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Priya Sivaraman

Senior Product Manager Hemoglobin Testing

Bio‐Rad Laboratories, Inc.

priya_sivaraman@@bio‐rad.com

June 2015  

Learning Objectives

Describe the importance of A1c testing by automated HPLC methods. 

Discuss the use of HbA1c for Screening and Diagnosis. 

Explain the limitations of various methods used for A1c testing. 

What we’ll cover Diabetes‐ Definition, Statistics

A1c – Chemistry, Historical perspective, importance

Methods for measuring A1c

Screening and Diagnosis

A1c on automated analyzer 

HPLC and Variant Detection

Conclusion

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Diabetes is a group of diseases marked by high levels of blood glucose resulting from defects in insulin production, insulin action, or both. 

Insulin Dependent or Juvenile (IDDM)

A result of the destruction of insulin secreting pancreatic cells by the body’s own immune system

Treatment Daily Insulin Injections

Diet and Exercise

Prevalence 10% of diabetic population

Type 1 Diabetes

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Non‐Insulin Diabetes (NIDDM) or Adult onset Diabetes

Insulin Resistance

Inadequate insulin secretion

Treatment Drug Therapy

Insulin Injections if necessary

Diet and Exercise

Prevalence 90% of diabetic population

Type 2 Diabetes

If untreated Diabetes can cause

Eye Disease (retinopathy) 

Kidney Disease (nephropathy)

Nerve Disease (neuropathy)

Heart Disease & stroke

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Global Diabetes Facts

U.S. Diabetes Facts (2012)

29.1M or 9.3% of the United States is diabetic

21M have been diagnosed

8.1M have not been diagnosed

http://www.cdc.gov/diabetes/index.htm

Once diagnosed, 2 methods are used to monitor diabetes disease and effectiveness of therapy

• Immediate (Many times a day)

‐ Blood Glucose Testing 

• Long term (90 to 120 day)

‐ HbA1c Testing 

Diabetes Monitoring

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RBC freely permeable to glucose

Glucose binds to hemoglobin in RBC

Glucose and hemoglobin

RBC & glucose in bloodstream

HbA1c is formed

Hemoglobin A1C

Red blood cells live for ~120 days

A1c represents the average blood glucose for the last 2‐3 months

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• Measures Hemoglobin A1c (HbA1c)

• Indicator of glycemic control over a 90 to 120 day period

• Performed 2 to 4 times a year

• Good Control 2 times/year

• Poor Control 4 times/year

• Ideal level is <6.5% HbA1c

Long term

HbA1c Monitoring

Multi‐center, randomized clinical trial

1,441 type 1 patients with diabetes participated

HPLC analyzer used for laboratory analysis

Subjects were randomly assigned to either intensive or conventional therapy

Overall cost: $165 million

Final report: ADA 1993 Annual Meeting

Ref: N Engl J Med 1993;329:977-86.

DCCT The Diabetes Control and Complications Trial

(1983 –1993)

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Glycemic Control

Ref: N Engl J Med 1993;329:977-86.

DCCT Study Results

Intensive therapy dramatically reduced risks for 

development and/or progression of microvascular 

complications of diabetes (eyes, kidneys, peripheral 

nerves).

Benefit was directly related to glycemic control as 

assessed by serial hemoglobin A1c determinations. 

Ref: N Engl J Med 1993;329:977-86.

Risk of Retinopathy

0

4

8

12

16

20

24

0 1 2 3 4 5 6 7 8 9

Rat

e of

Ret

inop

athy

Pro

gre

ssio

n

Time During Study (Yrs)

Mean A1c = 11%

10%

9%

8%

7%

The Diabetes Control and Complications Trial Research Group. N Engl J Med 1993;329:977-86.

9 Yrs

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Monitoring diabetes with HbA1c

Stratton IM, et al. BMJ 2000;321:405–12

HbA1c Advantages

No fasting

Better index of overall glycemic exposure and   risk for long‐term complications

Indicator of glycemic control over a 90 to 120 day period

Well standardized

HbA1c is more reproducible and less cumbersome than OGTT

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Methods

Standardize glycohemoglobin test results (HbA1c) so that clinical laboratory 

results are comparable to those reported in the DCCT where 

relationships to mean blood glucose and risk for vascular complications have 

been established

www.NGSP.org

NGSP Market Criteria ChangesYear Bias (95% CI of the difference

between method and NGSP)Precision

(CV)

2003 +/- 1.0% <5%

2005 +/- 1.0% <4%

2007 +/- 0.85% <4%

2010 +/- 0.75% N/A

2012 +/- 0.70% N/A

2014 +/- 6% N/A

2014 NGSP criteria corresponds to CAP Survey Grading criteria that has been +/- 6% since 2013A +/- 6% CAP Limit corresponds to +/-0.4% HbA1c at a target of 7% HbA1c

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Standardization Worked

www.NGSP.org

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

%H

bA

1c

1993 2012

Total GHB

HbA1

HbA1c

CAP Survey: Mean +/- 2sd

DCCTTarget

20041999

Method Groups

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

%H

bA

1c

1993 2012

Total GHB

HbA1

HbA1c

CAP Survey: Mean +/- 2sd

DCCTTarget

20041999

Method Groups

Paving The Way For DiagnosisHbA1c Ranges – Yesterday

% A1c Interpretation45 Non‐Diabetic67 Normal89 Action Suggested101112

HbA1c Ranges ‐ Today

Interpretation

Pre-diabetes

Diabetes

Paving The Way For Diagnosis

%A1c

5.7% - 6.4%

≥6.5%

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If I don’t know… it doesn’t exist!

Why screen for diabetes?

Diabetes represents a sizable burden to population

Diabetes has a pre‐clinical phase when it can be detected and the onset can be delayed

Diabetes has improved prognosis after diagnosis

$$$ saved by intervention

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Original Diagnostic Criteria

Fasting glucose of >126mg/dL 

Casual or random glucose of >200mg/dL, with symptoms

Symptoms – polyuria, polydipsia, weight loss

2 hour GTT >200mg/dL

Diabetes Diagnostic Criteria(American Diabetes Association)

Method HbA1c * FPG OGTTRandom

Plasma Glucose

Pre-diabetes

5.7% - 6.4%100-125

mg/dl

(IFG)

140-199 mg/dl (IGT)

n/a

Diabetes ≥6.5%≥126

mg/dl≥ 200 mg/dl

≥200mg/dl with

symptoms

Diabetes Care, Vol 35, January 2012

HbA1c* - If > 6.5%, Repeat to Confirm

Why screen for diabetes with HbA1c?

Main factors that support screening using HbA1c

HbA1c does NOT require patients to be fasting

HbA1c reflects a better index of overall glycemic exposure and risk for long‐term complications

HbA1c methods are now well standardized, reliable, and aligned to DCCT 

Errors caused by non‐glycemic factors affecting HbA1c are infrequent and can be minimized – greater pre‐analytical stability

HbA1c is more reproducible and less cumbersome than OGTT

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ADA Recommendations

Diabetes Care, Vol 33, January 2010

• Testing for diabetes should be considered in all adults who are overweight (BMI 25 kg/m2) and have additional risk factors:

• First-degree relative with diabetes

• Members of a high-risk ethnic population (e.g., African American, Latino, Native American, Asian American, Pacific Islander)

• Women who delivered a baby weighing 9 lb or diagnosed with GDM

• Hypertension, High Cholesterol, High Triglyerides

• Women with polycystic ovary syndrome

• A1C 5.7%, IGT, or IFG on previous testing

• other clinical conditions associated with insulin resistance

Diabetes Care, Vol 33, January 2010

• In the absence of the above criteria, testing diabetes should begin at age 45 years

• If results are normal, testing should be repeated at least at 3-year intervals, with consideration of more frequent testing depending on initial results and risk status

ADA Recommendations Con’t

NGSP Certified Methods Only

Point of care HbA1c assays are only used for monitoring diabetes not screening for diabetes

HbA1c can be used for patients with Hb variants, but only those with normal RBC turnover. Assay must be free of interference from the Hb variant

ADA Recommendations Con’t

Diabetes Care, Vol 33, January 2010

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MethodsHemoglobin A1c Methodologies

ChemicalSeparation

HPLCIon Exchange

CapillaryElectrophoresis

AffinityChromatography

Immunoassay Enzymatic

ChargeDifference

StructureDifference

Separates Hb species based on structure

Affinity Chromatography

Non Glycated

Glycated

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Immunoassay Principles

Capillary Electrophoresis

Rhea and Molinaro. AJCP. 2013

Ion Exchange 

Rhea and Molinaro. AJCP. 2013

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1:00 2:00 3:00

Retention time

Sig

nal

RT

= 0

.21

, A1a

RT

= 0

.29

, A1b

RT

= 0

.45

, F

RT

= 0

.62

, LA

1c

/CH

b-1

RT

= 0

.80

, A1c

RT

= 1

.36

, P3

RT

= 1

.44

, A0

HPLC Ion Exchange Chromatography

Peak Identity

A1a Fructose-1,6-diphosphate (+) Glucose-6-phosphateA1b Pyruvic acid F Hemoglobin FLA1c/cHB-1 Labile hemogobin A1c / carbamylated hemoglobinA1c GlucoseP3 Degraded hemoglobinA0 Non-glycated hemoglobin

1:00 2:00 3:00

Retention time

Sig

nal

RT

= 0

.21

, A1a

RT

= 0

.29

, A1b

RT

= 0

.45

, F

RT

= 0

.62

, LA

1c

/CH

b-1

RT

= 0

.80

, A1c

RT

= 1

.36

, P3

RT

= 1

.44

, A0

Peak R.Time Ht Area Area%

A1a 0.21 8208 31293 0.7A1b 0.29 13438 63839 1.4F 0.45 6231 36039 0.8LA1c/cHB-1 0.62 8665 64049 1.5A1c 0.80 19586 200449 5.9P3 1.36 42417 203234 4.6A0 1.44 970693 3812497 86.4

HPLC Ion Exchange Chromatography

Advantages of HPLC

Superior precision meets all requirements

Directly comparable to DCCT

Use for HbS screening

Most Hemoglobin variants visible

Not influenced by low Hb concentration

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With HPLC one can see the full patient picture

The laboratory can alert the physician to the 

presence of abnormal hemoglobin

Identifies disease or traits that would influence 

HbA1c

Give physician added information

Advantages of HPLC

Bio‐Rad A1c Product Offering

We GROW with YOU

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RML Profile• Reference Laboratory in Tulsa Oklahoma

• ~ 1,000 CBCs / day

• ~ 500 A1cs / day

• Provides laboratory testing for physicians and hospitals within a 4-state region

• Primary facility in Tulsa, satellite facilities through the region

XN hematology systems

Slide maker / stainer

Tube sorter/archiver

Bio-Rad VARIANT II TURBO Links – A1c testing

RML System- Lavender Top Management with A1c

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Lavender Top Efficiency Gain

Voice of the Customer• HPLC assay is “gold standard” for A1c

measurements

• HPLC is widely accepted as the best long-term marker of diabetes control (I, II)

• With….improved service the laboratory now receives requests for "discharge” A1c values on patients before they are released.

Lawrence Johnson MD,

Director of hematology, flow cytometry, urinalysis and coagulation,

Jennifer Starks MT (ASCP)

Hematology Supervisor

• Integrated A1c line has improved A1c turn around times from 24-48 hrs. to 4 hrs.

• “Smart sorter” has automated sample handling- isolates exceptions, duplicates

Sysmex WAM Rules Template

Pre‐defined

Editable

Standardizes review

Enables auto‐verification

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Automated Review

Automated Decision

A1c Rules

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Conditions that may increase HbA1c results

Iron deficiency

Hypertriglyceridemia

Hyperbilirubinemia

Uremia

Chronic alcoholism

Chronic ingestion of salicylates

Opiate Addiction

NACB 2011 Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus (HbA1c)

Conditions that shorten erythrocyte survival lower HbA1c results

Acute blood loss

Hemolytic anemia

High concentration of Vitamins C and E

Hemoglobinopathies

Hb Variants

NACB 2011 Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus (HbA1c)

Hb Variant Avg. RBC Lifespan in Days (n)

HbSS 17 (10)

HbSC 28 (14)

HbCC 29 (7)

HbS-β Thal 75 (2)

HbAC 82 (9)

HbAS 93 (3)

Wild-type 120

Blood. 1969 

Blood. 1970Blood. 1975 

Hb Variant RBC Lifespan 

Blood. 1969 

Blood. 1970Blood. 1975 

Lancet. 1943 

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A1c values from Different Methods

Arch Pathol Lab Med. 2013 Dec;137(12):1788-91. Rhea, J Emory Univ.

● Immunoassay can give incorrect HbA1c results

● HPLC can give incorrect HbA1c results

● Is there any advantage of using one method over another?

YES!  

With HPLC the interference can be seen 

and appropriate action taken.

Interferences From Hemoglobin Variants

Ion Exchange

A1c

7.0%

Physicians need more information!!

8.00% A1c

Immunoassay

Sickle CellTrait

Immunoassays for HbA1c – Just a Number

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52 Yr Old Female:

Symptoms: Hepatitis, hyperlipidemia, hypertension, anemia and depression

Case Study – Feb 2011

Prior Testing revealed: Impaired Fasting Plasma Glucose: 106 mg/dL

TURBO Link HbA1c: 115% which is not possible analytically.

Siemens Immunoassay HbA1c Results: 4.1% indicates normal glycemic status

VARIANT II Thal: Indicates No Hb A present

Conclusion: HPLC identified interference

Clinical Chemistry 57:2, 153-157 TURBO Link Customer

52 year old male of Thai origin

HPLC shows no HbA1c

Major Peak at ~ 1.10 minutes

HbA1c = 5.8% by immunoassay

Hemoglobinopathy investigation

shows homozygous HbE

HbA1c In Presence Of HbE

HbA1c In Presence Of HbD Punjab

● 63 year old male of Indian/Pakistani origin

Fasting glucose 6.2 mmol/L

HPLC shows a peak at 1.13 mins

Immunoassay gives a HbA1c “result” of 6.5%

Hemoglobinopathy investigation shows presence of homozygous hbD Punjab.

No clinical or hematological Features.

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For diagnosing diabetes it is important to select a method that:

Is reliable

Adapts to your testing needs

It is important to know the limitation of the method that you are using

Conclusion