Denney; Niall O'leary
Original assignee: Synermed, Inc.
Priority date: 1993-06-18
Filing date: 1994-06-17
Publication date: 1995-01-05
Also published as: AU7178194A

Abstract

An automated method for the assay of total bilirubin in bodily fluids, a solubilizing reagent for bilirubin and a diagnostic test kit for assay of total bilirubin. A first spectrophotometric measurement of the absorbance of bilirubin is made using two wavelengths, one of which is between about 450-480 nm and the other is between about 600-660 nm. The absorbance of bilirubin is destroyed by an oxidizing agent, and a second spectrophotometric measurement is made at the same two wavelengths. The assay of total bilirubin is calculated from the absorbance readings, along with the absorbance readings obtained from a test standard and a reagent blank.

Description

ASSAY FOR TOTAL BILIRUBIN The present invention relates to a method for the assay of total bilirubin in bodily fluids, and particularly to an automated method which makes use of a comparison of the absorbance of total bilirubin in a sample before and after oxidation of bilirubin by a suitable oxidizing agent. The invention also relates to a diagnostic kit for use in the assay of total bilirubin, and to a solubilizing agent useful in the method. Bilirubin is an orange-colored or yellowish substance found in bodily fluids such as blood serum, plasma and amniotic fluid. Bilirubin is formed as a product of the catabolism of hemoglobin. In most cases the catabolism of bilirubin is a normal bodily process, and only a small amount of bilirubin is present in the blood serum.

Bilirubin is not soluble in water, and is carried throughout the body in blood serum attached to serum albumin. The bilirubin is transported to the liver, where it is conjugated with glucuronic acid to form the diglucuronide. The bilirubin, in its water soluble conjugated glucuronide form, enters the biliary system for excretion in the bile.

Elevated serum bilirubin levels may be observed in a variety of conditions. These conditions include hemolytic disorders, biliary obstruction, cholestasis, hepatitis, cirrhosis and decreased conjugation (e.g. neonatal jaundice) . It is important that rapid and reliable methods for detection of such elevated serum bilirubin levels are available, so that a timely diagnosis of the condition may be obtained, and appropriate treatment measures can be taken in a timely fashion.

Total bilirubin assay must measure both the water soluble conjugated form, and the non-water soluble unconjugated form of bilirubin. Most bilirubin assays are based on the diazo reaction, first described by Ehrlich in 1883. A later diazo method originally described by Jendrassik and Grof in 1938 and later modified by Doumas et al. in 1973 is currently the method of choice for total bilirubin assay. This method, which employs an azosulfanilic acid, has been considered as the reference method by which new methods are compared.

Presently, most total bilirubin assays are performed in clinical laboratories using automated biochemical analyzers. The Jendrassik and Grof method is not widely used for these assays due to its poor suitability for automation. The Jendrassik and Grof method requires the use of as many as four separate reagents, while the most commonly used analyzers are designed to use only two reagents. Additionally, the azosulfanilic acid reagent used in the Jendrassik and Grof method is stable for only about one day and must be prepared regularly by the laboratory. Further, the ascorbic acid utilized in this method is unstable and must be prepared regularly. In U.S. Patent No. 3,569,721, Denney et al. attempted to improve the Jendrassik and Grof type method by replacing the ascorbic acid with hydroxyla ine salts. Although this method avoided the use of the unstable ascorbic acid, it was still necessary to use azosulfanilic acid. A number of other modifications of the diazo methods have been made involving different promoters and stabilizers in attempts to improve this method. In addition, several attempts have been made to simplify the diazo reaction for use with automated analyzers. Most of these attempts have used fast diazonium salts, such as

2,4-dichlorophenyl or 2,5-dichlorophenyl diazonium salts as diazo reagents. U.S. Patents 3,754,862 and 3,880,588 are representative of these attempts. R. Poon et al.. Clinical Chemistry. 31, pg. 92-94, (1985), noted severe interference in this type of diazo method due to the presence of indican in the serum of patients with renal failure. The interference from indican invalidates the results of the bilirubin test using these prior art methods for the analysis of the serum of such patients, and represents a serious problem in the use of these methods. The bilirubin content of infants has been measured by direct spectrophotometric estimation at 454 nm with correction at 540 nm. However, the method lacks specificity when applied to older children or adults due to the presence of carotene and other pigments in the serum, Tietz, N. Textbook of Clinical Chemistry. .B. Saunders Company, 1986 page 1386.

A device embodying direct spectrophotometric observation has been described in Goldberg, U.S. Patent No. 3,569,721. The specificity of the measurement of adult bilirubin by direct spectrophotometric observation has been improved by making observations before and after the destruction of bilirubin with the enzyme bilirubin oxidase. Although the enzyme treatment improves specificity, the enzyme reagents are unstable and do not solve the stability problems of the diazo methods. Additionally, enzymes are costly relative to diazo reagents.

Serious errors in prior art total bilirubin assays occur due to the presence of lipemia (fatty substances) and hemolysis (red blood cell contents) in the sample to be assayed. Glick has reported that a significant percentage of blood samples presented to clinical laboratories for assay contain these interferences. M. Glick, Interferocrraphs: User's Guide to Interferences in Clinical Chemistry Instruments. Second Edition, 1991, Science Enterprises, Inc. Indianapolis, IN. Glick et al. have reviewed the performance of most commonly used clinical chemistry automation in the presence of lipemia and hemolysis in the sample. Melvin R. Glick and Kenneth W. Ryder, Clinical Chemistry. 33, pg. 1453-1458, (1987) . The work of Glick indicates that bilirubin assays are among the most prone to interference from lipemia and hemolysis of all commonly performed diagnostic assays.

Accordingly, it is desired to provide a method for assay of total bilirubin in both adult and infant bodily fluid samples that may be performed utilizing automated chemical analyzers, that minimizes errors in the assay due to interferences and is capable of providing results comparable to present reference methods, and that is cost effective when compared to current reference methods. It is also desired to provide suitable reagents to carry out the process on automatic chemical analyzers.

It has been discovered that total bilirubin can be measured in bodily fluids by direct spectrophotometric means before and after chemically destroying the absorbance of bilirubin with a suitable oxidizing agent. Results can be achieved which correlate directly with the Jendrassik and Grof reference method.

The present invention comprises, in one form thereof, a method for assay of total bilirubin in bodily fluids. Only two stable reagents, a diluent and an oxidant are required to perform an assay. The sample is mixed with a suitable diluent which solubilizes both the conjugated and unconjugated bilirubin present in the sample. A first spectrophotometric measurement is made using two wavelengths, one of which is between about 450- 480 nm, and the other of which is between about 600-660 nm. The oxidant is added and, after a suitable incubation period, the measurement is again made at the two wavelengths. The absorbance at the primary wavelength is subtracted from the absorbance at the secondary wavelength for both a blank (prior to oxidant addition) and a test sample (after oxidant addition) . The absorbance change for the blank is subtracted from the absorbance change for the test sample to give a net absorbance change. A zero concentration blank and a sample of known bilirubin content are each similarly treated, and the respective absorbances are measured. The concentration of total bilirubin in the unknown is calculated proportionally from the difference in net absorbance between the blank and the standard, and the blank and the unknown. The present invention further comprises a reagent for solubilizing conjugated and unconjugated bilirubin for use in total bilirubin assay. The solubilizing reagent comprises, in one form thereof, a sufficient amount of a detergent to solubilize substantially all of the conjugated and unconjugated bilirubin in a solution of test sample, and a buffer sufficient to maintain a constant pH in the range of about 8.0 to 9.5 in the test sample solution.

The present invention further comprises, in another form thereof, a diagnostic kit for the assay of total bilirubin. The diagnostic kit comprises a solubilizing agent, comprising a sufficient amount of a detergent to solubilize substantially all of the conjugated and unconjugated bilirubin in a solution of test sample, and a buffer sufficient to maintain a constant pH in the range of about 8.0 to 9.5 in the test sample solution.

The kit further comprises a chemical oxidant including at least one of ferricyanide ion and hypochlorite ion.

The present invention provides a novel method for total bilirubin assay in a sample of a bodily fluid of unknown bilirubin content. Blood serum, plasma and amniotic fluid are generally used for bilirubin assay, although various other bodily fluids may also be utilized for analysis. The invention also provides a diagnostic kit useful for total bilirubin assay using automated chemical analyzers, and a solubilizing reagent useful for solubilizing both conjugated and unconjugated bilirubin present in a sample.

According to the present invention, total bilirubin is measured in a sample of bodily fluid, such as blood serum, by direct spectrophotometric means before and after destroying the absorbance of bilirubin with a suitable oxidizing agent. A suitable diluent is mixed with the sample of bodily fluid to solubilize both the conjugated and unconjugated bilirubin present in the sample. A first spectrophotometric measurement is made on this mixture of solubilized bilirubin at each of two separate wavelengths (i.e. the "primary" and the

"secondary" wavelengths) . A second measurement is made at these wavelengths after the absorbance of bilirubin has been destroyed by the oxidizing agent.

One of the wavelengths is selected to measure the characteristic bilirubin absorbance peak in the range of 450-480 nm, and the other wavelength is selected to be a blanking wavelength. The blanking wavelength is chosen from a range where bilirubin and the reagents utilized in the method exhibit no appreciable absorbance. This latter wavelength must be one at which hemoglobin exhibits a similar absorbance in both the solvent reagent only and after the addition of the oxidant, since the absorbance characteristics of hemoglobin change after the oxidizing agent is added. For example, hemoglobin has a significant absorbance at about 540-560 nm in the solvent reagent alone. After the oxidizing reagent is added, the absorbance at 540-560 is greatly diminished. The use of a secondary wavelength in this range would therefore cause overcorrection for hemolysis because the absorbance would be higher for a blank sample. By contrast, the absorbance of hemoglobin is more constant in the blank and test reagents between about 600-660 nm. Although the preferred absorbance is in the range of 600-660 nm, other wavelengths in which the reagents and the bilirubin exhibit no appreciable absorbance difference before and after the addition of the oxidant may also be suitable in a particular case.

A reagent blank, and a sample of known bilirubin content are treated in the same manner as the test sample. The absorbances are taken at the primary and secondary wavelengths, both before and after the addition of the oxidizing agent. The assay of total bilirubin in the test sample may then be calculated, in a manner to be described in greater detail hereinafter.

Unlike most analytes of interest in clinical chemistry, bilirubin exhibits a characteristic absorbance with its peak at about 450 nm. On oxidation of bilirubin to biliverdin, the optical peak at about 450 nm disappears. This absorbance change is the basis of several enzymatic bilirubin assays utilizing bilirubin oxidase. See, e.g. Perry et al. Measurement of Total Bilirubin by use of Bilirubin Oxidase, Clinical

Chemistry, 32, pg. 329-32, (1986). The present assay is predicated on the comparative absorbance of the test solution before and after the destruction of bilirubin by oxidation, rather than by enzymatic action. The method of the present invention is designed to measure total bilirubin, i.e. both the water soluble conjugated form and the non-water soluble unconjugated form. It is known in the art that unconjugated bilirubin must be solubilized in order to react in diazo reactions. See, e.g. Pearlman & Lee, Clinical Chemistry. 20, pg. 447-453, (1974). It is a new concept of the present invention that the bilirubin must be solubilized to have uniform absorbance between the conjugated and unconjugated forms of bilirubin, and also to participate in the oxidation reaction. The oxidation reaction in the present method is believed to be an electron transfer reaction. The molecules used are small inorganic molecules, in contrast to the relatively larger organic molecules used for diazotization reactions. The diazotization reaction forms new covalent bonding. Although it may not be surprising that such large molecules might not participate in chemical reactions due to stearic hindrance, etc., the non-reaction of small inorganic molecules is surprising. Solubilizing reagent

Having discovered that such solubilization is required, a number of solubilizing agents may be utilized in the present method. In order to achieve optimum results, an alkaline solution is desirable. It was discovered by the present inventors that a certain low level turbidity may be present in the wavelength range in nonalkaline solubilizing reagents used to detect the bilirubin absorbance (about 450-480 nm) , which turbidity may exhibit a significant absorbance in this range. When serum is added to the reagent, the turbidity may change. Water, as used in the blank, may behave differently as far as the clearing effect of the turbidity is concerned. This turbidity is prone to cause problems, particularly when an alkaline oxidizing reagent is utilized. If a non-alkaline solubilizing agent is utilized to solubilize the bilirubin, the initial readings taken (prior to addition of the alkaline oxidizing agent) include an additional absorbance factor due to this low level turbidity. If the sample is alkalinized upon addition of an alkaline oxidant, the turbidity clears. Thus, when the effect is present, there is a difference in background absorbance between measurements taken before and after oxidation, due to the clearing of the turbidity. The effect of this turbidity is believed to vary with the characteristics of the chemical instrument, and some instruments are more affected by the phenomenon than others.

Therefore, in order to avoid this phenomenon, in the preferred embodiment of the present invention, the solubilizing solution is alkaline. Thus, detergents which are insoluble under alkaline conditions are not useful in this preferred embodiment. For example, cationic detergents such as cetylperidium chloride and cetalkonium chloride, used in acidic medium as described in the Pearlman et a_l reference are not effective in alkaline embodiments. In contrast to Pearlman, et al, a zwitterionic detergent (having both cationic and anionic properties on the same molecule) cocoamido sulfobetaine is the preferred detergent for use in the solubilizing solution.

Certain substances in serum may form turbidity with cationic or anionic detergents. Free fatty acids, which are normally found in serum and plasma, and which may be present at very elevated levels in disease states such as diabetes, may precipitate cationic detergents and form turbidity. Additionally, cationic detergents may not be soluble in alkaline solution. Unconjugated bilirubin has free carboxylic acid functional groups which may complex with the cationic function of the zwitterionic detergent. The cationic function would then be necessary to solubilize the bilirubin-detergent complex. Cationic detergents might complex the bilirubin, but the bilirubin-detergent complex itself might be insoluble.

When selecting a detergent it is important that the detergent not contain reducing substances or antioxidants as preservatives. The inclusion of such compounds is common for certain detergents as supplied commercially, and is generally disclosed by a vendor, such as Sigma Chemical Company.

The preferred solubilizing reagent comprises a solution of the zwitterionic detergent cocoamido sulfobetaine at an alkaline pH. Other zwitterionic detergents such as CHAPS (3-[3-Cholamidopropyl)- dimethylamminonio]-l-propanesulfonate) and CHAPSO (3-[3- Cholamidopropyl)-dimethylamminonio]-2-hydroxy-l- propanesulfonate) are suitable and are used in a concentration in the solvent reagent of 15 grams per liter. However, these detergents are currently more expensive than cocoamido sulfobetaine.

Another suitable detergent is the zwitterionic detergent coco amido betaine, which has also been found to be useful and superior to the non-ionic detergents. Coco amido betaine is normally used in an amount of 20 ml per liter of solvent reagent. Both coco amido betaine and cocoamido sulfobetaine are believed to have amino groups as cationic group(s) . The sulfobetaine has sulfonic acid anionic group(s) , while the betaine has carboxy-anionic-functional groups. Both of these detergents are supplied by commercial sources, such as Lonza, Fair Lawn, New Jersey. The coco amido betaine is supplied by Lonza as Lonzaine^® C and the cocoamido sulfobetaine is supplied as Lonzaine^® CS.

Cocoamido sulfobetaine is also advantageous over many known solubilizing reagents because it does not form a heavy residue upon drying. Known bilirubin solubilizing reagents containing ingredients such as caffeine, sodium benzoate, sodium acetate and the like are prone to form crystalline deposits at the end of the dispense lines due to drying of the reagent at the tip, particularly after a prolonged period of disuse. Thus, automated chemical analyzers in which reagent lines remain filled with reagent can fail due to blockage of the lines with such deposits.

The preferred solubilizing reagent is described below:

Cocoamido sulfobetaine 15 ml

Tris 10.26 gm Tris hydrochloride 2.46 gm Pentachlorophenol 2 mg Tetrasodiu EDTA 1 gm

Sodium chloride 6 gm

The reagent may be prepared as follows: Add 15 ml of cocoamido sulfobetaine and the tetrasodium EDTA to about 800 ml of deionized water. Adjust the pH of the solution to about 8.5 with 10% NaOH or 10% HC1. After the EDTA is in solution and the pH has been adjusted as specified, add the remaining ingredients. The volume is then brought to one liter with deionized water. This reagent has a stability of at least one year when stored at 4°C.

The addition of the buffer salts maintains the pH at 8.5 at 37°C. The buffer has a concentration of tris which as about 0.1 molar. The use of the salt mixture simplifies preparation of the reagents as it makes temperature measurement and correction to pH 8.5 at 37°C unnecessary. The EDTA is added to avoid turbidity due to serum calcium and magnesium forming insoluble substances with serum phosphates, fatty acids and the like.

Pentachlorophenol is used as an antimicrobial agent to inhibit bacterial and fungal growth.

A variety of known antimicrobials can likewise be used, provided that they do not react with bilirubin or the oxidizing agent. ProClin^® 300 (a combination of 5- chloro-2-methyl-4-isothiazolin-3-one,2-methyl-4- isothiazolin-3-one, and alkyl carbonate) supplied by Rohm & Haas, Philadelphia, Pa., can be used in a concentration of about 0.5 ml per liter. Thimerosal in a concentration of 5 mg/liter and sodium azide in a concentration of 0.5 gm/liter may also be used. These latter compounds slightly decrease biliverdin formation in favor of a red compound, believed to be stercobilin. Thus, although the disappearance of bilirubin absorption can be measured, the assay may not be optimal with respect to sensitivity and specificity. Should the antimicrobial be acidic or basic, and be used in an amount which would affect the pH, then the antimicrobial should be added before initial pH adjustments are made. Anti-turbidity agents, such as the EDTA, oxalate or citrate are used since turbidity may occur in the absence of a complexing agent, due to the alkaline pH employed in certain of the embodiments of the invention.

Tris buffer systems are well known in the art. Tris(hydroxymethy)aminomethane is the basic salt of the buffer system, and tris(hydroxymethy)aminoethane hydrochloride is the acid salt. Tris buffers are known to exhibit different pH values at different temperatures. For example, a tris buffer which exhibits a pH of 8.51 at 37°C will show a pH of 8.8 at room temperature.

Automated biochemistry analyzers generally conduct assays at 37°C. Widely available buffer tables provide the relative amounts of tris(hydroxymethexy)aminomethane hydrochloride which will produce a desired pH at a desired temperature.

Although the solution described above is preferred, other solubilizing reagents may also be acceptable for use under certain conditions, and with certain chemical analyzers. These reagents do not generally exhibit the sensitivity shown by the preferred reagent under alkaline conditions, and they may cause blockage at the ends of the dispense lines upon dryness after prolonged periods of disuse in some instances.

Another preferred reagent includes boric acid (6.2 gm) , KC1 (7.4 gm) , tetrasodium EDTA (1.0 gm) , cocoamido sulfobetaine (15 ml) and pentachlorophenol (2 mg) . These ingredients are added to about 800 ml of deionized water and mixed. The pH of this solution is adjusted to pH 9.2 at room temperature with 50% NaOH. The KCl is added to "salt in" serum proteins and avoid the precipitation of certain abnormal serum proteins. A variety of salts having a concentration near isotonicity with serum are well known to be suitable, i.e. NaCl. It will be apparent to those of skill in the art that a variety of buffer salts having a pK near this chosen pH (9.2) could also be used. Although this reagent may be suitable for testing under a wide variety of conditions, it does not provide the overall sensitivity shown by use of the preferred reagent.

Many mixtures containing various combinations of caffeine, sodium benzoate and sodium acetate are known in the art to solubilize unconjugated bilirubin. These mixtures may also be used in the present method under some conditions, however the turbidity problem noted above must be considered when the results of tests utilizing these reagents are evaluated, particularly when an alkaline oxidizing agent is utilized.

Sodium benzoate may be used alone in concentrations of about 120 grams/liter, desirably with a non-ionic detergent or detergent mixture of mixed ionic detergent. A number of polyoxyethylene ether type detergents such as 23 Lauryl ether (Bridge 35) and Triton X-100, and block copolymers of propylene oxide and ethylene such as Prlunic L62 LF (BASF) may also be utilized. These non- ionic detergents appear to require the presence of a promoter, such as the sodium benzoate. In the absence of a promoter the reaction is slow. Additionally, the zwitterionic detergents, especially cocoamido sulfobetaine, appear to intensify the absorbance of the biliverdin reaction product. Caffeine used alone is not suitable as a solvent for the reaction, but it may act to accelerate the reaction. Oxidizing reagent The oxidizing solution comprises a chemical oxidizing agent having potential to oxidize virtually all of the bilirubin in the test sample. Oxidizing solutions including ferricyanide ions or hypochlorite ions are preferred, however other oxidants well known to those of ordinary skill in the art may be substituted.

The preferred oxidizing reagent includes ferricyanide ions, and is described below:

Potassium ferricyanide 1.65 grams Sodium chloride 9.0 grams The solution is prepared by mixing the above ingredients in 900 ml of deionized water until they are dissolved, then diluting to a volume of 1000 ml. The solution is stored under refrigeration in a light protected bottle. This reagent is stable for at least a year when stored at 4°C.

Another preferred oxidizing reagent is described below:

Sodium hypochlorite (5.25%w/v) 20 ml Sodium carbonate 15.0 gm Tetrasodium EDTA 0.1 gm

This solution is also prepared by adding the ingredients to 900 ml of deionized water and mixing until dissolved. The solution is then diluted to one liter. The solution is stored under refrigeration in a light protected bottle. It is also stable for at least a year at 4°C.

Test procedure Bichromatic chemistry analyzers are now in wide use in the art for assay of numerous analytes found in bodily fluids. According to the present invention, a bichromatic analyzer is utilized to calculate the total bilirubin in the test sample based upon the comparative absorbances of the bilirubin and its oxidation product. In the bichromatic assay using the reagents of the present invention, a wavelength is chosen in the range 450-480 nm, preferably 455 nm, and another wavelength is chosen in the range 600-660 nm, preferably 600 nm. The wavelength in the 450-480 nm range is selected to take advantage of the relatively strong bilirubin peak in this range.

The particular optimum wavelength within each range may be selected based upon certain criteria, which criteria may vary from test to test, and from instrument to instrument. For example, when the test reagents include ferricyanide and caffeine, a wavelength of 480 nm is preferred. Both ferricyanide and bilirubin exhibit a significant absorbance at 450 nm. The bilirubin absorbance declines only 30% at 480 nm, whereas the ferricyanide/caffeine absorbance is at a minimum at this wavelength, as shown in Figure 1. Figure 1 shows a spectral scan of icteric pool, 1/10, (bilirubin = 135 umol/L) and 1.0 mmol/L potassium ferricyanide in caffeine. The bilirubin comprised mised isomers from bovine gallstones, obtained from Sigma Chemical Co. Ltd. Poole, Dorset, U.K. The chemicals were of Analar grade, and supplied by BDH Chemicals, Poole, Dorset, U.K. The spectral scan was performed on the Shimadzu (Koyota, Japan) UV-VIS-160 recording spectrophoto eter. The 480 nm wavelength also has the advantage that oxidation of hemoglobin by hemolysed samples causes only a slight decrease at this wavelength.

Thus, it is seen that the wavelength chosen need not necessarily be the wavelength of maximum bilirubin absorbance, rather, other factors should also be considered. In addition, the absorbance of hemoglobin in the blank and test is more constant between about 460- 480 nm than at 450 nm. Some of the common analyzers, such as the Technicon

Axon™, have only the 455 nm wavelength available, but have a "blank correction factor" which can be used to adjust for these differences. 480 nm may be used as a primary wavelength on the Hitachi^® 704. Although hemoglobin has a higher absorbance after oxidation at this wavelength, the Hitachi system does not have a blank correction factor. Thus, there is a volume difference which causes a systematic overcorrection of the blank. The higher reading for hemoglobin after oxidation in the test is offset by this overcorrection by the blank. A determination of the optimum conditions for any one particular test is well within the knowledge of one skilled in the art, when combined with the teachings of the present invention. When ferricyanide ion is used as the bilirubin oxidant, the product of the reaction is believed to be biliverdin. Biliverdin has a broad absorbance from about

600 to 660 nm. ferricyanide ion bilirubin - > biliverdin

(yellow) (green)

(A_M„ 455 nm) (A_M„ 600-660 nm)

The optimum wavelength in this range for a particular analyzer, and with the use of particular test reagents may be determined by one of ordinary skill in the art without undue experimentation using the techniques described above with regard to the bilirubin adsorbance.

One of the wavelengths is designated as the primary wavelength, and the other wavelength is designated as the secondary wavelength in the set-up parameters for the analyzer. The choice of which wavelength is designated as the primary wavelength and which is designated as the secondary wavelength depends primarily on the ability of the instrument to correct for differences in volume in the dynamic serum blank.

The dynamic serum blank is performed by making an absorbance measurement on the mixture of solubilizing reagent (RI) and the serum sample, and subtracting this absorbance from the test which is made by adding the oxidizing reagent (R2) reagent to the RI plus sample mixture. The absorbance of background interferences tend to be lower in the test than in the blank because of the dilution of the sample produced by the addition of the R2 reagent. In general, the dynamic serum blank, if not corrected for this volume difference tends to overcorrect for background absorbance due to lipemia. The bichromatic correction using a higher wavelength tends to undercorrect for lipemia. Therefore, when using instruments lacking the ability to correct for volume difference, a wavelength in the range of 450-480 should be designated as the primary wavelength and a wavelength in the 630 region should be designated as secondary.

In order to minimize interference from hemoglobin, the following technique may be utilized. A solution of lysed red blood cells is prepared by centrifuging heparinized blood, decanting off the plasma and resuspending the cells in about four times their volume of deionized water. After a period of about five minutes during which the cells lyse, the mixture is again centrifuged to remove remaining cellular material. The resulting solution is tested to determine the effects of hemolysis. The prepared sample is treated as an unknown in the assay, and should give a value of near zero as a bilirubin result. If the value is significantly greater than 0, the volume of R2 (the oxidizing reagent) should be increased slightly. Correspondingly, if the value is 5 significantly less than 0, the volume of R2 should be decreased. Additionally, the wavelength in the operable range may be altered to minimize the effect of the hemoglobin absorbance. The preparation of a he olysate in this manner serves as a means to fine tune the method 0 to minimize this interference. It is not a routine process necessary to practice the invention.

The testing wavelength is selected between the range of about 450-480 and 600-660 nm on the basis of the available filters or wavelengths on the automated device, 5 the availability of a correction factor, and empirical results in assaying the hemolyzed sample thus prepared.

To practice the invention, 10 μl microliters of sample is added to 360 μl of solubilizing reagent RI. After five minutes, the absorbance of this solution was 0 measured at 480 nm and again at 600 nm. 90 μl of 5 mmol/L potassium ferricyanide reagent R2 is then added to the test sample, effecting a final concentration of 1.0 mmol/L. After an incubation period of five minutes, another absorbance reading was taken at each wavelength. 5 The incubation period may be varied with particular reagents and particular instruments, and need not necessarily be five minutes in all cases; however the present inventors have determined that a five minute period is preferred in most instances in order to obtain

30 a stable absorbance reading after oxidation.

A reagent test blank (tb) and a sample containing known bilirubin content (tst) are mixed, and absorbance readings are taken in the same manner as the test sample (ts) . A bilirubin concentration of about 5-10 mg/100 ml

35. is suitable. Bilirubin calibrators of this type are widely available to clinical laboratories. In accordance with general laboratory practice, a commercially supplied protein containing material, usually blood serum, which has been assayed by a reference method is used.

The instrument parameters for the automated assay on the Hitachi^® 704 are presented below: PARAMETER SETTING

Assay Code 2 point - 14 - 31

Sample vol 10 μl

Solubilizing Rgt Vol 360 μl Oxidizing Rgt Vol 90 μl Wavelength (analytical) 480 nm

Wavelength (blanking) 600 nm

Calibration method Linear (0) (0)

It is desirable that the biliverdin formed by the oxidation with ferricyanide be included in the measurement because the absorbance of biliverdin is only present after oxidation. Its absorbance is added to the absorbance of the bilirubin mathematically thereby increasing the sensitivity of the assay.

For analyzers such as the Axon™ having the capability to correct absorbance values for the dilution differences between the blank and test, greater sensitivity can be achieved at peak absorbance wavelengths for bilirubin and biliverdin. A wavelength combination of 455 and 620 nm has been found to be suitable for the Technicon Axon™ (Miles Inc.). The instrument parameters for use with the Axon™ automated blood chemistry system are provided below.

Calculation of Total Bilirubin

When the 480 is designated as primary, an automated device such as the Hitachi^® 704 measures the absorbance of the blank absorbance on the sample mixed with the RI reagent. The absorbance of this mixture at the 600 nm secondary wavelength is subtracted from that reading giving a net (sample) blank absorbance value. After the R2 is added to the mixture and incubated, the instrument again reads the absorbance at 480 nm and subtracts the absorbance at the 600 nm wavelength. When the bilirubin absorbing wavelength range (450-480 nm) is used as a primary wavelength, and the 600-650 nm biliverdin absorbing range is used as a secondary wavelength in the bichromatic assay with a dynamic serum blank, the reaction appears as a color loss reaction.

Other than serum chromatic substances such as hemoglobin and lipemia, the principal contribution to absorbance at the 450-480 nm wavelength is bilirubin. The principal contribution to the absorbance at the 600 nm wavelength is the oxidation product of the bilirubin and, if present, the oxidation product of hemoglobin. Thus, subtracting the sample blank value provides an indication of the amount of absorbance decrease due to the oxidation of bilirubin. The subtraction of the absorbance at the secondary wavelength of the final mixture causes a decrease in absorbance due to oxidation products. The combined decrease in absorbance is proportional to the bilirubin present. The net absorbance change (A^) is calculated as follows. A delta absorbance (ΔA) is derived by subtracting the absorbance at the primary wavelength (A„) from the absorbance at the secondary wavelength (A,) for both a sample blank (prior to oxidant addition) and test sample (after oxidant addition) . The term "sample blank" is used in this instance to refer to the test sample prior to addition of the oxidant. It does not refer to the reagent blank. The ΔA for the blank is then subtracted from the ΔA for the test to give a net absorbance change (A_NET) : A_NCT = Test ΔA - Blank ΔA = [A_p-A.]_τ.._t - [A_p-A.]„,.„„

The assay may be calibrated by treating water and the reagents as a zero concentration (reagent) blank, and utilizing a test standard consisting of protein solution containing about 7 grams per 100 cc. of protein and also containing a known amount of bilirubin. The concentration of total bilirubin in the unknown is calculated proportionally from the difference in net absorbance between the blank and the standard, and the blank and the unknown:

A_m* °f Unknown - A_H<S, of Blank

Cone, of unknown = x Cone, of Standard A_Ne, of Standard - A_Ne, of Blank

When the invention is applied to instruments having the ability to adjust for the difference in volume between the blank reading and the test reading, i.e. the Technicon Axon™, either wavelength may be used as the primary wavelength. When the 600-660 nm biliverdin absorbing range is designated as the primary wavelength (1°) , and the bilirubin absorbing wavelength range (450- 480 nm) is designated as the secondary wavelength (2°) in the bichromatic assay with a dynamic serum blank, the reaction appears as a color gain reaction. The absorbance due to bilirubin from the sample blank is in effect added to the absorbance due to the biliverdin in the final reaction (test) . It is a novel feature of the inventive method that the primary and secondary wavelengths can be reversed in a bichromatic, dynamic sample blank method.

It has been determined that there is virtually no deviation from linearity up to bilirubin concentrations of about 1000 umol/L with an absorbance change of 0.66 milli absorbance units/ umol/L of bilirubin in the method of the present invention. The inventive method is not significantly affected by increasing hemolysis and lipemia up to 5 g/L hemoglobin.

The mathematical manipulations used by bichromatic analyzers may be summarized as follows where A is absorbance, 1° is the primary wavelength and 2° is the secondary wavelength:

= Test Al° - Test A2° -[ Blank Al° - Blank A2°] = Test Al° - Test A2° - Blank Al° + Blank A2° The Test Al° is principally due to biliverdin. The Blank A2° is primarily due to bilirubin absorbance. All measurements of absorbance have a contribution from the background absorbance from lipemia or hemolysis. The Test A2° and Blank Al° principally measure background absorbance. Thus, the net absorbance change due to the presence of bilirubin is due to the combined absorbance of bilirubin and biliverdin, and the absorbance of the background tends to cancel as may be seen by substituting the principal contributors to the absorbance of each measurement:

Test Al° - Test A2° - Blank Al° + Blank A2° = (biliverdin absorbance + background absorbance) -

(background absorbance) - (background absorbance) + (bilirubin absorbance + background absorbance) = biliverdin absorbance + bilirubin absorbance +

2(background absorbance) - 2(background absorbance) = biliverdin absorbance + bilirubin absorbance

Appropriate care must be taken in applying the reagents of the present invention to various analyzers. The wavelength chosen is important also for optimal correction of interference from hemolysis when applying the invention to commonly used bichromatic analyzers lacking the ability to correct for different dilution of sample and reagent between the blank and test measurements. The absorbance of hemoglobin changes in the presence of the oxidants used in the present invention. The difference in absorbance can be minimized by selecting a primary and secondary wavelength within the range of 450-480 nm and 600-660 nm such that the absorbance of the oxidized hemoglobin and native hemoglobin are similar. Thus, although the maximum absorbance of bilirubin is at about 455 nm, the peak absorbance wavelength may not be optimal for the elimination of chromatic interferences. Thus, when using the Hitachi^® 704 analyzer, for example, the wavelength combination of 480 and 600 nm is satisfactory. Selection of a particular wavelength within the disclosed range to be used with any particular analyzer and with the appropriate reagents may be readily determined by one skilled in the art without undue experimentation when utilizing the principles of the present invention.

The bilirubin assay of the present method shows good agreement with the diazo/caffeine/benzoate method, and the all-method mean values quoted by the various manufacturers, in both human based and animal based tests. Close agreement with the animal based control methods with the diazo/benzoate/caffeine method indicates that carotenoids do not interfere with the ferricyanide method.

The inventive method has been successful in eliminating interference due to urinary indican. A 1 millimolar solution of 3-indoxyl sulfate, potassium salt (Urinary Indican) was prepared by dissolving 25.1 mg of Sigma 3-indoxyl sulfate, potassium salt (Urinary Indican) in 100 milliliter of deionized water. The mixture was then treated as an unknown sample and assayed using the present invention. No apparent bilirubin was detected. By contrast, R. Poon. et aJL. Clinical Chemistry, 31, pg. 92-94, (1985) demonstrated that as much as 80 mg/dl of apparent bilirubin was falsely indicated by similar levels with commercially supplied bilirubin reagents using diazo methods.

Claims

WHAT IS CLAIMED IS:

1. A method for total bilirubin assay in a sample, comprising the steps of: preparing a test sample (ts) by mixing a sample of unknown total bilirubin content with a solubilizing agent to solubilize the bilirubin present in the sample; preparing a test blank (tb) by mixing water with the solubilizing agent; preparing a test standard (tst) by mixing a sample of known total bilirubin content with the solubilizing agent; spectrophotometrically measuring the respective absorbances (A) of the test sample, test blank and test standard at a primary wavelength, and at a secondary wave1ength; calculating a delta absorbance (ΔA) (ts) for the test sample by subtracting the absorbance of the test sample at the primary wavelength (Al°) from the absorbance at the secondary wavelength (A2°) ; calculating a delta absorbance (ΔA) (tb) for the test blank by subtracting the absorbance of the test blank at the primary wavelength (Al°) from the absorbance at the secondary wavelength (A2°), calculating a delta absorbance (ΔA) (tst) for the test standard by subtracting the absorbance of the test standard at the primary wavelength (Al°) from the absorbance at the secondary wavelength (A2°) ; adding a sufficient amount of a chemical oxidizing agent to each of the test sample, test blank and test standard to oxidize substantially all of the bilirubin in the test sample; incubating each of the test sample, test blank and test standard for a period of time sufficient to oxidize substantially all of the bilirubin; after oxidation, spectrophotometrically measuring the respective absorbances (AOx) of the oxidized test sample, test blank and test standard at the primary wavelength, and at the secondary wavelength; calculating a delta absorbance (ΔAOx) (ts) for the oxidized test sample by subtracting the absorbance of the test sample at the primary wavelength (AOxl°) from the absorbance at the secondary wavelength (A0x2°) ; calculating a delta absorbance (ΔAOx) (tb) for the test blank by subtracting the absorbance of the test blank at the primary wavelength (A0xl°) from the absorbance at the secondary wavelength (A0x2°) ; calculating a delta absorbance (ΔAOx) (tst) for the test standard by subtracting the absorbance of the test standard at the primary wavelength (AOxl°) from the absorbance at the secondary wavelength (A0x2°) ; calculating the A_Nβt of the test sample by determining the difference between (ΔA) (ts) and (ΔAOx) (ts) ; calculating the A_Nβt of the test blank by determining the difference between (ΔA) (tb) and (ΔAOx) (tb) ; and calculating the A^ of the test standard by determining the difference between (ΔA) (tst) and (ΔAOx) (tst) ; calculating the concentration of total bilirubin in the test sample by the following equation:

^Aw-* of ^ts " A*-* of tb

Cone, of unknown = x Cone, of Standard

A,^, of tst - A^,, of tb

2. The method of claim 1, wherein the solubilizing agent has a pH of between 8 and 9.5.

3. The method of claim 2, wherein the pH is 8.5.

4. The method of claim 2, wherein the solubilizing agent includes cocoamido sulfobetaine.

5. The method of claim 3, wherein the solubilizing agent includes, per liter of reagent, 15 ml cocoamido sulfobetaine, 10.26 gm tris, 2.46 gm tris hydrochloride, 2 mg of a antimicrobial agent, and a sufficient amount of a strong acid or a strong base to bring the pH of the solubilizing agent to 8.5.

6. The method of claim 1, wherein the primary wavelength is in the range of one of (a) 450-480 nm and (b) 600-660 nm; and the secondary wavelength is in the range of the other of (a) 450-480 nm and (b) 600-660 nm.

7. The method of claim 1, wherein the solubilizing agent comprises a zwitterionic detergent.

8. The method of claim 1, wherein the chemical oxidizing agent includes at least one of ferricyanide ion and hypochlorite ion.

9. The method of claim 1, wherein the solubilizing agent comprises a sufficient amount of a zwitterionic detergent to solubilize substantially all of the bilirubin present in the test sample, and a sufficient amount of a buffer to maintain a constant pH in the test sample the range of about 8.0 to 9.5.

10. The method of claim 9, wherein the solution further includes an antimicrobial agent and an antiturbidity agent.

11. The method of claim 1, wherein the test sample, test blank and test standard are incubated at 37°C for at least five minutes.

12. The method of claim 7, wherein the zwitterionic detergent is at least one of cocoamido sulfobetaine and coco amido betaine.

13. The method of claim 1, wherein the solubilizing agent comprises sodium benzoate, EDTA and caffeine.

14. The method of claim 1, wherein the solubilizing agent comprises a non-ionic detergent in the presence of a reaction promoter.

15. A reagent for solubilizing conjugated and unconjugated bilirubin for use in total bilirubin assay, comprising: a sufficient amount of a detergent to solubilize substantially all of the conjugated and unconjugated bilirubin in a solution of test sample; and a buffer sufficient to maintain a constant pH in the range of about 8.0 to 9.5 in the test sample solution.

16. The reagent of claim 15, wherein the detergent is a zwitterionic detergent, further comprising an antimicrobial agent in an amount sufficient to inhibit bacterial and fungal growth in the test solution.

17. The reagent of claim 16, wherein the antimicrobial agent is pentachlorophenol.

18. The reagent of claim 16, wherein the zwitterionic detergent comprises at least one of cocoamido sulfobetaine and coco amido betaine.

19. The reagent of claim 15, wherein the buffer comprises a tris buffer system.

20. The reagent of claim 15, further comprising an antimicrobial agent and an antiturbidity agent.

21. The reagent of claim 15, wherein the solubilizing reagent comprises per liter of reagent, 15 ml cocoamido sulfobetaine, 10.26 gm tris, 2.46 gm tris hydrochloride, 2 mg of a antimicrobial agent, and a sufficient amount of a strong acid or a strong base to bring the pH of the solubilizing agent to 8.5.

22. The reagent of claim 15, further comprising a tris buffer in an amount sufficient to maintain a pH of 8.5 in a test sample.

23. A diagnostic kit for the assay of total bilirubin, comprising: a reagent for solubilizing conjugated and unconjugated bilirubin for use in total bilirubin assay comprising a sufficient amount of a zwitterionic detergent to solubilize substantially all of the conjugated and unconjugated bilirubin in a solution of test sample, and a buffer sufficient to maintain a constant pH in the range of about 8.0 to 9.5 in the test sample solution; and a chemical oxidizing agent in an amount sufficient to oxidize substantially all of the bilirubin present in the tst sample.