CLSC 4440 Clinical Chemistry

II - Glycohemoglobin

Kathleen Schulman M.S., MT(ASCP)

Susan T. Smith Ph.D., MT(ASCP)
Rev 2011

Property of CLS Department, East Carolina

University
Not to be reproduced or distributed without
written permission from the department.
Glycohemoglobin

This topic is covered on page 749 and 777

in K & P, and on the Elsevier website for
the text.
How do Glycated Proteins
Form?

Amine groups (NH2) of proteins can react with

and bind to glucose and other chemicals (urea,
salicylates, bilirubin, acetaldehyde from alcohol
metabolism) after the protein is formed in a cell
Major blood proteins affected are albumin and
hemoglobin
Binding of glucose to hemoglobin forms Hgb A1
Proteins that have glucose attached are said to
be glycosylated, or glycated. The preferred term
is glycated
Glycosylation of Protein
How do Glycated Proteins
Form?

By measuring levels of glycated proteins, the

physician can get an idea of what the patients
mean glucose level must have been over a
previous time period.
A. Hgb A1- reflects mean glucose for past
6-8 weeks
B. glycated albumin reflects mean glucose
for past 10 14 days
How do Glycated Proteins
Form?

One thing to keep in mind when interpreting the

results of glycated protein measurement is that
an acute, marked increase in glucose can
produce the same changes in the levels of
glycated proteins as moderate increases over 1-
2 weeks, or mild increases for many weeks.
Most of the time, the glycated protein result
reflects a consistent rather than an acute
increase
Chemistry and Synthesis of
Glycohemoglobin

Hgb A1 - is fast hemoglobin composed

of hemoglobin molecules to which glucose
has attached.
Glucose attaches to the NH2 group of the
terminal valine subunit on the beta chain
A1 forms- A1a1, A1a2, A1b, A1c
Chemistry and Synthesis of
Glycohemoglobin

A1a1- 1,6 FDP attached

A1a2- G6P attached
A1b- carbohydrate attached is
unknown
A1c- Glucose is attached to
terminal valine
Glycation process
HbA-B-NH2 + RCH=O ------>
Hb A valine Glucose
RCH=N-B- HbA (unstable aldimine )
(Schiff Base)

RCH=N=B HbA<------> RCH-NH-B- HbA

unstable aldimine ketoamine
Chemistry and Synthesis of
Glycohemoglobin

Reaction between glucose and Hgb A is

non-enzymatic, and occurs throughout
RBC life span
level of Hgb A1c is proportional to:
a. glucose concentration
b. RBC half-life - decreased in anemia or
recent blood loss
Forms of Glycated
hemoglobin

a. Labile - is the aldimine or Schiff base

its conc is affected by recent changes
in glucose conc, so it is removed before analysis
by pre-incubating the sample with a pH 5-6
buffer
b. Stable - ketoamine - this reflects mean
glucose level for past 2-3 mo. Its conc is NOT
affected by recent changes in plasma glucose
conc, unless they are acute increases
Clinical correlation of
results

a. reflects mean plasma glucose for previous 6-8

weeks. A 1% increase in glycohemoglobin is
equivalent to 30 mg/dL increase in glucose
levels
b. a direct relationship has been shown between
the mean plasma glucose level and the
development of diabetic complications
Clinical correlation of
results

It has been suggested that measurement

of Hgb A1c be used in the diagnosis of
diabetes in addition to its use in
monitoring diabetes.
Fall 2009- American Diabetes Association
recommends use of Hgb A1c assay to
diagnose diabetes.
Levels >/= 6.5% indicative of diabetes
Glycosylated hemoglobin
in other diseases-

a. hemolytic anemia - dec values due

to decreased RBC lifespan
b. liver disease - inc values, esp in
alcoholics (carbamyl derivatives)
c. patients on renal dialysis - may see
inc levels that dont correlate with
glucose, or may see dec levels - problem
is also due to carbamyl derivatives
Carbamylation of Hgb
Carbamylation of hemoglobin (Hb) and
other proteins occurs in uremia.
It results from the non-enzymatic
posttranslational modification of proteins
by isocyanic acid, the reactive form of
cyanate derived from the spontaneous
dissociation of urea.
Basically, urea breakdown products attach
to proteins in a manner similar to glucose.
Glycosylated hemoglobin
in other diseases-
D. Hemoglobinopathies - may bind glucose
differently, depending on # of lysine groups
in addition, life span of RBC is reduced, which
will cause falsely dec results.
For patients like these, the physician would
compare current results to patients
previous results rather than the reference
interval
In addition, physician may decide to measure
other glycated proteins, such as albumin
Fructosamine assay
Other Interfering Compounds
Other compounds can attach to the B
chain and interfere with A1c
measurement.
These compounds include: acetyl
groups, Vitamin C (ascorbic acid), E
or alcohol
The B chain altered by these compounds
may not be separated from Hgb A1c,
causing a falsely (Inc, Dec) result.
Answer
Estimated Average Glucose

The estimated average glucose result is

derived from the A1c result.
AGmg/dl = 28.7 A1C 46.7
Source: Diabetes Care August 2008 vol.
31 no. 8 1473-1478
This is an automated calculation that
would be programmed into the LIS.
Estimated Average Glucose

Providing eAGs regularly will put clinicians

and patients on the same page, give them
a common languagean Esperanto for
diabetesfor what are arguably confusing
numbers.
CAP Today, June 2009
Methods for measuring glycosylated
hemoglobin
Methods for measuring
glycosylated hemoglobin

Analytical techniques used include:

A. HPLC cation exchange
B. Immunoassay
C. Affinity Chromatography manual and
automated
D. Ion exchange manual
Glycosylated Hemoglobin
Fractions Measured

A. Hemoglobin A1c
B. Hemoglobin A1
C. Total Glycohemoglobin (Hgb A1, S1,
C1, F1, etc.)
Techniques and Fractions
Analytical Technique Glycosylated Fraction
Measured

HPLC- Ion exchange Hgb A1c

Affinity Total Glycohemoglobin

Chromatography- (Hgb A1, S1, C1)
manual or automated
Immunoassay Hgb A1c

Ion Exchange Stanbio Hgb A1

manual method
Methods for measuring
glycosylated hemoglobin -

Actual fraction measured (total A1, total

glycohemoglobin, hgb A1c) varies with
method
American Diabetes Association
recommends measuring Hgb A1c- shows
best correlation with changes in glucose
values
Methods for measuring
glycosylated hemoglobin -

Measurement of Total Hgb A1

This assay measures all Hgb A1 (Stanbio)
Advantages widely available, cheap, easy to
perform
Measure A1, but can convert result to Hgb A1c
Disadvantages false dec with Hgb S, C
False inc with Hgb F, other Hgb A1 formed by
alcohol abuse, salicylates, renal failure
Methods for measuring
glycosylated hemoglobin -

Measurement of Total Glycohemoglobin-

Measures any glucose modified hemoglobin
Advantages is not affected by Hgb variants,
can be automated, can be converted to true
Hgb A1c value
Disadvantages does not have same
correlation with glucose control in very poorly
controlled patients
Methods for measuring
glycosylated hemoglobin -

Measurement of True Hgb A1c

Measures only Hgb A1c
Advantages is not affected by Hgb
variants, is the assay recommended by
American Diabetes Assoc.
Disadvantages expensive, usually
harder to perform than other glycohgb
assays
Methods for measuring
glycosylated hemoglobin -

1. Chromatography on cation exchange

resin -
use Biorex 70 or carboxy methyl cellulose at pH
6.7
separation is based on charge - hgb are +
most kits contain mini-columns
Stanbio kit uses cation exchange resin in liquid
medium
Methods for measuring
glycosylated hemoglobin -

Procedure:
a. prepare hemolysate of whole blood,
apply to column, or mix with liquid resin
b. elute fraction of choice - Stanbio elutes
A1 fraction
c. make up total hemoglobin solution
d. read abs of both at 415 nm
e. calculate % glycohemoglobin
Methods for measuring
glycosylated hemoglobin -

Sources of error with cation

exchange columns or resin:
a. temperature critical
b. labile hgb fraction - inc results; must
remove
c. presence of abnormal hgb -
F- inc values elutes with Hgb A1
S, C - dec values bind tightly to resin
Methods for measuring
glycosylated hemoglobin -
Chromatography using HPLC -
uses the same resin and basic procedure
as previous method
shows less interference from temp and
better precision
advantage - allows separation and ID of
hgb variants - prev method does not
must incubate samples with pH 5-6 buffer
to remove labile fraction before analysis
Methods for measuring
glycosylated hemoglobin -
Procedure has been automated- can run
multiple samples with minimal operator
intervention
examples - BioRad, Variant, Diamat, Tosoh
Tosoh - used at PCMH; does not require pre-
incubation with acid buffer. Labile fraction is
removed on column
HPLC ion exchange method is considered
reference method- all other glycohemoglobin
methods are standardized to this method
This method can measure true Hgb A1c
Methods for measuring
glycosylated hemoglobin -

3. Affinity chromatography -
separation here is based on structural
differences, not charge
resin - phenylboronate linked to agarose
binding is based on affinity of boronate groups
for cis-diols
all glycosylated hemoglobins react with column,
not just A
this procedure measures total
glycohemoglobin (A1, S1, C1)
Methods for measuring
glycosylated hemoglobin -

Manual Assay Procedure:

a. prepare hemolysate and apply to column
b. elute non-glycated hgb first with buffer
containing asapragine, taurine, methionine,
MgCl2
elute glycohemoglobin with buffer containing
sorbitol- sorbitol has greater affinity for column
read abs of both fractions at 415 nm and
calculate % glycohemoglobin
Methods for measuring
glycosylated hemoglobin -

Interferences :
a. Hgb F - can falsely increase values
elutes with glycohemoglobin
Advantages of Affinity Method

1. labile hgb A1c, temp, sample storage,

variant hgbs - DO NOT affect results
2. procedure measures variants and
normal Hgb A
3. Results can be adjusted to Hgb A1c
values
Note: Reference interval is higher than
methods that measure A1c
Methods for measuring glycosylated
hemoglobin Affinity Chromatography

Glycated hgb on Abbott IMX -

is automated affinity chromatography method
glycohgb reacts with polyanionic affinity reagent
- negative complex forms with glycohemoglobin
complex is captured by a + charged solid matrix
flourophor color reagent added
Hgb quenches (reduces) flourescence
decrease in flour. Is proportional to conc
glycohgb
Methods for measuring
glycosylated hemoglobin -

Manufacturer has standardized this

method to HPLC Hgb A1c values -
instrument measures total glycohgb, but
reports Hgb A1c
Methods for measuring glycosylated
hemoglobin - Immunoassays

DCA 2000 - measures Hgb A1c

uses whole blood collected in glass
capillary and added to reagent cartridge
measures total Hgb using
cyanmethemoglobin method
measures A1c with mononclonal ab to
terminal portion of B chain
indicator system is latex agglutination
Methods for measuring
glycosylated hemoglobin -

DCA 2000-
Is competitive binding procedure
Hgb A1c in patient sample competes with A1c
polymer for binding sites on ab (limited amount)
patient A1c-ab complex is soluble; polymer A1c-
ab complex is not
polymer A1c-ab complex scatters light and
increases abs
Methods for measuring
glycosylated hemoglobin -

Formation of soluble patient A1c-

ab complex decreases abs
dec in abs is proportional to patient A1c
procedure does not measure non-
glycated hgb or hgb F1c. If hgb F conc
>10%, will get falsely low results
Procedure does measure other glycohgb,
including S and C
DCA 2000 Hgb A1C Procedure

A1C antibody

A1C polymer (reagent)

Patient A1C
 Abs. = 0.250

Light Scatter

Hgb A1C = 12%

 Abs. = 0.500

Light Scatter

Hgb A1C = 6%
Methods for measuring glycosylated
hemoglobin - Isoelectric focusing

Gives excellent results but needs special

equip- not good for routine analysis
Methods for measuring glycosylated
hemoglobin -

Manual glycohemoglobin methods are being

replaced by automated methods- see better
precision, control over interferences (temp,
labile fraction, variant hgbs)
Hospital labs originally used TOSOH HPLC
ion exchange method, but are now switching to
immunoassay
Physicians Office labs (POL)- many use
DCA 2000 instrument (immunoassay)
Reference Intervals
Reference intervals for
glycohemoglobin
very method dependent
patient results must be done by same lab
all the time to be of value
Diabetes Complications and Control Trial
(DCCT) recommends using range of 4-6%
American Diabetes Association
recommends maintaining
glycohemoglobin values of <7% for good
glycemic control
Reference intervals for
glycohemoglobin

National Glycohemoglobin Standardization

Project (NGSP)- has helped manufacturers to
standardize their methods to one set of values -
eg. Hgb A1c values
recommend that procedure have %CV of </=
5%
in 2007 most labs reported glycohemoglobin
values as %A1c
99% of labs reported using a method certified
by NGSP
Summary
Summary

Measurement of stable hgb A1c provides a

valuable monitor of efficacy/success of
treatment in diabetics.
Thanks to improved standardization of various
glycohemoglobin assays, Hgb A1c is now also
used to diagnose diabetes.
Serves as a predictor of long term complications
of diabetes
The clinical lab must provide results for Hgb A1c
which have good precision and accuracy
Summary

Physician must be familiar with what fraction of

glycohemoglobin is actually measured, and use
this info to interpret patient results.
Hgb A1C measurements are usually performed
4x/year.
Summary

When Hgb A1c measurements are used to

monitor diabetes treatment, it is
important that the same analytical
technique be used so that each new result
can be compared to the previous ones.
This allows doctor to see if A1c is
increasing, which would indicate
treatment failure and the need for more
aggressive treatment.
Summary

If the laboratory that is doing A1C testing

decides to change its analytical method, it
must run patient samples on both current
method and proposed new method and
report both results.
This allows doctor to compare the results
and become familiar with the new
methods results.
Summary

Patients with variant Hgbs (homozygous

S, C) may give falsely low results
compare current results to patients
previous results instead of reference
interval
Summary

The estimated average glucose result

(eAG) is now being reported along with
the Hgb A1c result.
It is thought to give patients a better
understanding of their long-term glucose
control, because they can relate this result
with the results of SBGM more easily.
Answer

Answer:
If a B chain structure altered by
substances other than glucose cannot be
separated from Hgb A1c, the result would
be falsely increased.

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