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Letters to the Editor |
1
Div. of Clin. Biochem., Dept. of Pediatr. Lab. Med., The Hosp. for Sick Children, Toronto, ON, and, Dept. of Clin. Biochem., Univ. of Toronto, 100 College St., Toronto, ON, Canada M5G 1L5
Div. of
2
Hematol., Dept. of Pediatr. Lab. Med., and,
3
Div. of Endocrinol., Dept. of Pediatr., The Hosp. for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8
a Address correspondence to this author, at: 17 Runnymede Rd., Toronto, ON, Canada, M6S 2Y1.
To the Editor:
When glucose reacts with hemoglobin (Hb), the first products are Schiff bases that slowly undergo Amadori rearrangement to produce stable ketoamines (glycohemoglobin). The relative fraction of the ketoamines to total Hb can be measured as hemoglobin A1c (Hb A1c), which constitutes ~60% of the bound glucose (1). The Schiff base (or labile fraction) is unstable and is fairly readily hydrolyzed, especially at acid pH. Effective removal of the labile fraction is essential for the accurate determination of Hb A1c because the labile fraction cannot be separated from the true (ketoamine) fraction by simple ion-exchange procedures and because its concentration varies acutely with the plasma glucose concentration (2). Partial and variable removal of the labile fraction adds to assay imprecision and reduces the correlation (3) between measured Hb A1c and mean plasma glucose.
The Bio-Rad Labs. Variant analyzer is a dedicated HPLC system designed to measure variant hemoglobins or Hb A1c. For Hb A1c the hemolyzing reagent is a pH 5.0 citrate buffer that also serves to remove the labile fraction. The Bio-Rad instrument manual advises users to "let samples stand at 18–28 °C for at least 10 min to allow Schiff base removal" (this was increased recently to 15 min). The manual also describes how to run the instrument essentially in batch mode. At start-up, or on restarting after running samples, the recommended protocol is an 8-min wash, a 3-min prime with hemolysate, followed by 3-min runs with two calibrators and two controls (total 23 min) before patients' samples are processed. With this protocol, patients' hemolysates are tested after variable periods of incubation at room temperature and at 8 °C (the temperature of the sample tray). A stat sample could be prepared, incubated for 10 min at room temperature and placed on the already started analyzer for almost immediate sampling, or at the other extreme, a second sample could be at position 100 and thus spend 10 min at room temperature followed by ~300 min at 8 °C before analysis.
Soon after we started to use the Variant and attempted to offer a quicker turnaround time for diabetes clinic patients, we received complaints from the diabetologists that some results were "unreliable" and "too high" compared with what they expected. Their expectations were based on observations of weekly mean clinic values (50–60 patients per week) and cumulative records for individual patients measured quarterly with our previous HPLC method (4) over a 12-year period. Yet the Bio-Rad Lyphocheck quality-control samples, Bilevel Diabetes Controls 1 and 2 (with claimed values of 5.6% and 9.7% of total Hb), run several times each day gave excellent precision with respective values for mean, SD, and CV of 5.68%, 0.11%, and 1.9% (n = 200) and 9.44%, 0.14%, and 1.5% (n = 209). We initially attributed the diabetologists' concerns to unfamiliarity with the Variant results, which we assumed included a slightly different fraction of Hb as Hb A1c, compared with that given by our previous in-house HPLC method (4). However, discussions with Randie R. Little of the University of Missouri–Columbia suggested that the labile fraction might be incompletely removed with a 10-min incubation at room temperature; thus, we decided to test the effectiveness of the Bio-Rad procedure.
We tested samples from inpatients and clinic patients at The Hospital
for Sick Children, Toronto: a tertiary-care, university-affiliated
hospital. We varied the incubation time for hemolysates prepared at
room temperature (21–23 °C) and 4–8 °C. For the latter, we
rapidly prepared hemolysates by using hemolyzing reagent straight from
the refrigerator at 4–8 °C and immediately placed the prepared
hemolysate in the Variant instrument with the sample tray at 8 °C.
For room temperature experiments, we prepared several hemolysates from
2 or 3 patients at periodic intervals, marking the time of preparation
on the hemolysate tubes. We then left them for various lengths of time
at room temperature before loading them on the Variant, one at a time
into a sample tray position that was to be sampled within the next
minute. The incubation time was determined from the time of injection
noted on the Variant printout and the time of preparation marked on the
hemolysate tube. Graphs were prepared of measured Hb
A1c against time (Fig. 1
). Complete removal of the labile fraction was assumed when the
Hb A1c had fallen to a plateau. Extrapolating the curve
back to zero incubation time gave the total value for "true" Hb
A1c plus the labile fraction.
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We found that the labile fraction, calculated as the difference, varied between patients. With eight poorly controlled patients whose mean Hb A1c was 11.9% of total Hb (range 9.0–16.1%), the mean labile fraction was calculated as 1.7% of total Hb (range 0.4–2.4%). The labile fraction was only partially removed after 10 min at room temperature (mean 40% removal, range 25–58%). Even at 30 min, removal was only 79% complete (range 67–100%). This resulted in overestimation of Hb A1c by 9.0% (range 2–13%) at 10 min and by 2.9% (range 0–5.2%) at 30 min (in comparison with plateau values = 100%). At 4–8 °C, the rate of removal of labile fraction was much less than that at room temperature. At 10 min, only 12% of the labile fraction had been removed; after 30 min, the corresponding value was 28% (n = 2).
In four routine batches of samples (n = 114) that were hemolyzed (in small groups) with either a 10-min (y) or 60-min (x) incubation with Bio-Rad hemolysis reagent, results (expressed as % of total Hb) correlated (r = 0.986), but were ~7% higher when the shorter incubation was used: y = 1.07x - 0.20.
We also tested 59 samples with an alternative procedure for removing the labile fraction (4). Samples (100 µL) were incubated for 45 min at 37 °C with 4 mL of 50 mmol/L acetate buffer, pH 5.5, containing 113 mmol/L NaCl. Afterwards, these samples were centrifuged at 1300g for 10 min and decanted. The wet erythrocyte button was gently mixed with the residual acetate buffer and the suspension was hemolyzed, as though it were blood, with the Bio-Rad hemolyzing reagent. Samples were then transferred to the Variant and tested without additional incubation. Comparison of results for these samples (y') with the results for those that had been incubated with Bio-Rad Hemolyzing reagent alone for at least 60 min at room temperature (x') showed excellent correlation (r = 0.991) for Hb A1c (expressed as % of total Hb): y' = 1.01x' + 0.15.
We therefore recommend that sample hemolysates to be run on the Variant be left 60 min at room temperature before sampling to completely remove the labile fraction.
References
Bio-Rad Labs., Hercules, CA
a Address correspondence to this author, at: 17 Runnymede Rd., Toronto, ON, Canada, M6S 2Y1.
To the Editor:
We have addressed the efficiency of Schiff base removal in the Performance Characteristics section of the current Variant Hemoglobin A1c Instruction Manual (March, 1997). Under conditions that simulate routine (batch) analysis, the reduction rate is 80–100%. The decrease in Schiff base occurs not only during the incubation time but also during the priming and calibration sequence, when the sample gradually cools from ambient temperature to the Variant's sample storage temperature (8 °C). Much of the data reported in this letter was produced by using a limited incubation (10 min) and "stat" analysis. Under the conditions described by the authors, Schiff base removal will be less effective. The Instruction Manual currently recommends a 30-min incubation for extremely hyperglycemic individuals; this approach will yield the most accurate results in tertiary-care centers where patients with type I diabetes are treated.
a Address correspondence to this author, at: 17 Runnymede Rd., Toronto, ON, Canada, M6S 2Y1.
To the Editor:
We welcome these comments, but are still unclear how Bio-Rad intends the instrument to be used in tertiary-care centers (or other laboratories). Must the technologist make a decision on whether the sample is from an "extremely hyperglycemic" patient, before deciding whether to incubate the hemolysate for 15 or 30 min? How is "extreme" hyperglycemia defined? Alternatively, does Bio-Rad now recommend a 30-min approach for all samples analyzed in tertiary-care centers, in case they are from extremely hyperglycemic individuals? As we have shown, even if analyzed "stat," samples after a 30-min incubation give results that are, on average, only 2.9% higher than the plateau values. The Variant is a very precise instrument and we see no merit in adding analytical imprecision by incomplete removal of the labile fraction.
The following articles in journals at HighWire Press have cited this article:
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  P. Colton, M. Olmsted, D. Daneman, A. Rydall, and G. Rodin Disturbed Eating Behavior and Eating Disorders in Preteen and Early Teenage Girls With Type 1 Diabetes: A case-controlled study Diabetes Care, July 1, 2004; 27(7): 1654 - 1659. [Abstract] [Full Text] [PDF]
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 I. Gibb, A. Parnham, M. Fonfrede, and F. Lecock Multicenter Evaluation of Tosoh Glycohemoglobin Analyzer Clin. Chem., October 1, 1999; 45(10): 1833 - 1841. [Abstract] [Full Text] [PDF]
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