AJCP / Review Article
Pathology Consultation on HbA1c Methods
and Interferences
Jeanne M. Rhea, PhD, and Ross Molinaro, PhD
From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA.
Key Words: HbA1c methods; Blood glucose concentrations; Diabetes; HbA1c interferences; HbA1c interpretation
DOI: 10.1309/AJCPQ23GTTMLAEVL
ABSTRACT
Objectives: To review the various hemoglobin (Hb) A1c
methods, with a focus on interferences resulting from Hb
variants.
Methods: HbA1c is a marker used for the diagnosis and
management of diabetes. Each available HbA1c method
has advantages and challenges, such as its susceptibility to
interferences.
Results: The presence of Hb variants and/or abnormalities in
RBC turnover cannot only interfere analytically with HbA1c
measurements but also may affect the clinical interpretation
of HbA1c values.
Conclusions: Familiarity with the advantages and
challenges of the various methods used for HbA1c testing is
essential when establishing therapeutic management and
goals based on HbA1c results, especially in populations with
a high prevalence of Hb variants.
© American Society for Clinical Pathology
Rhea_2013040215.indd 5
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CME/SAM
Upon completion of this activity you will be able to:
• discuss the clinical value of hemoglobin (Hb) A1c testing.
• describe interferences related to Hb variants that may be
encountered in HbA1c testing and interpretation.
• compare the following HbA1c methodologies: ion-exchange high-
performance liquid chromatography, capillary electrophoresis,
boronate affinity, immunoassay, and enzymatic.
The ASCP is accredited by the Accreditation Council for Continuing
Medical Education to provide continuing medical education for physicians.
The ASCP designates this journal-based CME activity for a maximum of
1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only
the credit commensurate with the extent of their participation in the activ-
ity. This activity qualifies as an American Board of Pathology Maintenance
of Certification Part II Self-Assessment Module.
The authors of this article and the planning committee members and staff
have no relevant financial relationships with commercial interests to disclose.
Questions appear on p 140. Exam is located at www.ascp.org/ajcpcme.
Case Scenario
A 36-year-old woman of Sicilian descent with a fam-
ily history of type 2 diabetes presented to her primary care
physician with a history of obesity and hypertension. Her
clinical examination findings were unremarkable. She had
a fasting glucose concentration of 124 mg/dL (7.0 mmol/L;
fasting plasma glucose cutoff, ≥126 mg/dL [7.0 mmol/L])
that was observed during her last physician’s office visit.
Her blood pressure was 140/80 mm Hg. She admitted to
feeling tired and getting up at night occasionally to urinate.
A hemoglobin (Hb) A1c test was ordered on two separate
occasions. The HbA1c method used in the laboratory was
immunoassay, and both results were reported as normal at
5.3% and 5.1% (cutoffs: prediabetes, 5.7%-6.4%; diabe-
tes, ≥6.5% ).1 The patient was notified of the results and
Am J Clin Pathol 2014;141:5-16 5
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12/10/13 10:40 AM
 Rhea and Molinaro / HbA1c Methods and Interferences
reassured that she did not have diabetes but was advised to
visit her physician again in 3 months for follow-up.
On relocating to a different state for employment rea-
sons, the patient presented to her new primary care physi-
cian approximately 4 months later for a workplace physical
examination. At this visit, her fasting glucose concentration
was found to be 142 mg/dL (7.8 mmol/L). Results of a
follow-up, 75-g, 2-hour oral glucose tolerance test were con-
sistent with a diagnosis of diabetes: 2-hour glucose of 220
mg/dL (12.2 mmol/L; 2-hour plasma glucose cutoff ≥200
mg/dL [11.1 mmol/L]).1 An HbA1c test was ordered and
performed in a laboratory using ion-exchange high-perfor-
mance liquid chromatography (HPLC), and results returned
with an abnormally high HbA1c value of 16.7%, remarkably
different from the patient’s previous HbA1c test result. Based
on the shape of the HPLC chromatogram, a comment was
appended to the HbA1c result indicating that a presumptive
Hb variant was suspected. Subsequent laboratory testing and
investigation by the patient’s physician revealed the pres-
ence of hemoglobin S (HbS) reduced β (β+)–thalassemia in
this 36-year-old woman.
Background
Hb is composed of four globin chains. HbA0, the most
abundant nonglycated form of adult Hb, consists of two α
and two β chains (α2β2) and accounts for approximately 95%
to 98% of Hb. Other normal Hbs present in adults include
HbF (α2γ2) and HbA2 (α2δ2), each making up approximately
2% of total Hb. In the presence of glucose, HbA0 is glycated
to form HbA1a1 (addition of fructose-1,6-diphosphate to the
amino terminus of the HbA β chain), HbA1a2 (addition of
glucose-6-phosphate to the amino terminus of the HbA β
chain), HbA1b (addition of a pyruvate to the amino terminus
of the HbA β chain), or HbA1c (nonenzymatic addition of
glucose to the amino-terminal valine residues of the HbA β
chains). HbA1c accounts for more than 80% of HbA1 and is
formed in two basic steps: glucose binds reversibly to Hb as
an aldimine or Schiff base, which in turn undergoes an Ama-
dori rearrangement to form a stable, irreversible ketoamine.
HbA1c forms in humans at a slow rate as a posttranslational
modification throughout the lifespan of the RBC.2
Several studies have established a direct relationship
between HbA1c and average blood glucose concentrations.3-5
The onset and progression of diabetic complications can
be delayed by maintaining near-normal HbA1c levels.3,4
Because HbA1c reflects the average blood glucose con-
centration over the previous 2 to 3 months (normal RBC
lifespan), it is currently the most widely used index of aver-
age glycemia for the routine monitoring and prevention of
long-term complications of diabetes. In addition to its use in
6 Am J Clin Pathol 2014;141:5-16
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Rhea_2013040215.indd 6
monitoring diabetic patients, the American Diabetes Asso-
ciation (ADA) and the World Health Organization (WHO)
have endorsed the use of HbA1c for the diagnosis of diabetes
and recommend that only NGSP (previously referred to as
the National Glycohemoglobin Standardization Program)-
certified HbA1c methods performed in clinical laboratories
be used for diagnosis.
When using HbA1c for diagnosing and/or managing
the care of diabetic patients, it is crucial to be aware of
the factors known to interfere with the HbA1c test. These
interferences may arise through analytic means as well as
through biologic variables affecting clinical interpretation.
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This report focuses on HbA1c interferences resulting from
the presence of Hb variants.
Common Hemoglobinopathies
Hb Variants
Hb variants arise from mutations and/or deletions in the
genes encoding for the α and β chains that result in amino
acid changes. Of the hundreds of Hb variants that have been
identified to date, the four most common worldwide are HbS,
hemoglobin E (HbE), hemoglobin C (HbC), and hemoglobin
D (HbD), in descending order of prevalence. Each of these Hb
variants, depending on the method used, may cause an inac-
curate HbA1c result because of analytic interference6 or, alter-
natively, an HbA1c value that is clinically misleading because
of biologic variables affecting the interpretation of the result.7
If the prevalence of conditions that alter the RBC lifespan is
high in a patient population, it may be beneficial to use an
HbA1c assay that can presumptively identify these conditions
to prevent reporting inaccurate HbA1c values in these cases.7
Hemoglobin S
HbS is produced when a valine is substituted for a glu-
tamic acid at position 6 in the β chain. Persons carrying a
single copy (heterozygous) of the mutated gene are described
as having sickle cell trait (HbAS); those with two defective
copies (homozygous) of the gene have sickle cell disease
(HbSS). It has been estimated that 2.5 million people in
the United States and 300 million people worldwide have
sickle cell trait. In the United States, approximately 90,000 to
100,000 individuals are affected by sickle cell disease.8 Sickle
cell disease is characterized by the formation of long, inflex-
ible chains of Hb that can facilitate RBCs to become caught
in small capillaries, causing significant amounts of organ
damage. In addition, HbSS is often associated with anemia
because of a decrease in the RBC lifespan. In patients with
sickle cell disease, the RBC lifespan is significantly shortened
(~10-20 days for HbSS vs ~120 days for HbAA).9 However,
© American Society for Clinical Pathology
12/10/13 10:40 AM

in patients with sickle cell trait, the RBC lifespan is approxi-
mately 93 days, which is currently considered to be normal.10
Hemoglobin C
The HbC allele can be found in up to 50% of individu-
als in West Africa. Migration has seen the spread of this Hb
variant to many other regions of the world, and it is carried
by approximately 3% of African Americans. This Hb vari-
ant arises when the glutamic acid found at position 6 of the
HbA β chain is replaced by lysine. Carriers of a single allele
of the HbC variant are reported to enjoy complete health,
whereas those with HbC disease commonly present with
mild hemolytic anemia. The RBC lifespan in patients with
the HbC allele is approximately 87 days.11
Hemoglobin E
The HbE variant arises from a mutation in the HbA β
chain that results in the substitution of a glutamic acid for
lysine at position 26. This mutation also results in reduced
synthesis of the β chain of HbE through the activation of a
cryptic messenger RNA splice site, producing a thalassemic-
like phenotype. The HbE allele is common in Southeast
Asia, and it can be found in some areas at a frequency equal
to that of HbA. Approximately 30% of Southeast Asians
living in the United States are carriers of the HbE variant.
Although HbE trait is clinically asymptomatic, the effect of
this variant on the RBC lifespan has not been investigated.11
Hemoglobin D
HbD is found in approximately 2% of North Indians
and descendants from Pakistan and Afghanistan living in the
United States. This variant is produced when the glutamic
acid at position 121 on the HbA β chain is changed to a glu-
tamine. Several HbD variants have been identified, with the
most common known as HbD-Punjab/Los Angeles. Persons
heterozygous for HbD are clinically asymptomatic. The
RBC lifespan of HbD trait has been reported in the literature
as 115 days.11
β-Thalassemia
The WHO estimates that approximately 1% to 5% of
the world’s population are carriers of β-thalassemia.12 Point
mutations, small deletions, or small insertions may result
in absent (β0) or β+ β-globin synthesis. Unlike HbS, which
produces a mutated form of β-globin, β-thalassemia affects
actual production of the β-globin chain. As a consequence,
the production of δ-globin and γ-globins may be increased,
resulting in higher levels of HbA2 and fetal Hb (HbF). The
clinical manifestations of these mutations can range from
asymptomatic (β-thalassemia minor) to completely transfu-
sion dependent (β-thalassemia major).13 In the more severe
forms of thalassemia, the loss of balance between α- and
© American Society for Clinical Pathology
Rhea_2013040215.indd 7
AJCP / Review Article
β-globin synthesis results in a toxic aggregation of unpaired
α chains early in RBC development, therefore leading to a
shortened RBC lifespan.14
Patients with HbS β-thalassemia have one HbS gene and
either a β+ or β0 gene. The clinical manifestation of the dis-
ease may be either in the form of severe sickle cell disease
(HbS β0- thalassemia) or a milder form of sickle cell disease
(HbS β+-thalassemia). Asymptomatic children and adults
have been reported to carry a specific β-thalassemia allele.15
Similar to HbSS, the lifespan of the HbS β-thalassemia RBC
is shortened.
In 2009, there were approximately 38.9 million non-
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Hispanic African Americans with HbS β-thalassemia living
in the United States.16 Of these, around 8% carry the HbS
allele. When combined with an estimated prevalence rate of
5% for β-thalassemia,17 it is estimated that approximately
156,000 non-Hispanic African Americans are affected by
HbS β-thalassemia. The presence of HbS β-thalassemia is
known to result in falsely elevated HbA1c measurements
because of analytic interferences using certain methods,18 as
seen in the case presented herein.
HbA1c Methods and Interference From Hb
Variants
Analytic interferences have been investigated for the
majority of the common, commercially available HbA1c
methods in the presence of heterozygous HbS, HbC,
HbE, and HbD19-24 and are summarized at www.ngsp.org
(accessed June 16, 2013). HbA1c can be measured in clinical
laboratories by means of immunoturbidimetric assay (immu-
noassay), enzymatic assay, boronate affinity, ion-exchange
HPLC, or capillary electrophoresis (CE). A list of currently
approved Food and Drug Administration assays, reagents,
and kits is shown in ❚Table 1❚.25 Reporting of analytically
inaccurate results most often can be avoided if the user care-
fully follows the manufacturer’s instructions.26 However,
some methods and/or manufacturers’ instructions alone do
not provide sufficient information for making the correct
decision about reporting analytically accurate or clini-
cally useful results. Some vendors of HbA1c methods have
multiple, different HbA1c assays commercially available
(see Table 1), each potentially having its own unique ana-
lytic interference issues. It is important that each laboratory
understands the analytic advantages and challenges associ-
ated not only with the general HbA1c method but also with
the specific assay they operate.
Immunoassay
Immunoassays are currently the most common type of
HbA1c method used in the clinical laboratory according to
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 Rhea and Molinaro / HbA1c Methods and Interferences

laboratories that enroll in the College of American Pathol- HbA1c immunoassays use an antibody that recognizes amino
ogy (CAP) proficiency testing program for HbA1c.2 These acids 4 to 10 on the β chain of HbA, resulting in an analytic
immunoassay methods use antibodies that recognize the interference in the presence of HbS as well as 26 other cur-
N-terminal glycated amino acids ❚Figure 1❚. First-generation rently identified Hb variants that may span this epitope

❚Table 1❚
List of Food and Drug Administration–Approved Assays/Reagents for Hemoglobin (Hb) A1c

Company Device Name Method

Abbott Laboratories Abbott IMx Glycated Hemoglobin II Boronate affinity

Alfa Wassermann Diagnostic Technologies S-Test % Hemoglobin A1c (HbA1c) Enzymatic
Alfa Wassermann Diagnostic Technologies ACE Hemoglobin A1c (HbA1c) Reagent Immunoassay
ARKRAY ARKIT HbA1C Enzymatic

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Axis-Shield Diagnostics ARCHITECT HbA1c Reagent Immunoassay
Axis-Shield Diagnostics AxSYM HbA1c Immunoassay
Axis-Shield PoC AS Afinion HbA1c Boronate affinity
Bayer Corporation A1CNow+ (Professional Use); A1CNow Self Check (Home Use) Immunoassay
Beckman Coulter AU Systems HbA1c (Hemoglobin) Test System Immunoassay
Beckman Coulter Olympus Hemoglobin A1c Test Immunoassay
Beckman Coulter Unicel DxC Synchron Systems Hemoglobin A1C-(HBA1C-) Reagent Immunoassay
Beckman Coulter Synchron LX Systems Hemoglobin A1c2 Reagent Immunoassay
Beckman Coulter SYNCHRON Systems Hemoglobin A1c Reagent Immunoassay
BioDiagnostic International Liqui-Heme Glycohemoglobin A1c Assay Immunoassay
Bio-Rad VARIANT II Turbo HbA1c Kit-2.0 Ion exchange HPLC
Bio-Rad Bio-Rad VARIANT II Turbo Link Hemoglobin A1c Program Ion exchange HPLC
Bio-Rad VARIANT II HbA1c Program Ion exchange HPLC
Bio-Rad Bio-Rad VARIANT Express Glycohemoglobin Program Boronate affinity
Bio-Rad D-10 Hemoglobin A1c Program Ion exchange HPLC
Ceragem International LabonaCheck A1c Boronate affinity
Clinical Data, Inc ATAC Hemoglobin A1c Reagent Kit Immunoassay
Diazyme Laboratories SMART HbA1c Assay Reagent Kit Enzymatic
General Atomics Diazyme HbA1c Enzymatic Assay Enzymatic
Horiba ABX ABX PENTRA HbA1c WB Immunoassay
Infopia Co HemoCue HbA1c 501 Glycosylated Hemoglobin Monitoring System Boronate affinity
Infopia Co Clover A1c HbA1c assay Boronate affinity
MEC Dynamics Corp Avie A1C System Immunoassay
Medica Corporation EasyRA HbA1c Reagent Immunoassay
Metrika DRx HbA1c Immunoassay
Metrika (Bayer) A1cNow (Home and Professional Use) Immunoassay
Microgenics DRI Hemoglobin A1c Assay Immunoassay
Olympus America Olympus Hemoglobin A1c Test Immunoassay
Ortho-Clinical Diagnostics VITROS Chemistry Products d%Alc Reagent Kit Immunoassay
Ortho-Clinical Diagnostics VITROS Chemistry Products %A1c Reagent Kit Immunoassay
Polymedco Polymedco HbA1c Test Immunoassay
Primus Corporation Primus A1care Assay Boronate affinity
Primus Corporation DBA Trinity Biotech Premier Hb9210 Boronate affinity
Primus Corporation NycoCard HbA1c Glycated Hemoglobin Assay Boronate affinity
Provalis Diagnostics G5 I HbA1c and G5 II HbA1c Test System Ion exchange HPLC
Provalis Diagnostics Glycosal II HbA1c Test Ion exchange HPLC
Roche Diagnostics INTEGRA Reagent Cassette for Hemoglobin A1c Immunoassay
Roche Diagnostics TQ HBA1C GEN3 Immunoassay
Roche Diagnostics Cobas Integra HbA1c Immunoassay
Roche Diagnostics Cobas Integra 800 Tina-quant HbA1cDx Immunoassay
Roche Diagnostics Tina-Quant Hemoglobin A1c Gen 2 Immunoassay
Sebia CAPILLARYS HbA1c kit Capillary electrophoresis
Seppim S.A.S. ELITech Clinical Systems HbA1c Immunoassay
Seradyn MULTIGENT Hemoglobin A1c Reagents Immunoassay
Siemens Siemens DCA 2000 Analyzer Immunoassay
Siemens Siemens DCA 2000+ Analyzer Immunoassay
Siemens Dimension HbA1c Assay Immunoassay
Siemens ADVIA Chemistry Hemoglobin A1c (A1c) Immunoassay
Sigma Diagnostics INFINITY HbA1c Immunoassay
Teco Diagnostics Hemoglobin A1c Reagent Set Immunoassay
Thermo Fisher Scientific HbA1c Test System Immunoassay
Tosoh Bioscience ST AIA PACK HBA1C Immunoassay
Tosoh Bioscience G8 Automated Glycohemoglobin Analyzer HLC-723G8 Ion exchange HPLC
Tosoh Medics A1c 2.2 Plus Automated Glycohemoglobin Assay Ion exchange HPLC
Wako Chemicals USA APOLOWAKO HbA1c Immunoassay

HPLC, high-performance liquid chromatography.

8 Am J Clin Pathol 2014;141:5-16 © American Society for Clinical Pathology

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 AJCP / Review Article

❚Table 2❚.27-59 This analytic interference from two of the most
common Hb variants worldwide (HbS and HbC) prompted
the development of second- and third-generation immuno-
assays in which the epitope of the antibody used spans the
first several amino acids closer to the N-terminal of the β
chain of HbA (Table 2). These next-generation antibodies
are believed to bind HbS similarly to HbA, providing ana-
lytically accurate HbA1c values in patients with HbS trait and
other heterozygous Hb variant conditions in which the RBC
lifespan may be normal. However, Hb variants exist that may
cause analytic interference when using these next-generation
antibodies (Table 2), although in most cases their prevalence
like the highly specific second- and third-generation immu-
noassay HbA1c methods, the end user is unable to discern the
presumptive presence of homozygous Hb variants in patient
samples.
Boronate Affinity Chromatography
Boronate affinity is a structurally specific method that
recognizes the cis-diol groups of glucose bound to Hb.26 It
tends to demonstrate the least analytical interference from
the presence of heterozygous Hb variants.60 Affinity separa-
tion of glycated Hb typically uses m-aminophenylboronic
acid and depends on a specific interaction between the glu-

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is extremely low, and any potential effects on different HbA1c
methods have not yet been evaluated. Although the improved
analytic specificity has eliminated assay interferences from
HbS and HbC, an unintended consequence of these highly
specific assays is the ability of the laboratory to report clini-
cally misleading HbA1c values for patients with no HbA (ie,
HbSS, HbCC, HbSC, or HbS β-thalassemia).7 This caveat of
analytic specificity is also observed for the enzymatic HbA1c
methods and presents the biggest concern when the end user
is unable to discern the presence of homozygous Hb variants
that alter the RBC lifespan, which can lead to reporting an
HbA1c value that is clinically misleading.7
Enzymatic Assay
For enzymatic methods, whole blood samples are lysed
and subjected to extensive proteolytic digestion. This pro-
cess releases amino acids, specifically the glycated N-ter-
minal valine from the Hb β chains. The signal produced by
the glycated valines in a subsequent chromagenic reaction is
used to calculate HbA1c ❚Figure 2❚. These methods are unaf-
fected analytically by the presence of Hb variants. However,
cose on glycated Hb and the immobilized boronic acid. Non-
glycated Hb and glycated Hb products elute separately off
the column through interaction of glycated Hb and boronic
acid immobilized on an agarose gel as well as by ionic and
hydrophobic forces ❚Figure 3❚. This method provides a visual
chromatographic separation of the glycated and nonglycated
Hb products. Although boronate affinity methods measure
total glycated Hb, results are reported as a corrected HbA1c
equivalent. However, as with immunoassay and enzymatic
HbA1c assays, the end user is unable to discern the presump-
tive presence of Hb variants when using boronate affinity,
which may affect the clinical interpretation of HbA1c values
in cases in which the RBC lifespan is altered.7
Ion-Exchange HPLC
Ion-exchange HPLC assays are currently the second
most common type of HbA1c method used in the clinical
laboratory according to statistics from the CAP proficiency
testing program.2 Ion-exchange HPLC separates Hb species
based on charge differences ❚Figure 4❚. Highly sophisticated
models of HPLC can be used to resolve various Hb products,

A B

No HbA1c HbA1c

Anti-HbA1c Polyhapten HbA1c

agglutinator

❚Figure 1❚ Turbidimetric inhibition immunoassay for hemoglobin (Hb) A1c measurement. A, In the absence of HbA1c,
polyhaptens (synthetic molecules containing multiple HbA1c epitopes) react with free anti-HbA1c antibodies that are bound to
microparticles to form an insoluble antibody-polyhapten complex. This results in significant light scattering. B, HbA1c in patient
RBC lysate reacts with the anti-HbA1c antibody, forming soluble antigen-antibody complexes and thereby reducing light scatter.
The rate of the reaction is measured turbidimetrically and is inversely proportional to the amount of HbA1c in the sample.

© American Society for Clinical Pathology Am J Clin Pathol 2014;141:5-16 9

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 Rhea and Molinaro / HbA1c Methods and Interferences

❚Table 2❚
Hemoglobin (Hb) Mutations Relative to HbA for the First 13 Amino Acids of the β Chain27

Name Hb Sequence Clinical Significance Comments

HbA MVHLTPEEKSAVTA N/A Wild-type Hb

Hb Niigata MLHLTPEEKSAVTA Clinically silent Seven cases reported (six from Japanese families, one
  from Romanian origin)28
Hb South Florida MMHLTPEEKSAVTA Clinically silent when heterozygous; produced Two cases reported (one from white family,29
  β-thalassemia carrier phenotype when   one of Malaysian origin30)
  combined with β0-thalassemia allele
Hb Raleigh MAHLTPEEKSAVTA Heterozygote clinically silent Found in white and Swedish families
Hb Doha MEHLTPEEKSAVTA Heterozygote clinically silent Reported in three members of a Qatari family22
Hb Watford MGHLTPEEKSAVTA Chronic mild anemia Reported in several members of a Jewish family31
Hb Fukuoka MVYLTPEEKSAVTA Heterozygote clinically silent Several cases reported in people of Japanese origin32
Hb Marseille MVPLTPEEKSAVTA Heterozygote clinically silent Occasionally found in Malta and Maltese descendants33
Hb Graz MVLLTPEEKSAVTA Heterozygote clinically silent Estimated at 0.36% in Australian population where it

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  was identified34
Hb Deer Lodge MVRLTPEEKSAVTA Heterozygote clinically silent Found in Welsh-Dutch-English and black families35
Hb Agrigente MVPLTPEEKSAVTA Not reported Not reported
Hb Okayama MVQLTPEEKSAVTA Heterozygote clinically silent Found in diabetic Japanese female36
Hb Niguarda MVHMTPEEKSAVTA Not reported Not reported
Hb Kamakura MVHVTPEEKSAVTA Heterozygote clinically silent Three reported cases in Japan37
Hb Santo Domingo MVHQTPEEKSAVTA Heterozygote clinically silent Not reported
Hb Jabalpur MVHPTPEEKSAVTA Not reported Not reported
Hb Benin City MVHLPPEEKSAVTA Not reported Not reported
Hb Würzburg MVHLNPEEKSAVTA Heterozygote clinically silent One case reported in a German woman38
Hb Görwihl MVHLTAEEKSAVTA Heterozygote clinically silent One case reported in a German woman39
Hb Tyne MVHLTSEEKSAVTA Heterozygote clinically silent Two cases reported in unrelated English male patients40
Hb Warwickshire MVHLTREEKSAVTA Heterozygote clinically silent Identified in a Scottish family41
Hb Aix-les-Bains MVHLTLEEKSAVTA Heterozygote clinically silent One case reported in an Italian woman42
HbC-Rothschilda MVHLTLEEKSAVTA Not reported Found in a Nigerian woman living in London, England
HbC MVHLTPKEKSAVTA Clinically silent in heterozygote; hemo- 40%-50% carrier rate in Burkina Faso, Côte d’Ivoire, and
  lytic mild anemia in homozygote   Ghana; ~3.5% of West African descendants in the
  Caribbean, Southern Europe, and in the US43
Hb Arlington Parka MVHLTPKEKSAVTA Heterozygote clinically silent Identified in an African American
Hb Kingsburya MVHLTPKEKSAVTA Anemia Found in people of African ethnicity
HbC-New Crossa MVHLTPKEKSAVTA Heterozygote clinically silent Occurs in people of Nigerian ethnicity
HbS-Cameroona MVHLTPVEKSAVTA Heterozygote clinically silent Ethnic background: Cameroonian
HbC-Harlema MVHLTPVEKSAVTA Heterozygote clinically silent Found in several black families
Hb Machida MVHLTPQEKSAVTA Heterozygote clinically silent Identified in a Japanese family44
Hb Lavagna MVHLTPGEKSAVTA Not reported Not reported
HbG-Makassar MVHLTPAEKSAVTA Clinically silent Found in Indonesian and Thai families
HbS MVHLTPVEKSAVTA Heterozygote clinically silent; hemolytic Affects approximately 25% of West African population
  anemia, jaundice, painful crisis, etc.   and 8.3% of African Americans11

  in homozygote
HbS-Providencea MVHLTPVEKSAVTA Heterozygote clinically silent Found in a West African female patient
HbS-Clichya MVHLTPVEKSAVTA Not reported Not reported
HbS-San Martina MVHLTPVEKSAVTA Heterozygote showed mild hemolytic anemia Found in people of Argentinian descent
HbS-South Enda MVHLTPVEKSAVTA Heterozygote clinically silent; compound Found in a Ugandan woman
  heterozygote with HbS shows severe
  sickle cell disease
HbS-Travisa MVHLTPVEKSAVTA Heterozygote clinically silent Found in members of a black American family
HbS-Omana MVHLTPVEKSAVTA Heterozygote has moderate sickle cell disease Found in a male from Oman
HbC-Ndjamenaa MVHLTPVEKSAVTA Compound heterozygote with HbS showed Found in a Canadian patient
  sickle cell disease
Hb Jamaica Plaina MVHLTPVEKSAVTA Heterozygote has moderate/severe sickle Found in a Puerto Rican child
  cell disease
HbS-Antillesa MVHLTPVEKSAVTA Heterozygote has moderate/severe sickle Found in a family from Martinique, French West Indies
  cell disease
HbS-Sao Pauloa MVHLTPVEKSAVTA Heterozygote has sickle cell disease Found in a Brazilian child
HbC-Ziguinchora MVHLTPVEKSAVTA Heterozygote clinically silent Found in members of an African family living in Senegal
HbG-Siriraj MVHLTPEKKSAVTA Heterozygote clinically silent Found in a few Thai and Chinese families45
Hb Bellevue III MVHLTPEQKSAVTA Not reported One case reported in an Indian boy46
  (Hb Vellore)
HbG-San José MVHLTPEGKSAVTA Clinically silent Mainly found in Italian and Mexican-American families
Hb Stockholm MVHLTPEDKSAVTA Not reported One case reported in a Swedish patient47
HbJ-Luhe MVHLTPEEQSAVTA Not reported Identified in a Chinese family
HbN-Timone MVHLTPEEESAVTA Heterozygote clinically silent Discovered in a French-Italian family48
Hb Rio Grande MVHLTPEETSAVTA Heterozygote clinically silent Discovered in a Mexican-American family49
Hb Lucknow MVHLTPEERSAVTA Not reported One case reported in an Indian patient50
Hb Nakano MVHLTPEEMSAVTA Heterozygote clinically silent One case reported in a Japanese woman51
Hb Limassol MVHLTPEENSAVTA Heterozygote clinically silent One case reported in a Greek Cypriot52
HbD-Agria MVHLTPEEKYAVTA Heterozygote clinically silent Found in a man from the Agri caste originating from
  Mumbai, India

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❚Table 2❚ (cont)

Name Hb Sequence Clinical Significance Comments

Hb Brem-sur-Mer MVHLTPEEKYAVTA Heterozygote clinically silent One case reported in a French white patient53
Hb Pôrto Alegre MVHLTPEEKCAVTA Clinically silent Found in families from Brazil, Cuba, and the
  Canary Islands54
Hb Belleville MVHLTPEEKSTVTA Not reported Identified in two Caucasians from Yorkshire, UK55
Hb Iraq-Halabja MVHLTPEEKSVVTA Not reported Found in members of an Iraqi family
Hb Ankara MVHLTPEEKSDVTA Heterozygote clinically silent Found in Turkish and Japanese families56
Hb Washtenaw MVHLTPEEKSAFTA Anemia and chronic cyanosis Identified in an Hungarian-American family57
Hb Hamilton MVHLTPEEKSAITA Heterozygote clinically silent Occurs at a frequency of 0.25% in Sardinians; also
  found in an Austrian and Chinese family
HbO-Tibestia MVHLTPEEKSAITA Compound heterozygote showed severe Found in a child of Chad-Sudan origin
  sickle cell anemia
Hb Windsor MVHLTPEEKSADTA Hemolytic anemia Found in an Anglo-Saxon female patient58

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HbJ-Lens MVHLTPEEKSAVTD Heterozygote clinically silent Identified in a French child59

NA, not applicable.

a Second mutation is present on the β chain in addition to the mutation shown in the table.

A.

such as HbA1a, HbA1b, HbF, labile HbA1c, HbA1c, HbA0, VAL HIS LEU THR PRO GLU GLU LYS
and HbA2 as well as other Hb byproducts or Hb variants.
Although it is an advantage to identify the presence of some
Proteolysis
of these Hb products, it is important to note that different
ion-exchange HPLC methods for HbA1c measurement,
even from the same vendor, have different chromatographic
resolving power. The advantage of being able to measure B.
VAL
HbA1c using ion-exchange methods that do not use high-
HIS GLU LYS
resolution chromatography (with longer runtimes) is the THR PRO
LE GLU
shorter time required for sample analysis. The challenge
with decreased chromatographic resolution, however, is
the increased risk of interference from the Schiff base, Deglycation
carbamylated Hb, or Hb variants, which may coelute with
the peaks of interest and cause an erroneous HbA1c result.
C.
There is an advantage to being able to visualize an analytic
VAL
interference that exists in patient samples (eg, whereby these
Color
Hb derivatives cannot be separated from HbA or HbA1c) so
H2O2
that inaccurate HbA1c results will not be reported. However,
some ion-exchange HPLC assays are analytically inaccurate
because of interference from common Hb variants.6

Capillary Electrophoresis Glucose Amino acid

CE separates Hb molecules on the basis of charge and
hydrodynamic volume ❚Figure 5❚.61 Charged molecules are
highly resolved by their electrophoretic mobility. Separa-
tion depends on electrolyte pH and electro-osmotic flow.
As with some other forms of highly sophisticated models
of chromatography, various Hb products, as well as other
Hb byproducts or Hb variants, can be presumptively identi-
fied using CE. Studies have demonstrated that none of the
most common heterozygous Hb variants (HbS, HbC, HbD,
and HbE) analytically interfere with HbA1c quantification
using CE.6
❚Figure 2❚ Enzymatic assay for hemoglobin (Hb) A1c
measurement. A, The first eight amino acids of the glycated
β chain of HbA are shown. In the presence of a protease
and patient RBC lysate, amino acids and small peptides
are released, including N-terminal glycated valines from
the HbA1c molecule (B). Enzymatic deglycation of the
glycated valine (C) produces hydrogen peroxide (H2O2). The
formation of H2O2 can be measured using a colorimetric
assay. The signal produced is proportional to the amount of
HbA1c in the sample.

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 Rhea and Molinaro / HbA1c Methods and Interferences

A
Standardization of HbA1c Assays
HbA1c standardization efforts have been largely success-
ful. In addition, the NGSP has tightened the criteria for HbA1c
manufacturer certification to further decrease measurement
variability (www.ngsp.org); the CAP has also continued to
tighten the grading criteria for proficiency testing over the
Flow of Buffer
B past several years. Because of this, manufacturers have been
urged to improve the accuracy and precision of HbA1c assays.
Despite the overall success of these HbA1c efforts, in some

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Flow of Buffer
C

Signal
– – – – – – – –
Time
Flow of Buffer

Signal
Flow of Buffer
D
– – – – – – – –
Time
Flow of Buffer

Signal
Flow of Buffer – – – – – – – –
Time
Boronate Glycated Hb Other nonglycated Hb Flow of Buffer
affinity resin
D HbA1c Other Hb
E

Signal
Nonglycated Hb
– – – – – – – –
Time
Flow of Buffer
Signal

Glycated Hb
E
HbA
Signal

– – – – – – – –
Time
Flow of Buffer

Time HbA HbA1c Other Hb

❚Figure 3❚ Boronate affinity chromatography for hemoglobin
(Hb) A1c measurement. A, Patient RBC lysate is injected
onto an analytic column containing a resin with bound
m-aminophenylboronic acid. At a pH above 8.0, the cis-diol
groups of glycated Hb react with the m-aminophenylboronic
acid (B) and are subsequently bound to the column while other
nonglycated Hb species flow through (C). An acidic buffer
is used to release the bound glycated Hb molecules from
the column (D). Panel E is a simplified chromatograph (blue
line) demonstrating separation of nonglycated and glycated
Hb species. The chromatogram trace for nonglycated Hb is
not drawn to scale. Boronate affinity methods measure total
glycated Hb but report results as a corrected HbA1c equivalent.
❚Figure 4❚ Ion-exchange high-performance liquid
chromatography (HPLC) for hemoglobin (Hb) A1c measurement.
Ion-exchange separates Hb molecules based on charge. A,
Patient RBC lysate is injected into a negatively charged column.
B, C, Positively charged Hb molecules move slower than
negatively charged molecules because of ionic interactions with
the negatively charged resin. D, HbA1c elutes first as it is more
negatively charged than HbA0. The chromatogram trace for
HbA is not drawn to scale (E). Other Hb species and some Hb
variants can at times be resolved and presumptively identified
by ion-exchange HPLC. These would appear as additional peaks
in the chromatograph.

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 AJCP / Review Article

cases patient samples may still have significantly different
HbA1c values when using different methods because of the
susceptibility of each method to various analytic interfer-
ences, including Hb variants. A list of the advantages and
challenges for each method is shown in ❚Table 3❚.
Biologic Variables Affecting HbA1c
Interpretation
HbA1c interferences that occur outside the realm of ana-
lytic testing can also affect HbA1c result interpretation. These
blood loss or transfusion, or some anemias, the diagnosis of
diabetes must employ glucose criteria exclusively.”1 Under-
standing the basis for this recommendation is important when
interpreting HbA1c values, especially in areas with a high
prevalence of Hb variants, because the presence of some Hb
variants is also known to affect the RBC lifespan (eg, HbS,
HbC, HbSC, and HbD disease).26,60 Because HbA1c concen-
tration is dependent on RBC survival to accurately reflect
average blood glucose concentrations, any changes in the
RBC lifespan may result in an HbA1c measurement that fails
to accurately reflect average glycemia for approximately the
previous 120 days. This issue is of particular concern when

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biologic variables include, but are not limited to, alterations using assays for HbA1c (eg, immunoassay) that will produce
in the RBC lifespan. The ADA states that “for conditions an HbA1c result for homozygous Hb variants, without provid-
with abnormal RBC turnover, such as pregnancy, recent ing information that an Hb variant is present in the sample.7 In

Capillary

+ – HbA
Detector HbA1c

Signal
Other Hb
Buffer
Time
Sample High voltage
Power supply

HbA HbA1c Other Hb

❚Figure 5❚ Capillary electrophoresis (CE) for hemoglobin (Hb) A1c measurement. CE separates Hb species based on their
electrophoretic mobility, defined by both their charge and hydrodynamic size (ie, mass). A high-voltage power supply is used
to generate an electric field that facilitates the migration of molecules through the capillary tube from the anode to cathode.
Positively charged Hb species in patient RBC lysate will be detected first, in decreasing order of their charge-to-mass ratios. For
example, two molecules with the same positive charge will be further resolved by size, with the larger of the two molecules
moving the fastest. These are followed by neutral species and finally by negatively charged Hb species. A representative
electrophoretogram (not drawn to scale) shows the separation of HbA1c from HbA0.

❚Table 3❚
Characteristics of HbA1c Methods and Analytic Impact of Hb Variants

Method Principle Advantages Challenges

Enzymatic Measures HbA1c using an enzyme that No analytical interference from Unable to detect Hb variants
  specifically cleaves the N-terminal valine   Hb variants
Immunoassay Uses an antibody targeted against the No analytical interference from the Unable to detect Hb variants; newer-
  glycated N-terminus of the β chain   most common Hb variants using   generation antibodies still susceptible
  newer-generation assays   to interference from rare Hb variants
Boronate affinity Glycohemoglobin binds affinity resin Minimal analytical interference Measures all glycated Hbs, not just
  while nonglycated hemoglobins   from Hb variants   HbA1c; unable to detect Hb variants
  pass through the column
Ion-exchange Separates Hb species based Ability to detect the most common Prone to interference by Hb variants that
  HPLC   on charge   Hb variants   coelute with peaks of interest
Capillary Separates Hb species based on High chromatographic resolution and Throughputa
  electrophoresis   charge and hydrodynamic volume   resulting ability to detect many Hb variants

Hb, Hemoglobin; HPLC, high performance liquid chromatography.

a Batch testing maximizes throughput for high-volume testing.

© American Society for Clinical Pathology Am J Clin Pathol 2014;141:5-16 13

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 Rhea and Molinaro / HbA1c Methods and Interferences
such cases, it is possible for the HbA1c test result to be clini-
cally misinterpreted. For example, in patients with conditions
that shorten the RBC lifespan, as in the case patient’s HbS
β+-thalassemia, the reported HbA1c value is likely to be low de
novo. Reporting results in these cases has the potential to give
a false impression of average glycemia over approximately
the preceding 120 days when in fact it is representing average
glycemia over a more shortened RBC lifespan.
Alternative methods of assessing average glycemia (eg,
glycated albumin and/or fructosamine) are recommended in
these populations. Unfortunately, these measurements are not
yet nearly as standardized as HbA1c. No guidelines or goals
are established on glycated protein values in these populations
that can be followed by clinicians or diabetic patients. Impor-
tantly, clinicians should be cautious when using glycated
albumin and fructosamine as an estimate of long-term aver-
age blood glucose. First, glycated albumin and fructosamine
assess the degree of glycemia over a period of approximately
2 to 3 weeks, as opposed to 2 to 3 months for HbA1c. Second,
glycated albumin and fructosamine have not been correlated
with the development of long-term complications resulting
from diabetes mellitus, as was shown with HbA1c.2
It is currently accepted that the most common Hb variant
traits (eg, HbAS, HbAC, HbAE, and HbAD) do not alter the
RBC lifespan to a level that would interfere with the clini-
cal interpretation of HbA1c. It is therefore recommended by
the ADA that HbA1c values be used in patients who harbor
Hb variant traits as long as the assay used is not analytically
affected by the presence of that Hb variant.1
Case Summary
HbA1c is the most widely used marker of average glyce-
mia and is now also a recommended criterion for diagnosing
diabetes. In situations in which HbA1c measurements do not
correlate with clinical impression and/or blood glucose val-
ues, as in the case presented here, the presence of an interfer-
ence, such as an Hb variant, should be considered. Using an
HbA1c assay that is unaffected analytically by the presence of
common Hb variants is important. One might also consider
using an alternative method that is capable of presumptively
identifying the presence of common Hb variants. The labora-
tory performing the HbA1c test has the discretion to comment
on both the specific method or assay used for analysis as well
as the presumptively identified Hb variant observed. Provid-
ing comments to clinicians about the limitations of the HbA1c
assay used in their patient population (specifically in regard
to analytic interference or biologic variables that affect HbA1c
levels) can assist in HbA1c interpretation. Some examples of
comments that accompany HbA1c results from different labo-
ratories have been previously described.62
14 Am J Clin Pathol 2014;141:5-16
14 DOI: 10.1309/AJCPQ23GTTMLAEVL
Rhea_2013040215.indd 14
Clinical laboratories should be aware of the limitations
of their HbA1c assays, which may preclude the clinical use-
fulness of HbA1c results, particularly in geographic locations
with populations who are likely to have a high prevalence
of Hb variants or conditions that would affect the RBC
lifespan. Laboratories serving patient populations with a
high prevalence of Hb variants may choose to (1) imple-
ment electronic hospital information system algorithms to
assist the physician in automatically choosing an appropriate
method for determining HbA1c based on medical history7
or (2) implement laboratory processes to identify analytic
interferences in such populations.63
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Address reprint requests to Dr Molinaro: Dept of Pathology and
Laboratory Medicine, Emory University School of Medicine,
Emory University Hospital Midtown, 550 Peachtree Ave NE,
Davis Fischer Bldg, Room 1239, Atlanta, GA 30308; rjmolin@
emory.edu.
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