1. MARK RIDGWELL, DIASUKE TANAKA & CCG WOOD Within run Imprecision
COBAS C111 – EVALUATION OF HBA1C PERFORMANCE AND SUITABILITY OF USE IN A CLINICAL SETTING
MARK RIDGWELL, DIASUKE TANAKA & CCG WOOD
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INTRODUCTION
Inter/Intra-assay variation of High pool HbA1c results measured on the Roche Cobas C111(DCCT/NGSP)
Haemoglobin A1c (HbA1c) also known as glycosylated or glycated haemoglobin, is a product of the non-enzymatic reaction of haemoglobin with glucose in blood. This reaction occurs to a small extent in normal blood (approximately 5% of total haemoglobin is in this form in normal subjects). Higher levels are found in patients with poorly controlled diabetes mellitus, due to higher glucose concentrations in their blood. Hence, the percentage of haemoglobin in the glycosylated form is a useful indicator of metabolic control in these patients. Due to the long half-life of haemoglobin (6 weeks approx) the percent HbA1c changes slowly and tends to indicate the "integrated" glucose concentration for a period of at least one month.
The Cobas C111 analyser is suitable for small workload laboratories and offers solutions for clinical chemistry and homogenous immunoassay testing. The analyser is a bench top unit with a width of 720mm and a weighty of 35kg and offers both photometric and ISE chemistry testing. HbA1c in whole blood is part of the test menu. We evaluated the analytical performance of the HbA1c method and the analysres suitability of use in a clinical setting.
Run
Date
Replicate 1
Replicate 2
Replicate 3 1
27/09/2010
8.40
8.20 2
29/09/2010
8.30 3
30/09/2010
8.00 4
1/01/2010
8.10 5
6/10/2010
Within run Imprecision
Total imprecision
%HbA1c
0.09
0.12 CV
1.10%
1.40%
RESULTS
Merhod comparison difference plot Biorad variant II v Roche Cobas C111(DCCT/NGSP)
Merhod comparison scatter plot with Passing and Bablok fit Biorad variant II v Roche Cobas C111(DCCT/NGSP)
r2=0.98
n=215
METHOD
This method uses TTAB* as the detergent in the hemolyzing reagent to
eliminate interference from leukocytes (TTAB does not lyse leukocytes).
Sample pretreatment to remove labile HbA1c is not necessary.
All hemoglobin variants which are glycated at the β-chain N-terminus and
which have antibody-recognizable regions identical to that of HbA1c are
measured by this assay. Consequently, the metabolic state of patients
having uremia or the most frequent hemoglobinopathies (HbAS, HbAC,
HbAE) can be determined using this assay.
The HbA1c determination is based on the turbidimetric inhibition
immunoassay (TINIA) for hemolyzed whole blood.
Sample and addition of R1 (buffer/antibody):
Glycohemoglobin (HbA1c) in the sample reacts with anti-HbA1c
antibody to form soluble antigen-antibody complexes. Since the
specific HbA1c antibody site is present only once on the HbA1c
molecule, complex formation does not take place.
Addition of R2 (buffer/polyhapten) and start of reaction:
The polyhaptens react with excess anti-HbA1c antibodies to
form an insoluble antibody-polyhapten complex which can
be measured turbidimetrically.
Liberated hemoglobin in the hemolyzed sample is converted to a derivative having a characteristic absorption spectrum which is measured
Bichromatically during the preincubation phase (sample + R1) of the above immunological reaction. A separate Hb reagent is consequently not necessary.
The final result is expressed as percent HbA1c and is calculated
from the HbA1c/Hb ratio as follows:
Protocol 1 (acc. to IFCC):
HbA1c (%) = (HbA1c/Hb) x 100
Protocol 2 (acc. to DCCT/NGSP):
HbA1c (%) = (HbA1c/Hb) x
HbA1c results obtained from the Cobas C111 analyser were compared with those obtained using the Bio-Rad Varaint II HbA1c testing system. Analytical performance was assessed using Analyse-It Passing Bablok comparison and Bland Altman difference plot. Imprecision was estimated using two samples run daily in triplicate over a period of five days.
A Passing-Bablok comparison equation C111 = Variant II was determined. The level of bias at the critical point of HbA1c of 8.0% was estimated to be and across the range of analysis.The total imprecision was determined to be 2.6% with an intra assay CV of 1.6%
Intra assay precision profileof the Cobas C111(IFCC)
Precision profile
Low pool mM/M
Medium pool mM/M
High Pool mM/M
34.75
82.28
119.80
34.53
81.66
122.22
34.71
81.33
120.87
34.94
81.03
122.32
35.57
81.36
121.11
35.42
81.99
119.36
35.48
83.02
119.58
35.38
83.08
119.73
Mean
35.10
81.97
120.62 SD
0.41
0.77
1.19 CV
1.2%
0.9%
1.0%
CONCLUSION
The HbA1C determination on the Cobas C111 is based on the turbidimetric inhibition assay for haemolysed whole blood. The analytical performance of the C111 is good and satisfies essential performance criteria. The analyser is relatively small with a comprehensive clinical chemistry test menu. A hospital scientist and a technical officer unfamiliar with Roche analysers were easily trained to operate the analyser. In our opinion the C111 would be best suited to low throughput laboratories, and would provide good analytical performance.
Inter/Intra-assay variation of Normal pool HbA1c results measured on the Roche Cobas C111(DCCT/NGSP)
Run
Date
Replicate 1
Replicate 2
Replicate 3 1
27/09/2010
6.000
5.800 2
29/09/2010
5.700
5.600 3
30/09/2010 4
1/01/2010 5
6/10/2010
5.900
References
Cobas C111 HbA1c Kit insert.
Within run Imprecision
Total imprecision
% HbA1c
0.09
0.15 CV
1.60%
2.60%