WO2016186521A1

WO2016186521A1 - Haemolysis stabilising composition

Info

Publication number: WO2016186521A1
Application number: PCT/NZ2016/050077
Authority: WO
Inventors: Maurice Cedric OWEN
Original assignee: Canterbury Scientific Limited
Priority date: 2015-05-15
Filing date: 2016-05-13
Publication date: 2016-11-24

Abstract

Described herein are haemolysis stabilising compositions and methods of use in the medical industry for the analysis of whole blood samples from patients. The compositions contain a combination of agents comprising at least one red blood cell membrane solubilisation agent, at least one chelating agent, at least one methaemoglobin binding agent, at least one pH buffering agent, at least one anticoagulant agent and at least one haemoglobin stabilising agent that contributes to improved stability of haemoglobin during transportation at ambient temperatures and improved ease of use in collecting and analysing the collected whole blood samples.

Description

HAEMOLYSIS STABILISING COMPOSITION

RELATED APPLICATIONS

This application derives priority from New Zealand patent application number 708194 incorporated herein by reference.

TECHNICAL FIELD

Described herein are haemolysis stabilising compositions and methods of use. More specifically, haemolysis stabilising compositions are described for use in the medical industry for the analysis of blood samples from patients. The compositions contain a combination of agents that provide various advantages.

BACKGROUND ART

Haemoglobin is the red blood protein that transports oxygen from the lungs to the tissues. It is the major component of the red blood cell (or erthyrocyte).

When whole blood is isolated outside the body and stored over a period of time it undergoes haemolysis where the haemoglobin is liberated from the red blood cell. This occurs due to changes within the red cell, membrane loss or a change in osmotic pressure between the internal contents of the red blood cell and the external environment.

Glycated haemoglobins

Glycated haemoglobins arise from the non-enzymatic attachment of glucose to haemoglobin. Because the life of a red cell averages some 120 days, the extent of haemoglobin glycation can be used as an indication of the average blood glucose level over the previous 2 -3 months and is thus useful as an indicator of diabetic control and as a diagnostic assay for diabetes.

A molecule of haemoglobin is made up of 4 protein chains. In the predominant adult form there are 2 alpha chains and 2 beta chains, usually denoted as α₂β₂- Glucose will react and bond to certain positively charged chemical groups on the haemoglobin. These are located at the start (N-terminal end) of each of the a and β chains and on some amino acid side chains within the protein such as the ε-amino group of certain lysine residues.

Glycated haemoglobin is defined as haemoglobin with glucose bound to any of these potential sites. HbAlc is a subset of glycated haemoglobins. It is defined as haemoglobin with glucose bound at the beginning (N-terminal) of the β-chain. The total glycated haemoglobin will include HbAlc plus all the other haemoglobins that have glucose bound to lysine side chains and/or to the N-terminal of the ct- chain.

The formation of HbAlc proceeds via a Schiff base adduct, or aldimine, (shown in step 1 above) followed by the Amadori rearrangement to form the stable ketoamine, HbAlc (shown in step 2 above). Glycation may occur on the Ale binding site (valine- 1 on the beta chain) and other non-Ale sites (valine- 1 on the alpha chain or lysine amino acid residues). The aldimine intermediate is often referred to as labile HbAlc and the rate of its conversion to the ketoamine is some 60 times slower than the reverse dissociation with release of the glucose.

HbAlc

The measurement of HbAlc is especially useful in insulin-dependent diabetic patients where blood glucose levels fluctuate widely and where the instantaneous blood glucose does not reflect the averaged situation. The formation of HbAlc occurs slowly and continuously during the 120-day lifetime of the red cell. Hence the measurement of HbAlc is useful to physicians as a long-term indicator of blood glucose concentration and thus as a measure of the degree of control or self-management by the diabetic patient.

The normal range for HbAlc is less than 5.8% of total haemoglobin, or in the IFCC units, less than about 40 mmol HbAlc/mol Hb. As a general rule for a diabetic subject HbAlc levels above 7.5% (or 58 mmol/mol) represent poor diabetic control, whereas values between 6.5% and 7.0% (48-53 mmol/mol) are indicative of good control. A level of less than 5.8% (40mmol/mol) virtually excludes diabetes, with 41-49 mmol/mol indicative of abnormal glucose tolerance and greater than 50 mmol/mol supportive of the diagnosis of diabetes. HbAlc is increasingly used as the diagnostic test of choice for diabetes.

HbAlc analytical instruments

There are many different types of instruments used for the analysis or quantitation of HbAlc. These instruments can be further classified as either laboratory based or as a point-of care analytical instrument.

In a first group, blood samples are collected, usually by venipuncture, from the subject and sent to the analytical laboratory for analysis (such as on a Tosoh Bioscience G7 or G8 HPLC analytical instrument). The result is usually delayed and is available to the doctor or nurse in 1 -2 days. In addition, there is an added cost in transporting the blood samples which are kept chilled to prevent degradation of the HblAc by conversion of haemoglobin to met-Hb when a heme iron is oxidised from the ferrous state (Fe2+) to the ferric state (Fe3+) with a resulting colour change from bright red to dark brown, or oxidation of the protein thiol group. This degradation may lead to inaccurate blood test results for HblAc due to the presence of an extra peak on some analytical instruments confounding the normal (desired) glycated Ale peak.

In a second group, the blood sample is taken from the subject, and immediately analysed on a point of care analytical instrument. The result is available to the doctor or nurse within a few minutes. Further the blood sample is from a finger prick or puncture, as used in a blood glucose test, and as such is less traumatic than a venipuncture. Also the blood sampling does not require a nurse with specific training in blood venesection.

Thus point of care analytical instruments has the advantage of delivering a test result whilst the subject is in the clinic. However the accuracy and precision of these instruments generally is not as good as the laboratory based equipment. Further controls and calibrators are used less frequently on point-of-care instruments.

Based on the above it should be appreciated that it may be useful to address at least some of the above disadvantages or at least provide the public with a useful choice.

Further aspects and advantages of the haemolysis stabilising composition and methods of use will become apparent from the ensuing description that is given by way of example only.

SUMMARY

Haemoglobin is inherently unstable and will readily oxidise or degrade if exposed to temperatures above 37 °C in diluted form for any length time.

The haemolysis stabilising composition and methods of use described herein broadly relate to haemolysis stabilising compositions comprising at least one chelating agent, at least one pH buffering agent and at least one anticoagulant that contributes to stabilising the haemoglobin.

In a first aspect, there is provided a haemolysis stabilising composition comprising:

a) a haemolysis stabilising effective amount of at least one red blood cell membrane solubilisation agent;

b) a haemolysis stabilising effective amount of at least one chelating agent;

c) a haemolysis stabilising effective amount of at least one methaemoglobin binding agent capable of modifying methaemoglobin to an oxy-haemoglobin equivalent form;

d) a haemolysis stabilising effective amount of at least one pH buffering agent;

e) a haemolysis stabilising effective amount of at least one anticoagulant agent; and

f) a haemolysis stabilising effective amount of at least one haemoglobin stabilising agent.

In a second aspect, there is provided a haemolysis stabilising composition comprising: a) approximately 0.001 to 0.05% w/v of at least one red blood cell membrane solubilisation agent; b) approximately 0.01 to 0.10% w/v of at least one chelating agent;

c) approximately 0.01 to 0.09 % w/v of at least one methaemoglobin binding agent capable of modifying methaemoglobin to an oxy-haemoglobin equivalent form;

d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent;

e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent; and

f) approximately 0.05 to 0.15 % w/v of at least one haemoglobin stabilising agent.

In a third aspect, there is provided a haemolysis stabilising solution comprising:

a) approximately 0.001 to 0.05% w/v of at least one red blood cell membrane solubilisation agent; b) approximately 0.01 to 0.10% w/v of at least one chelating agent;

d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent;

e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent; and

In a fourth aspect, there is provided a haemolysis stabilising composition comprising:

d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent;

e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent; and

f) approximately 0.05 to 0.15 % w/v of at least one haemoglobin stabilising agent

wherein the haemolysis stabilisation composition releases and stabilises the cellular haemoglobins, comprising HblAc, to achieve an extracellular haemoglobin to HbAlc ratio that is at least 95 % of the haemoglobin to HbAlc ratio of freshly collected red blood cells.

In a fifth aspect, there is provided a method of determining the HbAlc content in a blood sample from a patient, comprising the steps of:

(a) obtaining a whole blood sample container containing a quantity of the haemolysis stabilising composition as described above in a receptacle of the blood sample container;

(b) collecting the whole blood sample from the patient and transferring it into the receptacle of the whole blood sample container from step (a);

(c) mixing the collected whole blood sample from step (b) above with the haemolysis stabilising composition in the receptacle; and

(d) loading the mixture from step (c) above on an analytical instrument to measure the HbAlc level.

In a sixth aspect, there is provided a blood sample container comprising:

(a) a receptacle; and

(b) a quantity of haemolysis stabilising solution as described above contained in the receptacle.

In summary, advantages of the composition and methods of use described herein may comprise: · Improved stability of haemoglobin during transportation at usual ambient temperatures so that the analysed sample gives the same result as if fresh; and

• Improved ease of use in collecting and analysing whole blood samples without the need for a specialist venipuncture nurse and analysis on a more accurate laboratory-based blood analysis instrument.

BRIEF DESCRIPTION OF THE FIGURES

Further aspects of the haemolysis stabilisation solution and method of use thereof will become apparent from the following description that is given by way of example only and with reference to the accompanying drawing in which:

Figure 1 is a graph illustrating the relationship between HblAc levels (% of total haemoglobin) over time (hours) at a temperature of 20 to 25 °C.

DETAILED DESCRIPTION

As noted above, the application broadly relates to haemolysis stabilising compositions and methods of use comprising at least one chelating agent, at least one pH buffering agent and at least one anticoagulant. This combination of ingredients confers a haemolysis stabilisation effect with the advantages of improved stabilisation of haemoglobin and HbAlc such that it can tolerate ambient temperatures during transportation. In addition, haemolysis stabilisation composition prepares the whole blood sample for direct analysis on the analytical instrument without further processing. Further the ratio of the different constituent haemoglobins remains substantially unchanged relative to the haemoglobin levels in a freshly collected sample. In this way, the HbAlc level obtained is the same as if the sample were tested with freshly collected blood.

For the purposes of this specification, the term 'about' or 'approximately' and grammatical variations thereof mean a quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% to a reference quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length.

The term 'substantially' or grammatical variations thereof refers to at least about 50%, for example 75%, 85%, 95% or 98%. The term 'comprise' and grammatical variations thereof shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

The term 'haemolysis stabilising composition' broadly relates to a composition that has a stabilising effect on the total haemoglobins that are liberated from the red blood cells. 'Stabilising' as used here refers to inhibiting further haemoglobin degradation so as to limit its physical or chemical change.

The term 'HbAlc' refers to the subset of glycated haemoglobins with glucose bound at the beginning (N- terminal) of the β-chain. The total glycated haemoglobin will include HbAlc plus all the other haemoglobins that have glucose bound to lysine side chains and/or to the N-terminal of the a-chain. HbAlc is used as a measure of the degree of control or self-management of blood glucose by the patient and therefore as a indicator of diabetic control or as a diagnostic test for diabetes.

The term 'haemolysis stabilising effective amount' is intended to qualify the amount of composition that will inhibit further haemoglobin degradation by stabilising the structure of haemoglobin and glycated haemoglobins (HbAlc).

The term 'red blood cell' refers to erythrocytes which are the most common blood cell, the contents of which is rich in haemoglobin and delivers oxygen to the body tissues.

The term 'red blood cell membrane solubilisation agent' refers to a chemical, such as a detergent, capable of solubilising the cell wall of a red blood cell to liberate the internal contents of the red blood cell (such as haemoglobin) into solution. The term 'chelating agent' refers to a substance whose molecules can form multi-dentate ligands to single metal ions, such as calcium or magnesium ions in the blood.

The term 'methaemoglobin heme binding agent' refers to an agent capable of converting the heme iron from the ferric state (Fe³⁺) to the ferrous state (Fe²⁺) with a resulting colour change from dark brown to bright red . The term 'pH buffering agent' refers to a substance which is either a weak acid or base capable of maintaining the pH of a solution near a chosen value after the addition of a further acid or base to the solution.

The term 'anticoagulant' refers to a substance, which prevents the clotting (or coagulation) of blood which occurs within 10 to 20 minutes of exposure to ambient temperatures, for example, sodium citrate dihydrate forms citrate ions in solution which chelates calcium ions in the blood by forming calcium citrate complexes and thereby disrupting the blood clotting mechanism.

The term 'haemoglobin stabilisation agent' refers to agents such as sodium chloride capable of forming salt bridges between the two alpha and the two beta chains of the haemoglobin protein molecule.

The term 'analytical instrument' refers to a machine capable of determining the levels of HbAlc in a blood sample without further preparation of the blood samples after collection, such as a high performance liquid chromatograph (HPLC), such as a Tosoh Bioscience G7 or G8 analytical instrument, or the like.

b) a haemolysis stabilising effective amount of at least one chelating agent;

Typically the haemolysis stabilising composition stabilises the level of haemoglobin (including HblAc) after whole blood sample collection. That is after approximately 1, or 2, or 3, or 4, or 5 or more days, or after approximately 20, or 30, or 40, or 50, or 60, or 70, or 80, or 90, or 100, or 110, or 120 hours or more. In this way the whole blood sample is able to be transported at ambient temperatures without the need for transportation on ice or other freezing agents. Further, the container containing the whole blood sample in the haemolysis stabilising composition is able to be loaded directly onto the analytical instrument (TOSOH G7/G8) without further preparation.

The composition may release and stabilise the cellular haemoglobins, comprising HblAc, to achieve an extracellular haemoglobin to HbAlc ratio that is at least 85%, or 90 %, or 95 %, or 99 % of the haemoglobin to HbAlc ratio of freshly collected red blood cells. In one embodiment, the composition may decrease the number of microbes sufficient to substantially the same levels as present in a fresh blood sample. The composition may however be tailored to provide a lesser or greater stabilisation as desired by varying the concentration of the key active agents.

The pH of the composition may be between approximately 6.5, or 6.6, or 6.7, or 6.8, or 6.9, or 7.0, or 7.1, or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7, or 7.8 as measured at 25 °C. The variation in pH may depend on the amount of pH buffering agent used such as the ratio of disodium hydrogen phosphate dihydrate to potassium dihydrogen phosphate anhydrous. The inventor has found that this pH range provides the advantage of effective haemolysis stabilisation by mimicking the natural blood pH range. In one embodiment, the pH is in the approximate range 6.5 to 7.45 and is 7.30.

The osmolality of the composition may be between approximately 59, or 60, or 61, or 62, or 63, or 64 mOsmol/kg. In one embodiment the osmolality is in the approximate range 60 to 80 mOsmol/kg and is 62. The inventor has found that in this osmolality range provides the advantage of effective haemolysis without compromising the stability of haemoglobin.

The red blood cell membrane solubilisation agent is an anionic detergent selected from the group consisting of: tritors X-100, DDM, digitorsirs, tween 20, tween 80. In one embodiment the anionic detergent may be triton X-100, or the like. The red blood cell membrane solubilisation agent is approximately present in an amount 0.001, or 0.002, or 0.003, or 0.005, or 0.008, or 0.01, or 0.02, or 0.03, or 0.04, or 0.05 % w/v. In one embodiment the red blood cell membrane solubilisation agent is present in an amount between approximately 0.008 to 0.01 % w/v of the total composition.

The at least one chelating agent may be one or more members selected from the group comprising: EDTA (ethylenediaminetetraacetic acid), NTA (nitrilotriacetic acid), EDDA (ethylenediaminediacetic acid), CyDTA (trans- l,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate), DPTA-OH (1,3-Diamino- 2-hydroxypropane Ν,Ν,Ν',Ν'-tetraacetic acid), DTPA (Diethylenetriamine-N,N,N',N",N"-pentaacetic acid), EDDP (Ethylenediamine-N,N'-dipropionic acid, dihydrochloride), EDDPO [Ethylenediamine-N, N'- bis(methylenephosphonic acid), hemihydrate], EGTA [Ethyleneglycol-bis-( beta -amino-ethylether) tetraacetic acid], HBED [N,N'-bis(2-hydroxybenzyl) ethylenediamine-N,N-diacetic acid], HDTA (1,6-

Hexamethylenediamine-N,N,N',N'-tetraacetic acid), HIDA [N-(2-Hydroxyethyl) iminodiacetic acid], IDA (Iminodiacetic acid), NTP (Nitrilotripropionic acid), NTPO [Nitrilotris (methylenephosphonic acid), trisodium salt], TTHA (Triethylenetetramine-Ν,Ν,Ν',Ν'',Ν'", N"'-hexaacetic acid), etc., alpha -, beta -, gamma -CD (cyclodextrin), or these CD modified with polymer. A preferred chelating agent is EDTA. The EDTA can be a mixture of EDTA tetrasodium dihydrate and EDTA dipotassium dihydrate present at an amount of 0.01, or 0.02, or 0.03, or 0.04, or 0.05, or 0.06, or 0.07, or 0.08, or 0.09 or 0.1 % w/v and between 0.05 to 0.0423 % w/v of the total composition and the EDTA tetrasodium dihydrate is present at an amount 0.0285 % of the total composition and the EDTA dipotassium dihydrate is present at an amount of 0.0215 % w/v of the total composition. EDTA can also function as an anticoagulant agent as well as a chelating agent. The dual functionality of components can decrease the complexity of the haemolysis stabilising solution.

The methaemoglobin binding agent may be sodium azide. The methaemoglobin binding agent may be in an amount 0.01, or 0.02, or 0.03, or 0.04, or 0.05, or 0.06, or 0.07, or 0.08, or 0.09 % w/v of the total composition. In one embodiment the methaemoglobin binding agent may be present in an approximate range 0.0336 to 0.045 % w/v of the total composition. The oxy-haemoglobin form may be

azomethaemoglobin.

The at least one pH buffering agent may be either an acid or base capable of maintaining the pH of the haemolysis stabilising composition when in solution near a chosen value after the addition of a further acid or base to the solution. The pH buffering agent may be a mixture of disodium hydrogen phosphate dihydrate and potassium dihydrogen phosphate anhydrous. The pH buffering agent may be present in a total amount of approximately 0.005, or 0.01, or 0.02, or 0.03, or 0.04 or 0.05 % w/v of the total composition. In one embodiment the pH buffering agent may be present in an approximate range 0.032 to 0.038 % w/v of the composition. For example, the disodium hydrogen phosphate dihydrate may be present in an amount 0.0285 % w/v of the total composition and the potassium dihydrogen phosphate anhydrous is present in an amount of approximately 0.0095 % w/v of the total composition.

The at least one anticoagulant may be tri-sodium citrate dihydrate. The at least one anticoagulant may be present in an amount of approximately 0.05, or 0.10, or 0.15 % w/v of the total composition. In one embodiment the at least one anticoagulant may be present in an amount 0.05 to 0.10 % w/v of the total composition. For example, the at least one anticoagulant may be present in an amount 0.098 % w/v of the total composition. However a person skilled in the art will appreciate that other known anticoagulants (such as heparin or warfarin) could be used. Citrate ions in solution also provide a dual function as a haemoglobin stabilising agent.

The haemoglobin stabilisation agent may be a salt capable of making the haemolysis stabilising composition to have ions for salt bridges that are formed between the respective a and β globin chains. The salt may be sodium chloride and/or potassium chloride. The salt may be present in an amount of approximately 0.05, or 0.10, or 0.15, or 0.20 % w/v of the total composition. In one embodiment, the haemoglobin stabilising agent is present in an amount 0.09 to 0.102 % w/v of the total composition.

There also may be potassium chloride present in an amount of approximately 0.0027 % w/v of the total composition.

The haemolysis stabilising composition also comprises an antimicrobial agent capable of killing microbes in the blood sample such as bacteria, fungi or viruses. The antimicrobial agent may be sodium azide. The sodium azide is present in an amount of approximately 0.02 to 0.04 % w/v of the total composition and in one embodiment is present in an amount 0.0336 % w/v of the total composition. The antimicrobial agent may be effective in killing microbes in a 3, or 3.5, or 4, or 4.5, or 5-log reduction. The antimicrobial agent also modifies methaemoglobin to a form suitable for analytical purposes. So it has a dual function in the haemolysis stabilising composition.

In a second aspect, there is provided a haemolysis stabilising composition comprising:

d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent; e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent; and f) approximately 0.05 to 0.15 % w/v of at least one haemoglobin stabilising agent.

The inventor has un-expectantly found that these concentration ranges provides the advantage of effective haemolysis stabilisation.

d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent;

e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent; and

The solution should be sterile in order to prevent inadvertent introduction of microbes into the blood sample. The haemolysis stabilising composition may also be provided as a dry powder, except for the red cell membrane solubilisation agent which is a solution, although this component is a very small liquid amount which would be absorbed by the other solid components. The haemolysis stabilising composition may also be coated on the surface of plastic or glass such as inside a blood sample container such as a vial, and which would dissolve on the addition of water before use.

c) approximately 0.01 to 0.09 % w/v of at least one methaemoglobin binding agent capable of modifying methaemoglobin to a form detectable on an analytical instrument;

d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent;

e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent; and

g) a pH of the composition is substantially in the range 6.5 to 7.45; and

h) an osmolarity of the composition is of between about 40 to about 80 mOsmol/kg

The extracellular HblAc level may be at least 98 to 99.99 % of the HblAc level in a freshly collected whole blood sample over a time period of approximately 120 hours.

(a) obtaining a whole blood sample container containing a quantity of the haemolysis stabilising composition as described above;

The term "whole blood" typically means unmodified (not separated), arterial or venous blood, such as that drawn from a subject. But the term is also intended to encompass any blood drawn from a subject that has not been separated into component parts by means such as centrifugation. The whole blood sample may be human blood or a whole blood sample of non-human animal origin such as cow, pig, sheep, mouse, dog, monkey, rabbit, chicken or the like. Provided the analytical instrument is calibrated to analyse the particular whole blood sample.

A whole blood sample may be taken by a finger prick into a capillary tube, typically about 10 uL, and then transferred in a vial containing about 2 ml of the haemolysis stabilising composition that lyses the red cells and stabilizes the haemoglobin and HbAlc. The blood is "washed" out of the capillary tube by gentle mixing and dispersal in a solution of the haemolysis stabilising composition. The vial is then sent to a laboratory for the sample to be analysed. The whole blood sample container with the whole blood sample and used capillary tube is placed directly on the analytical instrument for analysis.

The analytical instruments that the haemolysis stabilising composition may be used on include laboratory based analytical instruments. In one embodiment the analytical instrument may be the TOSOH Bioscience G7 or G8 HPLC.

In a sixth aspect, there is provided a blood sample container comprising:

(a) a receptacle; and

The blood sample container could be manufactured out of a rigid material capable of being sterilised.

The container materials may be inert with respect to the blood and haemolysis stabilisation composition, examples including glass or plastic. The container may also be configured to be airtight. Examples include Vacutainer^® tubes that may contain additives or preservatives for stabilising the blood sample. WORKING EXAMPLES

The above described haemolysis stabilisation compositions and methods of use are now described by reference to a specific example.

EXAM PLE 1

A haemolysis composition was prepared by combining the components as set out in the below Table 1 below. The concentration of Triton X-100 is based on an average molecular weight of 625.

Ta b le 1

The above components are dissolved in water (ideally type 1 grade) and mixed. The solution has the following properties as shown in Table 2 below. Assay Target Result Range pH at room temperature (20 to 25°C) 7.30 6.5 to 7.8

Osmolality (mOsmol/kg) 62 40 to 80

Ta b le 2

If the osmolality is too high, it may be adjusted to the target result by adding extra water.

EXAM PLE 2

The haemolysis stabilising solution (HSS) as prepared in Example 1 above was mixed with a sample of human blood. The stability of HblAc was demonstrated by analysing HbAlc levels of a HSS-blood sample mixture over a 120 hour period (5 days). This result was obtained using a Tosoh Bioscience G8 analytical instrument. The HbAlc levels are shown in Table 3 below and represented in Figure 1.

Ta b le 3

These results show a high level of stabilisation of HblAc levels over a time period of 120 hours of 5.3% of total haemoglobin levels or 34.8 mmol of HbAlc/mol of haemoglobin at room temperature (20 to 25 oC). Therefore the HSS of the present invention enables accurate measurement of HblAc levels up to 5 days (depending upon ambient temperature) after blood sample collection and is therefore useful as a clinical laboratory test and in the pharmaceutical field. The embodiments described above may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features.

Further, where specific integers are mentioned herein which have known equivalents in the art to whic the embodiments relate, such known equivalents are deemed to be incorporated herein as of individually set forth

Aspects of the haemolysis stabilising composition have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the present invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A haemolysis stabilising composition comprising:

a) a haemolysis stabilising effective amount of at least one red blood cell membrane

solubilisation agent;

b) a haemolysis stabilising effective amount of at least one chelating agent;

e) a haemolysis stabilising effective amount of at least one anticoagulant agent; and f) a haemolysis stabilising effective amount of at least one haemoglobin stabilising agent.

2. A haemolysis stabilising composition comprising:

a) approximately 0.001 to 0.05% w/v of at least one red blood cell membrane solubilisation agent;

b) approximately 0.01 to 0.10% w/v of at least one chelating agent;

d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent;

e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent; and

3. A haemolysis stabilising composition comprising:

b) approximately 0.01 to 0.10% w/v of at least one chelating agent;

d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent;

e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent;

f) approximately 0.05 to 0.15 % w/v of at least one haemoglobin stabilising agent; and g) wherein the haemolysis stabilisation composition releases and stabilises the cellular

haemoglobins, comprising HblAc, to achieve an extracellular haemoglobin to HbAlc ratio that is at least 95 % of the haemoglobin to HbAlc ratio of freshly collected red blood cells.

4. The haemolysis stabilising composition as claimed in claim 3 wherein the extracellular haemoglobin to HbAlc ratio is at least 99 % of the haemoglobin to HbAlc ratio of freshly collected red blood cells.

5. The haemolysis stabilising composition as claimed in any preceding claim wherein the composition stabilises the cellular haemoglobins, comprising HblAc, over a time period of up to 120 hours at a temperature of up 25°C.

6. The haemolysis stabilising composition as claimed in any preceding claim wherein the red blood cell membrane solubilisation agent is present in an amount between 0.008 to 0.01% w/v.

7. The haemolysis stabilising composition as claimed in any preceding claim wherein the at least one chelating agent is EDTA.

8. The haemolysis stabilising composition as claimed in any preceding claim wherein the at least one methaemoglobin binding agent is present in an amount between 0.02 to 0.04% w/v.

9. The haemolysis stabilising composition as claimed in any preceding claim wherein the at least one methaemoglobin binding agent is sodium azide.

10. The haemolysis stabilising composition as claimed in any preceding claim wherein the at least one pH buffering agent is present in an amount between 0.03 to 0.04% w/v.

11. The haemolysis stabilising composition as claimed in any preceding claim wherein the at least one anticoagulant agent is present in an amount between 0.05 to 0.10% w/v.

12. The haemolysis stabilising composition as claimed in any preceding claim wherein the at least one haemoglobin stabilising agent is present in an amount between 0.09 to 0.10% w/v.

13. The haemolysis stabilising composition as claimed in any preceding claim wherein the at least one haemoglobin stabilising agent is potassium chloride.

14. The haemolysis stabilising composition as claimed in any preceding claim wherein the composition also comprises an anionic detergent.

15. The haemolysis stabilising composition as claimed in any preceding claim wherein the anionic

detergent is triton X-100.

16. A haemolysis stabilising solution comprising:

b) approximately 0.01 to 0.10% w/v of at least one chelating agent;

c) approximately 0.01 to 0.09 % w/v of at least one methaemoglobin binding agent capable of modifying methaemoglobin to an oxy-haemoglobin equivalent form; d) approximately 0.005 to 0.05 % w/v of at least one pH buffering agent;

e) approximately 0.05 to 0.20 % w/v of at least one anticoagulant agent; and

18. The haemolysis stabilising solution as claimed in claim 16 wherein the pH of the solution is between 6.5 and 7.8.

19. The haemolysis stabilising solution as claimed in claim 16 wherein the osmolarity of the solution is between 59 to 80 mOsmol/kg.

20. The haemolysis stabilising solution as claimed in claim 16 wherein the solution produces a 3 to 5 log reduction in microbes in a blood sample.

21. A method of determining the HbAlc content in a blood sample from a patient, comprising the steps of:

a) obtaining a whole blood sample container containing a quantity of the haemolysis stabilising composition as claimed in any one of claims 1 to 4;

b) collecting the whole blood sample from the patient and transferring it into the receptacle of the whole blood sample container from step (a);

c) mixing the collected whole blood sample from step (b) above with the haemolysis stabilising composition in the receptacle; and

d) loading the mixture from step (c) above on an analytical instrument to measure the HbAlc level.

22. A blood sample container comprising:

a) a receptacle; and

b) a quantity of haemolysis stabilising solution as claimed in any one of the preceding claims contained in the receptacle.