糖化血色素检验的品管课件

1、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 94-05-06Outlines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,

2、 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

3、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 HbA1cHemoglobin nomenclatureHbA: The major form of hemoglobin, a native, unmodified tetramer consisting of tw

4、o -chains and two 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;

5、 also called the “fast” hemoglobins.HbA1c: A specific GHb that is an adduct of glucose attached to the -chain terminal valine residueTotal GHb: A term used to describe all GHb species as measured by affinity chromatographic methods.Hemoglobin composition of non-diabetic adultA1C & mean blood glucose

6、 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)HbA1c(%) Approximate Mean Plasma Glucose*Interpretation mg/dL mmol/l4 65 3.5Non-DiabeticRan

7、ge 5 100 5.56 135 7.57 170 9.5ADA Target 8 205 11.5Action Suggested 9 240 13.510 275 15.511 310 17.512 345 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) a

8、nd 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 di

9、verge considerably, as do the accompanying clinical decision limits.HbA1c as a treatment targetThe 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 comp

10、arability 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 measurem

11、ent 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) Electrosp

12、ray mass spectrometry Cation exchange chromatographyTrivelli 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 defin

13、ed as the adduct of glucose and the N-terminal amino acid valine of the -chain of haemoglobin A0. This glycation process (old term glycosylation) is a non-enzymatic reaction. Ion exchange HPLCIon exchange high-performance liquid chromatography (HPLC) systems were the first automated systems totally

14、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 chrom

15、atography method by the Japanese Society of Clinical Chemistry (JSCC).Ion exchange HPLCAffinity chromatographyThe 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 mea

16、sures not only the specific glycation on the -N-terminal, but also on the -N-terminal and -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.

17、Boronate affinity HPLCAffinity HPLCImmune turbidimetric assaysImmune turbidimetric assays were developed, with the same glycation at the -terminal of haemoglobin as the antigenic property.Although most immune turbidimetric assays were more specific in nature, the lack of primary or secondary referen

18、ce materials meant that they had to be calibrated back to unspecific cation exchange HPLC systems.Chemical Reaction of DCA 2000TMFactors that interfere with HbA1c test results (I)Hemoglobin variants and derivatives: Genetic variants (e.g. HbS trait, HbC trait) and chemically modified derivatives of

19、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 C

20、hemistry 1999; 45: 438-440.Hb variants interfere with A1c valuesFactors 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

21、 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 result

22、s, 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

23、interfere with some assay methods, falsely increasing results.Factors that interfere with A1C testJ Clin Pathol 2004;57:346349.Comparative aspects of A1c assaysAffinity HPLCIon exchange HPLCImmunoassayAbnormal Hb variantsNo interferenceNegative or positive interferenceLittle but variableSalicylate a

24、dduct or uremic adductNo interferenceVariable interference depending on HPLCNo interferencePercision %CV* 2% CV 2.5% CV*Coefficient of variation (CV).Summary of GHb methods in TaiwanMethodNo. of Labs2000 / 2001 / 2004Ion exchange chromatography Bio-Rad HPLC/LPLC TOSOH HPLC 15 / 14 / 39 33 / 35 / 67A

25、ffinity chromatography Abbott Imx Primus Affinity HPLC 26 / 24 / 0 13 / 19 / 29Immunoassay Bayer DCA 2000 Fujirebio latex agglutination Roche Cobas Integra Beckmen system WAKO A1c reagent 7 / 2 / 0 8 / 6 / 20 5 / 4 / 15 4 / 5 / 1 1 / 0 / 0 施木青 Total: 112 109 171叁 Standardization of HbA1cThe three ma

26、jor 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

27、 results.Standardisation of different assaysHarmonisation-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 introd

28、uced, 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. Stand

29、ardisation of HbA1c assaysThe 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-

30、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, wh

31、ich 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

32、 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 ba

33、ckup 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 an

34、d immunoassay), provide excellent analytical performance in these laboratories and are calibrated to the CPRL method.CAP survey results of GHbCAP 2004 GHb surveyCAP survey of NGSP-certified NGSP-certified methods & Labs NGSP certification categoriesCertification Type # samples comparedPrecision Crit

35、eriaBias Criteria Monitoring Protocol Cost(basic cert.)Manufacturer 404% 95% CI within 1% GHB no$3500 Level I Lab 403% 95% CI within 0.75% GHB 10 samples quarterly $5000 Level II Lab 404%95% CI within 1% GHB no$3500 There were 41 NGSP Certified Laboratories worldwide and 4 in Asia in 2004-2005JDS/JS

36、CC standardisation schemeIn 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 method

37、s 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 2Swedish standardisation schemeThe Swedish standardisation scheme uses the MonoS HPLC m

38、ethod 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 u

39、sed 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 speci

40、fically measures the analyte in human samples; A global network of reference laboratories to guarantee that the reference method is performedElectrospray mass spectrometry (ES-MS)Firstly, haemoglobin from washed and lysed erythrocytes is cleaved into peptides by the proteolytic enzyme Endoprotease G

41、lu-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 -chain are separated from the crude peptide mixture by reversed phase HPLC.Thirdly, the glycated and non-glycated hexapeptides are quantifi

42、ed 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

43、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 i

44、n subjects of non-diabetic was 2.843.81% (n=120). Relationship between IFCC & DCMFive 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)

45、 = 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) + 0.884Relationship between IFCC & DCMA1C & mean glycaemia The higher specificity of the IFCC reference method has also influenced the relati

46、onship 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,

47、 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 tra

48、nslated into the new numbering system, yielding new reference ranges and new treatment goals. Clinical decision limits for HbA1cIFCC & 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 perc

49、ent of laboratories passed the survey based on the peer group mean +/- 3SD evaluation criteriaEighty-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 MethodsBioRadD-10BioRadVariant