Reagent for the determination of haemoglobin

A reagent for the determination of haemoglobin comprising an aqueous solution of potassium cyanide, potassium hexacyanoferrate-III, a buffer substance and a detergent, wherein the pH value is in the range of 7.1 to 7.3 and the potassium cyanide concentration is in the 90 to 120 mg/liter range. The reagent can be used with plastic throw-away cells, particularly polystyrene cells.

The invention relates to a reagent for the determination of haemoglobin, 
for use in plastic throwaway cells, which consists of a certain aqueous 
solution of potassium cyanide, potassium hexacyanoferrate-III, a buffer 
substance for adjustment of the pH value and a detergent. This reagent is 
based on the internationally recognised ICSH reference method, (see 
International Committee for Standardization in Haematology: 
Recommendations for Haemoglobinometry in Human Blood, British Journal of 
Haematology 13 (1967) Suppl. 71-75). 
The haemiglobin cyanide method is at present regarded as the most accurate 
process for the determination of haemoglobin (see W. G. Zijlstra and E. J. 
van Kampen: Standardization of Haemoglobinometry, I. The Extinction 
Coefficient of Haemiglobin Cyanide, Clinica Chimica Acta 5 (1960) 719-726; 
E. J. van Kampen and W. G. Zijlstra: Standardisation of Haemoglobinometry, 
II. The Haemiglobin Cyanide Method, Clinica Chimica Acta 6 (1961) 538-544; 
and Bestimmung des Hamoglobingehaltes im Blut (Determination of the 
Haemoglobin Content of Blood), Draft Standard Specification DIN 58,931, 
sheet 1, November 1970. 
In addition to the customary test packs, ready-to-use test kits which 
enable the user to carry out the determination rapidly and in a simple 
manner without having to make up the reagent himself are also available 
for the determination of haemoglobin. In these "ready-to-use test kits", 
which are also called mono-tests, single-tests or single-glass tests, 
small glass bottles are filled with the haemoglobin reagent and this 
reagent must still be transferred into cells for the measurement. 
In some cases, the reagent is also available in round cells in which the 
haemoglobin determination can be carried out directly. These round cells 
are also made of glass. 
Cells which can be sealed have recently been disclosed (see DT-OS (German 
Published Specification) No. 2,422,260); these are in the form of plastics 
throwaway measuring cells (preferably made of polystyrene) and are sealed 
air-tight with a plastics lid. 
In the case of mass production, the lid is attached to the cell by welding, 
for example ultrasonically. The lid has a prescribed breaking point, which 
can be broken open by means of a peg. The sample to be analysed can be 
introduced into the cell through the opening thus produced in the cell 
lid. 
The cells described in the abovementioned Offenlegungsschrift fulfil, to a 
large extent, the requirements of a "ready-to-use test kit". It is not 
necessary for the user either to dissolve reagents or mix different 
solutions exactly or transfer the finished reagent/sample mixture into a 
measuring vessel. He has only to introduce the sample to be analysed into 
the cell by exact metering, which can be effected, for example, simply by 
means of volume-calibrated glass capillaries, and to transfer the cell to 
the measuring unit (a photometer). 
Possible sources of error (e.g. in pipetting) are thereby excluded from the 
beginning, and the analysis operation is simplified to such an extent that 
even unskilled personnel can carry out the analysis correctly. 
However, using throw-away measuring cells has its problems. Glass is 
excluded as a material for throwaway measuring cells since glass cells of 
the quality required for photometric measurements are much too expensive. 
The use of plastics, for example polystyrene, indeed permits low-cost mass 
production of throw-away measuring cells, but frequently presents problems 
with respect to the life of the reagents. 
Thus, polystyrene is permeable to many gases. This has an adverse effect, 
for example, on the life of the reagent for the determination of 
haemoglobin as haemiglobin cyanide. As mentioned above, in addition to a 
buffer substance and a detergent for haemolysis of the erythrocytes, this 
reagent also contains, potassium hexacyanoferrate-III, which oxidises 
haemoglobin to haemiglobin, and KCN, which converts haemiglobin into 
haemiglobin cyanide. The haemiglobin cyanide is then determined 
photometrically. 
The KCN contained in the reagent is in equilibrium with HCN in the gas 
space above the liquid. However, HCN can diffuse through the polystyrene 
wall of the cell, so that the CN content of the reagent decreases with a 
half-life of about 4 weeks. This results in a life of a reagent which is 
insufficient for the reagent to be sold. 
DE-AS (German Published Specification) No. 2,721,942 describes a process 
which overcomes these problems of the life. 
In the process described, one reactant--KCN in the case of the 
determination of haemoglobin--is applied in the form of a solid to the 
inner surface of the glass capillary used for metering the sample. 
Long lives (18-24 months) are achieved by transferring the unstable 
component--the KCN--from the reaction solution into the capillary for 
metering the sample. 
The disadvantage of the process described is the fact that the capillaries 
coated with KCN on the inner surface must be manufactured with great 
accuracy, and the manufacture is correspondingly expensive. 
The object of the present invention is thus to provide a less expensive 
process in order to ensure the competitiveness of the haemoglobin 
ready-to-use cell, which the intended solution having an equivalent 
quality This applies, in particular, with respect to the precision and 
accuracy of the haemoglobin determination and also respect to the reaction 
time required. 
Various experiments have already been carried out to produce a haemoglobin 
reagent which is stable in plastics ready-to-use cells. 
Thus, for example, the pH value of the reagent has been shifted to the 
alkaline range in order to stabilise the cyanide in the reagent. Better 
lives are also thereby achieved. A decisive disadvantage of the process 
is, however, that the reaction time is considerably longer. Thus, the 
conversion of haemoglobin into haemiglobin cyanide with a reagent such as 
described, for example, in the abovementioned draft standard specification 
DIN 58,931, sheet 1, of November 1970, has ended after a reaction time of 
3 minutes. If the pH value of the reagent is now shifted from pH 7.2 to pH 
8.2 without changing other constituents, this reaction time is extended to 
12 to 15 minutes. This is a considerable disadvantage, for example for 
emergency determinations. 
Other manufacturers have attempted to obtain a reagent which is stable in 
plastic cells by increasing the phosphate buffer concentration from 2.5 
mmoles to 10 mmoles. However, these attempts have also been unsuccessful. 
A further disadvantage of increasing the phosphate buffer concentration is 
the fact that this has an adverse effect on the stability of the K.sub.3 
[Fe(CN).sub.6 ] complex. Attempts to achieve a sufficient life of the 
haemoglobin reagent in plastic cells by metering in an excess of KCN fail 
for two reasons. On the one hand, the half-life, of about 4 weeks, of the 
KCN in plastic ready-to-use cells is very short, so that 32 times the 
nominal concentration (which would be about 2,080 mg for a nominal 
concentration of 65.12 mg of KCN per liter of reagent) would have to be 
employed for a life of one year. On the other hand, the life of the 
K.sub.3 [Fe(CN).sub.6 ] complex is impaired by high KCN concentrations. 
For example, whilst the concentration of the K.sub.3 [Fe(CN).sub.6 ] 
remains constant at about 200 mg per liter for 1 year in the case of an 
initial concentration of 65 mg of KCN per liter of reagent, in the case of 
an initial KCN concentration of 860 mg per liter it falls from 200 mg per 
liter to 130 mg per liter in the course of 18 weeks, and is thus in a 
range in which significant deviations from the nominal value occur in the 
determination of haemoglobin. 
Various attempts have also been made to achieve a better life of the 
haemoglobin reagent by adding preservatives or stabilisers. Substances 
such as benzyl alcohol, p-chloro-m-cresol, solbrol, polyethylene glycol 
and collidone have been used for this. However, these attempts have also 
met with no success. 
Surprisingly, a reagent which is based on the ICSH reference method and is 
suitable for use in plastic throw-away cells and which has the required 
long-term stability has now been found. 
According to the present invention there is provided a reagent for the 
determination of haemoglobin for use in plastics throw-away cells, which 
consists essentially of an aqueous solution of potassium cyanide, 
potassium hexacyanoferrate-III, a buffer substance for adjustment of the 
pH value and a detergent, in which the pH value is adjusted within a 
narrow range of 7.1 to 7.3, with the aid of the buffer substance, and the 
potassium cyanide concentration is 90 to 120 mg/liter (1.38 to 1.85 
mmoles/liter). 
When this reagent is used in polystyrene ready-to-use cells, a quasi-stable 
state develops after a short time, so that a life of at least 12 months is 
achieved in polystyrene ready-to-use cells. The present invention thus 
also comprises plastics, especially polystyrene, throw-away cells 
comprising a solution of the present invention. 
In the stable state, the reagent corresponds to that of the ICSH reference 
method. There are no differences between the two reagents with respect to 
the rate of conversion of haemoglobin into haemiglobin cyanide (rate of 
reaction). The results of 431 haemoglobin determinations in which the 
haemoglobin content of in each case one blood sample was determined both 
using the ISCH reference method and using the reagent according to the 
invention show a very good agreement. An average haemoglobin value of 15.0 
g/100 ml was found with the ICSH reference method. An average value of 
14.8 g/100 ml was obtained with the reagent according to the invention. 
The pairs of values found were correlated. A correlation line was 
calculated as follows: 
EQU x=1.0139.multidot.y-0.0333, 
in which 
x corresponds to the values determined using the ICSH reference method and 
y corresponds to the values determined with the reagent according to the 
invention. 
The correlation coefficient was calculated as r=0.9934.

The following Example illustrates the preparation of a reagent according to 
the present invention. 
EXAMPLE 
A haemoglobin reagent of the following composition was prepared, for 
filling a polystyrene throw-away measuring cell: 1.54 mmoles/liter of 
potassium cyanide, 0.6 mmole/liter of potassium hexacyanoferrate-III, 2.5 
mmoles/liter of potassium phosphate buffer of pH 7.1 to 7.3 and 0.05% of 
detergent (for example saponin). 
1.25 ml of solution were obtained therefrom, using distilled water as the 
solvent. If appropriate, small amounts of NaCl (about 1.5 mmoles/liter) 
could also be added to this solution, in order to avoid cloudiness.