Method for eliminating hemolysis interference in an amylase analysis

A method for increasing the accuracy of photometric-based assays for .alpha.-amylase by subjecting a sample to a secondary interrogating beam of radiation at a wavelength distinguishable from a primary interrogating beam of radiation. The secondary interrogating beam of radiation is indicative of an interfering reaction occurring in the absence of analyte at the primary wavelength. The secondary wavelength is outside the absorption spectrum of the analyte of interest. This secondary radiation beam's absorption is proportional to the interfering reaction at the primary wavelength.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to photometric measurements useful in 
homogeneous medical diagnostic assays performed on an automatic chemical 
analyzer. More particularly, the invention pertains to the use of 
photometric reaction rate measurements made at a second wavelength to 
correct for the effect of non-specific reactions which interfere with 
reaction rate measurements made at a first wavelength of interest during 
an analysis for .alpha.-amylase. 
2. Description of the Related Art 
Amylases are a group of hydrolases that split complex carbohydrates 
constituted of .alpha.-D-glucose units linked through carbon atoms 1 and 4 
located on adjacent glucose residues. Animal amylases, including those 
present in humans, are .alpha.-amylases and can attack .alpha.-1 -4 
linkages in a random manner anywhere along the polyglucan chain. 
Qualitative kinetic determinations of .alpha.-amylase 
(n1,4-glucan-4-glucanohydrolase) activity in human serum and urine are 
helpful in diagnosis of diseases of the pancreas and in investigations of 
the pancreatic function. The diagnosis of acute pancreatitis is sometimes 
difficult since a distinction must be made from other acute 
intra-abdominal disorders with similar findings, such as perforated 
gastric or duodenal ulcer, intestinal obstruction, and mesenteric vascular 
obstruction. In addition, most common anticoagulants inhibit amylase 
activity because they chelate Ca(II); furthermore, citrate, EDTA and 
oxalate inhibit it by as much as 15%. As a consequence, amylase assays 
must be performed only on serum or heparinized plasma, however both of 
these may be imperfectly separated and other endogenous materials, such as 
red blood cells, may be present causing an inaccurate assay. 
Historical assay techniques were based on a change in the absorption maxima 
of a complex between starch and iodine as the .alpha.-amylase degraded the 
starch or on a measurement of the increase in reducing groups as the 
starch was hydrolyzed by the .alpha.-amylase. These methods are not as 
reliable and easy to quantitate as spectro-photometric methods using a 
defined substrate. A defined substrate, such as maltotetraose, is degraded 
by .alpha.-amylase to produce glucose which is then measured in a coupled 
enzyme assay. Although accurate, such methods also necessitate the 
complete removal of any endogenous glucose which can give an erroneous 
background reading within the assay. 
Synthetic substrates comprising nitro aromatic glycosides have been 
employed in .alpha.-amylase determinations, such as reported in U.S. Pat. 
No. 4,145,527. The .alpha.-amylase acts preferentially on the endo bonds 
to form smaller fragments and therefore in order to get complete action to 
generate the chromophore, e.g. nitrophenol, an additional supporting 
enzyme must be employed. The use of aromatic glycosides directly without 
the use of an additional supporting enzyme has generally proved to be 
impractical because of poor kinetics and/or poor rate of color release. 
One such assay involving a synthetic substrate has been described (Nature, 
182 (1958) 525-526) in which a p-nitrophenol derivative of maltose is 
used. The p-nitrophenol replaces the anomeric hydroxyl group of maltose. 
Amylase causes cleavage of the substrate to produce p-nitrophenol which 
can be monitored at 41 0 nm. However, the assay is 16 hours long and 
maltase also cleaves the substrate. 
In all of the above methods, it is generally recognized that 
spectrophotometric methods may be adversely affected by the presence of 
certain endogenous materials in body fluids. Typically this interference 
is attributed to absorbance of the interfering substance at the wavelength 
of interest. In many cases a sample blank is required to correct for the 
interference. Another approach is the use of rate techniques which, in 
effect, subtract out the absorbance attributable to the interfering 
compound. These approaches are limited to those situations where the 
absorbance due to the interference does not exceed the capacity of the 
spectrophotometer. In response to this concern, since many materials 
endogenous to body fluids absorb only at lower wavelengths (&lt;500 nm), 
there have been efforts to develop new substrates for enzyme 
determinations which absorb at wavelengths greater than 600 nm. Two such 
examples are disclosed in U.S. Pat. No. 4,933,277 and JP No. 2,306,990. 
Absorption measurements made at secondary or reference wavelengths 
(typically referred to as the blanking wavelength) are routinely used in 
spectrophotometric determinations to correct for errors that otherwise may 
arise due to gross environmental differences, such as air bubbles, debris, 
scratched surfaces, etc. Implicit in the selection of a reference 
wavelengths is the assumption that the gross environmental factors have 
similar effects across a wide absorbance range. Thus secondary or blanking 
wavelengths are normally chosen to be clearly outside the spectrum of the 
chromophore of interest and on the upper end of the absorbance spectrum. 
Unfortunately, these methods of the prior art fail to account for 
ancillary reactions which occur as a result of the presence of certain 
endogenous materials in bodily fluids. 
An endogenous substance may interfere with a spectro-photometric method by 
participating in a reaction, unrelated to the chromophore generating 
scheme, but which affects the absorbance of the assay matrix at the 
wavelength of interest. One .alpha.-amylase assay which encounters this 
interference reaction uses a synthetic substrate to measure activity, as 
described in U.S. Pat. 4,963,479. It is susceptible to such ancillary 
reaction interference by hemolyzed samples. The hemoglobin induces a 
negative bias in the absorption measurements which results in low 
estimates of .alpha.-amylase activity. The negative bias obtained with 500 
mg/dL hemolysate in the sample matrix has been observed to vary 
unpredictably from less than 10 International Unit/Liter (U/L) to greater 
than 40 U/L. In the absence of .alpha.-amylase the hemolysis effect is 
observed, after the hemolyzed serum is added to the reaction mixture, as a 
slow decrease in 405 nm. absorbance. The mechanism for the inhibition is 
not known, however it may be supposed that iron in the hemoglobin is 
reacting with either the thiocyanate or hydrazoic acid in the reaction 
mixture forming a product with a lower extinction coefficient at 405 nm. 
The lot to lot variability suggests that the rate of formation of this 
product is dependent on the presence of unknown trace substances in the 
reagent, such as metals or an as yet unidentified substance. 
SUMMARY OF THE INVENTION 
The present invention addresses a problem of the prior art analysis systems 
using photometric analysis in homogeneous assays, wherein absorption 
interference is caused by a spurious rate of change in absorbance due to 
ancillary chemical reactions between a substance or substances endogenous 
to body fluids, such as hemoglobin, and a component(s) of the reaction 
mixture. The present invention increases the accuracy of photometric-based 
enzyme directed assays by subjecting the reaction vessel to a secondary 
interrogating beam of radiation selected to have a wavelength 
distinguishable from a primary interrogating beam of radiation that has 
radiation absorption peak(s) peculiar to the chromophore of interest. The 
wavelength of the secondary interrogating beam of radiation is selected 
(1) to be out of the radiation absorbance peak(s) of the chromophore of 
interest, and (2) to provide a rate of absorbance change equal to or 
proportional to that absorbance rate observed in the absence of analyte at 
the primary wavelength. 
The assay reaction mixture, including the .alpha.-amylase and a chromogenic 
substrate, is subjected to a first or primary wavelength related to the 
primary reaction between .alpha.-amylase and the chromogenic substrate and 
the absorbance measured. Simultaneously, the assay reaction mixture is 
subjected to a second wavelength related only to the absorbance 
interference caused by the reaction of an endogenous substance with 
component(s) of the reaction mixture, and the absorbance measured. The 
rates of change in absorbance are substracted. This method thereby 
minimizes the effect of endogenous materials interfering with photometric 
determinations through unforeseen reactions that induce a spurious rate of 
change in the absorbance of the assay mixture. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
It is a common practice of the prior art to make spectro-photometric 
measurements at a single wavelength distinguishable from the primary 
interrogating beam of radiation so as to account for vagarite in the 
composition of the vessel 24 and/or spectral changes due to oddities in 
the reagent mixture, e.g. air bubbles, debris, etc. However, as mentioned 
previously, spectrophotometric measurements at a single wavelength do not 
correct for interference in homogeneous assays caused by an undesirable 
rate of change in absorbance due to unknown chemical reactions between a 
substance or substances endogenous to bodily fluids, such as hemolysis, 
and components of the reaction mixture, especially when such rates vary 
from one reagent lot to another. 
The present invention provides a method for increasing the accuracy of 
photometric-based assays by subjecting a uniform matrix (the assay 
reaction mixture) including .alpha.-amylase in a test sample, an 
interfering endogenous substance, and a chromogenic substrate for the 
.alpha.-amylase to a primary wavelength and to a second wavelength, 
measuring the rates of change in absorbance of the test sample matrix at 
each wavelength, and subtracting the rate of change in absorbance at the 
second wavelength from the rate of change in absorbance at the first 
wavelength. The secondary wavelength is selected to not be included within 
the absorption peak(s) of the chromophore of interest. The absorbance at 
the secondary wavelength also has a rate of change equal to or 
proportional to that observed in the absence of analyte at the primary 
wavelength. While it is known that endogenous materials may interfere with 
photometric determinations through unforeseen reactions that induce an 
undesirable rate of absorbance change within the assay mixture; the 
present invention corrects for the undesirable rate of change by 
subtracting the absorbance values, or a proportion thereof, preferably 
from 60 to 100 percent, most perferably 90 to 100 percent, of the assay 
mixture at a wavelength distinguishable from the primary interrogating 
beam of radiation. A key feature of this invention is that the interfering 
reaction must produce a change in the absorbance of the solution at a 
secondary wavelength which is outside the absorption spectrum of the 
component of interest. This change should be equal to or proportional to 
the absorbance change that is causing the interference at the primary 
wavelength. 
As shown below in Formula 1,the .alpha.-amylase assay involves the use of a 
chromogenic substrate preferably 2-chloro-4-nitrophenol linked with 
maltotriose in a fluid matrix. Other substrates which produce matrixes 
having a secondary reaction characterized by absorption outside the 
primary reaction's absorbance bandwidth are also contemplated within the 
bounds of the present invention. The direct reaction of .alpha.-amylase 
with the substrate results in the formation of 2-chloro-4-nitrophenol, 
which is monitored spectrophotometrically. The .alpha.-amylase hydrolyzes 
the 2-chloro-4-nitrophenyl-.alpha.-D-maltotrioside (CNPG3) to release 
2-chloro-4-nitrophenol (CNP) and form 2-chloro-4- 20 
nitrophenyl-.alpha.-D-maltoside (CNPG2), maltotriose (G3) and glucose (G). 
The rate of formation of the CNP can be detected spectrophotometrically at 
405 nm (the primary wavelength) to give a direct measurement of 
.alpha.-amylase activity in the sample. The reaction proceeds rapidly, and 
can be easily automated. No coupling enzymes are required however, the 
reaction has been observed to be inhibited by endogenous factors. 
##STR1## 
Based on the absorption spectra of CNP which peaks at 405 nm and drops to a 
low level at 500 nm, conventional "blanking" techniques practiced in the 
art would recommend a reference wavelength of 510 nm for the 
aforedescribed assay. As such and as discussed hereinbefore, the assay 
would inaccurately account for the .alpha.-amylase concentration in 
hemolyzed samples. In addition, lot-to-lot variations in the amylase 
content of individual samples are observed. According to this invention 
the second or "blanking" wavelength is selected to be 577 nm. It can 
actually fall in the range of about 565 nm to 585 nm. At this wavelength, 
all of the inhibiting effect of endogenous factors are experienced, the 
same as at 405 nm, and yet at this wavelength the method is essentially 
non-responsive to .alpha.-amylase. 
Table 1 shows a comparison of the results obtained when hemolysate spiked 
samples, lot GB5192,lot CD5084 and lot D50029, are assayed with three 
different lots of AMY Flex.TM. reagent cartridges (DF17A) using 510 nm as 
a blanking wavelength and using 577 nm as an ancillary reaction rate 
measurement. When performed according to the method of the present 
invention, accounting for the 577 nm ancillary reaction rate measurement 
significantly improves the accuracy of and the lot to lot consistency of 
the .alpha.-amylase measurements. 
TABLE 1 
______________________________________ 
Hemolysate 
510 nm 577 nm 
mg/dL GB5192 CD5084 D50029 
GB5192 
CD5084 
D50029 
______________________________________ 
0 50 50 50 50 50 50 
100 44 47 43 48 48 47 
200 42 46 40 48 49 47 
300 38 45 38 46 49 46 
400 37 44 36 47 49 46 
500 34 42 34 46 48 46 
______________________________________

EXAMPLE 
Enzymatic spectrophotometric assay for .alpha.-amylase with 
2-chloro-4-nitrophenyl-.alpha.-D-maltotrioside 
The reagent CNPG3 is available commercially under the tradename AMY 
Flex.TM. reagent cartridge (DF17A) which is intended for the detection of 
.alpha.-amylase in human specimens using the DuPont DIMENSION.RTM. 
Clinical Chemistry System. 
The assay protocol for .alpha.-amylase involves the use of 
2-chloro-4-nitrophenyl-.alpha.-D-maltotrioside reagent, hereafter 
designated CNPG3, being a solution of 2.25 millimoles/L 
2-chloro-4-nitrophenyl-.alpha.-D-maltotrioside in 0.5 molar 
2-N-(morpholino)-ethanesulfonic acid (MES) buffer, pH =6.0,containing 350 
millimoles/L of sodium chloride, 6 millimoles/L of calcium acetate and 900 
mM thiocyanate and/or 0.1 % azide. 
The following procedural method can be used to perform an .alpha.-amylase 
assay using the apparatus of this invention in the DuPont DIMENSION.RTM. 
Clinical Chemistry system. 
Procedure 1. Prior to testing specimens containing unknown concentrations 
of .alpha.-amylase, three verifier samples are normally tested in a 
"Verification" mode of the system. The "assigned values" of each verifier 
are manually entered into the Dimension.RTM. computer and appropriate 
verifiers and reagent cartridge are loaded on the system. After the tests 
are completed, the computer automatically performs a mathematical 
regression using the signals and assigned-values of all three verifiers. A 
verification slope between 0.90 to 1.10 indicates that the system is 
performing properly. 2. An amylase test is scheduled on computer. A sample 
specimen is placed in a sample well and the AMY Flex.TM. reagent cartridge 
is loaded onto sample transport means. The sequence of events by which the 
system performs the .alpha.-amylase test follow. 3. Upon receiving 
commands to perform an .alpha.-amylase test, the system forms a reaction 
vessel situated around the perimeter of the sample transport means. 4. A 
220 uL aliquot of CNPG3 is automatically withdrawn from the AMY Flex.TM. 
reagent cartridge and dispensed, followed by 130 uL water, into the 
reaction vessel and mixed. 5. After 60 to 102 seconds, a 14 uL sample of 
the specimen is withdrawn from the sample well and dispensed, followed by 
36 uL water, into the reaction vessel and mixed to provide a uniform 
matrix comprising the specimen and CNPG3 by thoroughly mixing the specimen 
with the CNPG3 solution. 6. After approximately 72 seconds, the absorbance 
at ten wavelengths are measured. The difference of absorbance between 405 
nm and 577 nm is computed and recorded as rA. 
The exact time of the first reading is recorded and designated r1. 7. 
Approximately 144 seconds after the initial reading, the absorbance of 
reaction vessel 23 at 10 wavelengths is measured again. The difference of 
absorbance between 405 nm and 577 nm is computed and recorded as rB. The 
exact time of the second reading is recorded and designated r2. 8. The 
rate of absorbance change is calculated as .DELTA.mA/min. 
EQU .DELTA.mA/min.=(rB-rA)/(r2-r1). 
The .alpha.-amylase activity of each sample and control is known to be 
determinable using the following formula: 
EQU .alpha.-amylase (U/L)=5.4 * .DELTA.mA/min. 
Where: 
.DELTA.A/min.=Change in milli absorbance units per minute for the sample or 
control, and 
5.4 =Conversion factor.