EDUCATION :: HEMOGLOBIN A1C


are also extreme cases such as sickle cell disease, where the RBC turnover is significantly impacted. Homozygous Hb S9


or


Hb S in combination with some other variants (Hb C, D-Los Angeles, O-Arab) results in rapid RBC turnover and cause low glycated hemoglobin fractions. To address this, manufacturer package inserts note that the assay should not be employed “in the absence of Hb A.” Further, some rare hemoglobin variants


can cause profound analytical interferences even in trait form.10- 14


Proven interferences such as these motivated the ADA to recommend the following: “Marked discordance between measured A1C and plasma glucose levels should raise the possibility of A1c assay interfer- ence due to hemoglobin variants (i.e., hemoglobinopathies) and consideration of using an assay without interference or plasma blood glucose criteria to diagnose diabetes.” It is further recommended that “In conditions associated


with increased red blood cell turnover, such as sickle cell disease… only plasma blood glucose criteria should be used to diagnose diabetes.”15 When identified, a clinician could consider a hemoglob-


inopathy in conjunction with the A1c result; common trait variants could be disregarded (except in borderline cases, calling for more careful assessment), while severe interfer- ences (compound hemoglobinopathies, or no Hb A) would warrant reflex to an alternate tests or analytes (fructosamine, glycated albumin, fasting blood glucose) as mentioned by the ADA. However, the practical considerations of the laboratory aren’t addressed; how does the lab know which patients have interfering hemoglobinopathies? Interference here is detected after the fact: an A1c result is discordant from the rest of the patient clinical profile, suggesting a variant or altered RBC turnover. While effective in cases of obvious discordance, it presumes access to additional results not always available to the lab, placing the impetus for interference detection on the clinician, a burdensome and roundabout approach to result validation. It further ignores the reality of results less obvi- ously discordant, such as the noted Hb S trait and Hb E trait influencing the A1c value.


Testing methodologies incapable of detecting hemoglobinopathies include those employing antisera (immunoassay) or enzymatic digestion.


This context surrounding HbA1c testing implies a pref-


erence for HbA1c techniques capable of incidental hemo- globinopathy discovery. If detected at the time of the A1c testing, then appropriate notice of minor interferences can be provided to the clinician, and extreme discordance (Hb A absence, sickling disease) identified by the lab can be blocked before release. This stratifies the current A1c testing meth- odologies; those able and those unable to visualize variants and thalassemias. Testing methodologies incapable of detecting hemoglob- inopathies include those employing antisera (immunoassay) or enzymatic digestion. These techniques, using highly specific antisera or enzymes, excel in finding the recognition sequence at the glycated N-terminal beta chains, but their limited scope means they can’t discern further distant mutations, “seeing” only the amino acids within their target region. Variants with mutations outside of this window are included in A1c calcula- tions and may significantly influence the reported value. The


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Many labs resort to the expediency of running non-separation, low-resolution techniques and simply “send out” samples with questionable results, hoping reference labs will solve the problem for them.


boronate affinity method similarly fails to detect variants, as it back-calculates A1c from measurements of all hemoglobins and glycated hemoglobins within the sample, regardless of mutations. The blinded nature of these methods’ results is true even in the extreme cases where no Hb A is present. Methods capable of detecting hemoglobinopathies (vari- ants and some thalassemias) include HPLC and capillary electrophoresis (CE). A more detailed profile of the patient’s hemoglobins is integral to these methods, as the different fractions are separated by either electrophoretic mobility or affinity to an anion exchange column, isolating the Hb A and HbA1c for measurement. As hemoglobin variants are, by their very nature, structurally different from Hb A or HbA1c, they typically migrate or elute differently on these “separation” tech- niques, generating distinct and obvious peaks or distortions. This indicates patients who have hemoglobinopathies for closer analysis, while normal patients will pass without note. All the common variants (and many rare variants) are distinctly visualized, and using a separation methodology significantly minimizes the chance of an undetected variant interfering with A1c measurement. This presents a seemingly obvious choice: the laboratory can employ either a “separation” technique, or a technique where hemoglobinopathies are undetected, leaving them uncertain of their result without additional testing. However, practical considerations of lab testing may not allow for the obvious choice. Many labs resort to the expediency of running non-separation, low-resolution techniques and simply “send out” samples with questionable results, hoping reference labs will solve the problem for them. Unfortunately, reference lab may not be employing the separation techniques either. Labs may assume that separation techniques have lower throughput, and result review may seem an intensive, specialized job, especially as staffing shortfalls are common in the modern laboratory. There simply are not technologists available to review every A1c result in even the moderate-volume laboratories. Fortunately, workflow improvements solve these challenges; for example, the capillary systems employ an auto-flagging software that detects and flags abnormal profiles. Normal profiles require no further review, with their results sent automatically to the LIS. The remaining samples, representing the significant but small minority of patients with hemoglobinopathies, can be retained for review, representing a more reasonable workload. Those samples exhibiting non-analytically interfering variants are reportable, and appropriate notice to the clinician could be provided. The remaining few, where A1c results are inappro- priate for diabetes diagnosis, could be referred for alternate analyte testing.


Further streamlining could rely on the genetic nature of hemoglobinopathies; a hemoglobin variant or thalassemia will not “suddenly develop” in a patient. Separation techniques could therefore be used for an initial screen and those pa- tients exhibiting no indication of hemoglobinopathies would be transferred for future monitoring on “non-separation”


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