Effect of Hemoglobin Variants - Clinical Chemistry

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Mosekilde L, Herman AP, Beck-Nielsen H, Charles P, Pors Nielsen S, Helmer. Sørensen .... CLC 385 analyzer (Primus Corporation) served as the comparison ...
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Technical Briefs

levels of serum Gc-globulin: predictive value in fulminant hepatic failure. Hepatology 1996;23:713– 8. Yamamoto N, Kumashiro R. Conversion of vitamin D3 binding protein (group-specific component) to a macrophage activating factor by the stepwise action of ␤-galactosidase of B cells and sialidase of T cells. J Immunol 1993;151:2794 – 802. Schneider GB, Benis KA, Flay NW, Ireland RA, Popoff SN. Effects of vitamin D binding protein-macrophage activating factor (DBP-MAF) infusion on bone resorption in two osteopetrotic mutations. Bone 1995;16:657– 62. Kew RR, Webster RO. Gc-globulin (vitamin D-binding protein) enhances the neutrophil chemotactic activity of C5a and C5a des Arg. J Clin Invest 1988;82:364 –9. Piquette CA, Robinson-Hill R, Webster RO. Human monocyte chemotaxis to complement-derived chemotaxins is enhanced by Gc-globulin. J Leukocyte Biol 1994;55:349 –54. Mosekilde L, Herman AP, Beck-Nielsen H, Charles P, Pors Nielsen S, Helmer Sørensen O. The Danish Osteoporosis Prevention Study (DOPS): project design and inclusion of 2000 normal postmenopausal women. Maturitas 1999;31:207–19. Thymann M. Gc subtypes determined by agarose isoelectrofocusing. Hum Hered 1981;31:214 –21. Solberg HE. International Federation of Clinical Chemistry (IFCC). Approved Recommendation (1987) on the theory of reference values. Part 5. Statistical treatment of collected reference values. Determination of reference limits. Clin Chim Acta 1987;170:S12–32. Haddad JG, Walgate J. Radioimmunoassay of the binding protein for vitamin D and its metabolites in human serum. Concentrations in normal subjects and patients with disorders of mineral homeostasis. J Clin Invest 1976;58: 1217–22. Bouillon R, Van Baelen H, DeMoor P. The measurement of the vitamin D-binding protein in human serum. J Clin Endocrinol Metab 1977;45:225– 31. Walsh PG, Haddad JG. “Rocket” immunoelectrophoresis assay of vitamin D-binding protein (Gc globulin) in human serum. Clin Chem 1982;28: 1781–3. Lee WM, Emerson DL, Werner PAM, Arnaud P, Goldschmidt-Clermont P, Galbraith RM. Decreased serum group-specific component protein levels and complexes with actin in fulminant hepatic necrosis. Hepatology 1985; 5:271–5. Constans J, Arlet P, Viau M, Bouissou C. Unusual sialilation of the serum DBP associated with the Gc 1 allele in alcoholic cirrhosis of the liver. Clin Chim Acta 1983;130:219 –30. Daiger SP, Miller M, Chakraborty R. Heritability of quantitative variation at the group-specific component (Gc) locus. Am J Hum Genet 1984;36:663– 76. Braun A, Brandhofer A, Cleve H. Interaction of the vitamin D-binding protein (group-specific component) and its ligand 25-hydroxy-vitamin D3: binding differences of the various genetic types disclosed by isoelectric focusing. Electrophoresis 1990;11:478 – 83. Kawakami M, Blum CB, Ramakrishnan R, Dell RB, Goodman DS. Turnover of the plasma binding protein for vitamin D and its metabolites in normal human subjects. J Clin Endocrinol Metab 1981;53:1110 – 6.

Effect of Hemoglobin Variants (Hb J, Hb G, and Hb E) on HbA1c Values as Measured by Cation-Exchange HPLC (Diamat), Li-Yu Tsai,1* Shih-Meng Tsai,2 Me-Nung Lin,1 and Shu-Fen Liu1 (1 Department of Clinical Biochemistry, School of Technology for Medical Science, and 2 Department of Public Health, School of Medicine, Kaohsiung Medical University, Kaohsiung 80702, Taiwan; * author for correspondence: fax 886-7-2370544, e-mail [email protected]) Hemoglobin A1c (HbA1c) is used for the long-term management of patients with diabetes mellitus (DM) (1, 2 ). Hb variants other than HbA1c and ⑀-N-lysine-glycated Hb A0 may cause analytical interference in determinations of HbA1c (3– 6 ). In one study, the authors estimated the prevalence of thalassemia in Taiwan as 7%; moreover, ⬃1% of the people in northern Taiwan are ␤-thalassemia

heterozygotes (7 ). The occurrence of 24 abnormal Hbs (13 ␣-chain variants and 11 ␤-chain variants), including Hb G-Taipei, in populations in the Silk Road area of Northwestern China has been presented in a review (8 ). The frequency of thalassemia has been estimated to be ⬃1 in 2350 in Japan (9 ) and even higher in North Africa (10 ). Hb E is the second most prevalent Hb variant worldwide and the third most prevalent variant in the US, after Hb S and C. Hb E is found primarily in Southeast Asia, especially among the Thai population (11 ). In the northeastern region of India, the gene frequency of Hb E is 10.9% (12 ). In a study of 222 000 blood samples in Canada, 23 cases of Hb J were identified (13 ). Given that the majority of hemoglobinopathic cases are from families of Asian, Southeast Asian, and Asian Indian ancestry (7–12, 14 –16 ), the aim of this study was to investigate the influence of selected Hb structural variants on HbA1c values measured by cation-exchange HPLC. We collected 17 EDTA-anticoagulated whole blood specimens from DM patients with Hb AJ (6 patients), Hb AG (10 patients), or Hb AE (1 patient had a fasting sugar of 10.4 mmol/L) to analyze HbA1c. The ranges and mean values for fasting sugar were 8.2–17.8 mmol/L and 12.6 ⫾ 3.9 mmol/L, respectively, in the DM patients with Hb AJ and 7.1–18.1 mmol/L and 9.8 ⫾ 4.1 mmol/L, respectively, in the DM patients with Hb AG. In addition, one specimen from a nondiabetic patient with the Hb AG variant (fasting sugar, 4.4 mmol/L) and another from a nondiabetic patient with Hb AE (fasting sugar, 5.2 mmol/L) were analyzed. HbA1c and glycated Hb were measured by cation-exchange HPLC (Diamat HbA1c program; BioRad Laboratories) and by boronate ion capture (IMx analyzer; Abbott Laboratories). Both methods had a CV ⬍5%, and both reported results as percentage of HbA1c. When Tiran et al. (4 ) comparatively evaluated five glycated Hb assay methods, including the Abbott IMx glycated Hb ion capture assay, they found that the methods showed generally acceptable precision and good accordance with the Bio-Rad Diamat system. Bon et al. (17 ) determined the accuracy of the IMx assay by comparison with a reference HPLC assay for 603 specimens; the correlation coefficients were 0.88 – 0.96. In addition, several investigators have shown that glucose, bilirubin, triglycerides, labile fraction, and Hb variants do not interfere with the Abbott IMx assay (18 ). Moreover, the IMx assay is not sensitive to interference by cyanate derived from spontaneous dissociation of urea. In the present study, a boronate-affinity analytical method on a CLC 385 analyzer (Primus Corporation) served as the comparison method because of the high specificity and the negligible interference of Hb variants in that method (1 ). The Hb variants were identified by electrophoretic separation of Hb on cellulose acetate membranes. Specimens for which HPLC chromatograms suggested the presence of abnormal peaks underwent hemoglobinopathy studies. The Abbott IMx boronate ion-capture method showed no important effects from any of the Hb variants tested, and its results for HbA1c agreed well with those of the

Clinical Chemistry 47, No. 4, 2001

comparison method (r2 ⫽ 0.93, n ⫽ 43 for HbA1c; r2 ⫽ 0.95, n ⫽ 43 for glycated Hb), whereas the results analyzed by cation-exchange HPLC method were falsely low (Fig. 1A). Three different chromatographic patterns were observed in blood specimens with HB J (6 samples), Hb E (2 samples), and Hb G (11 samples). An asymmetrical HbA1c peak with a “left shoulder” appeared in the specimens with Hb AJ (Fig. 1B, chromatogram A), and an asymmetrical HbA1c peak with a “right shoulder” appeared in the specimens with Hb AE (Fig. 1B, chromatogram B). For specimens with Hb AG, the HPLC chromatogram showed an additional peak at Hb A0 (Fig. 1B, chromatogram C). HbA1c values measured by affinity chromatography were appropriately increased for the patients’ blood glucose values, but HbA1c values measured by HPLC were lower than those measured by affinity chromatography, whether the patients were diabetic or not. Thus, the Hb mutations studied caused an abnormal HPLC chromatogram and falsely low HbA1c values when measured by HPLC. The effects of Hb variants on HbA1c values determined by the HPLC system have been evaluated previously (3, 5, 6, 19, 20 ). An earlier report by Oshima et al. (5 ) of a

Fig. 1. HbA1c (A) and glycated Hb (B) results in patients with various Hb variants. (A), M1 represents the Bio-Rad assay and M2 represents the Abbott IMx assay. (B), HPLC chromatograms of glycated Hb in DM patients with Hb variants Hb AJ (chromatogram A), Hb AE (chromatogram B), and Hb AG (chromatogram C). The HbA1c peak is shaded.

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study conducted in Japan described an abnormal chromatogram for HbA1c [as analyzed on an automated glycohemoglobin analyzer, HLC-723 Ghb V (Tosoh)] from a male DM patient with Hb J Lome. In a study performed in Singapore, Wong et al. (19 ) observed an asymmetrical HbA1c peak with a right shoulder in nine HbA1c blood specimens analyzed by cation-exchange HPLC (Variant HbA1c program; Bio-Rad) from diabetic patients with Hb AE. In a study performed in Austria, Schnedl et al. (20 ) reported an additional peak at Hb A0, as well as falsely low HbA1c values, measured by cation-exchange HPLC (Diamat HbA1c) from a diabetic patient with Hb O Padova. Our results not only agree with those of Schnedl et al. (20 ), but also deal with Hb J, G, and E variants in a single study. We conclude that Hb variants can contribute to mismanagement of patients with DM because of falsely low HbA1c values measured by HPLC. Careful interpretation of glycohemoglobin results is critical in populations with a relatively high prevalence of Hb variants, such as Hb AJ, AG, and AE. References 1. Nuttall FQ. Comparison of percent total GHb with percent HbA1c in people with and without known diabetes. Diabetes Care 1998;21:1475– 80. 2. Edelman SV. Importance of glucose control. Med Clin North Am 1998;82: 665– 87. 3. Chen D, Crimmins DL, Hsu FF, Lindberg FP, Scott MG. Hemoglobin Raleigh as the cause of a falsely increased hemoglobin A1c in an automated ion-exchange HPLC method. Clin Chem 1998;44:1296 –301. 4. Tiran A, Pieber T, Tiran B, Halwachs-Baumann G, Dobnig H, Grubelnig H, Wilders-Truschnig MM. Automated determination of glycated hemoglobin: comparative evaluation of five assay systems. J Clin Lab Anal 1994;8:128 – 34. 5. Oshima Y, Ideguchi H, Takao M, Okamura T, Arima F, Miyahara M, et al. A patient with a hemoglobin variant (Hb JLome) unexpectedly detected by HPLC for glycated hemoglobin (HbA1c). Int J Hematol 1998;68:317–21. 6. Roberts WL, Frank EL, Moulton L, Papadea C, Noffsinger JK, Ou CN. Effects of nine hemoglobin variants on five glycohemoglobin methods. Clin Chem 2000;46:569 –72. 7. Ko TM, Hsu PM, Chen CJ, Hsieh FJ, Hsieh CY, Lee TY. Incidence study of heterozygous ␤-thalassemia in northern Taiwan. J Formos Med Assoc 1989;88:678 – 81. 8. Li HJ, Zhao XN, Qin F, Li HW, Li L, He XJ. Abnormal hemoglobins in the Silk Road region of China. Hum Genet 1990;86:231–5. 9. Harano T. Hemoglobinopathy in Japan: detection and analysis. Jpn J Clin Pathol 1999;47:215–23. 10. Chami B, Blouquit Y, Bardadjian-Michau J, Riou J, Wajcman H, Rosa J, Galacteros F. Hemoglobin variants in North Africa. Hemoglobin 1994;18: 39 –51. 11. Tanphaichitr VS, Mahasandana C, Suvatte V, Yodthong S, Pung-amritt P, Seeloem J. Prevalence of hemoglobin E. Southeast Asian J Trop Med Public Health 1995;26(Suppl 1):271– 4. 12. Balgir RS. Genetic epidemiology of the three predominant abnormal hemoglobins in India. J Assoc Physicians India 1996;44:25– 8. 13. Vell F. Haemoglobins J in Canada. Hum Hered 1975;25:1–12. 14. Lorey FW, Arnopp J, Cunningham GC. Distribution of hemoglobinopathy variants by ethnicity in a multiethnic state. Genet Epidemiol 1996;13:501– 12. 15. Lorey FW, Cunningham GC. Impact of Asian immigration on thalassemia in California. Ann N Y Acad Sci 1998;850:442–5. 16. Heer N, Choy J, Vichinsky EP. The social impact of migration on disease. Cooley’s anemia, thalassemia, and new Asian immigrants. Ann N Y Acad Sci 1998;850:509 –11. 17. Bon C, Revenant MC, Sotta C, Mailliavin A, Bannier E, Goujon R. Multicenter evaluation of the Abbott glycosylated hemoglobin assay on IMx. Ann Biol Clin (Paris) 1996;54:151–7. 18. Wilson DH, Bogacz JP, Forsythe CM, Turk PJ, Lane TL, Gates RC, Brandt DR. Fully automated assay of glycohemoglobin with the Abbott IMx analyzer: novel approaches for separation and detection. Clin Chem 1993;39: 2090 –7.

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19. Wong SC, Aw TC. HbE1c as an indicator for the presence of HbAE phenotype in diabetic patients. Clin Chem 1998;44:660 –1. 20. Schnedl WJ, Reisinger EC, Katzensteiner S, Lipp RW, Schreiber F, Hopmeier P, Krejs GJ. Hemoglobin O Padova and falsely low hemoglobin A1c in a patient with type I diabetes. J Clin Pathol 1997;50:434 –5.

Errors Caused by the Use of D,L-Octanoylcarnitine for Blood-Spot Calibrators, Donald H. Chace,1* James C. DiPerna,1 Barbara W. Adam,2 and W. Harry Hannon2 (1 Neo Gen Screening, PO Box 219, Bridgeville, PA 15017; 2 Centers for Disease Control and Prevention, 4770 Buford Hwy. NE, Atlanta, GA; * author for correspondence: fax 412-220-0784, e-mail [email protected]) The use of tandem mass spectrometry (MS-MS) in the analysis of filter paper blood spots from newborns for acylcarnitines and amino acids has expanded significantly in recent years (1, 2 ). With estimates of 1 million specimens analyzed by MS-MS per year throughout the world, the demand is acute for assay standardization and harmonization. Programs exist at the CDC for amino acid standardization and quality assurance pertaining to newborn screening (3, 4 ). This program is being extended to include acylcarnitines, and the data in this report stem from that extension. Five metabolites are key in the diagnosis of several disorders of fat and organic acid metabolism. Preliminary results demonstrated excellent linearity for each of the five acylcarnitines added to blood. However, an extremely unusual result was observed for octanoylcarnitine: the recovery of octanoylcarnitine was significantly lower than that of other acylcarnitines. This observation is supported by Turner and Dalton (5 ), who report a 40% loss of octanoylcarnitine after addition to whole blood and plasma. We investigated the cause of this loss of octanoylcarnitine so that this serious error could be prevented or accounted for. The results of this study demonstrate the importance of assay standardization and the validation required in clinical screening that goes well beyond this particular quality-assurance/quality-control program for acylcarnitines. We obtained isotope-labeled internal standards (l-2H3propionylcarnitine, l-2H3-butyrylcarnitine, l-2H3-octanoylcarnitine, l-2H9-myristoylcarnitine, and l-2H3-palmitoylcarnitine) from Cambridge Isotope Laboratories. Unlabeled standards (d,l-octanoylcarnitine, d,l-myristoylcarnitine, and l-palmitoylcarnitine) were obtained from Sigma, and l-propionylcarnitine, l-butyrylcarnitine, and l-octanoylcarnitine were obtained from Life Sciences Resources. The unlabeled standards were used to prepare a series of blood specimens at the CDC using procedures described previously (3, 4 ) with the following modifications: l-propionylcarnitine, l-butyrylcarnitine, d,l-octanoylcarnitine, d,l-myristoylcarnitine, and l-palmitoylcarnitine were added to whole blood containing EDTA, whole blood containing heparin, and lysed cells at final concentrations of 0 –14 ␮mol/L. Blood (25 ␮L) was ap-

plied to S&S Type 903 filter paper (Schleicher & Schuell), dried, and sent to Neo Gen Screening for analysis by MS-MS. A smaller subset of blood specimens containing heparin were prepared at Neo Gen Screening by the addition of an equimolar solution (31 ␮mol/L) of d,loctanoylcarnitine or l-octanoylcarnitine to the internal standard, l-2H3-octanoylcarnitine. For specimens obtained from the CDC, a 4.8 mm diameter spot was punched from each dried blood specimen, extracted with methanol containing deuterated l-acylcarnitine internal standards, and derivatized using a procedure described previously (6 ) with the following modification: dry, derivatized sample extracts were reconstituted immediately before analysis in acetonitrilewater (50:50 by volume) containing 0.2 mL/L formic acid and analyzed using electrospray MS-MS as described below. Specimens that contained both the internal standard and its unlabeled analog were extracted using methanol without the internal standard. An Applied Biosystems/MDS Sciex Model API 3000 tandem mass spectrometer equipped with an electrospray ionization source was used for all analyses. A 10-␮L aliquot of each specimen was injected using a Gilson 215 sample handler fitted with a Rheodyne Model 7010 injector and a Perkin-Elmer LC Pump operating at a flow rate of 18 ␮L/min with acetonitrile-water (50:50 by volume) containing 0.2 mL/L formic acid as the mobile phase. Precursors of 85 Da scans and 103 Da scans were used, representing analyses for acyl and free carnitines (6 ). Concentration calculations were obtained by the following method: raw data (ion intensity data) were processed using an Apple script followed by its exportation to an Excel spreadsheet for further data reduction and calculations. Excellent linearity (R2 ⬎0.98) was obtained for acylcarnitine calibration curves from blood containing either EDTA or heparin or in which the red cells were lysed. The slopes and intercepts for these addition assays are provided in Table 1. The results for the addition containing d,l-octanoylcarnitine had a slope of 0.59, which suggests a significantly reduced recovery. Similar results were reported by Turner and Dalton (5 ), who reported a significant loss of octanoylcarnitine of 40%. The stereoisomeric forms used in their study for octanoylcarnitine, however, were not noted. In an experiment in which l-octanoylcarnitine was used in an addition analysis, no significantly reduced recovery of l-octanoylcarnitine was observed (slope ⫽ 0.93). Repeat preparation of the loctanoylcarnitine calibration curve using EDTA-treated blood did not show reduced recovery of l-octanoylcarnitine (data not shown). Experiments were designed to further clarify and confirm the loss of d,l-octanoylcarnitine in blood. An equimolar mixture of d,l-octanoylcarnitine with l-2H3octanoylcarnitine or of l-octanoylcarnitine with l-2H3octanoylcarnitine was first analyzed as pure compounds. The MS-MS analyses of these mixtures demonstrated molar equivalents of d,l- or l-octanoylcarnitine (Fig. 1, A and B). An aliquot of this equimolar mixture was then