Factors that interfere with GHB (HbA1c) Test Results
UPDATED 4/08

Information for physicians and patients regarding HbS and HbC traits can be found here. More about hemoglobin variants and HbA1c can also be found at the NIDDK web site: Sickle Cell Trait and Other Hemoglobinopathies and Diabetes: Important Information for Physicians For People of African, Mediterranean, or Southeast Asian Heritage: Important Information about Diabetes Blood Tests

Hemoglobin Variants and Derivatives: Genetic variants (e.g. HbS trait, HbC trait) and chemically modified derivatives of hemoglobin (e.g. carbamylated Hb in patients with renal failure, acetylated Hb in patients taking large amounts of aspirin) can affect the accuracy of HbA1c measurements.  The effects vary depending on the specific Hb variant or derivative and the specific HbA1c method.  Table 1 contains information for most of the commonly used HbA1c methods for some of the more common Hb variants and derivatives. Interferences from less common Hb variants and derivatives are discussed in Bry, et al (1).  All entries in Table 1 are based on published information.  In addition, if a product insert indicates clearly that there is inference from a particular factor, then the interference is entered as “yes” and the product insert is cited.   When selecting an assay method, laboratories should take into consideration characteristics of the patient population served, (e.g. high prevalence of hemoglobinopathies or renal failure).

Shortened Erythrocyte Survival: Any condition that shortens erythrocyte survival or decreases mean erythrocyte age (e.g., recovery from acute blood loss, hemolytic anemia) will falsely lower HbA1c test results regardless of the assay method used (23).  HbA1c results from patients with HbSS, HbCC, and HbSC must be interpreted with caution given the pathological processes, including anemia, increased red cell turnover, transfusion requirements, that adversely impact HbA1c as a marker of long-term glycemic control.  Alternative forms of testing such as glycated serum protein (fructosamine) should be considered for these patients.

Other factors: Vitamins C and E are reported to falsely lower test results, possibly by inhibiting glycation of hemoglobin (24, 25); vitamin C may increase values with some assays (25).  Iron-deficiency anemia is reported to increase test results (26).  Hypertriglyceridemia, hyperbilirubinemia, uremia (see carbamylated Hb in Table 1), chronic alcoholism, chronic ingestion of salicylates, and opiate addiction are reported to interfere with some assay methods, falsely increasing results (3, 5, 9, 11, 12, 14, 16-18, 21-22, 27-30).

Table 1: Effects of frequently encountered Hb variants and derivatives on GHB measurement

Method (listed in alphabetical order by manufacturer)	Interference (Yes/No)
	Hb C trait	Hb S trait	Hb E trait	Hb D trait	Elevated HbF	Carbamyl-Hb
*Abbott Architect (Seradyn Reagents)	Yes 35	Yes 35	-		-	-
*Axis-Shield Nycocard (Primus Nycocard)	No 4	No 4	-		-	-
*Axis-Shield Afinion	No 38	No 38	No 40	No 40	-	-
*Siemens (previously Bayer) Advia	Yes 34	Yes 34	-		-	-
*Siemens (previously Bayer) DCA 2000	Yes/No 1, 5, 6, 13	No 1, 5	No 1, 7, 8, 40	No 40	Yes 33, 39	No 3, 9
Beckman Diatrac	Yes 1, 2, 10	Yes 1, 2, 10	-		Yes 10	Yes 1,10
*Beckman Synchron	No 4	No 4	No 40	No 40	-	-
*Bio-Rad D-10 (short Program)	Yes/No 31, 38	No 31, 38	No 40	No 40	-	-
*Bio-Rad D-10 (extended   program)	No 38	No 38	No 40	No 40	-	-
*Bio-Rad DiaSTAT	No 31	Yes 31	-		-	-
*Bio-Rad Variant A1c	No 1, 2, 38	Yes/No 1, 2, 38	No 1,7, 40	No 40	-	Yes 1, 3, 11, 12, 14
*Bio-Rad Variant GHb	No 4	No 4	-	-	-	-
*Bio-Rad Variant II A1c	Yes/No 31, 4, 32, 38	Yes/No 31, 4 ,32, 38	No 40	No 40	No  33, 39	No 11
*Bio-Rad Variant II Turbo	No 38	No 38	Yes 40	Yes 40	-	-
*Dade Dimension	No 31	No 31	No 40	No 40	-	-
Diazyme Direct Enzymatic HbA1c	No 38, 43	No 38, 43	No 40, 43	No 40	-	-
Drew Scientific DS5	No 31	Yes 31	-	-	-	-
Helena Glyco-Tek	Yes 4, 5	No 4, 5	-	-	-	-
*Menarini HA8140	No 4	Yes 4	Yes 1, 15	Yes, 44	No 1, 15	Yes/No 1, 3, 16, 17
*Menarini HA8160 (Diabetes Mode)	No 31	No 31	Yes 40	Yes 40	-	-
*Menarini HA8160 (Thalassemia Mode)	-	-	No 40	Yes 40
Microgenics	No 38	No 38	-	-	-	-
*Bayer (previously Metrika) A1c Now	Yes 31	Yes 31	No 40	No 40	-	-
*Olympus	Yes 35, 36, 38	Yes 35, 36, 38	No 40	No 40	Yes (>10%) 36	-
Ortho-Clinical Vitros	No 32, 38	No 32, 38	No 40	No 40	-	-
Pointe Scientific Hemoglobin A1c	No 32, 38	No 32, 38	No 40	No 40	-	-
*Primus Boronate Affinity HPLC	No 1, 2, 31, 38	No 1, 2, 31, 38	No 1, 7, 40	No 40	Yes 33, 39	No 1, 3, 9, 18
*Bio-Rad Deeside (previously Provalis) MicroMat (also sold by Cholestech as GDX)	Yes4	No4	-		-	-
Randox Haemoglobin A1c	Yes 37	Yes 37	-	-	Yes (>10%) 37	-
*Roche Cobas Integra	Yes 4	Yes 4	-	-	-	-
*Roche Cobas Integra Gen2	No 38, 41, 42,	No 38, 41, 42	No 40	No 40	-	-
*Roche Tina-quant II	No 1, 2, 19	No 1, 2, 19	No 1, 19, 40	No 40	No (<30% HbF) 19	No 1, 3, 16
*Roche Unimate	Yes 1, 20	Yes 1, 20	-	--	-	No 1, 3, 11
*Tosoh A1c 2.2 Plus	No 1, 2, 31	No 1, 2, 31	Yes 1, 7, 40	No 40	Yes 33, 39	Yes/No 1, 3, 11, 14, 16, 21, 22
*Tosoh G7	No 31, 38	No 31, 38	Yes 40	No 40	No 33, 39	-
*Tosoh G8	-	-	Yes 40	No 40	-	-

* indicates NGSP certification at the time of this update (April 1, 2008, www.ngsp.org).
Yes/No indicates that there is conflicting data in the literature. The indicator in bold is the opinion of the NGSP based on review of the literature cited.

NOTE:

• Many other publications have been reviewed.  Only those with conclusions that are reasonably supported by data are included.
• For ion-exchange HPLC methods, interference from Hb variants and adducts may be dependent on the lot of reagents used (2).

References:

1. Bry L, Chen PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays for glycated hemoglobin. Clin. Chem. 2001;47:153-63. REVIEW
2. Frank EL, Moulton L, Little RR, Wiedmeyer HM, Rohlfing C, Roberts WL. Effects of Hemoglobin C and S Traits on Seven glycated hemoglobin Methods. Clin Chem 46:864-86-867, 2000.
3. Weykamp CW, Miedema K, de Haan T, Doleman C. Carbamylated hemoglobin interference in glycated hemoglobin assays. Clin Chem 1999;45:438-40.
4. Roberts WL, Barun KD, Brown D, Hanbury CM, Hoyer JD, John WG, Lambert TL, Lundell RB, Rohlfing C, Little RR. Hemoglobin C and S Trait on Eight glycated hemoglobin Methods. Clin Chem 2002;48:383-5.
5. Weykamp CW, Martina WV, van der Dijs F, Penders TJ, van der S lik W, Muskiet F. Hemoglobins S and C: reference values for glycated hemoglobin in heterozygous, double-heterozygotes and homozygous subjects, as established by 13 methods. Clin Chim Acta 1994;231:161-71.
6. Blakney G, Higgins TN, Holmes DJ. Comparison of hemoglobin A1c results by two different methods on patients with structural hemoglobin variants. Clin Biochem 1998;31:619-26.
7. Roberts WL, Frank EL, Moulton L, Papadea C, Noffsinger J, Ou C. Effects of nine hemoglobin variants on five glycated hemoglobin methods. Clin Chem 2000;46:569-72.
8. Musalmah M, Normah J, Mohamad WBW, Salwah ON, Fatah HA, Zahari NAN. Effect of Hemoglobin E on glycosylated hemoglobin determinations using different commercial kits. Med J Malaysia 2000;55:352-6.
9. Little R, Mathew AS, Tennill AL, Rohlfing CL, Goldstein DG. Measurement of glycated hemoglobin (GHB) in patients with chronic renal failure (CRF): are ion-exchange HPLC results really invalid? (abstract). Clin Chem 1997;43(suppl 1):S136.
10. Weykamp CW, Penders TJ, Muskiet FA, van der Slik W. Influence of hemoglobin variants and derivatives on glycated hemoglobin determination, as investigated by 102 laboratories using 16 methods. Clin Chem 1993;39:1717-23.
11. Little RR, Tennill AL, Rohlfing C, Wiedmeyer H, Khanna R, Goel S, Aggrawal A, Madsen R, Goldstein DE. Can Glycohemoglobin (GHB) be used to assess Glycemic Control in Patients with Chronic Renal Failure? Clin Chem 48:784-6, 2002
12. Chevenne D, Fonfrede M, Ducrocq R, Chauffert M, Trivin F. Uremia and HbA1c measured by high-performance liquid chromatography. Diab Care 1998;21:463-4.
13. Roberts WL, McCraw M, Cook CB. Effects of sickle cell trait and hemoglobin C on determinations of HbA1c by immunoassay method. Diabetes Care 1998;21:983-6.
14. Hansen KW, Wrlandsen E, Helleberg K, Danielsen H. Uremia and HbA1c. Diabetes Care 1997;20:1341-2.
15. John WG, Braconnier F, Miedema K, Aulesa C, Piras G. Evaluation of the Menarini-Arkray HA 8140 hemoglobin A1c analyzer. Clin Chem 1997;43:968-75.
16. Chachou A, Randoux, Millart H, Chanard J, Gillery P. Influence of in vivo hemoglobin carbamylation on HbA1c measurements by various methods. Clin Chem Lab Med 2000;38:321-6.
17. Thoma J, Stirn F, Kutter D. Influence of Urea on HbA1c-determinations by Menarini HA 8140 and on the difference between immunoturbidimetric and HPLC HbA1c results. Clin Lab 2000;46:261-8.
18. Philipov G, Charles P, Beng C, Philips P. Alternate site testing for HbA1c using the Primus CLC 330 GHb analyzer. Diabetes Care 1997;20:607-9.
19. Chang J, Hoke C, Ettinger B, Penerian G. Evaluation and interference study of hemoglobin A1c measured by turbidimetric inhibition immunoassay. Am J Clin Pathol 1998;109:274-8.
20. Roberts WL, Chiasera JM, Ward-Cook K.  glycated hemoglobin results in samples with hemoglobin C or S trait: a comparison of four test systems Clin Chem 1999;45:906-9.
21. Gibb I, Parnham A, Fonfrede M, Lecock F. Multicenter evaluation of Tosoh glycated hemoglobin analyzer. Clin Chem 1999;45:1833-41.
22. Holbrook I. Measurement of HbA1c by high-performance liquid chromatography in patients with renal failure. Ann Clin Biochem 1999;36:238-9.
23. Goldstein DE, Little RR, Lorenz RA, Malone JI, Nathan D, Peterson CM: American Diabetes Association Technical Review on Tests of Glycemia. Diabetes Care 18:896-909, 1995.
24. Davie SJ, Gould BJ, Yudkin JS. Effects of vitamin C on glycosylation of proteins. Diabetes 1992;41:167-73.
25. Ceriello A, Giugliano D, Quatraro A, Donzella C, Dipalo G, Lefebvre PJ. Vitamin E reduction of protein glycosylation in diabetes. New prospect for prevention of diabetic complications? Diabetes Care 1991;14:68-72.
26. Tarim O, Kucukerdogan A, Gunay U, Eralp O, Ercan I. Effects of iron deficiency anemia on hemoglobin A1c in type 1 diabetes mellitus. Pediatr Int 1999;41:357-62.
27. Goldstein DE, Little RR, Wiedmeyer HM, England JD, McKenzie EM: Glycosylated hemoglobin: Methodologies and clinical applications. In: Proceedings of the 9th Annual Arnold O. Beckman Conference.  Diabetes Mellitus: From theory to therapy. Clin Chem 32(l0b):B64-78, l986.
28. Nathan DM, Francis TB, Palmer JL. Effect of aspirin on determinations of glycosylated hemoglobin. Clin Chem 1983;29:466-9.
29. Stevens VJ, Fantl WJ, Newman CB, Sims RV, Cerami A, Peterson CM. Acetaldehyde adducts with hemoglobin. J Clin Invest 1981;67:361-9.
30. Ceriello A, Giugliano D, Dello Russo P, Sgambato S, D’Onofrio F. Increased glycosylated haemoglobin A1 opiate addicts: evidence for a hyperglycaemic effect of morphine. Diabetologia 1982;22:379.
31. Roberts WL, Safar-Pour S, De B.K., Rohlfing C, Weykamp CW, Little RR. Effects of hemoglobin C and S traits on glycohemoglobin measurements by eleven methods. Clin Chem 2005;51:776-78.
32. Mongia SK, Connolly S, Hanson S, Little RR, Rohlfing CL, Roberts RF, Roberts WL. Effects of hemoglobin C and S traits on five commercial glycated hemoglobin (HbA1c) assays. (abstract) ACLPS, 2006.
33. Rohlfing C, Connolly S, England J, Little R. Effect of Elevated Fetal Hemoglobin on HbA1c Measurements: Four Common Assay Methods compared to the IFCC Reference Method. Clin Chem 2006, 52, A108.
34. Bayer Advia, product insert
35. Torke NS, Boral L, Nguyen T, Chakrin A, Kimball D, Comparison of four methods for Glycohemoglobin (HbA1c) determination. Clin Chem 2005;51:A242-3.
36. Olympus Diagnostica GmbH, HbA1c (Hemoglobin A1c) product insert
37. Randox Laboratories Ltd, Haemoglobin A1c product insert
38. Mongia SK, Little RR, Rohlfing CL, Hanson S, Roberts RF, Owen WE, D’Costa MA, Reyes CA, Luzzi VI, Roberts WL. Effects of Hemoglobin C and S Traits on Fourteen Commercial Glycated Hemoglobin Assays. Amer J Clin Path 2008;130:136-140.
39. Rohlfing C, Connolly S, England J, Hanson S, Moellering C, Bachelder J, Little R. The effect of elevated fetal hemoglobin on HbA1c results: five common HbA1c methods compared to the IFCC reference method. Amer J Clin Path 2008;129:811-4.
40. Little RR, Rohlfing CR, Hanson S, Connolly S, Higgins T, Weykamp C, D’Costa M, Luzzi V, Owen WE, Roberts WL. Effects of hemoglobin E and D traits on glycated hemoglobin (HbA1c) Measurements by twenty-three methods. Clin Chem 2008 (in press)
41. Luzzi VI, McKenna M, Landry C, Karsteter L, Mongia SK,  Roberts WL, Rohlfing CL, Tennill A, Little RR.  Cobas Integra Tina-Quant Hemoglobin A1c Gen.2 Assay Accurately Monitors HbA1c in Patients with S, C and E traits. (abstract) Euromedlab June, 2007.
42. Luzzi VI, McKenna M, Landry C, Karstater L, Mongia SK, Roberts WL, Rohlfing CL, Tennill A, Little RR. Cobas Integra Tina-Quant Hemoglobin A1c Gen.2 Assay: Accurate and Precise with no interference from Hb Variants. (abstract) Clin Chem 2007, 53
43. Liu L, Hood S, Wang Y, Bezverkov R, Dou C, Data A, Yuan C. Direct enzymatic assay for %HbA1c in human whole blood samples. Clin Biochem 2008 (in press).
44. Schnedl WJ, Lipp RW, Trinker M, Hopmeier P. Hemoglobin D [b 121(GH4)Glu—Gln] Causing Falsely Low and High HbA1c Values in HPLC. Clin Chem 1998;44:1999-2000.
    

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