Download Report

Journal of Life Sciences 6 (2012) 456-460
Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC
Analyzer for HbA1C Determination
Eloísa Urrechaga
Laboratory of Clinical Analysis, Hospital Galdakao Usansolo, Galdakao 48960, Vizcaya, Spain
Received: July 09, 2011 / Accepted: September 20, 2011 / Published: April 30, 2012.
Abstract: Menarini ARKRAY ADAMS ™ A1C HA-8180 is a high pressure liquid chromatography (HPLC) system for the
measurement of HbA1C. The analysis time is 48 seconds per sample. The analytical performance was evaluated to verify quality of
analysis, according to the criteria established in the recently published documents of consensus on this analyte. Precision and linearity
studies were performed according to CLSI’s guidelines. Recovery, the effect of Hemoglobin (Hb) concentration and the presence of
coexistent interfering substances were evaluated. The drastic reduction in the time of analysis does not impair the overall quality of the
results which were found to be accurate and linear in the clinically significant analytical range and excellent precision (total coefficient
of variation bellow 0.6 %). HbA1C measurement is independent of the total concentration of Hb and the presence of Hb chemically
modified elutes in the labile A1C fraction. Given the short time of the analysis this is a suitable system for the control of diabetic patients
in laboratories with high workflow.
Key words: Diabetes, HPLC, HbA1C, HA-8180 analyzer.
1. Introduction
Glycohemoglobin
(HbA1C)
the
N-terminal
(1-deoxyfructosyl) valine glycation product of
hemoglobin (Hb) A0 measurement provides the most
important medium- to long-term marker of
time-averaged glycemic status. Its relationship to likely
clinical outcome in diabetes mellitus has been
convincingly demonstrated for both type I and type II
subjects in major clinical trials [1, 2].
The recently implemented consensus statement on
the worldwide standardization [3] and the International
Federation of Clinical Chemistry (IFCC) reference
method for the HbA1C measurement [4], have
contributed substantially to the quality of the HbA1C
measurements.
An extension of the role of this Hb fraction from
monitoring to the screening and diagnosis of diabetes
[5], leads to the possibility of even greater demand for
this analysis.
Various methods based on its physical, chemical or
immunologic characteristics have been used for the
measurement of glycohemoglobins [6].
The automation and precision of the high
performance
liquid
chromatography
(HPLC)
methods has favored their use and the technical
improvements has allowed the achievement of
shorter analysis times. Along with this, there have
come compromises in the chromatographic quality
and resolution.
Menarini ARKRAY ADAMS ™ A1C HA-8180
(HA-8180) is a fully-automated HPLC device for
HbA1C measurement. The most important new
feature of this instrument is that the results can be
obtained within 48 seconds. We have evaluated the
analytical performance of this system to assess
Corresponding author: Eloísa Urrechaga, Ph.D., consultant
for clinical analysis, research field: analytical hematology.
E-mail: [email protected].
whether the reduction in the analysis time impairs
the quality of the results.
Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination
2. Materials and Methods
2.1 Characteristics of the HA-8180 Analyzer
The HA-8180 is an automated bench top analyzer
[dimensions: 530 (W) × 530 (D) × 530 (H) mm]. The
instrument is designed to measure HbA1C (range 9-195
mmol/mol IFCC units; 3%-20% NGSP units), as well
as hemoglobins F. Hb variants are not detected. The run
time is 48 s per sample that is fast in comparison to the
170 s used by the previous generation (Model
HA-8160). The instrument has a capacity of 100
samples per run. Specimens are either primary tubes
with cap piercing (patient samples), or tubes for
hemolisates (calibrators, controls, patients with small
sample volume) placed in specific ARKRAY racks
(nine types). There is an option to insert urgent (STAT)
samples. The instrument can spin tubes in order to
prevent blood sedimentation, automated reagent
information codes, self-diagnostic functions, precision
controls function and a large colour LCD.
In total, 3.4 µL of automatically diluted (standard
1:100; anemic samples 1:50) whole blood is injected.
The stainless steel ARKRAY column, maintained at
40 °C in an oven, consists of a prefilter and an
analytical column packed with an ion exchange resin (a
hydrophilic polymer of methacrylate ester copolymer).
Sealing screws are made of PEEK (polyether ether
ketone). Elution is achieved in a five-step phosphate
buffered gradient with increasing ionic strength. There
are three buffers (80A, 80B and 80CV) in aluminium
foil packs placed on top of the instrument. Hbfractions
are detected with a dual wavelength (420-500 nm)
LED-photodiode. At this wavelength, the absorption of
oxy- and deoxyhemoglobin is equivalent and thus,
ensures a stable signal irrespective of the
oxy-/deoxyhemoglobin ratio in the sample.
The instrument is calibrated with two calibrators
(low and high concentration). Calibration can be
performed after power up, but is not required when
operation is started from stand by. The reported result is
derived from the ratio HbA1C/HbA total, adjusted for
457
calibration and expressed in both IFCC and National
Glycohemoglobin Standarization Program (NGSP)
units.
2.2 Samples and Controls
Blood specimens were obtained from patients whose
diabetic control was being assessed routinely and were
used in this study according to the hospital’s ethics
guidelines.
Whole blood samples were obtained in the course of
routine analysis and collected in EDTA anti coagulant
tubes (VacutainerTM Becton-Dickinson, Rutherford,
NJ, USA).
Quality-control materials used throughout the
evaluations were provided by Menarini Diagnostics
(Glyco Hb Control, Menarini Diagnostics, Firenze,
Italy).
2.3 Protocol of Evaluation
The analyzer was calibrated at the beginning of the
evaluation, according to manufacture’s instructions in
NGSP units (%).
Precision and linearity studies were performed.
Sample carryover, precision and linearity studies, the
effect of Hemoglobin (Hb) concentration and the
presence of coexistent interfering substances were
evaluated according to the Clinical and Laboratory
Standards Institute (CLSI) guidelines and the
manufacture’s recommendations.
Carryover between blood samples with high and low
HbA1C concentrations was checked using the method
of Broughton et al. [7], processing samples of low (4.6
%) and high (12.8 %) concentration in triplicate and
sequentially, examining the first and third results of
each tripletfor systematic deviation.
Within run precision (intra assay) was studied at two
concentrations, using pooled blood from non diabetic
and poorly controlled diabetic adults; these samples
were assayed 10 times. Mean value and coefficient of
variation (CV) of the measurements were calculated.
Total precision was investigated using CLSI EP-5
458
Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination
A2 protocol [8] with control materials (Glyco Hb
Control, Menarini Diagnostics, Firenze, Italy). With
this protocol, on 20 working days, a low and a high
samples are assayed in duplicate twice a day in an
analytical run, with at least 10 samples. EP-5 defines
four parameters for the precision, all listed in Table 1.
Linearity was estimated in accordance with protocol
CLSI EP6 [9]. Linear regression analysis was applied
to compare expected and observed analytical values
using results from proportional mixtures of high (12.5
% ) and low (5.0 %) HbA1C samples.
The manufacturer’s claim is that the optimized total
chromatographic area should be between 1,000 and
4,000 units. We therefore investigated the effect of
varying hemoglobin concentration on chromatographic
quantification.
One sample was centrifuged for 10 minutes at 300
rpm to separate blood cells from plasma. Blood cells
and plasma were mixed at the ratios 9:1, 8:2 … 1:9; the
set of samples obtained had a broad hematocrit range.
The labile fraction is separated as a distinct peak on
the column. The following steps were taken to confirm
the effective separation of the labile Schiff base peak.
Glucose incubation: pooled blood with a HbA1C
concentration 5.0% was poured into six tubes; a
glucose solution was added to these test samples at
increasing ratios to achieve final glucose
concentrations up to 55.5 mmol/L. These samples were
incubated at 37º C for two hours and then analyzed.
Carbamylated Hb elutes form column in the same
peak along with the labile Schiff base fraction. The
following steps were taken to confirm the effective
separation of this interfering chemically modified.
Sodium Cyanide incubation: pooled blood with a
HbA1C concentration 5.0% was poured into six tubes; a
Sodium Cyanide solution was added to these test
samples at increasing ratios up to 10 mmol/L Sodium
Cyanide concentrations). The samples were incubated
for 2 h at 37 ºC to achieve in vitro carbamylation of the
hemoglobin before analysis and then analyzed.
The effect of HbF concentration was investigated
adding blood cord to samples before the analysis.
Comparison of HA-8180 against our current routine
method (Menarini/ARKRAY ADAMS A1c HA-8160,
Menarini Diagnostics, Firenze, Italy) was carried out
applying Passing-Bablok Linear Regression [10] and
Bland-Altman Analysis [11], using samples from 170
diabetic and nondiabetic subjects being routinely
monitored (range 4.7 % - 14.3 % HbA1C).
3. Results
Precision within Laboratory study is summarized in
the Table 1; the results of the different analytical
characteristics are summarized in the Table 2.
4. Discussion
HbA1C is a high volume request test in the Clinical
Laboratory and therefore efficiency is required. Fully
automation, high turn around time for results,
robustness of the instrument and low costs are
prerequisites. Efforts of manufactures contributed to
Table 1 Precision study. Total precision was investigated using CLSI EP-5 guideline with commercial controls HbA1C 6.0% and
11.5%.
HbA1C 6.0%
Within run
Between run
Between day
Total
CV %
0.52
0
0.18
0.57
HbA1C 11.5%
Within run
Between run
Between day
Total
CV %
0.24
0.14
0.1
0.29
Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination
459
Table 2 Summary of the results. The procedures are described in Materials and Methods section; fresh whole blood samples from
non diabetic and poorly controlled diabetic adults were employed.
Carry over (HbA1C 12.4% and 4.9%)
Precision within run
Linearity
0%
Mean 4.2% CV 0.4%; Mean 10.5% CV 0%
y = 1.0 x - 0.1, r = 0.999; analytical range 5.0% - 12.5%
Total Hb concentration range 14 - 6 mmol/L
NO effect HbA1C 5.4%
Labile A1C fraction 4.5%
NO effect HbA1C 5.4%
Carbamylated Hb 5.3%
NO effect HbA1C 5.4%
HbF 6%
NO effect HbA1C 5.4%
Correlation with ADAMS HA-8160 results
y = 1.07 x - 0.11 (95 % CI intercept -0.2- 0.01; 95% CI slope 1.05-1.09;
P < 0.01); r = 0.997; mean difference -0.27
improvements of the analyzers creating faster systems.
On the other hand, therapeutic strategies rely on
reproducible and unbiased methods. These analytical
and clinical requirements become even more important
now, when HbA1C is on the threshold of becoming
applicable for screening and diagnosis of diabetes [5].
A high level of reproducibility in glycohemoglobin
measurement is a fundamentally important
requirement in the provision of laboratory support for
the process of diabetes monitoring. Changes in results
obtained between patient visits to the physician must
reflect the pathology of the disease and its response to
treatment rather than analytical uncertainty. In this
respect, biological variation generally has dictated the
desirable targets for analytical performance of
laboratory methods [12].
However, it has been shown that the situation in
persons with diabetes is more complex, being affected
by both clinical control and sampling time interval, and
a practical working CV for analytical reproducibility in
long-term monitoring of 2.1% has been proposed [13].
The analyzer achieved this stringent target in our
evaluations; between-run CVs of 0.2%-0.4%,
within-run CVs of 0.2%, and total CVs of 0.4%-0.7%
the evaluation revealed excellent reproducibility, far
below the most stringent requirements of 2% [14].
The results were reliable within a wide range of total
Hb values; this feature shows the robustness of the
system, so the patient could be controlled with
confidence in different clinical situations.
Labile A1C, the concentration of which varies with
blood glucose concentrations at the time of blood
collection, is an intermediate in the production of the
corresponding stable moiety. The isoelectric point of
the labile fraction closely approximates that of its
stable counterpart, which leads to little or no separation
of the two, giving falsely increased results in many
methods that rely on charge separation unless sample
pretreatment steps are taken to remove the labile
fraction [15, 16].
Technological advances have enabled modern
analyzers with high resolution capabilities to handle
these potentially interfering components, thus
obviating the need for pre- or on-column treatment to
destroy this fraction before analysis. The presence of
uremia in patients whose long-term glycemic status is
being assessed is not an uncommon finding in those
whose renal function has deteriorated; it can provide
yet another potential interference with some, although
not all, glycohemoglobin methods, because in most of
the commercially available systems carbamylated
hemoglobin elutes in the HbA1C labile fraction [17-19].
We have confirmed that the labile fraction is
separated on the Menarini 8180 system, irrespective of
its concentration and molecule Schiff base or
carbamylated hemoglobin so the HbA1C remains
reliable.
Nevertheless the presence of Hb variants or δβ
thalassemia (with HbF > 6 %), can affect the results.
5. Conclusion
The drastic reduction in the time of analysis does not
460
Evaluation of ADAMS ™ A1C Menarini HA-8180 HPLC Analyzer for HbA1C Determination
impair the overall quality of the results.
The results were accurate and linear in the clinically
significant analytical range.
The values were independent of the total
concentration of Hb and the presence of Hb chemically
modified Hb eluting in the labile fraction.
Given the short time of the analysis this is a suitable
system for the control of diabetic patients in
laboratories with high workflow, mainly in areas with
low incidence of hemoglobinopathies.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
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, New England
Journal of Medicine 329 (1993) 977-986.
UK Prospective Diabetes Study (UKPDS) Group,
Intensive blood glucose control with sulphonylureas or
insulin compared with conventional treatment and risk of
complications in patients with type 2 diabetes (UKPDS
33), Lancet 352 (1998) 837-853.
Consensus Committee, Consensus statement on the
worldwide standardization of the haemoglobin A1C
measurement: The American Diabetes Association,
European Association for the Study of Diabetes,
International Federation of Clinical Chemistry and
Laboratory Medicine, and the International Diabetes
Federation, Diabetes Care 30 (2007) 2399-2400.
C. Weykamp, W.G. John, A. Mosca, T. Hoshino, R. Little,
J.O. Jeppsson et al., The IFCC reference measurement
system for HbA1C: A 6-year progress report, Clinical
Chemistry 54 (2008) 240-248.
The International Expert Comitee, International Expert
Comitee Report on the role of the A1C assay in the
diagnosis of diabetes, Diabetes Care 32 (2009) 1327-1334.
C. Weykamp, W.G. John, A. Mosca, A review of the
challenge in measuring hemoglobin A1C, Journal of
Diabetes Science and Technology 3 (2009) 439-445.
P.M.G.
Broughton,
A.H.
Gowanlock,
J.J.
McCormack, D.W. Neill, A revised scheme for the
evaluation of automatic instruments for use in clinical
chemistry, Annals of Clinical Biochemistry 11 (1974)
207-214.
NCCLS (USA) National Committee for Clinical
Laboratory Standards, Evaluation Protocols, 1994, ISBN
1-56238-217-9.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
Evaluation of the linearity of quantitative measurement
procedures: A statistical approach; approved guideline.
NCCLS document EP5-A2, Wayne, PA, 2003.
H. Passing, W. Bablok, A new biometrical procedure for
testing the equality of measurements from two different
analytical methods, Journal of Clinical Chemistry and
Biochemistry 21 (1983) 709-720.
J.M. Bland, D.G. Altman, Statistical method for assessing
agreement between two methods of clinical
measurements, Lancet 1(1986) 307-310.
C. Ricós, N. Iglesias, J.V. Garcia-Lario, M. Simón, F. Cava,
A. Hernández, et al., Within subject biological variation in
disease: collated data and clinical consequences, Annals in
Clinical Biochemistry 44 (2007) 343-352.
F. Braga, A. Dolci, M. Montagnana, F. Pagani, R. Paleari,
G.C. Guidi, A. Mosca, M. Panteghini, Revaluation of
Biological variability of glycated hemoglobin using an
accurately designed protocol and an assay traceable to the
IFCC reference system, Clinica Chimica Acta 412 (2011)
412-1416.
I. Goodall, P.G. Colman, H.G. Schneider, M. McLean, G.
Barker, Desirable performance standards for A1C analysis
– precision, accuracy and standardisation, Clinical
Chemistry and Laboratory Medicine 45 (2007)
1083-1097.
D.M. Nathan, B.J. Avezzano, J.C. Palmer, A rapid method
for eliminating labile glycosylated hemoglobin, Clinical
Chemistry 23 (1982) 512-513.
P. Bannon, J.G. Jolly, F. Lessard, R. Lepage, Comparison
of three methods for the elimination of the labile fraction
of HbA1C, Clinical Biochemistry 18 (1985) 114-117.
D. Chevenne, M. Fonfrede, R. Ducrocq, M. Chauffert, F.
Trivin, Uremia and HbA1C measured by high-performance
liquid chromatography [Letter], Diabetes Care 21 (1998)
463-464.
C.W. Weykamp, T.J. Penders, C.W.M. Siebelder, F.J.
Muskiet, W. van der Slik, Interference of carbamylated
and acetylated hemoglobins in assays of glycohemoglobin
by HPLC, electrophoresis, affinity chromatography and
enzyme immunoassay, Clinical Chemistry 39 (1993)
138-142.
J. Szymezak, E. Lavalard, M. Martin, N. Leroy, P. Gillery,
Carbamylated hemoglobin remains a critical issue in
HbA1C measurements, Clinical Chemistry and Laboratory
Medicine 47 (2009) 612-613.