Abril - 2017

avares

342

ssoc

ras

2017; 63(4):341-346

For a reliable HbA1c result, some possible interferers

must be considered, including the labile fraction and sam-

ple preservation.

9,10

In addition to the variables mentioned

above, some factors such as hyperbilirubinemia, hypertri-

glyceridemia, hyperuremia, chronic alcoholism, chronic

ingestion of salicylates and opiate dependence can signifi-

cantly alter HbA1c results, producing falsely increased

results.

False-low results are observed in the presence of

large amounts of vitamins C and E, which are described as

inhibitory factors for hemoglobin glycation.

2,6,7,9,11,12

Hemoglobinopathies and hemoglobin variants are

also interfering factors, the latter being characterized by

changes in hemoglobin structure caused by differences

in the amino acid sequence of globin chains.

2,7,9

Hemoglobin (Hb) is a spheroidal molecule, which is

located inside the red blood cells and whose main func-

tion is the transport of oxygen to the tissues.

Its structure

is composed of four subunits formed by two pairs of

identical polypeptide chains, called globins, each bound

to a heme group. The hemoglobin profile of a normal

adult contains about 97% of HbA (two

-globin chains

and two

-globin chains, represented as

2), 2% of

HbA2 (

22) and 1% of fetal Hb (HbF, represented as

2),

which is the predominant Hb during intrauterine life.

HbA1c is a subtype derived from the binding of HbA1

by means of an irreversible non-enzymatic reaction be-

tween blood glucose and the N-terminal amino acid valine

of the beta chain.

The change in hemoglobin levels observed in hetero-

zygous individuals may interfere with test results, which

is the case of HbA1c.

Thus, our objective in the present

study was to verify A1c levels in patients heterozygous for

hemoglobin variants.

ethod

This is an experimental study, based on the comparison

of HbA1c test results from two different populations: a

test group represented by individuals heterozygous for

hemoglobin variants (AS and AC) and a control group

consisting of people with electrophoretic profile AA con-

firmed by hemoglobin electrophoresis at alkaline pH.

Both populations were required to meet the inclusion

criteria: Normal levels of fasting glucose, hemoglobin,

urea and triglycerides, bilirubin > 20 mg/dL, and non-use

of acetylsalicylic acid (ASA). All of the above factors, if

changed, can trigger falsely high or low results.

2,6,7,9,11,12

In addition to the HbA1c test, other tests were performed

in the two populations to contribute to the interpretation

of HbA1c, such as: fasting blood glucose and blood counts,

as well as tests to identify possible interferers: urea and

triglycerides. Still assessing interferences, we checked if the

samples were icteric, as bilirubin levels above 20 mg/dL can

cause falsely high HbA1c results. We paid special attention

to ascorbic acid, which influences the assessment of HbA1c.

It is a volatile compound and, after gathering sampling,

requires a period of 90 minutes to be separated from the

rest of the blood sample and proceed to dosing.

2,7,9

The heterozygous participants were selected from the

Clinical Laboratory (LAC, in the Portuguese acronym)

database at Pontifícia Universidade Católica de Goiás (PUC

Goiás), and invited to participate in the research. A Free

and Informed Consent Form with the Code of Approval

by the Ethics Committee (no. 4.1.1.00-0 PUC Goiás) was

promptly presented to them.

Whole blood samples were collected in three tubes stored

and distributed as follows: a tube containing EDTA (ethyl-

enediaminetetraacetic acid) for blood counts and HbA1c; a

fluoride tube for blood glucose dosing; and a tube for serum

separation and dosing of triglycerides and urea.

HbA1c was measured using a column method of ion-

-exchange resins to separate HbA1c, which was subsequent-

ly read on a CELM (Cia. Equipadora de Laboratórios Mo-

dernos) spectrophotometer, with the aid of the HbA1c

dosing kit Gold Analisa Diagnóstica Ltda

(Lot: 30031).

HbA1c levels read by column chromatography are

equivalent to those obtained with HPLC, which is certified

by the NGSP, and can be converted to standard HPLC

method from the International Federation of Clinical

Chemistry (IFCC) using the formula: IFCC=(10.93

NGSP)-23.50.

16,17

Blood glucose levels were measured using a glucose

oxidase method, ELITechGroup

(Lot: 13-0096), while

the blood counts were assessed with Sysmex

XE-2100

automated hematology analyzer.

To eliminate the interferers from the HbA1c test, urea,

triglycerides and bilirubin were dosed in the patients’ se-

rum. An enzymatic colorimetric test was used to estimate

serum urea and bilirubin, and peroxidase-oxidase for the

analysis of triglycerides, whose dosages were measured

using Selectra

XL – Vitalab. In order to validate the fast-

ing blood glucose, urea, triglycerides and bilirubin tests,

three control sera were used, namely positive and normal

controls branded ELITechGroup

(Lot: 5564 A and Lot:

3575 B, respectively) and the control serum of the Na-

tional Program of Quality Control (PNCQ) (Lot: 3575).

The study divided groups by age group according to

Ministry of Health criteria: children and adolescents aged

< 20 years, adults aged 20 to 60 years.

We evaluated 50 individuals heterozygous for hemoglo-

bin variants and 50 controls fromAugust 2013 toMay 2014.