Symposium of Quality Control of Glycosylated Hemoglobin by uif20236

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									Symposium of Quality Control of
   Glycosylated Hemoglobin
    糖化血色素檢驗的品管
       奇美醫院 內分泌科
          林文森
            94-05-06
               Outlines
壹   Introduction of HbA1c
貳   Methods of HbA1c measurement
叁   Standardization of HbA1c
肆   HbA1c survey in Taiwan
      壹 Introduction of HbA1c
 Diabetes is the most common cause of blindness in young
  adults, kidney failure, and nontraumatic limb amputation.
 Diabetes Control and Complications Trial (DCCT) showed
  that the risk for development and progression of the
  chronic complications is related to the degree of glycemic
  control, as measured by glycated hemoglobin (GHb).
 Haemoglobin A1c (HbA1c) or glycohaemoglobin is one of
  the most important parameters in the management of
  patients with diabetes mellitus, but to date there is no
  international standard for determining HbA1c.
Nonenzymatic glycosylation of HbA1c
       Hemoglobin nomenclature
 HbA: The major form of hemoglobin, a native, unmodified tetramer
  consisting of two α-chains and two β–chains.
 GHb A: general term for glucose bound nonenzymatically to
  hemoglobin and with a ketoamine structure.
 HbA1: GHb species that are more negatively charged forms of HbA
  detected by cation-exchange chromatographic and electrophoretic
  methods, which include HbA1a, HbA1b, and HbA1c; also called the
  “fast” hemoglobins.
 HbA1c: A specific GHb that is an adduct of glucose attached to the
  β-chain terminal valine residue
 Total GHb: A term used to describe all GHb species as measured by
  affinity chromatographic methods.
Hemoglobin composition of non-diabetic adult
    A1C & mean blood glucose
 MBG (mg/dl) = 33.3 x (%A1c) – 86
                       (NEJM 1984; 310:341-6)
 MBG (mg/dl) = 30.0 x (%A1c) – 57
                       (Clin Chem 1994; 40: 1317-21)
 MBG (mg/dl) = 30.9 x (%A1c) – 61
                        (Comtemp Int Med 1997; 9: 27-32)
A1C & mean plasma glucose (DCCT)
                     Approximate
                  Mean Plasma Glucose*
HbA1c(%)           mg/dL          mmol/l          Interpretation
    4                65             3.5
                                                  Non-Diabetic
    5               100             5.5
                                                    Range
    6               135             7.5
    7               170             9.5            ADA Target
    8               205             11.5
    9               240             13.5
    10              275             15.5         Action Suggested
    11              310             17.5
    12              345             19.5
* Mean blood glucose results are 10-15% lower.
        Introduction of HbA1c
 Most of the routine HbA1c assays are standardised against
  one of the local standardisation schemes like the NGSP
  (USA) and other schemes (Japan, Sweden).
 Besides the universal calibrator, it was found that materials
  prepared for use as calibrators, quality control materials,
  and proficiency-testing samples, may cause them to yield
  results that differ appreciably, i.e., matrix effects.
 Still, results of HbA1c tests diverge considerably, as do the
  accompanying clinical decision limits.
   HbA1c as a treatment target
 The amount of HbA1c reflects the mean glucose
  concentration over the previous two to three months and is
  as such an independent parameter of carbohydrate
  metabolism.
 Reproducibility, that is long-term comparability of the
  values within one individual patient, is therefore an
  absolute necessity.
 After publication of DCCT and UKPDS, HbA1c was
  introduced as a risk parameter for monitoring the potential
  development of late diabetic complications.
Diabetic complications in UKPDS
貳 Methods of HbA1c measurement
 At present, more than 20 different HbA1c methods are in
  use, based on three main different assay principles
  (1) Cation exchange chromatography-
       charge separation
  (2) Affinity chromatography-
       specific binding of cis-diol to boronic acid
  (3) Immunoassay-
       turbidimetry, enzyme or latex
  (4) Electrospray mass spectrometry
Cation exchange chromatography
 Trivelli et al used the resin Bio-Rex 70 (BioRad
  Laboratories, Hercules, Calif., USA) to separate HbA1c
  from HbA0 by cation exchange chromatography, the first
  commercial methods of measuring HbA1c.
 HbA1c, a peak in the chromatogram, was later defined as
  the adduct of glucose and the N-terminal amino acid valine
  of the β-chain of haemoglobin A0. This glycation process
  (old term glycosylation) is a non-enzymatic reaction.
          Ion exchange HPLC
 Ion exchange high-performance liquid chromatography
  (HPLC) systems were the first automated systems totally
  dedicated to HbA1c determination.
 The development of far more specific resins (less
  interfering substances under the HbA1c peak) and better
  separation of HbA1c and HbA0.
 The MonoS resin (strong methyl sulphonate cation
  exchanger on monobeads) in Sweden and the KO500
  high-resolution ion exchange chromatography method by
  the Japanese Society of Clinical Chemistry (JSCC).
Ion exchange HPLC
      Affinity chromatography
 The principle of specific and reversible binding of the
  1,2-cis diol groups of glycated haemoglobin to
  immobilised boronate (affinity separation).
 As affinity chromatography in principle measures not only
  the specific glycation on the β-N-terminal, but also on the
  α-N-terminal and ε-residues of the total haemoglobin
  molecule, the end-result is total glycated haemoglobin or
  glycohaemoglobin.
 Nevertheless, results of glycohaemoglobin are very
  frequently converted to and presented as „HbA1c‟.
Boronate affinity HPLC
Affinity HPLC
    Immune turbidimetric assays
 Immune turbidimetric assays were developed, with the
  same glycation at the β-terminal of haemoglobin as the
  antigenic property.
 Although most immune turbidimetric assays were more
  specific in nature, the lack of primary or secondary
  reference materials meant that they had to be calibrated
  back to unspecific cation exchange HPLC systems.
Chemical Reaction of DCA 2000TM
         Factors that interfere with
           HbA1c test results (I)
Hemoglobin variants and derivatives:
• Genetic variants (e.g. HbS trait, HbC trait) and chemically
  modified derivatives of hemoglobin (e.g. carbamylated Hb
  in patients with renal failure, acetylated Hb in patients
  taking large amounts of aspirin) can affect the accuracy of
  GHB measurements.
• The effects vary depending on the specific Hb variant or
  derivative and the specific GHB method.
HbA1c measured in uremia




            Clinical Chemistry 1999; 45: 438-440.
Hb variants interfere with A1c values
         Factors that interfere with
          HbA1c test results (II)
 Shortened erythrocyte survival:
• Any condition that shortens erythrocyte survival or
  decreases mean erythrocyte age (e.g., recovery from acute
  blood loss, hemolytic anemia) will falsely lower GHB test
  results regardless of the assay method used.
• Alternative forms of testing such as glycated serum protein
  (fructosamine) should be considered for these patients.
         Factors that interfere with
          HbA1c test results (III)
Other factors:
• Vitamins C and E are reported to falsely lower test results,
  possibly by inhibiting glycation of hemoglobin; vitamin C
  may increase values with some assays.
• Iron-deficiency anemia is reported to increase test
  results. Hypertriglyceridemia, hyperbilirubinemia, uremia,
  chronic alcoholism, chronic ingestion of salicylates, and
  opiate addiction are reported to interfere with some assay
  methods, falsely increasing results.
Factors that interfere with A1C test




                 J Clin Pathol 2004;57:346–349.
Comparative aspects of A1c assays

                    Affinity HPLC     Ion exchange HPLC      Immunoassay

Abnormal Hb         No interference   Negative or positive   Little but
variants                              interference           variable

Salicylate adduct No interference     Variable interference No interference
or uremic adduct                      depending on HPLC
Percision %CV* < 2% CV                < 2% CV                >> 2.5% CV


  *Coefficient of variation (CV).
  Summary of GHb methods in Taiwan
             Method                            No. of Labs
                                         2000 / 2001 / 2004
Ion exchange chromatography
  Bio-Rad HPLC/LPLC                       15 / 14 / 39
  TOSOH HPLC                              33 / 35 / 67
Affinity chromatography
  Abbott Imx                              26 /      24 / 0
  Primus Affinity HPLC                    13 /      19 / 29
Immunoassay
  Bayer DCA 2000                          7    /    2    / 0
  Fujirebio latex agglutination           8    /    6    / 20
  Roche Cobas Integra                     5    /    4    / 15
  Beckmen system                          4    /    5    / 1
  WAKO A1c reagent                        1    /    0    / 0
施木青                               Total: 112       109     171
   叁 Standardization of HbA1c
 The three major HbA1c harmonisation (standardization,
  comparability) schemes include:
  (1) the National Glycohemoglobin Standardisation
      Program (NGSP) in the United States,
  (2) the scheme of the Japanese Diabetes Society (JDS) and
  (3) the MonoS-method (Sweden),
 with considerable divergence existing between HbA1c
  results.
  Standardisation of different assays

 Harmonisation-by-calibration: a single sample, with an
  assigned value, is used to recalibrate all routine assays with
  much less divergence in results than without calibration.
 Harmonisation-by-method comparison: designated
  comparison method (DCM) is introduced, to which all
  other assays are recalibrated by means of extensive method
  comparison and regression analysis.
 Standardisation by introduction of a reference system:
  secondary reference materials are produced, to be used by
  all manufacturers of routine assays to calibrate their own
  methods.
   Standardisation of HbA1c assays

 The standardisation system of the Japanese Diabetes
  Society (JDS) uses a set of calibrators to harmonise results
  in Japan (harmonisation-by-calibration).
 All other known local standardisation schemes (NGSP in
  the US and the Swedish system) are based on the
  harmonisation-by-method comparison.
 The challenge of international standardisation was taken by
  the IFCC Working Group on HbA1c Standardisation, who
  developed a reference system for HbA1c.
     NGSP standardisation scheme

 The NGSP was established in the USA in 1994 after the
  publication of the results of the DCCT study, which
  showed the curve-linear relationship between HbA1c and
  risks for development and/or progression of diabetic
  complications.
 In their final report however, the American Association of
  Clinical Chemistry (AACC) Subcommittee stated that the
  method used in the DCCT study was not suitable as a
  primary reference method and that a purified standard for
  this method could not be prepared.
National Glycohemoglobin Standardisation Program
     NGSP standardisation scheme
 In the NGSP, the anchor is the very unspecific Bio-Rex 70
  ion exchange HPLC in the Central Primary Reference
  Laboratory (CPRL) and the backup PRL.
 A network of Secondary Reference Laboratories (SRL)
  was established to assist manufacturers with calibration to
  the DCCT value, as well as serving as comparison methods
  for NGSP certification.
 These SRLs use routine HbA1c methods of various
  method types (ion-exchange HPLC, affinity HPLC and
  immunoassay), provide excellent analytical performance in
  these laboratories and are calibrated to the CPRL method.
CAP survey results of GHb
CAP 2004 GHb survey
CAP survey of NGSP-certified
NGSP-certified methods & Labs
         45                                          41
                                             38
         40
         35                          30
                             28
         30
Number




         25          20
                                                          20
         20 15
         15                                       11
                                                        9
         10                     4       6       6
                        2
          5    0 1        1       1       2
          0
            1996-97 1997-98 1998-99 1999-00 2000-01 2001-02

               Methods      Labs (US)     Labs (Outside US)
      NGSP certification categories
  Certification   # samples   Precision     Bias        Monitoring    Cost
     Type         compared    Criteria     Criteria      Protocol  (basic cert.)
 Manufacturer        40         4%          95% CI          no         $3500
                                          within ± 1%
                                             GHB

  Level I Lab        40         3%          95% CI      10 samples     $5000
                                            within ±     quarterly
                                          0.75% GHB

  Level II Lab       40         4%          95% CI          no         $3500
                                          within ± 1%
                                             GHB

There were 41 NGSP Certified Laboratories worldwide and 4 in Asia in 2004-2005
  JDS/JSCC standardisation scheme

 In 1995 the JDS developed a first set of national calibrators,
  called JDS Calibrator lot 1, recommended to be used for
  the calibration of all routine HbA1c assays in Japan.
 The calibrator values were consensus values, assigned with
  the HPLC ion exchange chromatography methods of
  TOSOH and Kyoto Daiichi.
 In recent years, the JSCC developed a high-resolution ion
  exchange HPLC method named KO500 and a second set
  of national calibrators called JDS/JSCC Calibrator lot 2.
JDS Lot 1 and Lot 2
    Swedish standardisation scheme

 The Swedish standardisation scheme uses the MonoS
  HPLC method as designated comparison method for the
  harmonisation of HbA1c measurements.
 Split samples of fresh EDTA blood are distributed once a
  month to 40 hospitals using different HPLC methods. Five
  of them are contracted to run the MonoS reference method
  in a national network.
 These laboratories are used for calibration of all hospital
  and point-of-care instruments in Sweden every second year.
  International measurement system

 A clear definition of the analyte based on its
  molecular structure;
 A primary reference material containing the
  analyte in pure form;
 A validated reference method which specifically
  measures the analyte in human samples;
 A global network of reference laboratories to
  guarantee that the reference method is performed
Electrospray mass spectrometry (ES-MS)

 Firstly, haemoglobin from washed and lysed erythrocytes
  is cleaved into peptides by the proteolytic enzyme
  Endoprotease Glu-C, a serine protease which cleaves
  peptide bonds C-terminal at glutamic acid.
 Secondly, the resulting glycated and non-glycated
  N-terminal hexapeptides of the β-chain are separated from
  the crude peptide mixture by reversed phase HPLC.
 Thirdly, the glycated and non-glycated hexapeptides are
  quantified by electrospray mass spectrometry (ES-MS)
  or by capillary electrophoresis with UV detection.
Electrospray mass spectrometry
Electrospray mass spectrometry
       IFCC reference method
 In 2001, International Federation of Clinical Chemistry
  and Laboratory Medicine (IFCC) is supported by a global
  network of HbA1c Reference Laboratories from Europe,
  Japan and the US.
 The high specificity of the reference method results in
  lower values for HbA1c in patient samples since the
  unspecific components as HbA1c not measured by the
  reference method.
 In the Danish Population Study, the reference range of
  HbA1c in subjects of non-diabetic was 2.84–3.81%
  (n=120).
Relationship between IFCC & DCM
 Five method-comparison studies were organised from
  2001 to 2003 between the IFCC Network of HbA1c
  Reference Laboratories and the DCM (designated
  comparison method)
 The Master Equation:
  1. between IFCC and NGSP:
     y (NGSP) = 0.915 x (IFCC) + 2.15
  2. between IFCC and JDS/JSCC:
     y (JDS/JSCC) = 0.927 x (IFCC) + 1.724
  3. between IFCC and the Swedish MonoS:
     y (MonoS) = 0.989 x (IFCC) + 0.884
Relationship between IFCC & DCM
        A1C & mean glycaemia
 The higher specificity of the IFCC reference method has
  also influenced the relationship between HbA1c and mean
  glycaemia in the DCCT study.
 For the DCCT-aligned, the correlation is:
   y (mean glucose, mmol/l) = 1.98 x (% HbA1c) - 4.29.
 For the IFCC method, the correlation is:
   y (mean glucose, mmol/l) = 1.84 x (% HbA1c) - 0.01.
        IFCC reference system
 International organisations (IDF, EASD, ADA, IFCC and
  NGSP) support worldwide introduction of the IFCC
  reference system for HbA1c, reported as “long-term mean
  blood glucose”.
 IFCC HbA1c values: lower uncertainty, better
  comparability, same numbers worldwide.
 All clinical trials and existing targets for diabetes treatment
  must be translated into the new numbering system,
  yielding new reference ranges and new treatment goals.
Clinical decision limits for HbA1c
IFCC & DCM-based A1C
    肆 HbA1c survey in Taiwan
 A total 164 laboratories received 6 whole blood samples in
  2004 (four fresh whole blood and two lymphilized)
 Ninety-eight percent of laboratories passed the survey
  based on the peer group mean +/- 3SD evaluation criteria
 Eighty-seven percent of laboratories used NGSP-certified
  method with inter-laboratory CV ranged from 1.1%-6.1%
 The HbA1c results of 96.5% laboratories using certified
  methods and 63.5% using non-certified methods were
  concordant (+/-0.8%) with the target results determined in
  one NGSP-certified laboratory             (中華民國醫事檢驗學會)
          2004 HbA1c survey in Taiwan
Methods   BioRad   BioRad       Fujirebio   Primus   Roche    Tosoh   Tosoh    Tosoh
          D-10     Variant II   LA          Corp     Cobas    HLC-    HLC-     HLC-
                                                              723G7 S 723 G7 V 723 V


No.       16       18           21          29       13       33       10        23

Median    5.4      5.6          5.3         5.4      5.5      5.6      5.6       5.5

Mean      5.456    5.622        4.876       5.39     5.531    5.57     5.57      5.5

SD        0.1094   0.1215       0.7974      0.0817   0.3449   0.1212   0.08233   0.1732

CV        2.0%     2.2%         16.4%       1.5%     6.2%     2.2%     1.5%      3.2%

                                                                                 謝淑珠
Methods   BioRad   BioRad       Fujirebio   Primus   Roche    Tosoh     Tosoh      Tosoh
          D-10     Variant II   LA          Corp     Cobas    HLC-      HLC-       HLC-
                                                              723G7 S   723 G7 V   723 V

No.       16       18           21          30       13       23        10         20

Median    9.8      9.85         8.6         10.0     9.3      9.5       10.55      9.5

Mean      9.85     9.95         8.424       9.927    9.292    9.535     10.42      9.485

SD        0.1414   0.333        1.424       0.2504   0.7433   0.2838    0.3553     0.1631

CV        1.44%    3.35%        16.90%      2.52%    8.00%    2.47%     3.41%      8.80%



Methods   BioRad   BioRad       Fujirebio   Primus   Roche    Tosoh     Tosoh       Tosoh
          D-10     Variant II   LA          Corp     Cobas    HLC-      HLC-        HLC-
                                                              723G7 S   723 G7 V    723 V


No.       16       18           21          30       14       32        10          22
Median    11.4     11.5         7.9         10.8     9.9      11.2      11.4        11.2

Mean      11.68    11.94        10.64       11.19    11.53    11.51     11.54       11.52
SD        0.2082   0.3166       1.113       0.1954   0.7184   0.1973    0.1265      0.1816

CV        1.78%    2.65%        10.46%      1.75%    6.11%    1.98%     1.10%       1.58%
   2004 HbA1c survey in Taiwan
      BioRad BioRad       Fujirebio   Primus Roche     Tosoh         Tosoh      Tosoh
      D-10   Variant II   LA          Corp   Cobas     HLC-          HLC-       HLC-
                                                       723G7 S       723 G7 V   723 V



可接受 16        18          20          29     14        34            10         22
不滿意 0         0           1           1      0         0             1          1


         醫學中心        區域醫院             區域醫院        地區醫院           診所        檢驗所
                                      (教)

可接受      16          53               11          44             3         40

不滿意      0           1                0           2              0         3
     Primus CLC 385 奇美醫院

   利用 Boronate affinity HPLC 儀器分析出來的結果為
    total glycated hemoglobin, 或利用HbA1c standard 計
    算出HbA1c的值
   廠商用的儀器, 試藥和校正品都要取得NGSP的認證
   實驗室內的精密度 (CV) 品管不超過5%
   儀器最少每月校正一次, 校正液的標定值要能回溯
    到NGSP的標準值
   參加外部品管, 限制 Bias < 1% HbA1c
   加入CAP survey, 結果準確性有所依據
                 Conclusions
     Quality control of glycated hemoglobin (HbA1c):
1.   Type of assay (ion exchange, affinity, immunoassay,
     mass spectrometry).
2.   Reference interval (NGSP, JDS, Sweden, IFCC).
3.   Potential assay interferences (Hb variants, anemia,
     uremia, lipaemia).
4.   Assay imprecision (CV) & Bias.
     Take action according to HbA1c results !
  Better A1C test
 Better diabetes care

								
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