1. The CAPILLARYS Hb A1c kit performances evaluation
High resolution HbA1c separation
and Hemoglobinopathy detection by Capillary Electrophoresis

2. The analyte HbA1c
The new separation technique
Analytical performances
CE CAPILLARYS 2 Flex piercing® HbA1c kit
Two in one: HbA2

3. HbA1c an unique molecule
Erythrocytes are dead cells with no metabolism
Glycation of hemoglobin
non enzymatic binding of sugars on NH2 groups
Spontaneous, cumulative and irreversible process related to red blood cell lifespan (120 days) and glucose concentration
Measurement of HbA1c
retrospective and cumulative index of glycemic balance (6-8 weeks before sample)

4. Evidence Based Medicine : gold standard for monitoring metabolic control in DM
DCCT Study Group. The effect of intensive treatment of diabetes on
the development and progression of long-term complications in insulindependent diabetes mellitus: the Diabetes Control and Complications Trial
N Eng J Med 1993; 329: 977–986
UK Prospective Diabetes Study Group. UKPDS 33: intensive blood glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with Type 2 diabetes
Lancet 1998; 352 : 837–853
Relationship HbA1c /long term complications of DM

5. UK It is estimated that up to one in 20 people in England has diabetes
How common is diabetes?
• The estimated diabetes prevalence for 2010 is 285 million and is expected to affect 438
million people by 2030
• The International Diabetes Federation (IDF) estimates that in 2010 the five countries with
the largest numbers of people with diabetes are India, China, the United States, Russia and
Brazil
UK It is estimated that up to one in 20 people in England has diabetes
Diagnosed
• There are 2.6 million people who have been diagnosed with diabetes in the UK (2009)
• By 2025, there will be more than 4 million people with diabetes in the UK
• It is equivalent to around 400 people every day
Undiagnosed
It is estimated that there are up to half a million more people in the UK who have diabetes but
have not been diagnosed

6. Controversy in Medicine
Controversial molecule

7. 1995 IFCC working group for the standarization of HbA1C
Definition of the analyte to be measured
Glycation by glucose on N-terminal valine residue of βchains
β – N –(1-deoxi)-fructosil Hb (DOF Hb)
Units : mmol / mol HbA0
Preparation of pure HbA0 and HbA1c
Development of a reference method
Establishment of a reference Lab network
Preparation of secondary reference material

8. Approved IFCC Reference Method for the Measurement of HbA1c in Human Blood
Jeppsson, Jan-Olof / Kobold, Uwe / Barr, John / Finke, Andreas / Hoelzel, Wieland / Hoshino, Tadao / Miedema, Kor / Mosca, Andrea / Mauri, Pierluigi / Paroni, Rita / Thienpont, Linda / Umemoto, Masao / Weykamp, Cas
Clin Chem Lab Med 2002; 40: 78-83

11. Clinical Chemistry and Laboratory Medicine 2007; 45(8): 942-4
Diabetes Care 2007; 30 (9):
Diabetologia 2007; 50 (8):2042–3.

14. Capillary Electrophoresis
The CAPILLARYS 2 FLEX-PIERCING instrument uses the principle of capillary electrophoresis in free solution
Charged molecules are separated by their electrophoretic mobility in an alkaline buffer with a specific pH; Separation occurs according to the electrolyte pH and electroosmotic flow

15. Capillarys2 Flex Piercing® HbA1c
Full automation
Samples: whole blood primary capped tubes (minimal volume: 1ml) or specific tubes (minimalvolume:100μL)
Separation of all Hb fractions by capillary electrophoresis with detection at 415 nm
8 capillaries in parallel (8 samples analyzed simultaneously)
Throughput: 40 samples / hour
HbA1c results in IFCC units(mmol/mol) and/or NGSP units (%)
Optional display of calculated eAG (mg/dL)
Phoresis® software with unlimited data storage capacity

16. The CAPILLARYS Hb A1c procedure performed with the CAPILLARYS 2 2 Flex Piercing® instrument has been certified by the National Glycohemoglobin Standardization Program (NGSP)
The CAPILLARYS 2 Flex Piercing® strictly follows the recommendations of the international consensus held on 2010
The calibration is obtained by means of SEBIA calibrators traceable to IFCC measurement procedure giving results in mmol/mol. For the controls and samples analyzed, the software applies the master equation to move unit from IFCC mmol/mol to NGSP %

17. Capillarys2 Flex Piercing® HbA1c Electrophoretic separation profile

18. Normal sample
High HbA1c > 7 % 53 mmol/mol
Abnormal Profile

19. Technical Evaluation Precision Linearity
Correlation with HPLC method (HPLC Menarini/Arkray ADAMS A1c HA-8160, Menarini Diagnostics, Firenze, Italy).
Influence of hemoglobin concentration
Analytical interferences :
-Labile HbA1c
-Carbamylated Hb
-Acetylated Hb
-Bilirubin & Triglycerides
Hb variants (HbS, D, C, E) and HbF

20. Precision
Within run: 3 samples with 3 different HbA1c levels (high, medium and low) run simultaneously on the 8 capillaries
Mean % mmol/mol CV 1.4 %
7.2 % %
10.9 % %
Between run: 8 different samples presenting variable HbA1c levels simultaneously analyzed on the 8 capillaries, repeated 3 times
  Mean 4.6 % mmol/mol CV 1.35 %
4.9% %
5.5 % %
7.2 % %
8.5 % %
9.5 % %
10.3 % %
12.4 % %

21. Linearity protocol Linear regression analysis was applied to compare expected and observed values from serial dilutions of two samples with high (14.3 %, 133 mmol/mol) and low (5.1 %, 32 mmol/mol) HbA1C
y = 1.02 x , R = 0.997

22. Flex Peiorcing HbA1c (%)
Correlation Study
Menarini/ARKRAY ADAMS A1c HA-8160
n=107 range % ( mmol/mol)
R =
y = x
mean difference 0.2 % 2 4 6 8 10 12 14
Menarini/Arkray ADAMS A1c HA-8160 (%)
Flex Peiorcing HbA1c (%)
SEBIA Capillarys 2 4 6 8 10 12 14 16
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
-0.8
AVERAGE of Menarini and Sebia (%)
(% Menarini -% Sebia)
Mean
-0,20
-1.96 SD
-0,66
+1.96 SD
0,26
No reference method was used to conclude

23. Effect of varying hemoglobin concentration
One sample from a non diabetic non thalassemic subject was centrifugated 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

24. Known interferences
Lipemia was investigated by overloading samples of a pooled blood (HbA1C 5.0 %, 31 mmol/mol) adding increasing amounts of pooled bloods with high concentration of triglycerides (15 mmol/L)
The effect of bilirubin was evaluated similarly adding pooled bloods with high concentration of bilirubin (500 µmol/L)
Labile Hb
Blood with a HbA1c concentration 5.2 % (33 mmol/mol) was poured into six tubes; 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 2 hours and then analysed
Carbamylated Hb
Adding a Sodium Cyanide solution (up to 1 mmol/L Sodium Cyanide concentration)
Acetylated Hb
Adding Acetaldehyde solution (2- 10 mol/L Acetaldehyde concentrations )
The effect of HbF concentration was investigated adding blood cord to samples before the analysis

25. Labile fraction is formed by the unstable union of glucose to Hb
It presents an important biological variation due to recent fluctuations of blood glucose; most of this fraction releases to HbA0
The values are usually below 2%
Interference in HPLC: due to the incomplete separation of labile and stable peaks

27.  The carbamylation takes place in normal individuals and in a very pronounced way in patients with reduced renal function and high levels of urea in serum
• Levels of CHb can represent 2% of the total Hb in uremic patients
• Interference in HPLC: due to the incomplete separation of HbA1c and Carbamylated Hb , it elutes from column in the labile fraction

29. Acetylated Hb migrates in the Other Hb fraction
The in vivo acetylation of the N-terminal group of globin chains takes place in alcoholics, pregnant women and patients with high doses of aspirin
• Acetylation takes place mainly in the chains γ (HbF)
• The Acetylated Hb may represent even a 1.1% of Hb Total in patients treated with aspirin over a long period of time

30. HbA1C results were not modified for
HbF %
Bilirubin µmol/L
Triglycerides 18 mmol/L

31. Hemoglobin variants analysis
CAPILLARYSTM2 Flex Piercing HbA1c assay and the current technique(s) used in the laboratory in the presence of hemoglobin variants (S, C, E, D or other rare variants).
When a variant is suspected in one of the techniques used for HbA1c quantification, the sample must be analyzed using CAPILLARYS hemoglobin technique.
64 beta thalassemia; 12 delta beta thalassemia, 8 Hb Lepore, 1 HbE , 4 Hb D ; 6 Hb C ; 30 Hb S; 1 Hb Shelby

33. Phoresis®
Abnormal
Variant Hb Mode

35. Microcytosis Erythrocytosis Isocromy
HbA2
HIGH Beta Thalassemia
RR or LOW
MB alfa Thalassemia
Hospital Galdakao Usansolo
inhabitants
1000 hemograms/day
200 HbA1c /day
On average
2 β thalassemia carriers/ day
1 α thalassemia carrier/ week

36. HbA2 Performances Reproducibility
Within run Mean 2.2 % CV 1.9 % Mean 4.6 % CV 1.0 %
Linearity proportional mixtures of high (4.1 %) and low (2.4 %) HbA2 samples
R=0.995 slope ; intercept -0.08
Sensitivity
HbA2 4.0 % influence <0.2 %
Effect of varying hemoglobin concentration
HbA % range 19.5 – 3.9 g/dL Hb , 0.1 %

37. β thalassemia screening?
HbA2 was analysed by the HbA1C program on CAPILLARYS 2 Flex piercing® for a non-beta thalassemic population (279 samples). HbA2 1.4% - 2.8%
64 samples of beta-thalassemic patients were analyzed for HbA2 by using the same program.
Range 3.1 % %

38. β thalassemia screening?
The HbA2 concentration was assayed with the HbA1C program on CAPILLARYS 2 Flex piercing® in parallel using Hb program
60 normal individuals and 64 β thalassemia carriers
Correlation y = x , R =
mean difference %

39. Capillarys 2 Flex Piercing® HbA1c kit
Easy-to-use analyzer
Friendly software interface (Phoresis)
High throughput (8 capillaries in parallel) : 40 samples per hour
Quick validation step:
Mosaic screen displaying 48 results at a glance
Automatic discrimination using colour-coded profiles (normal, elevated and atypical)
Customizable results display and print-out:
HbA1c in mmol/mol and/or %
Estimated Average Glucose (mg/dL)

40. Conclusions
New separation method for HbA1c assay
First capillary electrophoresis assay for routine Laboratories
Enhanced analytical performance at a high throughput
No impact of the most frequent analytical interferences (labile A1c, carbamylated Hb, triglycerides ,bilirubin) on HbA1c measurement
HbA1c quantification possible in the presence of common Hb variants
Capillarys 2 Flex Piercing HbA1c kit is suitable for routine practice in Clinical Laboratories

41.
Hospital Galdakao Usasolo
48960 Galdakao, Vizcaya
SPAIN
43N03W