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Stabilization of abnormal hemoglobin standards with carbon monoxide for use in hemoglobin electrophoresis
Clinica Chimica Acta 317 (2002) 235 – 237 www.elsevier.com/locate/clinchim
Letter to Editors
Stabilization of abnormal hemoglobin standards with carbon monoxide for use in hemoglobin electrophoresis Keywords: Abnormal hemoglobins; Hemoglobinopathies; Electrophoresis of hemoglobin; Carbon monoxide
The diagnosis of hemoglobinopathies is of great value, as these abnormalities occur in several social and ethical segments in different parts of the world. The diagnosis of a hemoglobinopathy in neonates is of great importance, as special care can be taken earlier, and during the entire life of affected patients [1]. A very accurate hematoscopy is required to guide the clinician who has a suspicion of a hemoglobinopathy [2], and who would then request more specific blood tests for confirmation. Hemoglobin electrophoresis at pH 8.6 is the chosen method for the laboratory diagnosis in the vast majority of cases [3]. The procedure is in use mainly in large laboratories. Small and medium laboratories do not have the facilities for performing this technique because there is no stable commercially available variant hemoglobin standard for the electrophoresis analysis to be used in parallel with the blood sample of the patient. And because hemolysates are not stable for more than 1 week, laboratories cannot accumulate fresh hemolysates prepared from blood samples containing variant hemoglobins. Our preliminary studies showed that the addition of different concentrations of several preservative agents such as dimethylsulphoxide, sodium azide and butylated hydroxytoluene into the hemolysates before the storage, did not result in their stability for more than 2 months. However, when hemolysates were saturated with CO, suitable electrophoretic gels were obtained [4]. Since hemoglobin has a binding affinity of about 210 times greater for CO compared
to oxygen [5], the present work aimed to creating stable hemolysates, through the use of carbon monoxide saturation. In this study, stability was defined as a period of time in which hemolysate shows a suitable electrophoretic gel, with no qualitative and quantitative changes as compared to its corresponding electrophoretic gel from a fresh hemolysate. Five milliliters of venous blood samples were collected into Sistema Vacutainer tubes (Becton-Dickinson), containing EDTA, from normal subjects and patients with different variant hemoglobins. Normal blood samples were from volunteers of the Faculty of Pharmacy of the Federal University of Minas Gerais while the blood samples from patients were obtained at the Centre of Hematology and Hemotherapy of Minas Gerais (Hemominas Foundation). The protocol was approved by Local Ethical Committee and informed consent was obtained from all participants. Hemolysates were prepared according to Naoum [6] and their concentration was adjusted at 7 g/dl. Hemoglobin electrophoresis was carried out at pH 8.6 using fresh hemolysates to check the electrophoretic profile for all the participants. The electrophoretic system utilized was from Technow. Hemolysates containing normal (AA) and variant hemoglobins (SS, CC and SC) were treated to CO saturation, which was produced by reacting sodium formate and concentrated sulfuric acid. The first reagent was transferred to a filtering flask and the second one was dropped on the salt through a funnel thereby producing CO gas. This was passed through a rubber tube connected from kitasate to the vial containing the hemolysate [7]. Three-milliliter samples of hemolysate were saturated with CO for 20 min. After saturation, each hemolysate was divided into 10 aliquots, and sealed under vacuum. Five aliquots were stored in the freezer ( 20 °C) and the others, under refrigeration (4 –8 °C). These samples were electrophoresed following storage of 45, 90, 135, 180, 240, 300
0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 ( 0 1 ) 0 0 7 7 0 - 7
236
Letter to Editors
kept in freezer at the same temperature for 1 month did not provide a satisfactory electrophoretical run (Fig. 1b). Further work is needed to obtain hemoglobin standards with longer stability. Other experiments related to the methods development should include the use of a CO bottle as a CO source instead of the chemical method to produce CO gas, which may further improve the stability. After hemolysates saturation, it would also be desirable to assess the CO concentrations to use the same amount for all samples. We report for the first time, a method for producing stable hemoglobin standards. This methodology is useful for clinical laboratories conducting routine hemoglobin electrophoresis. It is easy to perform and is inexpensive.
Acknowledgements Research supported by Fundacßa˜o de Amparo a` Pesquisa de Minas Gerais (FAPEMIG-CDS 2153/96). Godoi and Ladeira are recipients of CNPq research fellowship.
References Fig. 1. (a) Comparison of electrophoretic profiles of fresh (above) and CO-saturated hemolysates stored for 12 months (below). (b) Electrophoresis profile using non-CO-saturated hemolysate control kept in freezer at 20 °C for 1 month.
and 360 days. Non-CO-saturated hemolysates were used as the control. AA, SS and CC hemolysate variants showed a maximum of stability up to 6 months, while the SC hemolysates was stable for up to 12 months. The control samples did not show stability for longer than 2 and 4 weeks when kept in refrigerator or freezer, respectively. Fig. 1a shows a comparison of electrophoretic profiles of hemolysate prepared from a recently obtained SC patient and a hemolysate from a normal subject (HbAA), and the same samples stabilized by CO saturation and stored for 12 months at 20 °C. In contrast, the non-CO-saturated samples (controls)
[1] Carvalho MG, Souza MO, Silva MBS, Oliveira JMC, Cardoso IRCA, Carvalho IP, et al. Hemoglobinas anormais: perfil estatı´stico em doadores de sangue do Centro de Hematologia e Hemoterapia de Minas Gerais. Revista Brasileira de Ana´lises Clı´nicas 1994;26:39 – 40. [2] Coelho EAF, Carvalho MG. Aspectos morfolo´gicos das hemoglobinopatias. Revista Brasileira de Ana´lises Clı´nicas 1999;31: 201 – 3. [3] Bernard J, Le´vy JP, Clauvel JP, Rain JD, Varet B. Manual de Hematologia. 9th edn. Medsi, Rio de Janeiro; 2000, 368 pp. [4] Carvalho MG, Silva ZL, Ladeira MV. Tentativa de estabilizacßa˜o de padro˜es de hemoglobinas anormais para uso em eletroforese de hemoglobina em pH alcalino. Congresso Brasileiro de Patologia Clı´nica 1997;31:201. [5] Lima AO, Soares JB, Greco JB, Galizzi J, Cancßado JR. Me´todos de laborato´rio aplicados a` clı´nica. 6th edn. Guanabara Koogan, Rio de Janeiro; 1985, 699 pp. [6] Naoum PC. Diagno´stico das Hemoglobinopatias. Sa˜o Paulo: Sarvier, 1987, 242 pp. [7] ACCGC Malheiro. Determinacß a˜o espectrofotome´trica de carboxihemoglobinemia em indivı´duos expostos ocupacionalmente ao Mono´xido de Carbono. Master’s thesis, Faculdade de Cieˆncias Farmaceˆuticas, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, 1991.
Letter to Editors
Lara Carvalho Godoi Eduardo Antoˆnio F. Coelho Zelaine Lima e Silva Lauro Mello Vieira Maria Virgı´nia Ladeira Luci Maria Sant’Ana Dusse Geralda de Fa´tima Guerra Lages Maria Das Gracßas Carvalho * Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Minas Gerais, Avenida Olega´rio Maciel, 2360/608, Lourdes CEP 30180-112, Belo Horizonte, MG, Brazil E-mail address: [email protected]
237
Vicente Conceicßa˜o Macedo Liege Ribeiro Lyra Center of Hematology and Hemotherapy of Minas Gerais (HEMOMINAS Foundation), Belo Horizonte, MG, Brazil 21 March 2001
* Corresponding author. Tel.: +55-31-339-7647; fax: +55-31339-7666.
Letter to Editors
Stabilization of abnormal hemoglobin standards with carbon monoxide for use in hemoglobin electrophoresis Keywords: Abnormal hemoglobins; Hemoglobinopathies; Electrophoresis of hemoglobin; Carbon monoxide
The diagnosis of hemoglobinopathies is of great value, as these abnormalities occur in several social and ethical segments in different parts of the world. The diagnosis of a hemoglobinopathy in neonates is of great importance, as special care can be taken earlier, and during the entire life of affected patients [1]. A very accurate hematoscopy is required to guide the clinician who has a suspicion of a hemoglobinopathy [2], and who would then request more specific blood tests for confirmation. Hemoglobin electrophoresis at pH 8.6 is the chosen method for the laboratory diagnosis in the vast majority of cases [3]. The procedure is in use mainly in large laboratories. Small and medium laboratories do not have the facilities for performing this technique because there is no stable commercially available variant hemoglobin standard for the electrophoresis analysis to be used in parallel with the blood sample of the patient. And because hemolysates are not stable for more than 1 week, laboratories cannot accumulate fresh hemolysates prepared from blood samples containing variant hemoglobins. Our preliminary studies showed that the addition of different concentrations of several preservative agents such as dimethylsulphoxide, sodium azide and butylated hydroxytoluene into the hemolysates before the storage, did not result in their stability for more than 2 months. However, when hemolysates were saturated with CO, suitable electrophoretic gels were obtained [4]. Since hemoglobin has a binding affinity of about 210 times greater for CO compared
to oxygen [5], the present work aimed to creating stable hemolysates, through the use of carbon monoxide saturation. In this study, stability was defined as a period of time in which hemolysate shows a suitable electrophoretic gel, with no qualitative and quantitative changes as compared to its corresponding electrophoretic gel from a fresh hemolysate. Five milliliters of venous blood samples were collected into Sistema Vacutainer tubes (Becton-Dickinson), containing EDTA, from normal subjects and patients with different variant hemoglobins. Normal blood samples were from volunteers of the Faculty of Pharmacy of the Federal University of Minas Gerais while the blood samples from patients were obtained at the Centre of Hematology and Hemotherapy of Minas Gerais (Hemominas Foundation). The protocol was approved by Local Ethical Committee and informed consent was obtained from all participants. Hemolysates were prepared according to Naoum [6] and their concentration was adjusted at 7 g/dl. Hemoglobin electrophoresis was carried out at pH 8.6 using fresh hemolysates to check the electrophoretic profile for all the participants. The electrophoretic system utilized was from Technow. Hemolysates containing normal (AA) and variant hemoglobins (SS, CC and SC) were treated to CO saturation, which was produced by reacting sodium formate and concentrated sulfuric acid. The first reagent was transferred to a filtering flask and the second one was dropped on the salt through a funnel thereby producing CO gas. This was passed through a rubber tube connected from kitasate to the vial containing the hemolysate [7]. Three-milliliter samples of hemolysate were saturated with CO for 20 min. After saturation, each hemolysate was divided into 10 aliquots, and sealed under vacuum. Five aliquots were stored in the freezer ( 20 °C) and the others, under refrigeration (4 –8 °C). These samples were electrophoresed following storage of 45, 90, 135, 180, 240, 300
0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 ( 0 1 ) 0 0 7 7 0 - 7
236
Letter to Editors
kept in freezer at the same temperature for 1 month did not provide a satisfactory electrophoretical run (Fig. 1b). Further work is needed to obtain hemoglobin standards with longer stability. Other experiments related to the methods development should include the use of a CO bottle as a CO source instead of the chemical method to produce CO gas, which may further improve the stability. After hemolysates saturation, it would also be desirable to assess the CO concentrations to use the same amount for all samples. We report for the first time, a method for producing stable hemoglobin standards. This methodology is useful for clinical laboratories conducting routine hemoglobin electrophoresis. It is easy to perform and is inexpensive.
Acknowledgements Research supported by Fundacßa˜o de Amparo a` Pesquisa de Minas Gerais (FAPEMIG-CDS 2153/96). Godoi and Ladeira are recipients of CNPq research fellowship.
References Fig. 1. (a) Comparison of electrophoretic profiles of fresh (above) and CO-saturated hemolysates stored for 12 months (below). (b) Electrophoresis profile using non-CO-saturated hemolysate control kept in freezer at 20 °C for 1 month.
and 360 days. Non-CO-saturated hemolysates were used as the control. AA, SS and CC hemolysate variants showed a maximum of stability up to 6 months, while the SC hemolysates was stable for up to 12 months. The control samples did not show stability for longer than 2 and 4 weeks when kept in refrigerator or freezer, respectively. Fig. 1a shows a comparison of electrophoretic profiles of hemolysate prepared from a recently obtained SC patient and a hemolysate from a normal subject (HbAA), and the same samples stabilized by CO saturation and stored for 12 months at 20 °C. In contrast, the non-CO-saturated samples (controls)
[1] Carvalho MG, Souza MO, Silva MBS, Oliveira JMC, Cardoso IRCA, Carvalho IP, et al. Hemoglobinas anormais: perfil estatı´stico em doadores de sangue do Centro de Hematologia e Hemoterapia de Minas Gerais. Revista Brasileira de Ana´lises Clı´nicas 1994;26:39 – 40. [2] Coelho EAF, Carvalho MG. Aspectos morfolo´gicos das hemoglobinopatias. Revista Brasileira de Ana´lises Clı´nicas 1999;31: 201 – 3. [3] Bernard J, Le´vy JP, Clauvel JP, Rain JD, Varet B. Manual de Hematologia. 9th edn. Medsi, Rio de Janeiro; 2000, 368 pp. [4] Carvalho MG, Silva ZL, Ladeira MV. Tentativa de estabilizacßa˜o de padro˜es de hemoglobinas anormais para uso em eletroforese de hemoglobina em pH alcalino. Congresso Brasileiro de Patologia Clı´nica 1997;31:201. [5] Lima AO, Soares JB, Greco JB, Galizzi J, Cancßado JR. Me´todos de laborato´rio aplicados a` clı´nica. 6th edn. Guanabara Koogan, Rio de Janeiro; 1985, 699 pp. [6] Naoum PC. Diagno´stico das Hemoglobinopatias. Sa˜o Paulo: Sarvier, 1987, 242 pp. [7] ACCGC Malheiro. Determinacß a˜o espectrofotome´trica de carboxihemoglobinemia em indivı´duos expostos ocupacionalmente ao Mono´xido de Carbono. Master’s thesis, Faculdade de Cieˆncias Farmaceˆuticas, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, 1991.
Letter to Editors
Lara Carvalho Godoi Eduardo Antoˆnio F. Coelho Zelaine Lima e Silva Lauro Mello Vieira Maria Virgı´nia Ladeira Luci Maria Sant’Ana Dusse Geralda de Fa´tima Guerra Lages Maria Das Gracßas Carvalho * Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Minas Gerais, Avenida Olega´rio Maciel, 2360/608, Lourdes CEP 30180-112, Belo Horizonte, MG, Brazil E-mail address: [email protected]
237
Vicente Conceicßa˜o Macedo Liege Ribeiro Lyra Center of Hematology and Hemotherapy of Minas Gerais (HEMOMINAS Foundation), Belo Horizonte, MG, Brazil 21 March 2001
* Corresponding author. Tel.: +55-31-339-7647; fax: +55-31339-7666.