Improving the Accuracy of Hemoglobin A 1c. : Your Help Is Needed

Transcription

1 Improving the Accuracy of Hemoglobin A 1c : Your Help Is Needed Kristina Jackson Behan, PhD, MT(ASCP) (Clinical Laboratory Sciences Program, University of West Florida, Pensacola, FL) DOI: /RTVTWPYPTPA5YAGU Clinical laboratory personnel are important members of the diabetes treatment team, from the technical staff, to the supervisor, to the pathologist. The technicians and technologists measure glucose, hemoglobin A 1c (A1c), lipids, microalbumin, and liver function tests for diabetic patients to help those patients to decrease the risk of chronic complications. The laboratory staff and supervisors monitor quality control with a critical eye. The laboratory management brings in better instrumentation and methodology as soon as it is feasible. Questionable laboratory results are investigated and corrected. Quality assurance is not just a catchphrase, it is a way of life. So it is a cause for concern to all laboratory professionals that A1c results may be inaccurate, yet reported, in patients who have variant hemoglobins. The nature of the A1c inaccuracy, whether it is a spurious increase or decrease, is specific to the method used and to the variant hemoglobin (eg, HbS, HbC, HbE, HbF). An analysis of the capabilities of available methods is listed at a Web site maintained by the National Glycohemoglobin Standardization Program (NGSP). 1 The 2 major types of testing currently in use are high-performance liquid chromatography (HPLC) and immunoassay. High-performance liquid chromatography has an advantage in that the latest generation of analyzers can resolve and identify several forms of hemoglobin, including most variants, and it provides a chromatogram that shows peaks where the forms have been resolved. High-performance liquid chromatography analyzers also provide quantitation of those peaks. Immunoassay accuracy may be affected by some variant hemoglobins, but there is no visual interpretation of the results other than a number to aid the laboratory professional in evaluating accuracy. Immunoassay analyzers are simpler to use and, as a result, are used as point-of-care tests (POCT) in physicians offices. This can be problematic as physicians may be unaware that a particular diabetic patient has a hemoglobinopathy. Hemoglobin electrophoresis is not a recommended laboratory test for a new diabetic, according to the American Diabetes Association (ADA) Standards of Medical Care. 2 Moreover, diabetic individuals may not be aware that they have a hemoglobinopathy or that a known hemoglobin variant can affect their A1c results. Diabetic patients with hemoglobinopathies may receive inaccurate A1c results several times before the error and cause is discovered. This is a serious quality issue for all laboratory professionals. This report will discuss this system error in A1c testing and recommend a method to eliminate it. The recommendation will ask you, the reader, to make it happen. Collective data on laboratory practices provided by you will be used to determine testing and reporting practices and be used Complete and this information to kbehan@uwf.edu, or mail to Kristina Behan, PhD, Clinical Laboratory Sciences Program, University of West Florida, University Parkway, Pensacola, FL Name of your institution, city, and state. 2. Zip code. 3. A1c methodology used in your laboratory (instrument name and manufacturer). 4. When you report A1c values, do you (select one): A. state only the A1c as a percentage (eg, Hemoglobin A 1c 6.5% ); B. include the value and the methodology (eg. Hemoglobin A 1c 6.5%, Acme HPLC analyzer ); C. include a statement if a hemoglobin variant has been detected (eg, Hemoglobin A 1c 7.4%; patient has elevated Hemoglobin F ); or D. other format (include details). 5. Has your laboratory changed A1c methodology in the last 2 years? Answer with yes, no, or don t know. 6. Comments, examples, and suggestions are welcome. Figure 1_Hemoglobin A 1c testing and reporting practices survey. labmedicine.com July 2008 j Volume 39 Number 7 j LABMEDICINE 389

2 to propose a standardized format. Please complete the survey in Figure 1 on reporting practices in your laboratory, and mail or it to the author s address listed. Whether you are a technologist, a supervisor, or a pathologist, your data is important. The article will demonstrate how the system works, how it fails, and how it can be corrected. How Hemoglobin A 1c Works The System Hemoglobin A is the major adult hemoglobin and is a tetramer of 2 alpha and 2 beta chains. Hemoglobin A picks up glucose at the N-terminal valine of the beta chain to form hemoglobin A 1c (Figure 2A), and the percentage of hemoglobin that is glycated (% HbA1c) is proportional to the amount of glucose in the patient s blood and the length of time that the glucose has been elevated. A nondiabetic has an A1c below 6% and the goal for a diabetic is to keep his or her A1c below 7%. 3 Because glycation is a dynamic process, and because red blood cells age and are turned over approximately every 4 months, an A1c result approximates the average blood glucose for the past 2 to 3 months. Hemoglobin A 1c correlates Figure 2_Hemoglobin A 1c correlates to average blood glucose. (A) The beta chain of hemoglobin is glycated by the addition of glucose (Glc) at the N-terminal valine. An antibody to this glycated pentapeptide allows identification of hemoglobins A 1c, S 1c, C 1c, and E 1c. (B) Hemoglobin A 1c correlates with average blood glucose. with average glucose according to the scale shown in Figure 2B, and a 1% increase in A1c correlates to an increase of approximately 32 mg/dl of glucose as measured by capillary whole blood. 4 Case 1 is an 83-year-old white female who provided results from glucose self-monitoring. The patient fingerstick data is shown in Figure 3. This patient had an average fasting glucose of 163 mg/dl, an average 2-hour post-prandial glucose of 183 mg/dl, and a combined average glucose of 173 mg/dl. Her A1c of 7.8% correlated very well to her average glucose (compare using Figure 2B). How Hemoglobin A 1c Fails The System Error Case 2 is a 78-year-old African American female who provided 3 months worth of glucose self-monitoring, shown in Figure 4. This patient reported that she had sickle cell trait. Her self-monitoring revealed an average fasting glucose of 112 mg/dl and an average 2-hour post-prandial glucose of 150 mg/dl. The average of both, 130 mg/dl predicts an A1c of 6.3%. A POCT analyzer gave a result of 4.7%, with no error flag, so the result was reported. Subsequent hemoglobin electrophoresis showed that this patient actually had sickle beta + thalassemia with an elevated amount of hemoglobin F, hemoglobin S, and a decreased amount of hemoglobin A. The manufacturer of the POCT analyzer used in this case reports that results are accurate for patients with sickle cell trait but inaccurate for patients with hemoglobin F over 10%. The methodology uses an antibody to detect the glycated hemoglobin and compares the glycated result with the total hemoglobin. This particular method is also able to measure glycated hemoglobins S, C, and E because of the location of the mutant amino acid in these variants. The amino acid sequence of hemoglobin S and hemoglobin C is identical to hemoglobin A, except for amino acid 6. The sequence of hemoglobin E is identical to hemoglobin A except for amino acid 26. The reagent antibody is specific for the glycated form of the first 5 amino acids, which is identical for all 4 forms (Figure 2A). Hemoglobin F, however, is not a variant of hemoglobin A but a tetramer of 2 alpha and 2 gamma chains; as such, it has a different amino acid sequence from hemoglobin A, as shown in Figure 5. Glycated hemoglobin F does not react with the reagent antibody, so the A1c result is lower than expected. Figure 3_Case 1: One-month results from self-monitoring of blood glucose. Blue line from fasting glucose, pink line from 2-hour postprandial glucose. Figure 4_Case 2: Three-month composite results of self-monitoring of blood glucose. Blue line from fasting glucose, pink line from 2-hour post-prandial glucose. 390 LABMEDICINE j Volume 39 Number 7 j July 2008 labmedicine.com

3 The same patient blood sample run by an HPLC method reported an A1c of 7.0%, and the chromatogram and its interpretation identified increased levels of hemoglobin F (~16% of the total) and hemoglobin S, as shown in Figure 6. This HPLC manufacturer also reports that its results are inaccurate when hemoglobin F is more than 10%; however, the error is identified and the result would not be reported. Impact on Patient Care Patient treatment depends on the A1c result. The recommended goal for hemoglobin A 1c as <7% is based on the finding of the Diabetes Control and Complications Trial Research Group that diabetics who were treated intensively to keep blood glucose as close to normal as possible had a statistically and clinically significant lower incidence of retinopathy, nephropathy, and neuropathy than diabetics who were treated with conventional therapy. 5 There is a risk, however, of hypoglycemia when patients are treated with intensive therapy. If a diabetic patient has a lower than normal A1c, it is an indication that he or she has had episodes of hypoglycemia. In that case, it is prudent therapy to lower the dosage of his or her medication. Consider the consequence of lowering the dosage of medication for a patient whose result is falsely lower than normal. The consequence is that the blood glucose level will increase, and the patient is at higher risk for the chronic complications of diabetes, including retinopathy, nephropathy, heart disease, and stroke. The patients who are most likely to be affected by system errors due to hemoglobin variants are non-caucasians. Eight percent of African Americans have hemoglobin S trait and 3% have hemoglobin C trait. 6 Hemoglobin E is found in up to 30% of individuals in Southeast Asia, 7 and, in the United States, in immigrants from Vietnam, Cambodia, Indonesia, Thailand, and Laos. Although the POCT immunoassay is reported to produce accurate results in these Figure 5_N-terminus of glycated hemoglobin F gamma chain (nonalpha chain). Circled amino acids are different from hemoglobin A. individuals, a review of the NGSP Web site shows that many other methods give inaccurate results, including some HPLC methods. Hemoglobin F is a normal form of hemoglobin in the fetus from 3 months of gestation and is produced in infancy as well. By 2 years of age, hemoglobin F is less than 2% of the total hemoglobin. 6 Hemoglobin F is elevated in patients with sickle beta 0 thalassemia, sickle beta + thalassemia, and in hereditary persistence of fetal hemoglobin, 6 with levels high enough to interfere with A1c testing. Thalassemias are linked to particular geographic locations, such as the Mediterranean, India, and Southern China; however, immigration and slave trades introduced individuals with these mutations into other areas, including the United States. 6 Hemoglobinopathies and thalassemias can lead to falsely increased or decreased A1c values depending on the test methodology. 7 Errors in either direction that cause a change in patient therapy produce a negative patient outcome. Clinical Practice Recommendations Clinical practice recommendations from the ADA state that a new patient should have the following laboratory tests: A1c, lipid profile, liver function tests, urine albumin, and Figure 6_Case 2 HPLC chromatogram. Peaks for hemoglobins F, A, and S are clearly resolved and identified. labmedicine.com July 2008 j Volume 39 Number 7 j LABMEDICINE 391

4 creatinine, and that select patients should also have a TSH. 2 It is an ADA recommendation that patients be tested at the point-of-care for timely patient management. 3 Hemoglobin electrophoresis is not recommended as part of the testing, even for non-caucasian patients. The National Institutes of Health (NIH) do recommend checking a patient for a variant hemoglobin if patient glucose results do not agree with A1c values or are unexpected, if the A1c is more than 14%, or if A1c results are very different when the test methodology has changed. 8 These recommendations address the issue after the fact, after inaccurate results have been reported, and after therapy has been instituted. The last recommendation that concerns a change in methodology assumes that the physician is aware when a methodology has changed. This will be true if the physician office changes its methodology, or if the physician refers a patient to an outside laboratory and compares the result with his or her own POCT. It will not be true if a clinical laboratory changes its methodology, unless that laboratory routinely reports its method on patient results. The Plan for Improvement Point-of-care testing has been shown to improve patient outcomes as the physician can alter the treatment plan immediately and stress its importance to the patient. 9 Many laboratory professionals have reservations about POCT, but POCT is not going away. All laboratory professionals have a responsibility to the patient who is getting a false result. The obvious remedy to this problem is that each patient should be evaluated by hemoglobin electrophoresis prior to his or her initial A1c testing and then referred to a laboratory that has the technology to correctly measure A1c. It may be possible to convey the same information to the physician without hemoglobin electrophoresis. Most analyzers in hospital laboratories have the capability of detecting variant hemoglobins, but it is not standard practice to report their presence. It is also not standard practice to publish the methodology and its capability to detect variants on patient reports. This would not be difficult to accomplish, and it would not be costly. The model proposed here is this: the newly diagnosed patient is screened for hemoglobinopathy during A1c HPLC, and the presence or absence of variants is reported as part of the result. Furthermore, the methodology and instrument name is reported on the patient result. This will require (1) a small change in the standards of care for the physician, and (2) a small change in how the clinical laboratory reports results. I. Revision to the Standards of Care During the initial evaluation of the patient, the A1c should be performed by a clinical laboratory that uses a method capable of detecting the most common variant hemoglobins. If it is determined that the patient does not have a hemoglobin variant, he or she can subsequently be tested by POCT. II. Revision to Clinical Laboratory Reporting Patient results should include all of the following: the A1c result and reference range, a statement about the presence or absence of hemoglobin variants, the name of the A1c analyzer used, and the capability of the test methodology to detect hemoglobin variants. For example, the patient in case 2 would read: Hemoglobin A 1c 6.3% (reference range <6.5%) by Acme 2.2 HPLC analyzer. Hemoglobin S and hemoglobin F detected. Results acceptable according to the National Glycohemoglobin Standardization Program. The benefit of a standardized approach to reporting is well recognized. A plan for international standardization of A1c results with respect to unit reporting (% HbA1c versus mmol HbA1c/mol Hb) has recently been recommended by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). This proposed change is controversial, as it may take several years and considerable educational efforts and cost to accomplish. 10,11 The proposal described in this report adds a different facet to results standardization, is valuable regardless of the unit used, and is minimal in cost. Other preanalytic factors, such as the presence of iron deficiency anemia and hemolytic anemia, also cause inaccurate A1c results. These inaccuracies cannot be addressed by this proposal. Changing the Policy To make the strongest impact on patient care, result formatting should be standardized. Two organizations must approve this recommendation: the NGSP and the ADA. The NGSP has the ability to recommend a change in the reporting requirements for hemoglobin A 1c and the ADA has the ability to recommend initial A1c testing in a clinical laboratory as part of its standards of care. The ADA reviews and updates its policies annually and uses an evidence grading system to determine when to change a recommendation. To convince the ADA to recommend initial A1c testing at a clinical laboratory, there must be a clear benefit to patient outcome, which is the ability to identify the patient with a variant hemoglobin. Demonstration of the ability and availability to do so will be provided by a density map of the locations of the laboratories that have that capability. The evidence to support the change to initial testing in a clinical laboratory will be provided from the survey that you complete today. The survey will also provide information to the NGSP about the current format of results. The responses to the survey will be collated to determine how clinical laboratories actually report their results, as well as their ability to discriminate variant hemoglobins. The more laboratories that complete this survey nationwide, the stronger the evidence will be for changing the policy. This report, as well as your comments, will be shared with the ADA, the NGSP, and with you, the readers. With your help, we can prevent a diabetic patient from receiving the wrong A1c result and, with it, inappropriate treatment. LM Acknowledgments: This project has been funded by a grant from the American Society for Clinical Laboratory Sciences. Patient data is from a study approved by the Institutional Review Board at the University of West Florida. The author thanks Dr. Mark Kummer for critical review of the manuscript. 1. Factors that interfere with GHB Test Results. Available at: org/prog/factors/htm. Accessed: Feb 9, American Diabetes Association. Standards of medical care in diabetes Diabetes Care. 2008;31:S12 S LABMEDICINE j Volume 39 Number 7 j July 2008 labmedicine.com

5 3. American Diabetes Association. Executive Summary: Standards of medical care in diabetes Diabetes Care. 2008;31:S5 S Rohlfing CL, Weidmeyer HM, Little RR, et al. Defining the relationship between plasma glucose and HbA1c. Diabetes Care. 2002;25: The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. NEJM. 1993;329: Harmening DM. Clinical Hematology and Fundamentals of Hemostasis. 4th edition. Philadelphia, PA: FA Davis; Bry L, Chem PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin. Clin Chem. 2001;47: Sickle Cell Trait and other Hemoglobinopathies and Diabetes: Important Information for Physicians. Available at: pubs/hemovari-a1c/sicklecell-fact.pdf. Accessed: Feb 9, Cagliero E, Levina EV, Nathan DM. Immediate feedback of HbA1c levels improves glycemic control in type 1 and insulin-treated type 2 diabetic patients. Diabetes Care. 1999;22: Pizzi R. The quest for glycohemoglobin standardization. Clin Lab News. 2006;32: Levenson D. Groups issue consensus statement on HbA1c standardization. Clin Lab News. 2007;33:14. labmedicine.com July 2008 j Volume 39 Number 7 j LABMEDICINE 393