Restoration of Hemoglobin Function in Stored EDTA Blood Application in Identification of Hemoglobin Variants with Abnormal Oxygen Affinity ABRAHAM SUMOZA, M.D., VIRGIL F. FAIRBANKS, M.D., AND ALVARO A. PINEDA, M.D.
Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
have evaluated a "rejuvenation" method 16 and have confirmed that application of this simple method results in a return to normal values of 2,3-DPG and of the dissociation curve and P 50 for blood anticoagulated with ethylenediaminetetraacetic acid (EDTA) and stored as long as 19 days. Thus, blood specimens can be collected in ordinary EDTA anticoagulant tubes, packed in ice, and mailed to a central laboratory, and satisfactory whole-blood 0 2 affinity studies can be done one or two weeks later. This rejuvenation method now makes 0 2 affinity studies accessible to any physician or clinical laboratory.
ERYTHROCYTOSIS AND CYANOSIS may be clinical expressions of a hemoglobin with altered 0 2 affinity. Such hemoglobinopathies may not be demonstrable by present conventional technics, such as electrophoresis, which depend on alteration in the number of charged groups. Measurement of 0 2 affinity of hemoglobin has become essential for the identification of such hemoglobinopathies. However, such facilities are not presently generally available. It has been difficult to measure 0 2 affinity on mailed blood samples because of the rapid decline in the concentration of 2,3-diphosphoglycerate (2,3-DPG) that occurs during storage and in turn leads to a leftward shift in the dissociation curve and a reduction in the P 50 . We
Materials and Methods To determine normal control values in the 0 2 studies, 10 ml of blood were obtained from 25 healthy adult donors of both sexes. Blood was anticoagulated with approximately 100 units of heparin. This was deoxygenated in a tonometer that was continually flushed with a 96% N 2 - 4 % C 0 2 mixture. When deoxygenation was complete, 7 ml of blood were transferred to a dissociation curve analyzer (DCA-1, Radiometer Corp.), and the complete dissociation curve was plotted for the Po 2 range 0 to 200 mm Hg by the method of Duvelleroy and co-workers. 8 These studies were completed within 2 hours after the blood was collected. Specimens for assay of 2,3-DPG were chilled and lysed immediately by transferring 0.1 ml of whole blood to 2 ml of distilled water at 4 C. Either assays were performed within two hours or the hemolysates
Received May 10, 1976; received revised manuscript August 2, 1976; accepted for publication August 2, 1976. Address reprint requests to Dr. Sumoza: c/o Section of Publications, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55901. Dr. Sumoza's present address is Department of Medicine, University of Carabobo Medical School, Valencia, Venezuela. 53
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Sumo/.a, Abraham, Fairbanks, Virgil F., Pineda, Alvaro A.: Restoration of hemoglobin function in stored EDTA blood. Application in identification of hemoglobin variants with abnormal oxygen affinity. Am J Clin Pathol 68: 53-56, 1977. The incubation of stored blood in a mixture of inosine, pyruvate, glucose, and phosphate restores the 0 2 affinity of hemoglobin to physiologic levels, as measured by the configuration of the dissociation curve and the P50. This regeneration of normal hemoglobin function not only is consistent for samples anticoagulated with EDTA and stored eight days at 4 C but also is demonstrable for at least 19 days of 4 C storage of EDTA-anticoagulated whole blood. This regeneration procedure is simple to perform and makes it possible to measure reliably 0 2 affinity in blood samples transmitted by mail. (Key words: Abnormal hemoglobins; Hemoglobin dissociation curve; Hemoglobin function; Restored hemoglobin function.)
A.J.C.I'. • July 1977
SUMOZA, FAIRBANKS AND PINEDA
54
Table I. Stock Solutions Used for Restoration of Hemoglobin Function in Stored EDTA Blood Solution A lnosine 3.48 mmol 935 mg NaCI 15.40 mmol 900 mg Glucose 5.55 mmol 1.000 mg H20 Add to 100 ml Store at 4 C Solution B Sodium pyruvate 0.54 mmol 60 mg H20 Add to 1 ml May be frozen in 0.05-ml volume and stored at -22 C Solution C Na3P04- 12H.,0 H20 Store at 4 C
0.005 mmol Add to 100 ml
1,900 mg
Results The effects of the rejuvenation procedure on 36 stored blood specimens are indicated in Tables 2 and 3, and results of serial studies of rejuvenation of blood from the patient with hemochromatosis are shown in Figure 1. The mean P 50 value of eightday-old specimens after rejuvenation was 27.3 mm Hg, compared with 27.0 mm Hg for freshly drawn whole
Table 2. Comparison of 0 2 Affinity of "Rejuvenated" EDTA Blood Specimens with That of Fresh, Heparinized Blood Specimens
Specimen
No. Tested
Normal control (fresh, heparinized) Five-day-old (rejuvenated)
25
27.0
1.1
8
28.4
2.2
6 22
16.1 27.3
2.3 2.6
Eight-day-old Untreated Rejuvenated
t
I1 (Approximate)
2.4
Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
have evaluated a "rejuvenation" method 16 and have confirmed that application of this simple method results in a return to normal values of 2,3-DPG and of the dissociation curve and P 50 for blood anticoagulated with ethylenediaminetetraacetic acid (EDTA) and stored as long as 19 days. Thus, blood specimens can be collected in ordinary EDTA anticoagulant tubes, packed in ice, and mailed to a central laboratory, and satisfactory whole-blood 0 2 affinity studies can be done one or two weeks later. This rejuvenation method now makes 0 2 affinity studies accessible to any physician or clinical laboratory.
ERYTHROCYTOSIS AND CYANOSIS may be clinical expressions of a hemoglobin with altered 0 2 affinity. Such hemoglobinopathies may not be demonstrable by present conventional technics, such as electrophoresis, which depend on alteration in the number of charged groups. Measurement of 0 2 affinity of hemoglobin has become essential for the identification of such hemoglobinopathies. However, such facilities are not presently generally available. It has been difficult to measure 0 2 affinity on mailed blood samples because of the rapid decline in the concentration of 2,3-diphosphoglycerate (2,3-DPG) that occurs during storage and in turn leads to a leftward shift in the dissociation curve and a reduction in the P 50 . We
Materials and Methods To determine normal control values in the 0 2 studies, 10 ml of blood were obtained from 25 healthy adult donors of both sexes. Blood was anticoagulated with approximately 100 units of heparin. This was deoxygenated in a tonometer that was continually flushed with a 96% N 2 - 4 % C 0 2 mixture. When deoxygenation was complete, 7 ml of blood were transferred to a dissociation curve analyzer (DCA-1, Radiometer Corp.), and the complete dissociation curve was plotted for the Po 2 range 0 to 200 mm Hg by the method of Duvelleroy and co-workers. 8 These studies were completed within 2 hours after the blood was collected. Specimens for assay of 2,3-DPG were chilled and lysed immediately by transferring 0.1 ml of whole blood to 2 ml of distilled water at 4 C. Either assays were performed within two hours or the hemolysates
Received May 10, 1976; received revised manuscript August 2, 1976; accepted for publication August 2, 1976. Address reprint requests to Dr. Sumoza: c/o Section of Publications, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55901. Dr. Sumoza's present address is Department of Medicine, University of Carabobo Medical School, Valencia, Venezuela. 53
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Sumo/.a, Abraham, Fairbanks, Virgil F., Pineda, Alvaro A.: Restoration of hemoglobin function in stored EDTA blood. Application in identification of hemoglobin variants with abnormal oxygen affinity. Am J Clin Pathol 68: 53-56, 1977. The incubation of stored blood in a mixture of inosine, pyruvate, glucose, and phosphate restores the 0 2 affinity of hemoglobin to physiologic levels, as measured by the configuration of the dissociation curve and the P50. This regeneration of normal hemoglobin function not only is consistent for samples anticoagulated with EDTA and stored eight days at 4 C but also is demonstrable for at least 19 days of 4 C storage of EDTA-anticoagulated whole blood. This regeneration procedure is simple to perform and makes it possible to measure reliably 0 2 affinity in blood samples transmitted by mail. (Key words: Abnormal hemoglobins; Hemoglobin dissociation curve; Hemoglobin function; Restored hemoglobin function.)
A.J.C.I'. • July 1977
SUMOZA, FAIRBANKS AND PINEDA
54
Table I. Stock Solutions Used for Restoration of Hemoglobin Function in Stored EDTA Blood Solution A lnosine 3.48 mmol 935 mg NaCI 15.40 mmol 900 mg Glucose 5.55 mmol 1.000 mg H20 Add to 100 ml Store at 4 C Solution B Sodium pyruvate 0.54 mmol 60 mg H20 Add to 1 ml May be frozen in 0.05-ml volume and stored at -22 C Solution C Na3P04- 12H.,0 H20 Store at 4 C
0.005 mmol Add to 100 ml
1,900 mg
Results The effects of the rejuvenation procedure on 36 stored blood specimens are indicated in Tables 2 and 3, and results of serial studies of rejuvenation of blood from the patient with hemochromatosis are shown in Figure 1. The mean P 50 value of eightday-old specimens after rejuvenation was 27.3 mm Hg, compared with 27.0 mm Hg for freshly drawn whole
Table 2. Comparison of 0 2 Affinity of "Rejuvenated" EDTA Blood Specimens with That of Fresh, Heparinized Blood Specimens
Specimen
No. Tested
Normal control (fresh, heparinized) Five-day-old (rejuvenated)
25
27.0
1.1
8
28.4
2.2
6 22
16.1 27.3
2.3 2.6
Eight-day-old Untreated Rejuvenated
t
I1 (Approximate)
2.4
