WO1994029730A1

WO1994029730A1 - METHOD FOR DETERMINING HbA1c, AND COMPOSITION FOR IMPLEMENTING THE METHOD

Info

Publication number: WO1994029730A1
Application number: PCT/SE1994/000564
Authority: WO
Inventors: Jan O. Jeppsson
Original assignee: BOHERINGER MANNHEIM SCANDINAVIA AB; Boehringer Mannheim Scandinavia AB
Current assignee: BOHERINGER MANNHEIM SCANDINAVIA AB; Roche Diagnostics Scandinavia AB
Priority date: 1993-06-11
Filing date: 1994-06-10
Publication date: 1994-12-22
Anticipated expiration: 1995-12-11
Also published as: SE9302013L; SE504914C2; AU7012394A; SE9302013D0

Abstract

A method for determining the HbA1c content in blood which is dried in an absorbing material is disclosed. The dried blood is treated with a cysteine solution for eliminating the interfering HbA3 fraction and reducing the amount of methaemoglobin, whereupon the HbA1c content is determined. According to one embodiment, the absorbing material with the dried blood is pretreated with a buffer solution. A composition for the treatment is also disclosed. The composition comprises a buffered cysteine solution.

Description

METHOD FOR DETERMINING HbA_l , AND COMPOSITION FOR IMPLEMENTING THE METHOD

The present invention relates to a method for deter¬ mining the HbA-]__c content in blood which is dried in an absorbing material, and a composition for use in the method. HbA]__c is an established marker when controlling dia¬ betes. The use of the glycated form of haemoglobin in these contexts has been known since 1975. The reaction between glucose and haemoglobin is an irreversible, non- enzymatic process in which the amount of HbA]__c formed in- creases over time and with a high concentration of glu¬ cose in the blood. Red blood corpuscles has a life of 120 days, and haemoglobin is the protein which exists longest in the blood circulation.

On the one hand, glucose may be attached to the N- terminal amino acid valine in the haemoglobin molecule via the a ino group thereof and form a so-called keto- a ine. Glucose can also be bound to one or more binding sites on some ε-amino groups on the amino acid lysine. By definition, the HbA]__c fraction represents glycating at the N-terminal amino acid valine, and HbAg represents haemoglobin without any N-terminal valine adduct. By gly¬ cating the N-terminal amino acid valine, a charge differ¬ ence arises between HbAg and HbA]__c, which may be used to separate the HbA]__c fraction from the HbAø fraction (main fraction) by various chro atographic, i munologic or electrophoretic methods.

All glucose adducts, i.e. also haemoglobin glycated on the lysine residues, are designated glycated haemoglo¬ bin and may be determined by using phenylboronate affin- ity chromatography. The levels will be higher, but there is excellent correlation between HbA]__c levels and gly¬ cated haemoglobin levels.

There is excellent agreement between the present HbA*]__c and the average blood sugar level 2-6 weeks back. Normally, HbA]__c constitutes 4-5% of the total amount of haemoglobin of a non-diabetic. In an untreated diabetic, Hb ic ^may amount to 10-18% of the total amount of haemo¬ globin. So-called glycating expresses a general phenomenon. Thus, most proteins in the body, including eye lens pro¬ teins and membrane proteins in blood-vessel walls, are glycated, which may affect their function.

The predominant types of methods for determining HbA_]__c internationally is ion-exchange chromatography

(e.g. HPLC) , affinity chromatography (e.g. phenylboro- nate) and electrophoretic methods in about equal por¬ tions. In Sweden, the HPLC technique according to the method developed by Jan 0. Jeppsson et al. is predominant (Clinical Chemistry, Vol. 32, No. 10, 1986, pp. 1867-

1872) . In this method, use is made of a "Mono S" ion-ex¬ changer from Pharmacia Biotechnology, Sweden. Recently, equipment for measuring the total amount of glycated hae¬ moglobin has been developed by ABBOT Diagnostica (USA) (phenylboronate affinity chromatography) ("Vision" or "IMX") . The results cannot be compared with those ob¬ tained by HPLC technique.

The third principle is based on polyclonal or mono¬ clonal antibodies which recognise the specific structure in glucose bound to the amino group in the valine of the β chain. At present three methods are commercially avail¬ able. The ELISA technique (Novo-Dakopatts) is based on microplate technique and thus is suitable for large series. An instrument (doctor's office instrument) sup- plied by Ames Bayer is based on similar principles, but is intended for separate samples or small series. Boehringer Mannheim has recently introduced a polyclonal antibody (Tina-Qvant HbA-]__c) by which HbA__c can be ana¬ lysed specifically in so-called multichannel analysers which are available in any large hospital laboratory. The choice of technique is always a compromise be¬ tween the complexity of the analysis, costs and avail¬ ability to doctors/patients. Merely HPLC ion-exchange chromatography, the ELISA technique and Boehringer Mannheim's antibody technique can use the filter paper technique.

The sampling procedure may be effected in various ways. Generally, use is made of venous blood (EDTA-blood) which has a life of 7-10 days when cooled. The drawback when testing HbAχ_c is that the patient must appear 5-10 days in advance for special taking of samples for HbA_]__c to make the result available at the planned visit to the doctor. The sample is often taken when visiting the doctor. Thus, no result is available at the visit, but must be sent to the patient by post or handed over at a subsequent visit.

Therefore there is a great need for a method of sampling which means that the patient does not have to go to a care centre/diabetes clinic or laboratory for sampling before visiting the doctor.

So far, two methods have been available in which capillary blood from a prick in the finger is collected on a filter paper. In one method, the blood is allowed to dry on the filter paper, whereupon it is eluted and ana- lysed by phenylboronate chromatography (R. R. Little et al., Clin. Chem. 1986; 32:869-871). Comparative studies with analysis of common venous blood demonstrated very poor agreement and, therefore, the method is not accept¬ able in Sweden. The poor agreement between the analysis of dried blood and the analysis of fresh venous blood is caused by, inter alia, the following.

When blood is dried, Fe is oxidised from 2⁺ to 3⁺, thereby forming methaemoglobin. The colour of the blood passes from cherry-red to brownish-red. Normally this does not occur in fresh blood since red blood corpuscles contain several enzymes with a reducing capacity (methaemoglobin reductase) . One more product is formed since the haemoglobin molecule (β-chains) exposures a free thiol group (-SH) . Red blood corpuscles normally contain large amounts of the tripeptide glutathione: gly- cine-cysteine-glutamic acid (about 2 mmol/1) . Since the reducing enzyme activity ceases in the dried blood, a disulphide of the SH group forms in the cysteine of the tripeptide and the free thiol of the haemoglobin mole- cule. This disulphide is present in the absorbing mate¬ rial, as well as in the test tube when the blood has been stored for a long time. The product, called HbA3, has a further charge since glutamic acid in the tripeptide has an additional carboxyl group in its side chain. Thus, the product has a further charge which permits separation of this product from the main component in ion-exchange chromatography or electrophoresis.

Both the methaemoglobin fraction and HbA3 interfere in connection with ion-exchange chromatography (HPLC) . This implies that HbA]__c cannot be determined in a suffi¬ ciently accurate manner (Fig. 2) .

In a newer method (Solveig Eckerbom et al., Ann. Clin. Biochem. 1989; 26:148-150), blood is applied to filter paper which is then placed in a special stabilis- ing solution in a test tube which is sealed to be sent to the laboratory. However, many patients find it compli¬ cated to handle a solution, seal the tube and ensure that it keeps tight during transport to the laboratory. More¬ over, the life of the stabilising solution is limited. According to the present invention, the problems arising in the prior art methods have been solved.

Thus, the invention relates to a method for deter¬ mining the HbA]__c content in blood which is dried in an absorbing material, the dried blood being treated with a buffered cysteine solution for eliminating the interfer- ing H A3 fraction and reducing the amount of methaemoglo¬ bin, whereupon the HbA-[_C content is determined.

Preferably, the absorbing material with dried blood is pretreated with a buffer solution. When performing the inventive method, capillary blood is applied to an absorbing material which prefer¬ ably is combined with a physician's referral. This referral is then posted by the patient himself about 4-5 days before visiting the doctor. In the laboratory, a little piece of the absorbing material with the dried blood spot is punched by means of a punch, and the dried blood product is preferably treated with a buffer solution and then with a buffered solution containing cysteine. The eluate is then analysed by some suitable technique, for example HPLC. The absorb¬ ing material with dried blood has a life of 5-7 days. A specific buffered cysteine solution has been developed to eliminate haemoglobin changes occurring in the drying process. The results of the analysis correspond entirely to those obtained in venous blood sampling.

The advantages of the invention are as follows:

- the patient does not have to appear at a hospital/care centre for sampling - more benefit out of a visit to the doctor since the re¬ sult of the sampling is already available

- the treating doctor may avoid sending a historical HbA-j__c result to the patient

- fewer working routines in the laboratory - the analytical result corresponds to that obtained in venous blood sampling.

According to the invention, a composition for elimi¬ nating the interfering HDA3 fraction and reducing the amount of methaemoglobin in eluate of dried blood from the absorbing material is also provided. The composition contains a buffered solution of cysteine. The cysteine reduces the amount of methaemoglobin by reducing Fe³⁺ to Fe2⁺ and reduces the Hb 3 fraction since the thiol binding to glutathione disappears. Then the blood returns to normal conditions like in fresh blood. The cysteine solution should suitably be buffered to a pH of about 7.4. A suitable cysteine solution contains cysteine, glycine and EDTA.

A suitable buffer solution for the pretreatment, if any, contains phosphate, citrate and a wetting agent. Its pH preferably is about 5.4.

In ion-exchange chromatography (HPLC) after treat¬ ment according to the invention of dried blood, a chroma- togram is obtained which is identical with that of fresh blood. The results of the analysis of dried blood and fresh blood, respectively, will therefore be comparable. As absorbing material for sucking up capillary blood, different types of filter paper may be used. Es¬ pecially good results are obtained with an absorption material which does not cause any irreversible changes of the blood spot.

The invention will now be described in more detail with reference to the accompanying Figures and Examples. Brief description of the Figures

Fig. 1 illustrates an example of the result of ion- exchange chromatography (HPLC) of HbA]__c. The surface of the HbA*]__c peak is calculated and expressed as % of the total haemoglobin surface.

Fig. 2 shows the results of ion-exchange chromatog¬ raphy (HPLC) of HbA__c with different sample processing techniques.

Fig. 3 shows the result of a comparative study between fresh venous blood and filter paper with the elu- ting solution according to the invention. Example 1 A dry filter paper with dried blood, which was sent by a patient, is placed on a clean surface. A piece (diameter 3 mm) is punched from the filter paper and placed in a small test tube. 150 μl of the cysteine solu¬ tion mentioned below are added to the test tube. The tube is shaken slowly for 5 minutes. Subsequently it is placed (covered with a film) in a heating cabinet at 37°C for 30 minutes.

The HbA]__c content is determined by means of HPLC equipment provided with a Mono S cation-exchanger. The result of the HPLC analysis is illustrated in Fig. 1. Cysteine solution glycine, 1 mol/1 0.75 g

EDTA, 12 mmol/1 0.045 g cysteine-HCl, 250 mmol/1 0.39 g

Glycine, EDTA and cysteine-HCl are mixed and diluted to 8 ml with distilled water. Subsequently, 100 μl of 10% Triton X-100 are added, and the pH is adjusted to 7.4 with 2 M NaOH. The adjusting of the pH is started before the cysteine has been completely dissolved. The solution is then diluted to 10 ml. Example 2

The experiment in Example 1 was repeated, except that the punched filter paper was first pretreated for 30 minutes at 37°C with a buffer solution containing phos¬ phate, citrate and a wetting agent. The solution had a pH of about 5.4.

Subsequently, the cysteine solution according to Example 1 was added and the pH adjusted to about 7.4. The treatment continued for 15 minutes at room temperature. The result corresponded to that in Example 1. Example 3

A comparative study was made, ion-exchange chroma- tograms being taken from fresh blood (EDTA-blood) , dried blood on filter paper eluted and treated with the compo¬ sition according to the invention, and blood which had been sucked up by filter paper and eluted with phosphate buffer. The chromatograms exhibit excellent agreement between fresh blood (EDTA-blood) and dried blood treated with the composition according to the invention. The result after eluting with phosphate buffer demonstrates a disturbing peak of HbA3 and influence by methaemoglobin formed. Example 4

A comparison was carried out between two different methods for sampling HbA^*]__c, viz. fresh venous blood and dried capillary blood on a filter paper treated according to the invention. The analysis was carried out by ion-ex¬ change chromatography (HPLC) . Fig. 3 illustrates the ex¬ cellent agreement between the HbA]__c contents in the two methods.

The analysis according to the invention thus pro- duces results which completely correspond to the ana¬ lytical results from fresh blood.

Claims

1. Method for determining the HbA-j__c content in blood which is dried in an absorbing material, c h a r a c - t e r i s e d in that the dried blood is treated with a buffered cysteine solution for eliminating the interfer¬ ing HbA3 fraction and reducing the amount of methaemoglo¬ bin, whereupon the HbA]__c content is determined.

2. Method as claimed in claim 1, c h a r a c t e r - i s e d in that the absorbing material with dried blood is pretreated with a buffer solution.

3. Method as claimed in claim 1 or 2, c h a r a c ¬ t e r i s e d in that the cysteine solution is buffered to a pH of about 7.4.

4. Method as claimed in one or more of claims 1-3, c h a r a c t e r i s e d in that the cysteine solution contains cysteine, glycine and EDTA.

5. Method as claimed in one or more of claims 2-4, c h a r a c t e r i s e d in that a solution of phosphate, citrate, and a wetting agent is used as buffer solution for the pretreatment, said buffer solution preferably having a pH of about 5.4.

6. Method as claimed in one or more of the preceding claims c h a r a c t e r i s e d in that the HbA]__c con- tent is determined by HPLC.

7. Composition for eliminating the interfering HbA3 fraction and reducing the amount of methaemoglobin in eluate of dried blood from an absorbing material when de¬ termining the HbA]__c content, c h a r a c t e r i s e d in that it comprises a buffered solution of cysteine.

8. Composition as claimed in claim 7, c h a r a c ¬ t e r i s e d in that the pH of the cysteine solution is about 7.4.

9. Composition as claimed in claim 7 or 8, c h a r a c t e r i s e d in that the cysteine solution contains glycine, EDTA and a wetting agent.