California
Association
for
Medical Laboratory Technology
Distance Learning Program
|
Hemoglobin
A1c Testing of Patients with Hemoglobinopathies Course
Number: DL-973 © California Association
for Medical Laboratory Technology. CAMLT is approved by the California Department
of Health Services 1895 Mowry Ave, Suite 112 Notification of Distance Learning Deadline This is a reminder that all the continuing education units required to renew your license must be earned no later than the expiration date printed on your license. If some of your units are made up of Distance Learning courses, please allow yourself enough time to retake the test in the event you do not pass on the first attempt. CAMLT urges you to earn your CE units early! |
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Hemoglobin A1c Testing of Patients with Hemoglobinopathies
OBJECTIVES:On completion of this course the participant will be able to:
-
1. describe hemoglobin A1 structure and function
2. discuss the formation of hemoglobin A1c (HbA1c) and its relationship to blood glucose levels
3. give the normal HbA1c range and the recommended percentage for diabetics
4. outline the chronic complications of diabetes due to long term increase in blood glucose
5. list the names of three HbA1c testing methodologies
6. state examples for each of the HbA1c methodologies
7. describe how hemoglobinopathies may give erroneous results in HbA1c testing
INTRODUCTION
Hemoglobin A1c is the most useful single index of blood glucose control available
to diabetics. Increased HbA1c is closely linked to risk of long-term microvascular
diabetic complications (1). HbA1c is measured in the laboratory using a variety
of methods. The presence of hemoglobinopathies in a patient presents a confounder
to HbA1c testing, yielding erroneous laboratory test results. Beginning with
a description of HbA1c and its relationship to blood glucose followed by methods
of testing for HbA1c in the laboratory, this course focuses on the problematic
aspects of testing for HbA1c in a patient with a hemoglobinopathy using each
of these methods. General conclusions, limitations, and recommendations for
testing are given.
DESCRIPTION OF HbA1c
Hemoglobin, found in red blood cells, carries oxygen to the tissues and facilitates
removal of carbon dioxide from the body. Hemoglobin is a tetrameric molecule
made up of four globin chains attached to four heme groups. The majority of
hemoglobin in normal adults is designated as hemoglobin A, or A1, which contains
two alpha and two beta chains (Figure 1). Hemoglobin A1c is an in vivo glycosylated
form of hemoglobin A1 with a glucose molecule irreversibly attached to the N
terminal amino group of the beta chain.
RELATIONSHIP OF GLUCOSE AND HbA1c
In the erythrocytes, the relative amount of HbA1 converted to stable HbA1c increases
with the average concentration of glucose in the blood. The conversion to stable
HbA1c is limited by the erythrocyte’s life span of approximately 100 to
120 days. The level of HbA1c at any time is contributed to by all circulating
erythrocytes, from the oldest to the youngest. As the older RBCs die off, the
younger ones contribute more to the level of HbA1c, meaning that the blood glucose
levels in the preceding 30 days contribute more to the HbA1c (approximately
50%) than the levels from 90-120 days. As a result, HbA1c reflects the blood
glucose level during the preceding two to three months. HbA1c is thus suitable
to monitor long-term blood glucose control in individuals with diabetes mellitus
(3).
The correlation between HbA1c and mean plasma glucose over the past two to three
months is shown in Table I.
TABLE I. Correlation
Between HbA1c and Mean Plasma Glucose |
||
HbA1c (%) |
Glucose (mg/dL) |
Glucose (mmol/L) |
4 |
65 |
3.5 |
5 |
100 |
5.5 |
6 |
135 |
7.5 |
7 |
170 |
9.5 |
8 |
205 |
11.5 |
9 |
240 |
13.5 |
10 |
275 |
15.5 |
11 |
310 |
17.5 |
12 |
345 |
19.5 |
USE OF HbA1c IN MANAGEMENT OF DIABETES
The normal range of HbA1c is 4 to 5.9% of the total hemoglobin. In diabetics
the higher the average blood glucose level over a two to three month period,
the higher the percentage of HbA1c. Measuring HbA1c levels gives a view of the
blood sugar control over that period of time, whereas day to day glucose levels
may fluctuate widely. With HbA1c as a guideline, the physician can better evaluate
the diabetic’s glucose control and can make adjustments in treatment.
The American Diabetes Association recommends that diabetics have a goal of HbA1c
less than 7.0%. The International Diabetes Federation and the American College
of Endocrinology suggest a lower goal of 6.5% (4).
Control of glucose levels is important to help decrease chronic complications
of diabetes. These complications are related to blood vessel diseases. These
vascular diseases are divided into microvascular diseases and macrovascular
diseases.
Microvascular diseases affect the eyes, kidneys and
nerves. High blood glucose causes thickening of capillary walls. The capillary
walls become weaker and more permeable.
Eye Complications–Diabetic Retinopathy:
Retinopathy occurs in about 13% of diabetic patients after five years; 50% to 80% after ten to fifteen years respectively. Weakened defective capillaries, release of vasoproliferative factors and increased intraluminal pressure cause microaneurysms to form in the retina. Microaneurysms lead to increased vascular permeability and leaking of fluid and red cells, causing macular edema and intraretinal hemorrhages. This condition threatens central vision. The next stage of eye complications is formation of new, brittle blood vessels (neovascularization). Spontaneous bleeding from these vessels leads to vitreous hemorrhages, further impairing vision. Further disease includes retinal scarring and retinal detachment eventually causing blindness
.
Kidney Damage–Diabetic Nephropathy
Endothelial damage in the kidney leads to increased glomerular permeability to macromolecules. Further damage results in glomerular sclerosis. The first sign of kidney disease is hypertension, coincident with or shortly followed by microalbuminuria. Later the kidneys lose their ability to cleanse and filter the blood, eventually requiring kidney dialysis or kidney transplant
.
Nerve Damage–Diabetic Neuropathy
In diabetes the blood flow to the nerves is limited, leading to damage that includes demyelination. Weakness, burning, pain and diminished sensation occur in the extremities. Eventually complete loss of sensation in the feet may lead to the patient’s being unaware of injuries. Because of poor circulation these injuries may not heal, become infected, and lead to gangrene which requires amputation. Nerve damage also affects the ability of men to get an erection. Diabetic neuropathy may affect nerves to the stomach and intestine, causing nausea, weight loss and diarrhea.
Macrovascular Disease affects the heart and larger blood vessels. Diabetes accelerates atherosclerosis, leading to coronary heart disease, strokes and pain in the lower extremities due to decreased blood supply.
VALUE OF DECREASING BLOOD GLUCOSE LEVELSHbA1c is a valuable tool used by the physician and patient to prevent chronic complications of diabetes. Studies have shown a 10% decrease in relative risk for microvascular disease for every 1% reduction in HbA1c. The Diabetes Control and Complications Trial (4) included 1,441 people with type 1 diabetes (formerly called insulin dependent diabetes mellitus or juvenile onset diabetes). Of these subjects half had no retinopathy, normal albumin excretion and diabetes for less than five years. The other half had mild-to-moderate retinopathy with normal kidney tests or only microalbuminuria. The subjects were randomly divided into conventional or intensive therapy groups. The conventional treatment consisted of no more than two insulin injections a day with blood glucose monitoring twice a day. They were seen every two to three months. The intensive treatment group either had insulin pumps or three or more injections per day. Blood sugar was done three to four times a day. They were seen every month. The conventional group averaged an HbA1c of 9.1%; the intensive group averaged 7.2%. The intensive groups had 70% reduction of retinopathy; 60% less microalbuminuria; and 64% reduction in clinical neuropathy compared to the conventional therapy group. Other studies have corroborated these results.
HbA1c TESTING METHODOLOGIES (5)
Currently, there are five available methods of testing for HbA1c in the laboratory.
The methods use separation based on:
- charge differences–ion exchange chromatography, HPLC, electrophoresis and isoelectric focusing
- structural differences–affinity chromatography and immunoassay
- chemical analysis–photometry, spectrophotometry
- Charge differences:
1. Boronate affinity chromatography is used by greater than 50% of laboratories in the United States. The boronic acid reacts with the cis-diol groups of glucose bound to hemoglobin to form a reversible complex, thus selectively holding the glycated hemoglobin on the column. The non-glycated hemoglobin does not bind. Sorbitol is then added to dissociate the complex and elute the glycated hemoglobin. Absorbance of the bound and unbound fractions is used to calculate the percentage of glycated hemoglobin.
2. Cation or ion exchange high performance liquid chromatography (HPLC) is used by 30% of laboratories. Phosphate buffers of increasing ionic strength are used for stepped elution of the hemoglobins, which are detected by absorbance at 415 and 690 nm, enabling calculation of the percentage of HbA1c in the sample.
3. Electrophoresis is used by less than 5% of laboratories.
- Structural differences
4. Immunoassay is used by 15% of laboratories. An antibody to a specific antigen on a glycated hemoglobin molecule is used to detect the amount of HbA1c. An antibody to the glycated amino terminus of beta chains is one example.
- Chemical analysis
5. Electrospray mass spectrometry is rarely used by laboratories due to high cost of the test system (5).
Hemoglobinopathies are a group of diseases resulting from a defect in structure of the hemoglobin molecule. Over 700 hemoglobin variants have been discovered to date; most are the result of point mutations in the globin chains. There are 16 million diabetics in the United States, of which more than 150,000 have a hemoglobin variant, the most common of which are Hemoglobin S and Hemoglobin C (6). Hemoglobinopathies are of concern because the presence of some variants will affect the accuracy of HbA1c measurements. This concern is discussed below. The more common hemoglobinopathies, HbS and HbC do not interfere with assays for HbA1c. However, HbA1c levels are affected by severe anemias. If patients with these hemoglobinopathies have significant anemia, the HbA1c levels could be low.
RESULTS OF TESTING FOR HbA1c IN THE PRESENCE OF A HEMOGLOBINOPATHY
The five methods of testing for HbA1c and their results in the presence of a hemoglobinopathy appear below:
1. Boronate affinity: Boronate affinity has shown the least interference from hemoglobin variants.
2. Cation Exchange Chromatography: Three situations exist. The first situation occurs when the native hemoglobin variant (non-glycated) co-elutes with HbA1c resulting in a gross overestimation of HbA1c values. These excess values may be as high as 54%. Examples include Hb Raleigh (beta 1 Val‡Ala) and Hb Sherwood Forest (beta 104 Arg‡Thr). The second situation occurs when the glycated hemoglobin variant co-elutes with HbA1c and the non-glycated hemoglobin variant is separated from HbA resulting in an overestimation of HbA1c, but to a lesser degree than the first situation. The third situation occurs when the hemoglobin variant co-elutes with HbA1, whereas the glycated hemoglobin variant separates from HbA1c. This results in an underestimation of HbA1c. Hb D is an example of a hemoglobin variant which causes this.
3. Electrophoresis: The co-migration of hemoglobin variants or derivatives with either HbA or HbA1c interferes with the determination of HbA1c. For example, HbF migrates with HbA1c resulting in an increased HbA1c value.
4. Immunoassay: HbF, Hb Graz and Hb Raleigh have been shown to decrease levels of HbA1c. Immunoassay tests use an antibody specific for the glycated amino terminus of b globin. When the amino terminus is altered by an amino acid substitution, the antibody may not recognize the altered structure (Hb Graz and Hb Raleigh). Fetal Hb (HbF) contains g chains instead of b chains. The amino terminal end, being different from HbA, is not recognized by the antibody.
5. Electrospray Mass Spectrometry (ES-MS) ES-MS appears to provide a means of measuring total glycated hemoglobin unaffected by the genetic chemical modifications to the hemoglobin molecule. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) proposed using it as a reference method for the detection of glycated hemoglobin (1, 6).
Case Study
A 45-year-old Cambodian male with a five-year history of diabetes had been tested
three times a year for HbA1c. The range of measurements by immunoassay technique
varied between 5.5 and 6.6%. The laboratory initiated a cation exchange HPLC
procedure. His HbA1c by the new method was 7.5%. The HbA1c on the same specimen
by the old method was 6.6%. Other blood values on the patient were hematocrit
= 40%, MCV = 78 fl, MCH = 25 pg. The laboratory suspected a hemoglobinopathy.
Electrophoresis of the patient’s blood sample revealed 18 % HbF. The cause
of this HbF (a2g2) elevation is most likely due to hereditary persistence
of fetal hemoglobin (HPFH). HPFH can be due either to deletion of the d
or b globin genes on chromosome 11 or to point mutations in the promoter
of one of the g-globin genes.
Discussion: Immunoassay uses an antibody specific for the glycated amino terminal
end of b chain. Since the antibody does not recognize the glycated amino
terminal end of the g chain, the HbA1c level is falsely low. HPLC method
determines HbA1c levels by comparing the areas of the HbA1c and HbA peaks in
the HPLC chromatogram. The glycated and non-glycated forms of HbF migrate differently
from HbA1c and HbA so the assay is not affected by the presence of high levels
of HbF. Using the falsely low results of the immunoassay for HBA1c meant that
the patient’s diabetes had not been adequately treated or controlled.
CONCLUSION AND RECOMMENDATIONS
Based on the above discussion, it appears that boronate affinity chromatography
and ES-MS are the best methods for accurately detecting HbA1c. However, there
are limitations. ES-MS is expensive and cannot detect mean glycemia in the presence
of those hemoglobin variants that have altered rates of glycation (7) or a shortened
erythrocyte life span (6). Boronate affinity chromatography cannot detect that
a variant hemoglobin is present (8). In both methods, results are unlikely to
accurately reflect long-term glycemic control due to pathological conditions
that affect the formation and turnover of glycated hemoglobin in vivo (6).
General recommendations regarding testing for HbA1c include evaluating samples
with a glycated hemoglobin value of greater than 15%. This includes examining
chromatographs manually and obtaining the clinical history of the patient (6).
Samples with clinically silent hemoglobin variants should be analyzed by a second
method with a different assay principle, preferably boronate affinity (9).
REFERENCES
1. Saudek CD, Kalyani RR, Derr RL. Assessment of Glycemia in Diabetes Mellitus:
Hemoglobin A1c. Journal of the Association of Physicians of India. April 2005;53:299-305.
2. Bishop ML, Duben-Engelkirk JL, Fody EP. Clinical Chemistry (4th ed., p281).
Philadelphia: Lippincott Williams & Wilkins.
3. Roche. (2004-12). HbA1c II (9th vol.) [Package Insert]. Indianapolis, IN.
4. http://www.clevelandclinicmeded.com/diseasemanagement/endocrinology/microvascular/microvascular.htm
5. Lee KF, Szeto YT, Benzie IFF. Glycohaemoglobin measurement: methodological
differences in relation to interference by urea. Acta Diabetol 2002;39:35-9.
6. Bry L, Chen PC, Sacks DB. Effects of Hemoglobin Variants and Chemically Modified
Derivatives on Assays for Glycohemoglobin. Clinical Chemistry. 2001;47:2:153-163.
7. Bisse E, Schauber C, Zorn N, Epting T, Eigel A, Van Dorsselaer A, et al.
Hemoglobin Görwihl [_2_25(a2)Pro‡Ala], an electrophoretically silent
variant with impaired glycation. Clinical Chemistry. 2003;49:137-143.
8. Sacks, DB. Hemoglobin Variants and Hemoglobin A1c Analysis: Problem Solved?
Clinical Chemistry. 2003;49:1245-1247.
9. Schnedl WJ, et al. Determination of glycated hemoglobin in clinically silent
hemoglobin variants. Diabetes/Metabolism Research and Reviews. 2004;20:460-465.
Link to On-line REGISTRATION, PAYMENT and QUIZ to submit for credit
1. HbA1c is used to
a. monitor long-term blood glucose control in individuals with
diabetes mellitus
b. monitor the course of a hemoglobinopathy
c. monitor an individual’s erythrocyte life span
d. monitor long-term blood fructose control in individuals with
a pathologic condition
2. HbA1c is produced when
a. hemoglobin loses one of its globin chains
b. glucose attaches to the N-terminal amino group of the beta chain
of hemoglobin
c. hemoglobin binds oxygen to its four heme groups
d. there is a defect in the structure of the hemoglobin molecule
3. Which three laboratory test methods are used most commonly to test for HbA1c
in the United States?
a. electrospray mass spectrometry, immunoassay, boronate affinity
chromatography
b. electrophoresis, boronate affinity, high performance liquid
chromatography
c. boronate affinity chromatography, high performance liquid chromatography,
immunoassay
d. electrophoresis, immunoassay, boronate affinity chromatography
4. The presence of a Hemoglobinopathy may confound a HbA1c test because
a. individuals with a hemoglobinopathy do not have hemoglobin
b. individuals with diabetes and a hemoglobinopathy do not undergo
HbA1c testing
c. a vast majority of individuals with diabetes have a hemoglobinopathy
d. the presence of a hemoglobin variant may affect the accuracy
of HbA1c measurements
5. Which laboratory test method appears to provide a means of measuring total
glycated hemoglobin unaffected by the genetic chemical modifications to the
hemoglobin molecule?
a. electrospray mass spectrometry
b. immunoassay
c. electrophoresis
d. cation exchange chromatography
6. A patient has a Hemoglobin D hemoglobinopathy. Which of the following would
not be an appropriate HbA1c test?
a. boronate affinity
b. cation exchange chromatography
c. immunoassay
d. electrospray mass spectrometry
7. The American Diabetes Association recommends that HbA1c be below
a. 6.5%
b. 7.5%
c. 5.5%
d. 7.0%
8. Microvascular diseases associated with long term increased glucose levels
include all the following organs except
a. kidney
b. brain
c. eye
d. nerves
9. HbF is not recognized in immunoassay because
a. there is an amino acid substitution at the amino terminal end
of the beta globin
b. the antibody is directed against the alpha chain terminus
c. HbF has gamma chains instead of beta chains
d. the folding of HbF hides the amino terminal end of the globin
chain
10. Testing for HbA1c reflects the mean blood glucose for the previous
a. two to three weeks
b. 100 to 120 days
c. six months
d. two to three months