CS238020B1 - Method of polarographic blood analysis - Google Patents

Abstract

Method of polarographic analysis of blood consists in the serum filtrate and two sera of serum Ε ^ θθ and Ε ^ θθ obtained after separation by ion exchange chromatography dextran gel ion exchangers by elution solutions containing the final concentration chloride anions 320-330 mmol / l and 500-520 mmol / L is determined by polarographic double-wavelength and double-wavelength of said eluates and whole blood serum calculated! ratios as quotient and percentages quotient, distinguishing inflammatory and tumorous diseases, according to the formulas Brdičkov serum index --------------------------- - quotient K, Brdick's eluate index Ε ^ θθ Brdičkov eluate index Ε5θθ —------------------------- x 100 = percenBrdičkov serum index solid quotient.

Description

The subject of the invention is a method of blood polarographic analysis by determining a polarographic double-wave serum filtrate.

Elevated values of the polarographic double-wave serum filtrate (Brdičky index) can be found in both cancer and inflammatory diseases. It is a sensitive reaction, but its disadvantage is its non-specificity in relation to these diseases. It is not possible to determine from the Brdička index whether it is a cancer or an inflammatory disease.

This disadvantage is overcome by the method of blood polarographic analysis by determining the polarographic activity of the blood serum filtrate according to the invention, which consists in that the serum filtrate and serum eluates obtained after separation by elution ion exchange chromatography on dextran gel ion exchanger with elution solutions containing up to 320 330 mmol / l and 500-520 mmol / l are determined by a polarographic binary wave and from the observed binary values of the indicated eluates and whole blood serum the ratios as quotient and percentage quotient that differentiate inflammatory and tumor diseases are calculated according to

Brdičkov Index Serum Brdičkov Index of eluate Ε ^ θθ quotient K,

Brdička index eluate v

500

—.- x 100 r percentage quotient.

Brdička's serum index

To determine the clinical condition of the patient, they are used! graphs empirically obtained according to the above formulas.

Example 1 a d

Working solutions

1. Tris- / hydroxymethyl / aminomethane basic buffer: 50 mmol / l Tris-HCl buffer, adjusted to pH 7.0 with concentrated HCl

2. Elution Solution A: 10 mM NaCl solution in Tris base buffer

3. Elution solution B: 300 mmol / l Nation solution in Tris base buffer. The resulting chloride anion concentration must be in the range of 320 to 330 mmol / l

4. Elution Solution C: 500 mM NaCl solution in Tris base buffer. The resulting chloride anion concentration must be in the range of 500-520 mmol / l

5. Sephadex DEAE A-50 gel: 0.5 g of substance is swelled 12 hours before use in 100 ml of Tris base buffer.

6. 2 N ammonia (accurate)

7. Stock solution of hexamocobalt trichloride: 0,267 5 g of Co / NH4 / Clg and 5,35 g of NH4Cl are dissolved in 500 ml of distilled water

8. Brdička's trivalent cobalt solution: Mix solution no. 6 and 7 in a 1: 1 ratio

9. 20% aqueous sulfosalicylic acid solution

10. 0.1 N KOH

Solution no. 5 lasts about 2 weeks

3.5 ml of Sephadex DEAE A-50 swollen gel is pipetted into a glass or plastic column (used and cleaned porphyrin column) terminated with a frit having an internal diameter of about 8 mm.

The gel volume required is most preferably indicated on the column. The column thus filled is carefully washed with 2 ml of elution solution A. After the last drop has been dripped, 0.5 ml of blood serum is carefully pipetted onto the gel surface and allowed to soak.

Place a beaker marked as eluate A under the column. 1 ml of elution solution A is pipetted onto the gel surface and allowed to flow. 3 ml of elution solution A is then pipetted.

After dropping the last drop, eluate A is obtained.

Place a beaker, called eluate B, under the column. Pipette 1 ml of elution solution B onto the gel surface in the column and allow to run. 2 ml of elution solution B is then pipetted. After the last drop is dripped, eluate B is obtained and mixed.

Place a beaker labeled eluate C under the column. 1 ml of elution solution C is pipetted onto the gel surface in the column and allowed to flow. 1 ml of elution solution C is then pipetted.

After dropping the last drop, eluate C is obtained and mixed. Then, 0.4 ml of the serum to be examined is pipetted into the well marked S, into the well marked 0.6 ml of eluate B, into the well marked 0.6 ml of eluate C.

To these slides was added 1 ml of 0.1 N KOH solution, mixed and allowed to stand for 45 minutes. After this time, 1 ml of sulfosalicylic acid is added dropwise to these slides, mixed well and allowed to stand for 10 minutes. The precipitates are then filtered through Filtrak 391 filter paper.

The filtrate of eluates B and C must be clear after filtration. If it is not clear, 10 to 6 000 to 7 000 rpm must be centrifuged. Pipette 0.2 ml of the supernatant from the slide labeled S, from the slides labeled as Ε ^ θθ and Ε ^ θθ with 0.8 ml of the supernatant.

If _E3QQ and _{E5Q0 slides have been} centrifuged, the required amount of supernatant must be aspirated carefully to prevent the sedimentation of the sedimented proteins. Add 2.0 ml Brdička solution successively to all the slides, first slide S, then slide Ε ^ θθ and Ε ^ θθ and finally carry out a blank test consisting of 2,0 ml Brdička solution in the range 1,3 to 1 , 4 V.

For polarography, the range is: 0 to 4 V, polarograph of 0.8 V, sensitivity 1/1000, cathodic polarization, anode can also be realized with a silver wire, Coarse 0.1, Fine 0.04. The Brdicka index is calculated according to the formula

Corrected wave height of serum or eluate experiment = Brdicka index / Bl / Average wave height of normal serum

According to the above calculation, serum B1 and serum eluates ^Ε 30θ and ^E 500 'are descended

The detected B1 is inserted into the patterns

Bl sera

B1 eluate E _30q quotient K,

B1 eluate Ε _5θθ

- X 100 = percentage of the quotient / BI eluate _{E_-voltage} in percentage.

Bl serum ⁵⁰⁰

The graphs of FIG. 1 and FIG. 2. Both graphs were constructed from a set of 852 patients; 531 patients were diagnosed with verified cancer, 321 patients were diagnosed with acute and chronic inflammatory diseases. Serum B1 and quotient K1 values are shown in FIG. 1, where the quotient value K is calculated on the x-axis and the serum analyzed B1 on the y-axis, the serum B1 values and the percent heights B1 Ejqq in percent are shown in FIG. 2, where on the x-axis is expressed the height B1 Ejqq in percent and on the y-axis B1 is the serum analyzed.

Area A represents normal values, area B acute inflammatory processes, area C represents a group of chronic inflammatory and malignant diseases. Areas C and FIG. They represent areas of chronic inflammatory diseases and malignant diseases. Area A was determined by analysis of 84 blood donors, Area B, C, and a group of 852 patients.

The graph of FIG. 1 is used to distinguish acute inflammatory processes from the group of malignant and chronic inflammatory diseases. Chronic inflammatory processes are distinguished from malignant tumors by the graph of FIG. 2 when the clinical condition of the patient falls in the graph of FIG. 1 to area C.

The advantages of the method are in speed and reliability. The method is able to detect still clinically symptomless stages of malignant diseases. The cost per examination is low. The method is suitable for general biochemical laboratories in the field. Its sensitivity and specificity are statistically up to 90 4.

Claims (2)

OBJECT OF THE INVENTION A method polarografiekého analysis of the blood by determining the polarografiokej DvoJvlna filtrate of blood serum, characterized in that the determination DvoJvlna transferred in the sera and the two eluates serum Ε ^ θθ, obtained after separation by ion-exchange chromatography, the ion exchange type dextran gel eluting solution, the eluent E _{Q3 ( )} was obtained with an elution solution containing a final chloride anion concentration of 320 to 33U mmol / l and eluate E, qq was obtained with an elution solution containing a final chloride anion concentration of 500 to 520 mmol / l and from the determined polarographic double values of said eluates and whole blood sera are calculated as quotients distinguishing inflammatory and tumor diseases according to formulas Brdlček's serum index * quotient K, Brdička eluate index Ε _30θ Brdička eluate index E „____ ^x Ιθθ - percentage quotient. Brdička's serum index