GB2396913A

GB2396913A - A cyanide-free reagent for measuring haemoglobin in blood and a method for measuring haemoglobin

Info

Publication number: GB2396913A
Application number: GB0327866A
Authority: GB
Inventors: James Walsh; Caoimh Ronan Patrick O; Brendan Kevin Farrell; Arcy Marie D
Original assignee: Trinity Research Ltd
Current assignee: Trinity Research Ltd
Priority date: 2002-12-03
Filing date: 2003-12-02
Publication date: 2004-07-07
Also published as: IES20020936A2; GB0327866D0

Abstract

A cyanide-free reagent and method for stabilising and measuring the total haemoglobin concentration in a blood sample. The reagent contains a surfactant capable of lysing erythrocytes and releasing haemoglobin and a compound in a sufficient amount capable of oxidising haemoglobin and its derivatives to methemoglobin and forming a stable chromogen for haemoglobin concentration measurement. This compound is selected from the group consisting of nitrite and nitrate salts. The reagent is mixed with a blood sample and the optical density of the chromogen formed is measured at the corresponding absorption wavelength.

Description

! 2396913 "A cyanide-free reagent for measuring haemoglobin in blood, and

a method for measuring haemoglobin" The present invention relates to a cyanide- free reagent for measuring haemoglobin s in blood, and the invention also relates to a method for measuring haemoglobin in blood using the cyanide- free reagent.

Measurement of haemoglobin concentration in a blood sample is useful for clinical diagnosis of diseases such as leukaemia, anaemia, polycythemia and other haematological disorders. Drabkin's method, described in an article entitled "Spectrophotometric Studies" in the Journal of Biological Chemistry, 1935, Volume 112, page 57 is the approved reference method for total haemoglobin measurements in whole blood samples. In this method, an erythrolytic agent containing a surfactant is added to a specimen to haemolyse red blood cells to release haemoglobin. The released haemoglobin is then oxidised by the action of potassium ferricyanide to produce me/hemoglobin. Cyanide ions are then added to the mix and methemoglobin is converted to a more stable chromogen, cyanomethemoglobin. The haemoglobin concentration of the sample is determined by measuring the absorbance of the chromogen at a specific wavelength, 540nm.

Although low concentrations are used, cyanide is a lethal chemical and constitutes significant hazard to laboratory personnel. Moreover, this method does not lend itself easily to automation, and furthermore, can yield erroneous results.

U.S. Patent Specification No. 4,853,338 of Benezra, et al discloses a cyanide-free reagent comprising hydroxide anions as heme oxidising and binding ligands and a surfactant at very high concentration (2% to 5% v/v) to Iyse cells and release haemoglobin. However, for the chromogen to be stable, large amounts of hydroxide anions have to be used.

U.S. Patent Specification No. 5,612,223 of Kim and Stroupe discloses a cyanide-free reagent and method for determining haemoglobin concentrations in blood samples.

The reagent consists of a heme-binding ligand, imidazole or its derivatives, and a surfactant from the group of lauryidimethylamine oxide to Iyse cells and release lo haemoglobin. However, for imidazole to bind the heme molecule, the pH of the reagent needs to be adjusted to from 11 to about 14. In other words, a high concentration of hydroxide ions is required, as is the case in the method of Benezra.

Accordingly, neither of the reagents of Benezra or Kim when used with the Drabkin's Is method lend themselves to easy automation.

There is therefore a need for a reagent for measuring haemoglobin in blood which is cyanide-free, and which lends itself to ready automation, and also and importantly produces accurate results. There is also a need for a method for measuring haemoglobin in blood.

The present invention is directed towards providing such a reagent and a method.

According to the invention there is provided a cyanide-free reagent for measuring haemoglobin in blood, the reagent comprising a surfactant with an erythrolytic capability, the surfactant being at a concentration for haemolysing erythrocytes in a blood sample and releasing haemoglobin, and an oxidising and ligand-forming agent selected from a nitrite or nitrate salt at a concentration capable of simultaneously s oxidising haemoglobin and binding home sites to form stable chromogens.

In one embodiment of the invention the oxidising and ligand-forming agent is selected and at a concentration capable of oxidising the haemoglobin and its derivatives to methemoglobin to form the stable chromogen.

In one embodiment of the invention the oxidising and ligand-forming agent is selected from the group of nitrite and nitrate anion.

In another embodiment of the invention the surfactant comprises at least one anionic lS surfactant selected from the group of oclylphenoxy ethanol.

In another embodiment of the invention the cation is selected from the group of sodium, potassium, magnesium, amyl and butyl, although needless to say, the cation may be selected from any compound capable of forming a salt with nitrite or nitrate. In another embodiment of the invention the concentration of the oxidising and ligand-forming agent in the reagent is in the range of 0.05M to 2M.

In another embodiment of the invention the concentration of the surfactant in the reagent is in the range of 0.1% to 3%.

Additionally, the invention provides a method for measuring haemoglobin in blood, the method comprising adding a blood sample to the reagent to form a chromogen and scanning the chromogen with light of wavelength 400nm to 700nm for determining the absorbance of the chromogen.

In one embodiment of the invention an aliquot of blood sample is added to the reagent according to the invention, and the optical density of the chromogen formed is read using a spectrophotometer for reading absorbance of the chromogen.

lo Preferably, the wavelength at which the optical density is monitored is a function of the pH of the solution, and for a solution of pH below about 9, the wavelength at which the absorption of the chromogen is monitored is in the range of 540nm to 570nm.

Is In another embodiment of the invention for a solution of pH above 9, the wavelength at which the optical density of the chromogen is monitored is in the range of 570nm to 600nm.

The advantages of the invention are many. By virtue of the fact that the reagent is cyanide-free, it is safe to use in laboratories and elsewhere. However, a particularly important advantage of the invention is that pH adjustment of the reagent is not required, and thus, high concentrations of hydroxide anions are not required.

Accordingly, the reagent readily lends itself to automatic measurement of haemoglobin in blood. Additionally, the reagent provides accurate results.

The invention will be more clearly understood from the following description of some non-limiting examples thereof, which are given with reference to the accompanying drawings, in which: Fig. 1 illustrates a waveform representing a spectrum of a sample prepared with a cyanide-free reagent according to the invention, Fig. 2 illustrates waveforms of spectra of a sample prepared with the cyanide o free reagent at various pH values, Fig. 3 illustrates waveforms of spectra of a sample prepared with the cyanide- free reagent at various times after mixing, Fig. 4 illustrates a correlation plot of haemoglobin data obtained using a cyanide-free reagent according to the invention with a pH of 7 using the method according to the invention plotted against a known method, and Fig. 5 illustrates a correlation plot of haemoglobin data obtained using a 2() cyanide-free reagent according to the invention with a pH of 12 using the method according to the invention plotted against a known method.

The preparation of a cyanide-free reagent according to the invention is prepared and described in Example 1.

Example 1 i

The cyanide-free reagent of this example is prepared by mixing an oxidising and ligand-forming agent, in this case nitrite salt, and a surfactant provided by s octylphenoxy polyoxyethanol sold under the trade name Triton X-100 at an appropriate concentration in water. Approximately 700 mL of deionised water is collected in a calibrated container. With constant mixing, 699 (1M) of sodium nitrite is added. Mixing continues until dissolution is complete. Triton X-100 is then added to the mix at a concentration of 1% v/v (1 ml/L). Once Triton is completely dissolved, the volume of the solution is adjusted to 1L with deionised water and filtered through; a 0.2 1lm filter into a clean container. The pH of this solution measured 7 and 8.

The method according to the invention for measuring haemoglobin in blood using the cyanide-free reagent prepared in Example 1 will now be described with reference to

Is Example 2. t

Example 2

An aliquot (10 pL) of a whole blood sample is added to 2.5mL of the reagent of Example 1 and is thoroughly mixed with the reagent. The absorbance of the chromogen formed is scanned from 700nm to 400nm on a Beckman DU-7 spectrophotometer (Fig. 1, nitrite me/hemoglobin). The mixture immediately turns to a dark green colour indicating that red blood cell Iysing and hence oxidisation and ligation is complete. As a control, the absorbance of sample mixed with a reagent prepared in the same manner as in Example 1 except that sodium nitrite was omitted was scanned (Fig. 1, oxyhaemoglobin). Fig. 1 clearly shows that the reagent of this invention converts oxyhaemoglobin to me/hemoglobin. This oxidation occurs only when sodium nitrite is present in the reagent.

Example 3

The absorbance curve for methemoglobin is markedly influenced by changes in pH.

The spectrum shown in Fig. 1 was obtained at a pH of 7.4. Reagents were prepared in the same manner as in Example 1 except that pH was adjusted to various levels (pH 8, 9, 10, 12) with sodium hydroxide. Fig. 2 shows spectra of whole blood lo sample mixed with the above reagents in the same proportion (sample to reagent) as in Example 2. At lower pH values, the absorbance shows peaks at approximately 540nm and 570nm. At higher pH values the absorbance peaks are at 570nm and 600nm.

Example 4

Whole blood sample (1 OuL) is mixed with 2.5mL of the reagent prepared in Example 1, and the absorbance is monitored with time to test the completeness and stability of the conversion to chromogen. Fig. 2 shows the spectra at various incubation times. As shown, the conversion is complete within ten seconds and the chromogen remains stable for a few hours after formation.

Example 5

Two different formulations of the reagent according to the invention are prepared as set out below under the headings "Formula 1" and "Formula 2" using a method similar to that of Example 1. The performance of the two formulations of the reagent are compared with a haemoglobin measurement method published by Oshiro I, Takenaka T. Maeda J., "New method for haemoglobin determination by using sodium lauryl sulfate (SLS), Clinical Biochemistry volume 15 (1982) at page 83. The reagent of the published method of Oshiro contains an anionic surfactant, sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS), and a nonionic surfactant such as Triton X-100. The red blood cells are Iysed by the anionic surfactant and a chromogen is formed within ten minutes, with absorption peaks at 539nm and 572nm. Triton is added to keep the anionic surfactant in solution at temperatures l0 below SAC.

Formula 1 1 M sodium nitrite (69g/L) 1% Triton X-100 (1mL/L) pH 7.4 Formula 2 1 M sodium nitrite (69g/L) 1% Triton X-100 (1mL/L) pH 12 (adjusted with 1M NaOH) Samples were prepared in the same manner as in Example 2 for all three methods.

Table 1 represents results obtained with twelve whole blood samples analysed on a Beckman DU-7 Spectrophometer (Beckman Instruments, Fullerton, CA). All three assays were calibrated with a whole blood material assigned by Drabkin's method.

Table 1

Sample Oshiro SDS Hb Formulation 1 Formulation 2 method 1 O O O 2 12.6 12.6 12.6 _.... _.

3 9.5 9.5 9.3 _. . . 4 11.2 10.3 10.5 12.3 _ 12.2 10.7 6 14.1 13.4 12.7 7 13.7 13.6 13.9 8 15.0 14.7 13.6 9 15.6 15.7 14.7

_

15.9 15.8 15.5

_ _ _

11 19.2 18.0 17.8 _ 12 16.2 15.5 14.6 s As can be seen in Fig.4 and 5, the performance of Formulations 1 and 2 is essentially equivalent to that of the published method, as indicated by the linear regression analyses (slope=0.96 and intercept=0.2, r=0.996) and (slope=0.92 and intercept=0.2, r=0.991), respectively.

From the above it can be seen that the cyanide-free reagent according to the invention provides excellent results, which are quite unusual and unexpected.

Nitrites and nitrates are often added as preservatives and texture enhancers to various foods, such as meat products and cheeses, but they are also a natural part of the diet and can be found in vegetables. In the 1980s nitrites and nitrates became common substances of abuse. Although these agents are safe when inhaled, ingestion of large amounts have been implicated as causes of both symptomatic and fatal methemglobinaemia which occurs when blood haemoglobin is oxidised to lo me/hemoglobin, see for example Steiner R.W., Manoguerra AS. Butyl Nitrite and Methemoglobinemia, Ann Inter Med 1980 Apr; 92(4):570; Dudley MJ and Solomon T., A case of methemoglobinemia. Archives of Emergency Medicine 101117-119, 1993; Ellis G. et al Nitrite and Nitrate Analyses: A Clinical Biochemistry Perspective.

s Clin Biochem 31, 195-220, 1998; Guss DA, Normann SA, Manoguerra AS Am J Emerg Med 3(1):46-7, 1985. This reaction interferes with the ability of haemoglobin to bind oxygen and transport it to cells. However, from the above it can be seen that! nitrates and nitrites can not only completely oxidise haemoglobin to methemoglobin but also stabilise it for a period long enough to accommodate automated analysers lo to determine haemoglobin concentrations in blood samples.

While the cyanide-free reagent according to the invention and the method according to the invention have been described for measuring haemoglobin in blood, the cyanide-free reagent and method may be used for other measurements requiring the measurement of haemoglobin, for example, measurements for white blood counts and glycated haemoglobin.

While a specific surfactant has been described, any surfactant or combination of surfactants may be used, however, at least one of the surfactants, in general, would be a strong detergent and typically would be present at concentrations in the range of from approximately 0.1 % to approximately 3%.

While specific oxidising and ligand-forming agents have been described, any other suitable nitrite and nitrate salts capable of simultaneously oxidising the haemoglobin and binding heme sites to form stable chromogens.

Claims

Claims 1. A cyanide-free reagent for measuring haemoglobin in blood, the

reagent comprising a surfactant with an erythrolytic capability, the surfactant being at a concentration for haemolysing erythrocytes in a blood sample and releasing haemoglobin, and an oxidising and ligand-forming agent selected from a nitrite or nitrate salt at a concentration capable of simultaneously oxidising haemoglobin and binding home sites to form stable chromogens.
2. A reagent as claimed in Claim 1 in which the oxidising and ligandforming lo agent is selected and at a concentration capable of oxidising the haemoglobin and its derivatives to methemoglobin to form the stable chromogen.
3. A reagent as claimed in Claim 1 or 2 in which the oxidising and ligand forming agent is selected from the group of nitrite and nitrate anion.
4. A reagent as claimed in any preceding claim in which the surfactant comprises at least one anionic surfactant selected from the group of octylphenoxy ethanol.
5. A reagent as claimed in any preceding claim in which the cation may be selected from any compound capable of forming a salt with nitrite or nitrate.
6. A reagent as claimed in Claim 5 in which the cation is selected from the group of sodium, potassium, magnesium, amyl and butyl.
7. A reagent as claimed in any preceding claim in which the concentration of the oxidising and ligand-forming agent in the reagent is in the range of 0.05M to 2M.

s
8. A reagent as claimed in any preceding claim in which the concentration of the surfactant in the reagent is in the range of 0.1% to 3%.
9. A cyanide-free reagent for measuring haemoglobin in blood, the reagent being substantially as described herein with reference to and as illustrated in the lo accompanying drawings.
10. A method for measuring haemoglobin in blood, the method comprising adding a blood sample to the reagent as claimed in any preceding claim to form a chromogen and scanning the chromogen with light of wavelength 400nm to 700nm for determining the absorbance of the chromogen.
11. A method as claimed in Claim 10 in which an aliquot of blood sample is added to the reagent, and the optical density of the chromogen formed is read using a spectrophotometer for reading absorbance of the chromogen. 2()
12. A method as claimed in Claim 10 or 11 in which the wavelength at which the optical density is monitored is a function of the pH of the solution, and for a solution of pH below about 9, the wavelength at which the absorption of the chromogen is monitored is in the range of 540nm to 570nm.
13. A method as claimed in any of Claims 10 to 12 in which the wavelength at which the optical density of the chromogen is monitored for a solution of pH above 9 is in the range of 570nm to 600nm.
14. A method for measuring haemoglobin in blood, the method being substantially as described herein with reference to and as illustrated in the accompanying drawings.