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HPLC conditions may affect D10


Several reasons may lead to false low or zero A1c values - especially on HPLC.
From the chromatogram, it appears the analysis is done on Bio-Rad D10 HPLC in short mode.   The method is based on principle of cation exchange chromatography and formal charge differences between hemoglobin variants.   Although, it is highly rare that a whole blood sample would give a false result, but possibilities can not entirely ruled out that the alteration of the HPLC reagents (polarity or ionic nature of the mobile phase) or change in serum polarity cause false lowering of A1c.   The inconsistency in polarity simply may have caused A1c to elute either early or late co-elution with other fractions.   A clue can be found from the distribution of the overall peak area.  Usually a zero A1c  value is obtained when the actual A1c is already low in hypoglycemic condition.     In this case, the reagents on HPLC should be changed, or calibration should be done.    Also, I noticed the CO2 levels are low in the patient  sample which may contribute to the blood polarity resulting in discrepancy in the normal chromatographic interaction leading to disappearance of A1c.  In this case, immunoassay based analysis should provide proper values.
Another possibility is also reported in literature is presence of other abnormal hemoglobin traits such as Hb-D, Hb-E, Hb-C or Hb-S. (Clin Chem, 2008, 130, 136 and Clin Chem, 2008, 54, 1277)   Presence of these traits has different effects on the value of A1c (Hb-A1c).  Usually modern HPLC based A1c analyzers have a longer mode which allows to detect this traits.  If they are present, enzyme assay (not immunoassay) can be performed to obtain correct A1c ( 
Another possibility could be low lifespan of erythrocyte.  Patient has slightly lower RBC and Hgb indices.  This may contribute to lower A1c than normal.
I hope this information will help to get some clue.

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Where Did the A1c Go?





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Created at 5/2/2011 10:31 AM by  
Last modified at 3/5/2012 3:35 PM by AACC\Administrator