DE3212795A1 - Method for the rapid determination of total and direct bilirubin - Google Patents

Description

3 April 5th

P 8765 - res

Dr. Augusta B r e g a, Brescia, Italy

Method for the rapid determination of total and direct bilirubin

The invention relates to a method for the rapid determination of the total and direct bilirubin in the biological Liquids with monoreagents (reactants). Since the introduction in 1883 due to the merits of Ehrlich, the diazotization reaction and the subsequent Representation (demonstration) of the direct and indirect reactions due to the work of Van den Berg are numerous attempts have been made to provide a quick and easy method for the determination (representation) of bilirubin in biological fluids.

Many decoupling reagents are suggested from ethanol, caffeine, benzoate to tension-active substances; there are several suggestions for the determination or definition of the reading time of the direct bilirubin has been formulated, whereby finally

a time of 5 minutes has been set after the vote; the problem of choosing the Final pH debated, whereby one has finally accepted or accepted essentially the alkaline environment.

In any case, the procedures proposed so far are quite complicated as they require the following: Prepare of white for each sample, different or diversified Treatment for the two types of reaction, alkalization, precise, strict control of the times in the various analytical passages and finally instability of the di-azotization reagent.

A method (DPD method) has recently been proposed using a monoreagent (monoreagent) for the dosing of the total bilirubin, whereby one has a stabilizing diazotizing agent used, but also in this case the preparation of the white sample ("bianco campione ") necessary.

In contrast, the aim of the present invention is to provide a stable diazotizing agent Fast red B (CI 37125) BF ₄ -SaIz (and the synonyms: l-äminö-2-methoxy-4-nitrobenzene diazotate tetrafluoroborate; azoik diazo N ₅ , BF ₄ ; 5-nitro-2 aminoanisole diazotate tetrafluoroborate; 5-nitro-2 amino-methoxybenzene diazotate tetrafluoroborate) with the formula C ₇ Hg N ^ 0 ^ · BF. and with a molecular

weight of 266.9 for the determination of both total and direct bilirubin without preparing the white sample also for sera with obvious hemolysis for both the Use by hand as well as with the help of automatic systems of any kind.

The method according to the invention is characterized in that that one uses Fast-red-B.BF.-Salζ in an acidic solution, the decoupling or decoupling means adapted to the goal or the task, z. B. urea and methanol, contains to determine and display the bilirubin, further thereby characterized in that the white sample ("bianco campione") is eliminated, and that the simultaneous determination is made des (total and direct) bilirubin with the same single reactant (reagent) made possible by simply use sodium azide (or other reducing substances) in a suitable concentration as an inhibitor of the diazotization reaction of indirect bilirubin, and also by interfering (superimposing) with the Prevents hemoglobin (in sera with hemolysis) by adding potassium iodide in a suitable concentration.

Another feature of the proposed with the present invention Method consists in the fact that there is a stable monoreacting agent (monoreagent) for both Forms of bilirubin used and not required white sample, creating a stable colored mixture in this way

(Composition) arises with a high absorption coefficient in a short time (about 15 minutes).

According to the present invention, the total and react in the presence of urea and methanol in an acidic environment direct bilirubins with a salt of the stabilized diazon (Fast-red-B.BF * -Salζ), with a green-blue colored Azo connection is formed. The indirect bilirubin response is enhanced by pretreating the sample with a reducing agents inhibited. The developed color, stable for at least two hours and read at 592 nm (580 to 610 nm), is proportional to the concentration of the Bilirubins up to 25 mg / dl.

In order to achieve the aim that the present invention has set, it is necessary to use two reactants for the to use both types of bilirubin, which are present in the solution of the dye and the decppler in an acidic environment (reactant A) and in the solution of the reducing agent with a well-defined concentration (reaction agent B) exist. Incidentally, a third reactant (Reagent C) which is from a solution may sometimes be useful of potassium iodide to eliminate the interference (superimposition) by the hemoglobin.

A typical composition is as follows: Reagent A: Fast-red-B.BF.- salt 0.4 mmol / 1,

Carbamide 4 mol / 1; Methanol 1 mol / 1; pH

Reagent B: sodium azide 0.15 mol / 1 Reagent C: KI 0.6 mol / 1

Example:

Pipetting
in the reagent
Glass White Sample for
total
BiIirub in Sample for
direct
BiIirub in H ₂ O 0.2 ml - - serum - 0.2 ml 0.2 ml Reaction
medium B - - 20 jul Reaction
medium A 2.0 ml 2.0 ml 2.0 ml

Mix; wait at least 15 minutes; at 592 (580 - 610) nm read the absorbance of the various samples with the setting at zero against the white.

invoice

Bilirubin (total or direct) mg / dl = Ax 10 Sr bilirubin (total) or (ester) cz.sz. u mol / 1 = A χ 171

Reference intervals

Total bilirubin: 1 mg / dl

Sr bilirubin (total) cz.sz. = 4-17 µmol / 1 Direct bilirubin: <0.35 mg / dl

Sr-BiIirübin (Ester) cz.sz. = < 6 µmol / 1

The method for achieving the purpose according to the present invention can be summed up in the following points:

1. Use fresh serum and avoid direct Light on the sample;

2. the analysis can be carried out with micro-methods, using the same calculation factor and proportional the volumes decreased;

3. The samples with hemolysis are treated by directly to the serum 10 ju 1 of the reagent C / 0.2 ml of serum adding, thus eliminating interference (superimposition) by hemoglobin up to 400 mg / dl;

4. To control the quality, sera with bilirubin in the development stage (statu nascendi), without that preservatives or the like are added (normal Roche brand serum, Clinicontrol N Sclavo, Seronorm Bracco etc.) or one compares or confronts the data obtained by using the classic method used.

The experiments according to the method according to the invention were

I P

I '"""''"' * £ "» · ■ · - 321279ο

performed with sera from hyperbilirubinemic patients, comparing the data obtained with the Jendrassik method.

The · diazotization reaction is caused by reducing substances inhibits, which is why sera from patients who are undergoing massive treatment with ascorbic acid, Can result in yards that are wrong and low.

Results obtained

Lin'earita't: A hyperbiotic rubinemic serum with an egg concentration of total bilirubin of 30 mg / dl was used, which was used with the Jendrassik method, following the instructions and indications of Schellong and; Has followed the turn. Using the indicated volumes, the method is linear up to at least 20 mg / dl ^ standard of commercial bilirubin (e.g. bilirubin standard Behring). There will be no suitable results unless at very high dilutions.

Repeatability: The analysis (n = 40) of a normal serum (bilirubin = 0.7 mg / dL) shows a coefficient for the percentage variation of 2.5.

Correlation: Parallel dosages of total and direct bilirubin are in 70 normal and pathological sets with the present method and with the procedure

to Jendrassik.

The correlation is optimal for both forms of biirubin (direct bilirubin: η = 70; r = 0.9974; a = -7.067; b = 0.9480; total bilirubin: η = 70; r = 0.9886; a = 0.7358; b = 0.8828).

Development of the color and effect of the reducing substances : The development of the color of the reaction mixture is obtained in two to three minutes if samples with a high concentration of bilirubin are also used; in any case, it reaches the maximum intensity within 15 minutes.

However, when using sera from patients who are being treated with reducing substances (ascorbic acid etc.), the color develops more slowly or even partially depending on the concentration of the reducing substance.

Use of sodium azide in the determination and representation of direct bilirubin: The phenomenon of the inhibition of the diazotization reaction due to the action of reducing substances is exploited in the present method by using sodium azide in the determination of direct bilirubin. In fact, if one uses 100 ^ g of NaNg / l.lml of a reaction mixture

AA

det, values for the direct bilirubin that perfectly correspond to and correlate with those obtained with the Jendrassik method (r = 0.9986).

Characteristics of the chromogenic reactant: The chromogenic reactant has a light, delicate yellow color and is stable at 4 degrees Celsius for at least 6 months. The colored complex has a molar absorbance or absorbance

3 -1 -1 tion of approx. 80 χ 10 1st mol. cm at 592 nm, stable for a few hours. It has an absorbance spectrum with a maximum between 590 and 600 nm.

Sodium azide and potassium iodide change in concentrations The use in the present process graded the reactant, however changing it when it was just added not the absorbance spectrum.

Interference due to the effects of potassium iodide: The procedure not only senses the reducing substances but also the hemoglobin, even in low concentrations. The addition of potassium iodide to the serum in the final concentration of 0.5 to 5.0 μl allows correct dosing even in the presence of hemolysis up to 400 mg / dl of hemoglobin. Hyperlipemic sera do not give any significant interference.

Accuracy: Various commercial control sera have been analyzed (Table 1), some show values that are considerably below the predetermined value, probably for the type of preservative added.

Intervals for reference: The study related to both forms of bilirubin was carried out on normal people from outpatient departments as well as on blood donors. It should be noted that the blood donors show values that tend to be higher.

Table 1 - Results of the analysis of the control sera:

Controls company value Theoretical
value H Serum control N Roche 1.30 1.25 104 Serum control A Roche 2.70 3.2 84 Control boxes; L Ist.Behring 1.37 1.30 105 Seronorm Nyegaard 1.72 1.77 97 SVRS Welcome 3.50 3.78 92 Clinicontrol N Sclavo 1.15 1.20 96 Precinorm U Bi ochemia 1.05 1.61 65

Table 2 - Reference intervals of total and direct bilirubin dosed with two methods

have been (mg / dl):

- 11 -

ι
object Total bilirubins Direct bilirubins Procedure
ren jen-
drassik invented
dunqsgem.
procedure procedure
Jendrassi k Normal
persons
Blood donor erfi η -
appropriately
procedure 0.68 ± 0.26 0.21 ^ 0.12
0.25 ^ 0.09 0.18-0.11 0.69 ± 0.25
0.83 ^ 0.22

Claims (4)

Ψ m · · P 8765 - res Dr. Augusta B r e g a, Brescia, Italy, method for the rapid determination of total and direct bilirubin Claims: 1. j Method for quickly determining the total and direct bilirubins in the biological fluids with monoreagents, characterized in that "Fast-Red-B.BF ₄ " salt is used in an acidic solution which contains decoupling agents or decoupling agents adapted to the objective or task, e.g. B. urea or methanol contains. 2. The method according to claim 1, characterized in that the white sample ("bianco campione") is eliminated. 3. The method according to claim 1 or 2, characterized in that the determination of the total and direct Bilirubins with the same single reactant (reagent) made possible by simply taking sodium azide or other reducing agents Substances in a suitable concentration as an inhibitor the diazotization reaction of indirect bilirubin. 4. The method according to any one of claims 1 to 3, characterized in that that interference (overlaying) by the hemoglobin present in sera with hemolysis is prevented, by adding potassium iodide in a suitable concentration,