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HbA1c A Global Standardized Laboratory Test

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1 HbA1c A Global Standardized Laboratory Test
中國醫藥大學附設醫院 檢驗醫學部 施木青

2 為什麼要談這個主題:HbA1c Hot topic : International ; Medical ; public health
Standardization : How to do it ? M.T. responsibility ? Traceability : NGSP or IFCC ?

3 內 容 大 剛 為什麼要重視糖尿病? 什麼是糖化血紅素? 糖化血紅素的應用? 檢測糖化血紅素的方法有幾種? 國際標準化是怎麼做到的?
內 容 大 剛 為什麼要重視糖尿病? 什麼是糖化血紅素? 糖化血紅素的應用? 檢測糖化血紅素的方法有幾種? 國際標準化是怎麼做到的? NGSP 標準亦或IFCC標準?

4 為什麼要重視糖尿病? 糖尿病 醫療面向 最常導致視網 膜病變及失明 最常導致 非外傷性截肢 心血管致死率 是一般人的 2-4倍
糖尿病的臨床影響 糖尿病的併發症包括: 心血管疾病(CVD), 腎病變(Nephropathy), 視網膜病變(Retinopathy)及神經病變(neuropathy), 其中糖尿病腎病變是造成末期腎病(ESRD)最主要的原因。 Slide source: 最常導致 末期腎臟 病及洗腎 治療

5 79 Ref.NHRI

6 台灣現況(健保局資料) 全民中有4.0 % 被歸類為糖尿病,但醫療支出佔11.5 % 每位糖尿病患的平均給付金額為其他疾病的3.3倍
調降1/3的嚴重個案比例,可減少20 %的總支出

7 糖尿病併發症可以預防嗎? DCCT : Diabetes Control and Complication Trial
Functional Group of the American Diabetes Association (ADA)


9 HbA1c in Intensive vs Conventional Treatment (DCCT study)
11 n: 1441 A1C% 10 Mean glucose : 23155 mg/dL 9.0 9 Quantitative HbA1c (%) 8 7.0 7 15530 mg/dL 6 5 1 2 3 4 5 6 7 8 9 1984 Study Time in Years

10 Results of DCCT Report (DCCT Report in NEJM 329:977-86, 1993)
Retinopathy 發生率減少 76% Nephropathy發生率減少 56% Neuropathy 發生率減少 60% PS. Type I and Type II DM 有相同結果。

11 DCCT HbA1c levels and the risk of complications in type 1 diabetes
Improved glycemic control following intensive diabetes therapy delayed the onset and slowed the progression of diabetic retinopathy, nephropathy and neuropathy in patients with type 1 diabetes. [1] :DCCT. Diabetes 1995;44:968–83.


13 ADA recommendations for Diabetes care quality (2005)
Perform the A1C test at least 2 times per year in stable patients. Achieve A1C < 7.0% as goal of glycemic control. Lower A1C is associated with lower risk of cardiovascular diseases. A1C < 6.0% can be considered in individual patients.

14 什麼是HbA1c? 血糖代謝的指標 糖尿病併發症的危險參數
糖化血紅素是葡萄糖和血紅素長期接觸所形成,HbA1c只是眾多糖化血紅素中(HbA1a , HbA1b ….)的一種。 血糖代謝的指標 糖尿病併發症的危險參數 β-chain N端Valine Glucose 糖化作用也會發生在α鍵的N端和α,β鍵上的lysin上

15 Formation of Glycated Hemoglobin
Hemoglobin + Glucose labile HbA1c (Schiff Base) stable HbA1c (ketoamine) Non-enzymatic reaction Fast Slow, irreversible Amadori rearrangement

16 Hb名詞解釋 HbA or HbA0 最主要的血紅素,由兩個α球蛋白與兩個β球蛋白組成,沒 有任何化學修飾
GHb (Glycated Hb) 葡萄糖以非催化性的連接上血紅素,所得的一種酮胺結構的血 紅素 HbA1 以HPLC法或免疫法測得陰電性較強的血紅素,速度較HbA為 快,包括HbA1a, HbA1b, and HbA1c等, 亦稱“快速血紅素 HbA1c (A1c) 葡萄糖接上血紅素β鏈之Valine Total GHb 利用親和力法測得的所有醣化血色素。

17 HbA1c的定位: 1.全世界以 DCCT Lab 為primary reference lab。
2.Cation - exchange HPLC 法是 reference method。 3.HbA1c值與Glucose值的相關性已建立且穩定。 4.Affinity 法和 Immunoassay 和 Ion-exchange HPLC 相關性己建立。 5.Total Gly-Hb 值必須轉成 HbA1c。

18 NIH Guidelines for A1C Test
1.Specific measurement of HbA1c. 2.Narrow non-diabetic range (4.0%-6.0%). 3.Highly precise measurement (<2% CV) 4.Removal of interferences. 5.Hemoglobin variant detected when present. 6.NGSP certified method and calibrator. 7.Within ± 0.5% HbA1c bias from the reference value.

19 糖化血紅素與血糖的關係 形成速率與血中葡萄糖濃度成正比。 良好的血糖標記,可反應過去2-3個月來血糖平均值。
較客觀、方便、不受飯後血糖升高的影響。 RBC第0-30天的前段生命期反應50%的糖化血 色素值。 美國DCCT已建立各種解讀A1C的臨床數據,可供引用。

20 We need translation to eAG

21 Correlation of eAG With HbA1c
AG (mg/dl) = 28.7 x HbA1c – 46.7 R2 = 0.84 P < eAG (mg/dl) The overall results of the ADAG study are shown here. There was a highly significant correlation between average glucose and A1C. The regression equation is shown here. The R-squared was 0.84, meaning that 84% of the variance in one measure is explained by the variance of the other, with the R or correlation coefficient being 0.91. HbA1c (%) Diabetes Care 2008;31:1-6 ADAG:A1C derived average glucose eAG : Estimated Average Glucose

22 How does HbA1c relate to (eAG)?
1. eAg is a mean value of 7-8 points blood glucose determination in 24 hrs. 2. The relationship between eAG and HbA1c was: eAG(mg/dl)= (28.7*HbA1c)-46.7, r2=0.84 3. Only post-lunch and bedtime single glucose value showed relationships to HbA1c 4. Single fasting glucose value was underestimated the level of HbA1c

23 ADAG Study: “Translation” of HbA1c into eAG
Blood glucose HbA1c (%) (mg/dl) (mmol/l) We can use the ADAG results to “translate” A1C into average glucose, with values shown here. Diabetes Care 2008;31:1-6

24 Consensus Statement IFCC, EASD, IDF, ADA ,Sept 2007)
HbA1c assay to be standardized worldwide using the new IFCC standard and expressed as: % as currently used (DCCT values) mmol HbA1c/mol HbA (IFCC units ) eAG in mmol/l or mg/dL This paved the way for reporting both HbA1c and eAG on lab reports There is a complicated “back-story” to all this that involves the potential a few years ago that the DCCT-standardized A1C as we know it might have gone away. This actually did not come to pass, in part because ADA and other international diabetes groups forged an agreement with the International Federation of Clinical Chemists in 2007 to: Standardized HbA1c assays worldwide using a new IFCC standard and expressed as: % as currently used (DCCT values) IFCC units in mmol HBA1c/mol HbA eAG in mmol/l or mg/dL (if ADAG study meets its data acceptability goals…which it did) This paved the way for reporting both HbA1c (in %) and eAG (in mg/dl or mmol/l, depending on country) on lab reports Diabetes Care and Diabetologia, 2007

25 Diagnostic Criteria for Diabetes
臨床應用 Diagnostic Criteria for Diabetes PG > 200 mg/dl or FPG > 126 mg/dl 2-h PG (OGTT,75 gm) > 200 mg/dl

26 Does HbA1c cam be used for Diabetes Screening
HbA1c cutoff Sensitivity Specificity 5.6% * % % 6.1% * % % *(1SD and 2SD above normal mean) Normal population : 5.1 +/- 0.5 % Diabetes Care 23:187-91, 2000

27 ADA Endorses HbA1c for Diabetes Diagnosis (2010 Feb.)
Pre-diabetes : 5.7%–6.4%

28 監測 1)自我血糖監測 (self-monitoring of blood glucose, SMBG)理想情形是維持飯前血糖在70-140,飯後≦140 mg/dl。但應依 病童 的年紀和處理低血糖的能力酌情調整。 2)醣化血紅素 (HbA1c) 反映最近2個月來血糖的平均值。目標值: 7.0 或6.5 % 3)尿酮:必須為陰性。 4)血脂:total Cholesterol, Triglyceride, LDL-Chol要保持 正常。 5)微尿白蛋白(microalbuminuria):發病5-6年後,必須 開始測定,期能早期檢出腎病變。 6) AGEs (Advanced Glycosylation End Products )最終糖化蛋白(AGEs)是引起糖尿病併發症的元兇

29 ADA recommendations for Diabetes care quality (2005)
Perform the A1C test at least 2 times per year in stable patients. Achieve A1C < 7.0% as goal of glycemic control. Lower A1C is associated with lower risk of cardiovascular diseases. A1C < 6.0% can be considered in individual patients.

30 Monitoring Diabetes Self Monitoring Blood glucose test (SMBG)
Hemoglobin A1c (HbA1c) testing AGE ?

31 2004年 流 行 病 學 調 查 (I) 總人數;1347 平均值;8.0% ≦7.0%,449個(33.3%)
≦6.5%,235個(17.4%) ≧10.0%,145個(10.8%), ≧9.5%,226個(16.8%), ≧8.0%,572個(42.5%),

32 流 行 病 學 調 查 (II)

33 ADA recommendation: Real-time results. Better outcomes.
Use A1C point-of-care testing for timely decisions on therapy changes . Feedback from A1C number real time may result in a 1% reduction in their A1C.

34 測定糖化血紅素的方法 陽離子交換層析法(Cation exchange HPLC)
親和力層析法(Boronate affinity HPLC) 免疫比濁法(Immuno-turbidity assay) 直接酵素法 (Direct enzymatic method) 毛細管電泳法(Capillary electrophoresis) Interference by Hb variants was method dependent

35 Cation Exchange HPLC No.3 No.2 No.1 使用階段梯度方式分離
A1a, A1b, F L-A1c, s-A1c A0


37 Hb J-meinung Non-diabetic Hb E Hb Phnom-Penh

38 Boronate affinity 將血紅素分成”糖化”與”非糖化”兩部份 Cis-diol Hb Hb CH NH CO Resin NH
2 CO Resin NH B OH HCOH Cis-diol HOCH HOCH CH OH 2 CH 2 NH- Hb CO HCOH OH O CH B O CH Resin NH CH2OH 將血紅素分成”糖化”與”非糖化”兩部份



41 Direct Enzymatic HbA1c Assay principle
rFVO:recombinant fructosyl valine oxidase (FVO) enzyme POD:horseradish peroxidase

42 Capillary electrophoresis CVs < 1,5% over 8 capillaries
New method for HbA1c test HbA1c Next Generation High resolution Capillary electrophoresis CVs < 1,5% over 8 capillaries NGSP-IFCC Certified

43 Complete separation of Hb Kaoshiung

44 Immuno-turbidimetric Method
檢測方法的特性比較 分析原理 Ion Exchange Method Affinity Method Immuno-turbidimetric Method Enzymatic Method Capillary – EP Method 分析標的 HbA1c Total GHb 測試原理 離子交換 HPLC 分子親和力HPLC 免疫抑制 比濁法 酵素法 電位差電泳 再現性(%) 1.5~3.8% 2~4% 5~10% 與國際標準化的方法比較 NGSP驗證 NGSP & IFCC 驗證 變異型血紅素的干擾 +++ +/- ++ G6PD 的干擾 降低A1c%值 降低GHb值 降低A1c% CAP-PT家數 >800 19 >1200 NA


46 1993

47 HbA1c global standardization
Ref. m. : cation ex. HPLC Ref. std. : human blood stored in CPRL Traceability to CPRL or DCCT value

48 NGSP證書 National Glycohemoglobin Standardization

49 NGSP國際標準化後的成效

50 歐、美、日的標準一樣嗎? Patient data graph

51 (International Federation of Clinical Chemistry)
IFCC Standardization (International Federation of Clinical Chemistry) The IFCC WG was formed in 1994 with its aim to establish a Global Reference System for HbA1c. Specific aims were: (a) To define the heterogenous HbA1C (b) To prepare pure HbA0 and HbA1C (c) To develop a Reference Method (d) To establish a Reference Laboratory Network (e) To prepare secondary reference calibrators and controls

52 Difference between IFCC and NGSP(2001)
NGSP %HbA1c IFCC %HbA1c Difference %HbA1c 4 2.1 1.9 5 3.2 1.8 6 4.3 1.7 7 5.4 1.6 8 6.4 9 7.5 1.5 10 8.6 1.4 11 9.7 1.3 12 10.7

53 The relationship between the IFCC and NGSP

54 The IFCC Reference System
Definition of the analyte: glycated and non- glycated N-terminal peptides of the beta-chain Primary Reference Material:mixtures of pure HbA1c and pure HbA0 Reference Method: HPLC and then Mass spectrometry / Capillary electrophoresis Traceability :calibrators and/or control materials must be "traceable" to the IFCC Reference Method

55 HbA1c reporting的共識宣告 The IFCC results are accuracy-based
NGSP results can be directly related to diabetes care goals. Although the IFCC/NGSP correlation is excellent, the absolute numbers are different. The IFCC number scale can not be related to clinical outcomes without conversion to DCCT (NGSP) -aligned numbers. HbA1c result can be expressed in IFCC units, derived NGSP units, and eAG.

56 國 際 現 況 與 我 們 的 選 擇 國際現況雨

57 Interference of Test results
by Hb variants & G-6-P.D ?

58 Relative Electrophoretic Mobility on Agarose Gel
( Fig. Locolization of seven Hb Variants on agarose gel electrophoresis ) H Bart’s J(?) , JMeinung A0,Kaohsiung F GTaichung A2,,E CS 在agarose gel electrophoresis上, 定位點以 HbA0 為“1”,每一pattern 與HbA0的相對移動距離分別如下: Hb-CS: 0.40 Hb-A2 : 0.48 Hb-E : 0.50 Hb-G Taichung: 0.80 Hb-F : 0.92 Hb-Kaohsiung: 1.12 Hb-J(),J Meinung: 1.28 Hb-Bart’s:1.36 Hb-H:常常偵測不到,但在HbA2~Hb-F間會有擴散的現象. 58


60 NGSP website

61 如何面對其他變異型血紅素? 全世界已發表的變異型血紅素,超過1000種。
台灣常見的變異型有Hb-GTaichung、Hb-JMeinung、 Hb-E、 Hb-Manitoba、Hb-H……等。發生率(0.04~0.76%) Ion-exchange HPLC方法受到的干擾最大,但仔細研究 HPLC圖形,很容易可發現變異型血紅素的存在。 選擇干擾最小的方法是上策。 建議採用個人的參考值作為比對基礎。 建立糖化蛋白 (Fructosamine ) 的檢驗方法。

62 HbA1c in G-6P.D deficiency blood
HbA1c(%) G-6P.D IU/gmHb Sex M. M. M. M. F. F. F. F. Normal range:4.0~5.7% HbA1c in G-6P.D deficiency blood

63 Distribution of HbA1c in G-6PD def. group
Group n Mean Min Max. Normal G-6PD def

64 QA Activity in HbA1c test in Taiwan
1.使用NGSP/IFCC認證過的檢測方法 2.Tracible to DCCT /IFCC value 的校正 3. CV< 3% and Bias < 0.5 A1C% 4. 鑑別變異型Hb/G-6-P.D.等干擾因素

65 Conclusion 1. Reference range of HbA1c is 4.0 ~6.0 %
2.HbA1c 6.5% is a New Diagnostic Criteria for Diabetes 3.Achieve A1C < 7.0% or < 6.5% as goal of good glycemic control. 4.“Translation” of HbA1c into eAG in laboratory report. 5.Real time report of HbA1c have good outcome 6.Traceability to NGSP unit(%) in Taiwan

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