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Hemoglobinometry: evaluation of a new method with a stable primary standard
Pathology (1988), 20, pp. 152-155
HEMOGLOBINOMETRY: EVALUATION OF A NEW METHOD WITH A STABLE PRIMARY STANDARD B. R. WYLIEAND V. A. LOVRIC Red Cross Blood Transfusion Service, Sydney
Pathology Downloaded from informahealthcare.com by University of Otago on 07/10/15 For personal use only.
Summary Using purified chlorohemin as a stable standard, a newly described hemoglobinometry method was evaluated. All blood samples as well as chlorohemin gave identical absorption spectra in an alkaline-detergent solution with a broad peak at 575 nm. Both the reagent mixture and the chlorohemin standard were stable for at least 250 days when stored between 8OC and 32OC in brown glass bottles. The new method showed close correlation when compared to the cyanmethemoglobin technique. Compared to the cyanmethemoglobin method the advantages included a stable standard, shorter conversion time, reduced plasma background and improved hemoglobin ligands conversion. We conclude that the new method confers significant advantages over the cyanmethemoglobin method and in particular provides a stable primary standard with a long shelf-life.
Key words: Hemoglobinometry, chlorohemin, cyanmethemoglobin, alkaline hematin, primary standard. Accepted November 29, 1987
INTRODUCTION Numerous methods have been used for the quantitation of hemoglobin, but many have significant drawbacks.’ The International Committee for Standardisation in Haematology (ICSH) has recommended the cyanmethemoglobin (HiCN) method as a reference method.2v3The advantages of HiCN over other methods are that it involves a single chemical reaction, it measures all hemoglobin species and derivatives of clinical relevance, it provides a stable reaction product with a broad extinction peak at 540 nm, it conforms to the BeerLambert law and a stable standard can be manufactured. However, the HiCN method still has disadvantages. The reagent mixture must be monitored carefully. The mixture is light-labile and has a shelf life of under 6 mth and even less in tropical settings. The reaction times for various hemoglobin species and derivatives vary; in particular, the conversion time of carboxyhemoglobin is long and sulphemoglobin is not converted completely into HiCN. Further, standardization is based on HiCN solutions which are analysed for their exact hemoglobin content. Finally, commercially obtained HiCN standards are expensive, have limited shelf life and require controlled refrigeration for prolonged storage. Once these
standards are removed from their sterile ampoule, the shelf life is at best only a few days. It was therefore deemed advisable to evaluate a recently published method, stated to be free from the above objections.4s5It is named alkaline hematin D-575,where D stands for the detergent ingredient and 575 for the maximum (nm) absorption wave length. In the presence of alkali and detergent, hemoglobin is released from the lysed erythrocytes and converted to alkaline hematin, resulting in a stable reaction product with an absorption peak at 575 nm. A stable standard, composed of purified chlorohemin, provides the reference material with a long shelf life.
MATERIAL AND METHODS Blood samples were obtained from blood donors attending the N.S.W. Red Cross Blood Transfusion Service, Sydney. Cord blood samples and others with elevated fetal hemoglobin levels were from the Haernatology Department, Royal Prince Alfred Hospital, Sydney. Carboxyhemoglobin was produced by bubbling carbon monoxide gas through the anticoagulated blood samples. Purified chlorohemin powder (MW 651.95) was kindly supplied by The standard reagent mixutre (AHD) was a 0.1M NaOH solution with 25 g of Triton-X-100 (Sigma)/]. Additional reagents were made by varying the concentrations of components and/or substituting KOH for NaOH. The chlorohemin standard solution was made by dissolving 0.0931 mmol of purified chlorohemin in 10 ml of the AHD reagent; this yielded the extinction coefficient corresponding to a hemoglobin concentration of 150 g/l. The HiCN reagent was that described by Van Kampen and Zijlstra.6 Tests were performed by adding 20 PI of anticoagulated blood to 3 ml of AHD or 5 ml of the HiCN reagents. Absorbances were measured at 575 nm or 540 nm respectively. All measurements were done in at least triplicate and recorded on a Beckman DU-50 recording spectrophotometer. Hemoglobin concentrations were calculated from absorbance values at 575 nm using the constant derived from the millimolar extinction coefficient of chlorohemin (E mmolar =6.960) and the fact that 1 mmol of chlorohemin corresponds to 0.25 mmol of hemoglobin, together with the dilution factor (1:151) giving Hb (mmol/l)
.‘. Hb (g/l)
=
151 -x AS7’ 27.84 5.424 X AS7’
=
66,458 x
=
10,OOo
5.424 x AS7’
= 34.96 X AS7’
NEW HEMOGLOBINOMETRY METHOD
0.8000
1 ,I
153
TABLE1 Comparison of paired hemoglobin estimates (with cyanmethemoglobin and AHD tests) in smokers and non-smokers. Smokers v non-smokers
0.6400
I
Correlation coefficient
Total (22) Smokers (12) Non-smokers (10)
I Slope
0.9974 0.9975 0.9970
0.9858 0.9827 0.9813
0.4800.
e,
a 0 op
m
Pathology Downloaded from informahealthcare.com by University of Otago on 07/10/15 For personal use only.
<
600
640
420
680
WAVELENGTH
680
700
lnml
Fig. 1 Absorption spectrum of the chlorohemin standard in the AHD reagent.
RESULTS All blood samples as well as the chlorohemin standard recorded characteristic absorption curves in the AHD reagent (Fig. 1). Blood samples were converted rapidly to alkaline hematin, the reaction product being olive-green in colour, and stable for at least 7 d (compared to HiCN which gave increasing absorbance values from 48 h onwards). Consistent and stable AHD results were obtained with samples kept at 4°C for up to one week. Almost identical results were obtained when the concentration of NaOH and/or Triton-X-100 were halved or the NaOH replaced by KOH.
The Beer-Lambert law was observed over a wide range of hemoglobin concentration (Fig. 2) with a correlation coefficient of 0.9998 and a slope of 1.028. Additional studies confirmed that the relationship remained linear even with hemoglobin concentrations as low as 0.1 g/l. Comparison of results between the AHD and HiCN methods over a wide range of hemoglobin concentrations also showed close correlation (Fig. 3). There were no significant differences in calculated hemoglobin values between the two methods when using 36.77 as the conversion factor for the absorbance of HiCN at 540 nm. No significant differences were found when comparing blood taken from smokers and nonsmokers, probably reflecting low carboxyhemoglobin levels in this donor population (Table 1). When blood samples were saturated with carbon monoxide gas, significant differences were observed between the two methods (Table 2). The HiCN method initially recorded falsely high readings which decreased with time to the corresponding true hemoglobin concentration. The AHD method recorded correct measurements almost immediately, indicating rapid and
AHD v HI C N
C o r r r l a t i o m e o * f f i e i * m t 0.9999
0 Hb
(41611
Fig. 2 Plot of hemoglobin concentration against absorbance values.
4
8
12
16
HiCN
10
14
iq/dll
Fig. 3 Comparison of hemoglobin measurements utilizing cyanmethemoglobin and AHD techniques.
28
154
Pathology (1988),20, April
WYLIE AND LOVRIC
TABLE 2 Difference in recorded hemoglobin values when measuring carboxyhemoglobin over set time periods using cyanmethemoglobin and A H D tests.
Pathology Downloaded from informahealthcare.com by University of Otago on 07/10/15 For personal use only.
AHD HiCN
5 mins
30 mins
60 mins
120 mins
159 168
159 162
159 159
159 158
complete conversion of carboxyhemoglobin into alkaline hematin. There were no significant differences when measuring hemoglobin concentrations in cord blood and other samples with raised fetal hemoglobin levels with the two methods (data not shown). Plasma background absorbance was measured by mixing 100 p1 of freshly prepared plasma with either 3 ml of AHD or 5 ml of HiCN reagent. The plasma background absorbance values were consistently and significantly lower in the AHD reagent in each of the 10 specimens tested, the mean AHD value being 18.2% of that obtained with HiCN reagent (range 5-47070). The chlorohemin standard had an initial absorbance 1 diluted into 3 ml of AHD of 0.428 k 0.004 when 2 0 ~ was reagent. This standard solution when stored in a brown glass screw top container at between 8-32 "C was stable after 250 days (Table 3). The chlorohemin standard followed the Beer-Lambert Law. Dilutions of samples of known hemoglobin concentration recorded identical points and slopes when plotted against dilutions of the standard using a range of photometers with appropriate filters. Similar comparable results were obtained when dilutions of the standard were measured with various photometers using appropriate filters (data not shown). DISCUSSION The D-575 alkaline hematin method appears to be an important advance for accurate measurements of hemoglobin concentration and provides for simplified hemoglobin standardization. Hemoglobin concentrations measured as acid-hematin are imprecise, whilst the drawback of oxyhemoglobin methodology is the lack of a stable primary standard. Although the HiCN method provides a primary standard, this is measured indirectly, and the standard is expensive and has a rather limited,
TABLE 3 Stability of chlorohernin standard
Standard solution
I Day Day Day Day Day
1 2
3 50 250
Abs at 575 nm
0.428f0.004 0.428k0.003 0.429f0.003 0.427f 0.005 0.428k 0.005
Stored at room temperature in a brown bottle
I
Hb (&I)
150 150 I50 149 150
refrigeration-dependent shelf life. The major advantage of the AHD method is the availability of chlorohemin as a primary, albeit indirect, standard with long-term stability. Chlorohemin is the chloride form of hemin which is the product of oxidized heme. Although chlorohemin is used as an indirect standard this is more than compensated for by its ready availability and extreme stability. We have confirmed stability for at least 250 days with continual use whilst Wolf4 has reported a shelf life of at least 2 years. Furthermore, chlorohemin is cheap and easily available commercially. Although commercial chlorohemin generally may not be pure enough and as such unsuitable for use as a standard, it can be readily purified by simple methodology within the capabilities of most laboratories .7 For convenience we used the purified chlorohemin supplied by Wolf who made it from a modification of a standard m e t h ~ dAs . ~ chlorohemin is available in pure form it allows for convenient preparation of standards from which the equivalent hemoglobin concentration may be determined exactly. Although Wolf reported some initial variation in extinction considered to relate to peroxide contamination of some batches of non-ionic detergents, we did not encounter this problem with Triton X-100, used locally and distributed commercially in large volumes. We also believe that the new method is superior to the current HiCN reference method when measuring carbo~yhemoglobin.~The measurement of carboxyhemoglobin has been a major limiting factor with the HiCN method and necessitated additional tests to overcome this problem. The AHD method demonstrates rapid and complete conversion of carboxyhemoglobin and allows for rapid assays, for example in automation. The presence of sulphemoglobin, although rarely a clinical problem, has also been shown to have rapid and complete conversion using this t e c h n i q ~ e . ~ The finding of reduced plasma background absorbance with the AHD reagent has further important implications. Whilst we could demonstrate no significant difference between hemoglobin values when examining normal blood samples by the two methods, we have not specifically selected specimens with turbidity due to hyperlipidemia or with elevated bilirubin levels, where the AHD method may confer an advantage. Reduced plasma background absorbance would also suggest a role for the AHD method in the measurement of plasma hemoglobin, particularly in stored blood. However, preliminary studies suggest that the method lacks sufficient sensitivity with hemoglobin concentrations of less than 0.1 g/l. Thus, for measurements of plasma hemoglobin concentrations we would prefer the method developed by Raftos.' The AHD method is simple to use, precise and provides for a stable primary standard. The simplicity, low cost of reagent materials, and stability of both reagent and the primary standard would make it ideal for routine application in developing countries, where accurate and precise hemoglobinometry is still an unresolved problem due to unavailability of standards. In the longer term, the D-575 method overcomes all major deficiencies of the current HiCN reference method and should be viewed as a significant advance in hemoglobinometry.
NEW HEMOGLOBINOMETRY METHOD
Address for correspondence: B.W., N.S.W. Red Cross Blood Transfusion Service, 153 Clarence Street, Sydney, N . S . W . 2000
References ~~~i~ J ~ Lewis , SM. Practical Haematology, 6th ed. Churchill Livingstone, 1984. International Committee for Standardisation in Haematology of the European Society of Haernatology. Recommendations and requirements for haemoglobinornetry in human blood. J Clin Pathol 1965; 18: 352-5.
4. Zander R , Lang W, Wolf HU. Alkaline haernatin D-575. A new tool for the determination of haemoglobin as an alternative to the cyanmethaemoglobin. I. Description of the method. Clin Chim Acta 1984; 136: 83-93. 5 . Wolf HU, L a w W, Zander R. Alkaline haematin D-575. A new
tool for the determination of haemoglobin as an alternative to the cyanmethaemoglobinmethod 11. Standardisation of the method using pure chlorohaemin. Clin Chim Acta 1984; 136: 95-104. 6. Van Kampen EJ* ZiJ1stra W G . Of haemoglobinometry 11. The haemoglobincyanidemethod. Clin Chim Acta 1961: 6: 538-44. 7. Labbe RF, Nishida G . A new method of hemin isolation. Biochim
Biophys Acta 1957; 26: 437. 8. Raftos JE, Stewart IM, Lovric VA. Supernatant hemoglobin determinations after prolonged blood storage. Pathology 1986; 18: 123-6.
Pathology Downloaded from informahealthcare.com by University of Otago on 07/10/15 For personal use only.
International Committee for Standardisation in Haematology; Expert Panel on Haemoglobinometry. Recommendations for reference method for haemoglobinometry in human blood (ICSH Standard 1986). Specifications for an -international haemoglobincyanide reference preparation (3rd edition) Clin Lab Haemat 1987; 9, 73-9.
155
THE ENGLISHMAN ABROAD Gracious I feel not in myself those common antipathies that I can discover in others: those national repugnances do not touch me, nor do I behold with prejudice the French, Italian, Spaniard or Dutch; but where I find their actions in balance with my Countrymen’s, I honour, love, and embrace them in the same degree. Sir Thomas Browne. Religio Medici. Works. ed. by Simon Wilkin. Pickering. 1836. Vol. 2. p. 86.
HEMOGLOBINOMETRY: EVALUATION OF A NEW METHOD WITH A STABLE PRIMARY STANDARD B. R. WYLIEAND V. A. LOVRIC Red Cross Blood Transfusion Service, Sydney
Pathology Downloaded from informahealthcare.com by University of Otago on 07/10/15 For personal use only.
Summary Using purified chlorohemin as a stable standard, a newly described hemoglobinometry method was evaluated. All blood samples as well as chlorohemin gave identical absorption spectra in an alkaline-detergent solution with a broad peak at 575 nm. Both the reagent mixture and the chlorohemin standard were stable for at least 250 days when stored between 8OC and 32OC in brown glass bottles. The new method showed close correlation when compared to the cyanmethemoglobin technique. Compared to the cyanmethemoglobin method the advantages included a stable standard, shorter conversion time, reduced plasma background and improved hemoglobin ligands conversion. We conclude that the new method confers significant advantages over the cyanmethemoglobin method and in particular provides a stable primary standard with a long shelf-life.
Key words: Hemoglobinometry, chlorohemin, cyanmethemoglobin, alkaline hematin, primary standard. Accepted November 29, 1987
INTRODUCTION Numerous methods have been used for the quantitation of hemoglobin, but many have significant drawbacks.’ The International Committee for Standardisation in Haematology (ICSH) has recommended the cyanmethemoglobin (HiCN) method as a reference method.2v3The advantages of HiCN over other methods are that it involves a single chemical reaction, it measures all hemoglobin species and derivatives of clinical relevance, it provides a stable reaction product with a broad extinction peak at 540 nm, it conforms to the BeerLambert law and a stable standard can be manufactured. However, the HiCN method still has disadvantages. The reagent mixture must be monitored carefully. The mixture is light-labile and has a shelf life of under 6 mth and even less in tropical settings. The reaction times for various hemoglobin species and derivatives vary; in particular, the conversion time of carboxyhemoglobin is long and sulphemoglobin is not converted completely into HiCN. Further, standardization is based on HiCN solutions which are analysed for their exact hemoglobin content. Finally, commercially obtained HiCN standards are expensive, have limited shelf life and require controlled refrigeration for prolonged storage. Once these
standards are removed from their sterile ampoule, the shelf life is at best only a few days. It was therefore deemed advisable to evaluate a recently published method, stated to be free from the above objections.4s5It is named alkaline hematin D-575,where D stands for the detergent ingredient and 575 for the maximum (nm) absorption wave length. In the presence of alkali and detergent, hemoglobin is released from the lysed erythrocytes and converted to alkaline hematin, resulting in a stable reaction product with an absorption peak at 575 nm. A stable standard, composed of purified chlorohemin, provides the reference material with a long shelf life.
MATERIAL AND METHODS Blood samples were obtained from blood donors attending the N.S.W. Red Cross Blood Transfusion Service, Sydney. Cord blood samples and others with elevated fetal hemoglobin levels were from the Haernatology Department, Royal Prince Alfred Hospital, Sydney. Carboxyhemoglobin was produced by bubbling carbon monoxide gas through the anticoagulated blood samples. Purified chlorohemin powder (MW 651.95) was kindly supplied by The standard reagent mixutre (AHD) was a 0.1M NaOH solution with 25 g of Triton-X-100 (Sigma)/]. Additional reagents were made by varying the concentrations of components and/or substituting KOH for NaOH. The chlorohemin standard solution was made by dissolving 0.0931 mmol of purified chlorohemin in 10 ml of the AHD reagent; this yielded the extinction coefficient corresponding to a hemoglobin concentration of 150 g/l. The HiCN reagent was that described by Van Kampen and Zijlstra.6 Tests were performed by adding 20 PI of anticoagulated blood to 3 ml of AHD or 5 ml of the HiCN reagents. Absorbances were measured at 575 nm or 540 nm respectively. All measurements were done in at least triplicate and recorded on a Beckman DU-50 recording spectrophotometer. Hemoglobin concentrations were calculated from absorbance values at 575 nm using the constant derived from the millimolar extinction coefficient of chlorohemin (E mmolar =6.960) and the fact that 1 mmol of chlorohemin corresponds to 0.25 mmol of hemoglobin, together with the dilution factor (1:151) giving Hb (mmol/l)
.‘. Hb (g/l)
=
151 -x AS7’ 27.84 5.424 X AS7’
=
66,458 x
=
10,OOo
5.424 x AS7’
= 34.96 X AS7’
NEW HEMOGLOBINOMETRY METHOD
0.8000
1 ,I
153
TABLE1 Comparison of paired hemoglobin estimates (with cyanmethemoglobin and AHD tests) in smokers and non-smokers. Smokers v non-smokers
0.6400
I
Correlation coefficient
Total (22) Smokers (12) Non-smokers (10)
I Slope
0.9974 0.9975 0.9970
0.9858 0.9827 0.9813
0.4800.
e,
a 0 op
m
Pathology Downloaded from informahealthcare.com by University of Otago on 07/10/15 For personal use only.
<
600
640
420
680
WAVELENGTH
680
700
lnml
Fig. 1 Absorption spectrum of the chlorohemin standard in the AHD reagent.
RESULTS All blood samples as well as the chlorohemin standard recorded characteristic absorption curves in the AHD reagent (Fig. 1). Blood samples were converted rapidly to alkaline hematin, the reaction product being olive-green in colour, and stable for at least 7 d (compared to HiCN which gave increasing absorbance values from 48 h onwards). Consistent and stable AHD results were obtained with samples kept at 4°C for up to one week. Almost identical results were obtained when the concentration of NaOH and/or Triton-X-100 were halved or the NaOH replaced by KOH.
The Beer-Lambert law was observed over a wide range of hemoglobin concentration (Fig. 2) with a correlation coefficient of 0.9998 and a slope of 1.028. Additional studies confirmed that the relationship remained linear even with hemoglobin concentrations as low as 0.1 g/l. Comparison of results between the AHD and HiCN methods over a wide range of hemoglobin concentrations also showed close correlation (Fig. 3). There were no significant differences in calculated hemoglobin values between the two methods when using 36.77 as the conversion factor for the absorbance of HiCN at 540 nm. No significant differences were found when comparing blood taken from smokers and nonsmokers, probably reflecting low carboxyhemoglobin levels in this donor population (Table 1). When blood samples were saturated with carbon monoxide gas, significant differences were observed between the two methods (Table 2). The HiCN method initially recorded falsely high readings which decreased with time to the corresponding true hemoglobin concentration. The AHD method recorded correct measurements almost immediately, indicating rapid and
AHD v HI C N
C o r r r l a t i o m e o * f f i e i * m t 0.9999
0 Hb
(41611
Fig. 2 Plot of hemoglobin concentration against absorbance values.
4
8
12
16
HiCN
10
14
iq/dll
Fig. 3 Comparison of hemoglobin measurements utilizing cyanmethemoglobin and AHD techniques.
28
154
Pathology (1988),20, April
WYLIE AND LOVRIC
TABLE 2 Difference in recorded hemoglobin values when measuring carboxyhemoglobin over set time periods using cyanmethemoglobin and A H D tests.
Pathology Downloaded from informahealthcare.com by University of Otago on 07/10/15 For personal use only.
AHD HiCN
5 mins
30 mins
60 mins
120 mins
159 168
159 162
159 159
159 158
complete conversion of carboxyhemoglobin into alkaline hematin. There were no significant differences when measuring hemoglobin concentrations in cord blood and other samples with raised fetal hemoglobin levels with the two methods (data not shown). Plasma background absorbance was measured by mixing 100 p1 of freshly prepared plasma with either 3 ml of AHD or 5 ml of HiCN reagent. The plasma background absorbance values were consistently and significantly lower in the AHD reagent in each of the 10 specimens tested, the mean AHD value being 18.2% of that obtained with HiCN reagent (range 5-47070). The chlorohemin standard had an initial absorbance 1 diluted into 3 ml of AHD of 0.428 k 0.004 when 2 0 ~ was reagent. This standard solution when stored in a brown glass screw top container at between 8-32 "C was stable after 250 days (Table 3). The chlorohemin standard followed the Beer-Lambert Law. Dilutions of samples of known hemoglobin concentration recorded identical points and slopes when plotted against dilutions of the standard using a range of photometers with appropriate filters. Similar comparable results were obtained when dilutions of the standard were measured with various photometers using appropriate filters (data not shown). DISCUSSION The D-575 alkaline hematin method appears to be an important advance for accurate measurements of hemoglobin concentration and provides for simplified hemoglobin standardization. Hemoglobin concentrations measured as acid-hematin are imprecise, whilst the drawback of oxyhemoglobin methodology is the lack of a stable primary standard. Although the HiCN method provides a primary standard, this is measured indirectly, and the standard is expensive and has a rather limited,
TABLE 3 Stability of chlorohernin standard
Standard solution
I Day Day Day Day Day
1 2
3 50 250
Abs at 575 nm
0.428f0.004 0.428k0.003 0.429f0.003 0.427f 0.005 0.428k 0.005
Stored at room temperature in a brown bottle
I
Hb (&I)
150 150 I50 149 150
refrigeration-dependent shelf life. The major advantage of the AHD method is the availability of chlorohemin as a primary, albeit indirect, standard with long-term stability. Chlorohemin is the chloride form of hemin which is the product of oxidized heme. Although chlorohemin is used as an indirect standard this is more than compensated for by its ready availability and extreme stability. We have confirmed stability for at least 250 days with continual use whilst Wolf4 has reported a shelf life of at least 2 years. Furthermore, chlorohemin is cheap and easily available commercially. Although commercial chlorohemin generally may not be pure enough and as such unsuitable for use as a standard, it can be readily purified by simple methodology within the capabilities of most laboratories .7 For convenience we used the purified chlorohemin supplied by Wolf who made it from a modification of a standard m e t h ~ dAs . ~ chlorohemin is available in pure form it allows for convenient preparation of standards from which the equivalent hemoglobin concentration may be determined exactly. Although Wolf reported some initial variation in extinction considered to relate to peroxide contamination of some batches of non-ionic detergents, we did not encounter this problem with Triton X-100, used locally and distributed commercially in large volumes. We also believe that the new method is superior to the current HiCN reference method when measuring carbo~yhemoglobin.~The measurement of carboxyhemoglobin has been a major limiting factor with the HiCN method and necessitated additional tests to overcome this problem. The AHD method demonstrates rapid and complete conversion of carboxyhemoglobin and allows for rapid assays, for example in automation. The presence of sulphemoglobin, although rarely a clinical problem, has also been shown to have rapid and complete conversion using this t e c h n i q ~ e . ~ The finding of reduced plasma background absorbance with the AHD reagent has further important implications. Whilst we could demonstrate no significant difference between hemoglobin values when examining normal blood samples by the two methods, we have not specifically selected specimens with turbidity due to hyperlipidemia or with elevated bilirubin levels, where the AHD method may confer an advantage. Reduced plasma background absorbance would also suggest a role for the AHD method in the measurement of plasma hemoglobin, particularly in stored blood. However, preliminary studies suggest that the method lacks sufficient sensitivity with hemoglobin concentrations of less than 0.1 g/l. Thus, for measurements of plasma hemoglobin concentrations we would prefer the method developed by Raftos.' The AHD method is simple to use, precise and provides for a stable primary standard. The simplicity, low cost of reagent materials, and stability of both reagent and the primary standard would make it ideal for routine application in developing countries, where accurate and precise hemoglobinometry is still an unresolved problem due to unavailability of standards. In the longer term, the D-575 method overcomes all major deficiencies of the current HiCN reference method and should be viewed as a significant advance in hemoglobinometry.
NEW HEMOGLOBINOMETRY METHOD
Address for correspondence: B.W., N.S.W. Red Cross Blood Transfusion Service, 153 Clarence Street, Sydney, N . S . W . 2000
References ~~~i~ J ~ Lewis , SM. Practical Haematology, 6th ed. Churchill Livingstone, 1984. International Committee for Standardisation in Haematology of the European Society of Haernatology. Recommendations and requirements for haemoglobinornetry in human blood. J Clin Pathol 1965; 18: 352-5.
4. Zander R , Lang W, Wolf HU. Alkaline haernatin D-575. A new tool for the determination of haemoglobin as an alternative to the cyanmethaemoglobin. I. Description of the method. Clin Chim Acta 1984; 136: 83-93. 5 . Wolf HU, L a w W, Zander R. Alkaline haematin D-575. A new
tool for the determination of haemoglobin as an alternative to the cyanmethaemoglobinmethod 11. Standardisation of the method using pure chlorohaemin. Clin Chim Acta 1984; 136: 95-104. 6. Van Kampen EJ* ZiJ1stra W G . Of haemoglobinometry 11. The haemoglobincyanidemethod. Clin Chim Acta 1961: 6: 538-44. 7. Labbe RF, Nishida G . A new method of hemin isolation. Biochim
Biophys Acta 1957; 26: 437. 8. Raftos JE, Stewart IM, Lovric VA. Supernatant hemoglobin determinations after prolonged blood storage. Pathology 1986; 18: 123-6.
Pathology Downloaded from informahealthcare.com by University of Otago on 07/10/15 For personal use only.
International Committee for Standardisation in Haematology; Expert Panel on Haemoglobinometry. Recommendations for reference method for haemoglobinometry in human blood (ICSH Standard 1986). Specifications for an -international haemoglobincyanide reference preparation (3rd edition) Clin Lab Haemat 1987; 9, 73-9.
155
THE ENGLISHMAN ABROAD Gracious I feel not in myself those common antipathies that I can discover in others: those national repugnances do not touch me, nor do I behold with prejudice the French, Italian, Spaniard or Dutch; but where I find their actions in balance with my Countrymen’s, I honour, love, and embrace them in the same degree. Sir Thomas Browne. Religio Medici. Works. ed. by Simon Wilkin. Pickering. 1836. Vol. 2. p. 86.