CH648128A5 - METHOD FOR DETERMINING VALUE AS A DIAGNOSTIC INDICATOR OF A PERSON'S BLOOD SUGAR STATE. - Google Patents

Description

The invention relates to a method for determining a value as a diagnostic indicator of a person's blood sugar level.

In biochemistry, hemoglobins are the amphoteric protein molecules of the red blood cells that serve to deliver oxygen to the tissue. There are various chromatographically separable subordinate hemoglobins in the hemolysates of the red blood cells of normal people. Certain subordinate hemoglobins are called Hb-A |. "Hb-A | b, Hb-A | C, Hb-Aw and Hb-A | e. The hemoglobin type Hb-Alc is the most prominent type and represents the majority of the subordinate hemoglobins. It is known that the level of hemoglobin Hb-A] C is related to the average blood sugar level of a patient or person. In normal people, 3 to 6% of Hb-Aic is expected relative to the total hemoglobin content. Untreated diabetics can have 6 to 12% Hb-A | C relative to total hemoglobin, depending on whether the disease is of the type that develops in adolescence or of the type that forms in adults. It is further understood that the levels or amounts of the hemoglobin types Hb-A) a c as a separate and identifiable subgroup over a longer period of time are an indicator of the degree of diabetes, i.e. an excess of sugar in the blood.

The literature regarding the diagnosis of abnormal blood sugar levels by determining and measuring the value or level of the hemoglobin types Hb-A | C includes:

(1) The Relation Between the Minor Components of Whole Normal Human Adult Hemoglobin as Isolated by Chromatography and Starch Block Electrophoresis, Schnek and Schroeder, Journal of the American Chemical Society, Vol. 83, pp. 1472-1478, March 1961;

(2) Hemoglobin Components in Patients with Diabetes Mellitus, Trivelli, et al, New England Journal of Medicine, Vol. 84, pages 353-357, February 1971;

(3) The Biosynthesis of Human Hemoglobin Aic, Bunn, et al, Journal of Clinical Investigation, Vol. 57, pp. 1652-1659, June 1976;

(4) Correlation of Glucose Regulation And Hemoglobin A | C in Diabetes Mellitius, Koenig, et al, New England Journal of Medicine, Vol. 295, pp. 417-420, August 1976;

(5) Red Cell Age-Related Changes of Hemoglobins Aia + b and A) c in Normal and Diabetic Subjects, Fitzgibbons, et al, Journal of Clinical Investigation, Vol. 58, pp. 820-824, October 1976;

(6) Glycosylated Hemoglobins and Long-Term Blood Glucose Control in Diabetes Mellitus, Gabbay, et al, Journal of Clinical Endocrinology and Metabolism, Vol. 44, pp. 859-864, 1977; and

(7) Rapid Estimation (2xli Hours) of Glycosylated Hemoglobin For Routine Purposes, Kynoch and Lehmann, The Lancet, p. 16, July 1977.

The literature on the chromatography of hemoglobins using miniature columns is mentioned: Horton and Chernoff, J. Chromatog., Vol. 47, pp. 493-498 (1970).

To date, the clinical techniques and procedures for determining the level of hemoglobin type Hb-A) c have the disadvantage that they require complicated equipment and a test period of several hours or even days.

It has been found that the ratio of the sub-

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Group of hemoglobin types Hb-Aia_c to total hemoglobins Hb can be determined quickly, cheaply and precisely as a numerical percentage. Such a numerical percentage is available for use as a diagnostic indicator of the blood glucose properties of a person under investigation and considered a possible diabetic.

It is an object of the invention to provide a method by which a value is determined which can serve as a diagnostic indicator of the blood sugar status of a particular person.

Another purpose of the invention is to provide a method by means of which the ratio of the subgroup of hemoglobin types Hb-A] a_ic to the total hemoglobin Hb present in the blood of a certain person is determined, measured, quickly, cheaply and precisely.

It is a further purpose of the invention to provide a method which, although it requires a number of consecutive steps, is of a type which enables the use of procedures and protocols which become a standard and routine so that people who are experienced in clinical chemistry technology can repeatedly and accurately test the blood of large groups of people to create a database for use by qualified, specialized, and medically trained personnel to diagnose the blood sugar level of certain individuals, that are considered possible diabetics.

The inventive method is characterized by the features 30 of claim 1.

The invention is explained below with reference to the drawings, for example, in the figures of which various embodiments of the invention are illustrated and explained.

Fig. I is a schematic representation of the first general embodiment of the method according to the first particular embodiment, the chromatographic column being shown essentially in the actual size and with corresponding dimensions.

40 FIG. 2 is a schematic illustration, similar to that of FIG. 1, and shows the second particular embodiment of the method.

FIG. 3 is a schematic illustration similar to that of FIGS. 1 and 2 and shows the third, fourth and fifth special embodiment of the method.

There are at least two procedures for preparing a suitable test sample, generally designated by the reference numeral 10, the test sample containing a hemolysate solution of red blood cells from a whole blood sample. A method of operation 10a is used to prepare a test sample 10, which consists predominantly of the hemoglobin content of the whole blood sample. A procedure 10b is used to prepare a test sample 10, which may include the plasma proteins, lipids, and the residues of the white 55 blood cells and the red blood cells in addition to the hemoglobin content of the whole blood sample.

Operation 10a may include centrifuging the red blood cells from a whole blood sample that has been treated with a conventional anticoagulant such as EDTA (ethy-6o-lendiaminetetraacetic acid). After decanting the supernatant, the precipitate in the centrifuge tube must be washed thoroughly using a physiological saline. Preferably, the washing process is repeated three times using 0.85% saline and using centrifugation. The washed precipitate or deposit is then dissolved, for example by vigorous mixing with a preferably equal volume of distilled water.

648 128 4

tem water and for about 2 min. The lysate obtained is of a size which is predominantly a hemolysate through a sieve with a clear mesh size, although small amounts of from 0.30 to 0.60 mm (100 to 400 mesh) pass through. That contains proteins, lipoids and cell wall residues. The Ly resin consists of a common copolymer of meth-

sat can be mixed with a solvent, acrylic acid and divinylbenzene, which are negatively charged. A volume of 1 of the 5 carboxyl groups is preferably contained. A resin of this type is under the

Lysates available with a volume of 0.2 carbon tetrachloride called "Amberlite CG-50".

(CCI4) strongly mixed, then for 30 min at room temperature- The commercially available forms of ion exchange-

and then centrifugal resin particles 27 for the last time must be used in the tube 22

yaws. The supernatant obtained (after dilution) is that of the microcolumn 20 between the disks 25 and 26 prepa-

Test sample 10 of the hemolysate solution from red blood cells is treated or treated. Such treatment could be carried out for the purpose of separating, determining and measuring the amount when the resin particles 27 are in their group of existing hemoglobin types, namely in the column tube 22. However, it is

according to the invention. prefers that the resin particles 27 for a number of identical

A method of operation 1 Whether involves collecting a relatively large number of columns 20 to be treated, using a batch small volume of whole blood sample, for example 15 technique that allows the use of columns 20, using a microhaematocrit. One volume of the predetermined microchromatographic properties of an entire blood gas sample is obtained with four volumes of water.

mixes. Preferably, one drop of whole blood is used. The resin particles 27 are used in the tube 22 of the sample and four drops of distilled water in a small le 20 in the form of a suspension which is shaken vigorously with 27 (S) test tubes. Then the test tube is drawn and a predetermined pH or one is left for 5 min at room temperature. According to an initial pH. The particles 27 are first converted to a further and last mixing comprising the Susner sodium form obtained, the H + ions on ih-pension (without dilution) being shifted to a test sample 10 of a haemolene, namely by means of complete mi-sat solution from red blood cells for separation, detection and see with an excess amount of sodium hydroxide (when measuring the amount of a group of hemoglobin present, for example 1N NaOH). The excess NaOH solution is binary according to the invention. and any remaining unscreened or fine resin

It is to be understood that the previously described working particles are considerably easier and cheaper to remove than the working water by repeated washing with distilled 10b. The sodium form of the resin particles 27 becomes 10a. It has been found that the subsequent ones are suspended and adjusted to a pH of essentially 6.8

Steps which are set for carrying out the method according to Er-30 at 22.5 ° C., specifically by a treatment and finding, are of such a type that by vigorous mixing with a buffer solution in the form of a

Separation, detection and measurement of the amount of a Grup phosphate cyanide treatment solution 28.

The phosphate cyanide resin treatment solution 28 can be made possible to hemoglobin types, even though others have the following formula: 4.59 g NaH2P04-H20 (0.033 mol),

Blood components present in the test sample at least 351.28 g Na2HOP4 (0.008 mol) and 0.65 g KCN (0.010 mol)

theoretically make the intended measurement unclear or with 0.10 g NaN3 (0.01%) as a preservative in a

can disturb. a liter of H20. The solution 28-b can also according to

Formed according to a first particular formula described below; 4.53 g KH2P04 (0.033

In the embodiment of the invention, the ions consist of moles), 1.45 g of K2HP04 (0.008 mol) and 0.65 g of KCN (0.010

exchange particles made of an ion exchange resin. 40 mol) with 0.10 g NaN3 (0.01%) as a preservative

In Fig. 1 is a chromatographic micro column with 20 tel in one liter of water.

designated. Column 20 includes a container 21 which is placed in an after mixing with treatment solution 28

Tube 22 merges, which in a discharge end 23 ends the sodium form of the resin particles 27 with an acidic solvent.

Det, which are closed by means of a cap 24, solution 29 (for example 4 mol of H3OP4) is further treated in order to The connection or transition between loading to a pH of essentially 6.8 at 22.5 ° C.

container 21 and the tube 22 is through a cross plate or place. Treatments with solution 28 and with the acidic

Cross disk 25 closed. The connection or the other solution 29 should be repeated a sufficient number of times to obtain a suspension 27 (S) between the tube 22 and the delivery end 23, which is also pH-adjusted to the desired one by means of a cross plate or a cross disk. Value is set. Then the tubes 22 are

26 closed. The particles of the ion exchange resin, including one or more microcolumns 20, with the resin particle suspension.

put the column bed between the discs 25 and 26, and pension 27 (S) filled, and the suspension is between

they are identified by reference numeral 27. see the permeable discs 25 and 26 arranged or

Each separating or holding disk 25 or 26 is kept permeable.

and has a network of micropores that allow an amount of a test sample 10 to be prepared either by means of a hemolysate solution of red blood cells from the container ss mode 10a or by mode 10b

21 into the tube 22, and which have been enabled, is inserted into one end of a column 20 which has an elution fraction from the sleeve 22 through the exhaust column bed which contains the suspension 27 (S) made of resin part.

end 23 to remove through, while the column bed made of chen 27. A manufactured according to the method of operation 10a

Particles 27 and the material adsorbed to them in the test sample 10 require a 1: 4 dilution by means of

Tubes 22 are held. Discs 25 and 26 can contain 60 distilled water.

NEN from a conventional flexible, resilient, linear poly- Preferably, the column 20 is arranged vertically. The

be made of high density ethylene of the Ziegler type. A sol cap 24 at the discharge end 23 is then removed and the polyethylene usable for filtering is manufactured to a predetermined quantity or volume and sold commercially under the name “Vyon”, the test sample 10 is placed in the container 21 . The size

According to the first particular embodiment of the first part of the test sample 10 runs easily through the disk 25

invention, the ion exchange particles 27 consist of a resin particle 27 through and onto the column bed

Resin of a weak and cationic type of exchange. In particular, a smaller part of the test sample 10, which is on or in the disk 25

in particular, the resin consists of a fraction with a part that remains if the column bed made of resin particles 27 is

rinsed or dispensed. For this purpose, a small volume, for example 0.2 ml, of solution 28 is preferably added to container 21. The solution 28 quickly runs through the disc 25 and onto the column bed of resin particles 27, carrying the last part of the test sample 10 with it.

The next step in accordance with the embodiment just described is to elute from the resin particle 27 column bed a fraction of the test sample 10 which, as will be understood, consists predominantly of Hb-A] a c. The first elution fraction, generally designated 30, is collected in a receptacle 31 located near the discharge end 23 of column 20 after a predetermined or aliquot of phosphate cyanide solution 28 is added to column container 21 has been. For example, 4 ml of the solution 28 are placed in the column container 21. After a certain period of time, for example 20 to 30 minutes, there is an elution fraction 30 with a volume of essentially 4 ml in the receiving container 31. The volume of the first eluting fraction 30 in the receiving container 31 is then ready to carry out the subsequent steps according to the invention.

In the first general embodiment of the invention, the next step is to elute substantially all of the remaining blood components of the test sample 10 from the column bed of resin particles 27. This second elution fraction, indicated generally by reference numeral 32, is collected at the column discharge end 23 in a receptacle 33 after a preferably predetermined or aliquot amount of a solution 34 having a washing solution or solution has been added to the column container 21 Solution is that completely repels or removes the remaining blood components from the column bed.

The exact composition or formula of wash solution 34 is not critical as long as its use does not change or modify the spectrometric absorption characteristics ("color") of elution fraction 32. A compatible wash solution 34 has either an ionic strength or a relative pH that is sufficient to completely expel or remove all remaining blood components of the test sample 10 from the column bed of resin particles 27. For example, 4 ml of 4 mol NaCl can be added to the column containers 21. After a period of 20 to 30 minutes, for example, an elution fraction 32 with a volume of essentially 4 ml is collected in the receiving container 33. The volume of the elution fraction 32 in the receptacle 33 requires an appropriate dilution using distilled water before the subsequent steps according to the invention are carried out.

The amount of Hb-Ala c present in the whole blood sample is determined and measured using a spectrometric device 40, using techniques and procedures used in micro-chromatography with liquid columns, and using a test sample 10.

The spectrometric analysis is carried out by means of the device 40, which measures the absorption of light, as caused by the types of hemoglobin present in the test sample 10. It is known that the visible part of the spectrum for determining the presence of a hemoglobin is in the violet region, in particular at essentially 415 nm or 4150 A. It is not necessary

that exactly these values are used, however, if somewhat different values are used, the same values should always be used in a test series.

The device 40 can be an optical spectrometer,

5 648128

which is set to the selected wavelength of 415 nm. The device 40 may also be a spectrophotometer, i.e. a form of a spectrometer with associated equipment, which provides the ratio or a function of the ratio of the radiation energy of two beams as a function of an adjustable spectral wavelength. Since the spectrometric analysis according to the invention is carried out for the purpose of determining and measuring types of hemoglobin in the test sample 10 by io light absorption characteristics, other forms of a device 40 can be used, for example visual comparison devices, for example a set of Nessler tubes.

The contents of the receptacles 31 and 33 are individually brought into 15 appropriate cuvettes for the spectrometric device 40. It has been found that the light absorption characteristics of the second elution fraction 32 are of such a size that a dilution of the fraction 32 is required for a conventional spectrometer or spectrophotometer for optimal effectiveness of the sensing photo cell. For example, a volume of 4 ml of elution fraction 32 requires a 1: 5 dilution using demineralized water.

Spectrometric analysis of the two elution fractions 30 and 32 provides natural numbers which express, represent or display the amounts of the types of hemoglobin present in test sample 10. If a conventional spectrometer or spectrophotometer is used for the device 40, the numerical value shown is a function of the specific absorption A, i.e. a measurement of the amount of light of the spectral wavelength of 415 nm that is absorbed by the hemoglobin species as they pass through the cuvette and travel towards the sensing photo cell.

35 If the device has 40 Nessler tubes, a number of standard solutions are prepared in a set of tubes with a capacity of 50 or 100 ml. The color of each tube in the row or set is assigned an arbitrary numerical value that is related to the presence of hemoglobin. After appropriate dilution, elution fractions 30 and 32 are transferred to duplicate tubes, if necessary, after which a visual comparison is carried out with the tubes of the standard solutions. By means of color vision comparison, the elution fractions 30 and 32 can be assigned to two tubes of the standard solutions with regard to their relative concentration.

The numerical values expressing the amounts of hemoglobin types present in the elution fractions 30 and so 32, as determined and measured by analysis in the spectrometric device 40, are then used in a calculation as factors or quantities. In particular, the amount of hemoglobin types present in the first elution fraction 30 is compared with the 55 sum of the amounts of hemoglobin types in both elution fractions 30 and 32 according to the following mathematical equation:

60

numerical value for the elution fraction 30 x 100

numerical value for elution fraction 30 + numerical value for elution fraction 32 = a numerical percentage value.

65

The first fraction 30 from a test sample 10 which has been eluted from the column bed of resin particles 27 can also be used. The amount of hemoglobins present in the elution fraction 30 is increased with the amount of

648 128

Compare hemoglobins present in an amount of test sample 10 containing a red blood cell hemolysate fraction. 1 does not contain any information regarding this second embodiment.

In this embodiment, a test sample 10 is made by operation 10a or operation 10b. A quantity of test sample 10 is introduced into one end of column 20, which contains suspension 27 (S) of resin particles 27 as before. An aliquot of the first elution fraction 30 is eluted from the column 20 by means of a corresponding amount of the phosphate cyanide solution 28 and collected in the receiving container 31.

Furthermore, an amount of a test sample 10 is produced as a hemolysate fraction at an earlier point in time, in succession or in succession, which can expediently be analyzed by a spectrometric device 40. It can be seen that the light absorption properties of test sample 10, without significant dilution, would be of such an order of magnitude that the effectiveness of the sensing photocell of conventional spectrometric device 40 would be compromised. Accordingly, for example, prior to eluting the first fraction 30, an amount of test sample 10 equal to the amount of test sample 10 placed in the end of column 20 should be diluted using a ratio of substantially 1: 480 using distilled water to prepare a hemolysate fraction for analysis by spectrometric device 40.

After individual analysis of the elution fraction 30 and the hemolysate fraction by detection and measurement in the spectrometric device 40, the numerical values which express the amounts of hemoglobins present in each fraction are used as factors or sizes in a calculation. In particular, the amount of hemoglobins present in the first elution fraction 30 is compared to the amount of hemoglobins present in the hemolysate fraction in accordance with the following mathematical equation:

numerical value for the elution fraction 30 x 100 _

numerical value for the hemolysate fraction = a numerical percentage.

In a second particular embodiment (FIG. 2), the ion exchange resin can be the same as in the first particular embodiment. However, the size of the mesh size of a sieve is from 0.30 to about 0.45 mm (200 to 400 mesh). In this embodiment, the treatment solution 28 is a bistris-cyanide chloride solution, for example a 2-hydroxyethyl - ("to") - trihydroxy-iminomethane - ("tris") - cyanide chloride solution. In particular, the resin treatment solution 28 in this embodiment can be formed as follows: 6.28 g (HOCH2CH2) 2NC (CH2OH) 3 (0.03 mol) and 0.10 g KCN (0.01%) with 0.10 g NaN3 (0.01%) as a preservative in one liter of H20. The pH of solution 28 is then adjusted to essentially 6.8 at 22.5 ° C. using concentrated hydrochloric acid.

After mixing with the treatment solution 28, the sodium form of the resin particles 27 is treated with an acidic solution 29, for example 4 moles of HCl, in order to adjust it to a pH of substantially 6.8 at 22.5 ° C. The treatments with the solution 28 and with the acid solution 29 should be repeated enough times to obtain a suspension 27 (S) which has the desired pH.

In the second particular embodiment, the small part of the test sample 10 that is on or in the disk 25

remains, rinsed or brought onto the column bed of resin particles 27, using a small amount, for example, 0.2 ml, of an elution solution 128 which is brought into the column container 21. The Löhsüng 128 runs quickly through the disk 25 and onto the column bed made of resin particles 27, carrying the last part of the test sample 10 with it.

The elution solution 128 can have the following composition: 6.28 g (HOCH2CH2) 2NC (CH2OH) 3 (0.03 mol), 10 0.10 gKCN (0.01%) and 7.02 gNaCl (0.12 mol) with 0.10 g NaN3 (0.01%) as a preservative in one liter of H20. The pH of this bis-tris-cyanide chloride solution 128 is then adjusted to a pH of substantially 6.8 at 22.5 ° C. using concentrated hydrochloric acid.

The next step in this embodiment is to elute a fraction of the test sample consisting predominantly of Hb-Aia ^ from the resin particle column 27 bed. This first elution fraction 30 is collected in 20 the receiving container 31 after a predetermined or aliquot of the bis-tris-cyanide chloride solution 128 has been added to the column container 21. For example, 5 ml of the elution solution 128 is placed in the column container 21. After a certain period of time 25 of, for example, 20 to 30 minutes, there is an elution fraction 30 of essentially 5 ml in the receiving container 31. The volume of the first eluting fraction 30 in the receiving container 31 is ready for carrying out the subsequent steps according to the invention.

In a third particular embodiment of the invention (FIG. 3), the column bed particles 27 consist of a weak and anionic microgranular ion exchange cellulose, which is modified by removing the amorphous areas and stabilized by crosslinking, and contains positively charged functional diethylaminethyl groups. The particles 27 have a size which is smaller than corresponds to a sieve with a mesh size of 0.30 mm (400 mesh). The particles 27 are adjusted in such a way that they have a pH value of 22.5 ° C. in have a substantial 8.5. This Cellulo-40 type is manufactured and sold under the name "Whatman DE-52".

In this third particular embodiment, the treatment solution 28 is a trihydroxymethylaminomethane “tris” cyanide solution. The treatment solution 28 for the Cellu-45 loose can have the following composition: 6.06 g H2N.C (CH2OH) 3 (0.05 mol) and 0.10 g KCN (0.01%) with 0.10 gNaN3 (0 , 01%) as a preservative, made in one liter of H20. The pH of solution 28 is then adjusted to essentially 8.5 at 22.5 ° C. using concentrated hydrochloric acid. The cellulose particles 27 are then mixed with the treatment solution 28. After mixing with the treatment solution 28, the cellulose particles 27 are treated further with an acidic solution 29 (for example 4 mol of HCl) in order to readjust the pH to essentially 55 8.5 at 22.5 ° C. The treatments with the solution 28 and with the acidic solution 29 should be repeated enough times to obtain a suspension 27 (S) which is adjusted to the desired pH.

As with the first particular embodiment, the smaller 60 portion of test sample 10 remaining on or in disk 25 should be rinsed or placed on the cellulosic particle bed 27 using a small amount, for example, 0.2 ml Elution solution 128, which is placed in the column container 21. The solution 128 quickly runs through the disk 25 and onto the column bed of cellulose particles 27, carrying the last part of the test sample 10 with it.

In this particular embodiment, the eluent

7 648128

Solution 128 have the following composition: seeing consists of Hb-A] a_ c. The first elution fraction 30

6.06 gH2N.C (CH2OH) 3 (0.05 mol) and 0.10 g KCN (0.01%) is in the receptacle 31 near the discharge end 23

with 0.10 g NaN3 (0.01%) as a preservative, collected from column 20 after having been in the column

det in one liter of H20. Thereafter, the pH of this tris container 21 is a predetermined or aliquot of the Ph-

Cyanide solution 128 using concentrated hydrochloric acid solution 5. For example, re set to substantially 7.7 at 22.5 ° C. 18 ml of solution 128 are gradually added to the column

The next step of this particular embodiment is given container 21. After a period of time, for example, the column bed of cellulose particles 27 and 60 to 90 min contains essentially 18 ml of the eluent.

to elute a fraction of the test sample 10, which, as for the control fraction 30, in the receptacle 31.

is essentially all other hemoglobins or hardening fraction 30 in the receptacle 31 is now for moglobin types than the types Hb-A] a c contains. This first execution of the subsequent steps according to the inventions

Elution fraction 30 is ready for use in the receptacle 31.

he collected at the outlet end 23 of the column, specifically after- In a fifth special embodiment, the column bed particles 27 have a predetermined or aliquot in the column container 21 of a weak cationic,

amount of the tris-cyanide solution 128 has been given. 15 microgranular ion exchange cellulose, which

for example, 20 ml of solution 128 are modified step by step in the direction of the amorphous areas and

Column container 21 given. After a period of time of etching is stabilized and there is negatively charged, functional, for example 60 to 90 min, an elution fraction contains carboxymethyl groups and has a particle size,

30 of a volume of essentially 20 ml in the opening which is smaller than the inside mesh size of a sieve container 31. The first elution fraction 30 in the opening 20 would correspond to 0.30 mm (400 mesh). The cellulose receptacle 31 is now used to carry out the subsequent pH of essentially 6.1 at 22.5 ° C.

steps according to the invention. In this embodiment, the treatment solution is 28

In a fourth particular embodiment (FIG. 3), a bis-tris-cyanide solution, for example a 2-hydroxy, the column bed particles 27 consist of a weak and ethyl-bis-trihydroxyiminomethane-tris-cyanide solution. The cationic microgranular ion exchange cellulose, the treatment solution, can modify and use the following composition by removing the amorphous areas: 6.28 g (H0CH2CH2) 2NC (CH20H) 3 (0.03 mol) and stabilized by crosslinking and negative charged, radio-0.10g KCN (0.01%) with 0.10g NaN3 (0.01%) as a conventional carboxymethyl group and contains a particle serving agent formed in one liter of H20. After that, it has a size which is smaller than it would correspond to the clear mesh size and the pH value of the solution 28 using a concentrated sieve of 0.30 mm (400 mesh). The hydrochloric acid triturated to essentially 6.1 at 22.5 ° C. cellulose is adjusted to a pH of 6.8 at 22.5 ° C. The cellulose particles 27 are then treated with the. This type of cellulose is mixed under the name «Whatman solution 28. After mixing with the Treat-CM-52 »manufactured and sold. solution 28, the cellulose particles 27 are treated with an acid

In this fourth particular embodiment, the berry solution 29, for example 4 mol of HCl, is further treated.

treatment solution 28 is a phosphate cyanide solution, the following 35 to bring the pH back to essentially 6.1

The composition can be: 1.38 g NaH2P04.H20 22.5 ° C. Treatments with solution 28

(0.01 mol) and 0.10 g KCN (0.01%) with 0.10 g NaN3 and the acidic solution 29 should be repeated enough times

(0.01%) as a preservative, formed in one liter, to obtain a suspension 27 (S) which is in the

H20. Thereafter, the pH of solution 28 is in the compound equilibrium and has the desired pH.

1 mol NaOH to essentially 6.8 at 22.5 ° C. as in the first particular embodiment should be adjusted. The cellulose particles 27 are then covered with the small part of the test sample 10 that is on or in the disk 25

action solution 28 mixed. After mixing with the remaining, put 27 on the column bed of cellulose particles.

treatment solution 28, the cellulose particles are rinsed or brought with a, using a

acidic solution 29, for example 4 moles of H3P04, further gently a small amount of, for example, 0.2 ml of an elution

delt to turn the pH back to essentially 6.8 with solution 128 which is placed in the column container 21. The

22.5 ° C. Treatment with solution 28 and solution 128 passes rapidly through disc 25 and with acidic solution 29 should be repeated on the cellulosic particle bed 27 a sufficient number of times to become suspension 27 (S) which is the carries the last part of test sample 10 with it.

has the desired pH. In this particular embodiment, the eluent

As with the first particular embodiment, the solution 128 should have the following composition:

small portion of test sample 10, which on or in disk 25 6.28 g (HOCH2CH2) 2NC (CH2OH) 3 (0.03 mol), 0.10 g KCN

remained on the column bed made of cellulose particles (0.01%) and 2.34 g NaCl (0.04 mol) with 0.10 g NaN3

rinsed or brought using egg (0.01%) as a preservative formed in one liter of a small amount of, for example, 0.2 ml of an eluting H20. Thereafter, the pH of this bis-tris-cyanide solution 128 is added to the column container 21. This 55 128 using concentrated hydrochloric acid on im

Solution 128 runs quickly through the pane 25 and is essentially set at 6.1 at 22.5 ° C.,

onto the column bed of cellulose particles 27, carrying the next step in this particular embodiment, the last part of test sample 10. form consists of the column bed made of cellulose particles

In this particular embodiment, the eluent 27 can elute a fraction of a test sample 10 which, as for the solution 128, has the following composition: 60 is understood to consist predominantly of Hb-Ala ^. This first 1.38 g of NaH2P04.H20 (0.01 mol) and 0.10 g of KCN (0.01%) elution fraction 30 is mixed in the receptacle 31 with 0.10 g of NaNj (0.01%) as one Preservative, collected at the discharge end 23 of column 20, in one liter of H20. Thereafter, using a predetermined or 1 mol NaOH in the column container 21, the pH of the phosphate cyanide solution 128 is added in an aliquot amount of the bis-tris-cyanide solution 128, followed by substantially 7.0 at 22.5 ° C. 65 den is. For example, 20 ml of solution 128 are

The next step according to the invention is to be placed in the column container 21. After a period of time

from the column bed made of cellulose particles 27 a fraction of the de of, for example, 60 to 90 min is the eluent

Elute test sample 10 which, as can be understood, predominant fraction 30 in an amount of substantially 20 ml in

648 128

the receptacle 31. The elution fraction 30 in the receptacle 31 is now ready to carry out the subsequent steps according to the invention.

It should be noted that the flow of the liquid stops in the microcolumn as soon as the liquid level reaches the upper transverse disk 25, so that the ion exchange bed is prevented from drying out.

It should also be noted that in the microcolumn, the liquid flows through the column under the effect of gravity. The elution is carried out under equilibrium conditions such that the pH of the elution liquid and the particles of the ion exchange bed are the same. Furthermore, the pH does not change during elution.

In connection with all general and special embodiments of the invention as described above, various steps, techniques or working methods are described or disclosed in which diluents

8th

tion using distilled water is either required or suggested. It is understood by a person skilled in the art of clinical chemistry that the best ways to carry out the invention using a microcolumn as described above require careful adjustment and consistent adherence to routine procedures when practiced by the invention to be a reliable method of determining the presence of diabetes and monitoring the level of diabetes control. It is further understood by a person practicing the invention that a procedure or protocol for repeated testing of a large number of people who are both diabetic and non-diabetic includes: As standard amounts of test samples and of the solutions and fractions used , stable and compatible dilution ratios as well as careful selection and control of the spectrometric device.

C.

3 sheets of drawings

Claims (11)

648128 PATENT CLAIMS 1. A method for determining a diagnostic indicator of a person's blood sugar condition, in which a blood sample is prepared into a test sample containing a hemolysate solution of red blood cells, characterized in that an amount of the test sample is inserted into one end of a column bed which comprises a suspension of ion exchange particles, then an amount of an elution solution is introduced into one end of the column bed in order to elute a fraction of the test sample therefrom, after which an aliquot of a first elution fraction is collected at the other end of the column bed, either an amount of Wash solution is introduced into one end of the column bed in order to desorb essentially all remaining blood components of the test sample from the column bed particles, an aliquot of a second elution fraction is collected at the other end of the column bed, and then that the hemoglobins or H present in each elution fraction Hemoglobin species are separated, determined and measured using spectrometric analysis and the amounts concerned are expressed as numerical values and the amount of hemoglobin or hemoglobin species in the first elution fraction is compared to the sum of the amounts of hemoglobin or hemoglobin species in each fraction in accordance with the mathematical equation below : numerical value for the first elution fraction x 100 numerical value for the first elution fraction + numerical value for the second elution fraction = a numerical percentage value, this numerical percentage being a diagnostic indicator of the subject's blood sugar characteristics, or a substantial amount of the test sample is diluted to provide a red blood cell hemolysate fraction that can be analyzed by spectrometric analysis that is present in the first elution fraction and in the hemolysate fraction Hemoglobins are separated, determined and measured by spectrometric analysis and the relevant amounts of hemoglobins are expressed as numerical values and that the amount of hemoglobins or hemolobin types present in the first elution fraction is in agreement with the amount of hemoglobins or hemoglobin types contained in the hemolysate fraction is compared to the following mathematical equation: numerical value for the first elution fraction x 100 numerical value for the hemolysate fraction = a numerical percentage value, this numerical percentage being a diagnostic indicator of the person's blood sugar characteristics. 2. The method according to claim 1, characterized in that resin particles of a copolymer of methacrylic acid and divinylbenzene are used as ion exchange particles for the column bed, which contains negatively charged carboxyl groups and a particle size corresponding to an internal mesh size of 0.3 to 0.6 mm (100 to 400 mesh) and has a pH of substantially 6.8 at 22.5 ° C. 3. The method according to claim 2, characterized in that the resin particles have a size corresponding to a mesh size of 0.30 to 0.45 mm of a sieve (200 to 400 mesh). 4. The method according to claim 1, characterized in that the ion exchange particles of the column bed consist of a weak and anionic, microgranular ion exchange cellulose, which is modified by removing the amorphous areas and stabilized by crosslinking and contains positively charged, functional diethylaminoethyl groups, a particle size which is smaller than the size which corresponds to a clear mesh size of a sieve of 0.30 mm (400 mesh) and which is adjusted to a pH value of essentially 8.5 at 22.5 ° C. 5. The method according to claim 1, characterized in that a weak and cationic microgranular ion exchange cellulose is used for the ion exchange particles, which is modified by removing the amorphous regions 15 and is stabilized by crosslinking and contains negatively charged functional carboxymethyl groups, a particle size which is smaller than the size which corresponds to a clear mesh size of a sieve of 0.30 mm (400 mesh), and to a pH of substantially 6.8 is set at 2o 22.5 ° C. 6. The method according to claim 1, characterized in that a weak and cationic, microgranular ion exchange cellulose is used for the column bed particles, which is modified by removing the amorphous areas and 25 is stabilized by crosslinking and contains negatively charged functional carboxymethyl groups and has a particle size which is smaller than a size which corresponds to a clear mesh size of a sieve of 0.30 mm (400 mesh), and to a pH of substantially 6.1 is set at 30 22.5 ° C. 7. The method according to claim 1, characterized in that the particle suspension for the column bed is formed in that the resin particles are converted to sodium form and the sodium form with a phosphate cyanide solution. 35 solution is treated, the treated sodium form is treated further with an acidic solution, and the treatments with the phosphate cyanide solution and the acidic solution are repeated until the suspension has a pH of substantially 6.8 at 22.5 ° C. . 8. The method according to claim 3, characterized in that the particle suspension for the column bed is prepared by converting the resin particles to sodium form and then treating the sodium form with a bis-tris-cyanide solution, the treated sodium form with a 45 acidic Solution is treated further, and that the treatment with the bis-tris-cyanide solution and with the acidic solution are repeated until the suspension has a pH of substantially 6.8 at 22.5 ° C. 9. The method according to claim 4, characterized in that the particle suspension is produced in that the Cellulose particles are treated with a tris-cyanide solution, the treated particles are treated further with an acidic solution and the treatments with the tris-cyanide solution and the acidic solution are repeated until the suspension has the desired pH. 10. The method according to claim 5, characterized in that the particle suspension is prepared in that the cellulose particles are treated with a phosphate cyanide solution, the treated particles are further treated with an acidic solution fio and that the treatments with the phosphate cyanide solution and with the acidic Solution are repeated until the suspension has the desired pH. 11. The method according to claim 6, characterized in 65 that the particle suspension is produced in that the Cellulose particles are treated with a bis-tris-cyanide solution, the treated particles continue to be treated with an acidic solution and that the treatments with the bis-tris-cyanide solution and repeated with the acidic solution until the suspension has the desired pH.