C(

STORE AT 15.3OOC

HEMOGLOBIN 41C

R*s"BjiJffiX,@
pRfNCtpLE
OF THE IT,|ETHOD
-where rhe raoire rraction rs eriminated,

Chromatographic

HEII4OGLOBIN 41C
_

spectrophotometric ION EXCHANGE

I|:ill,ilff"'. j'fl h1l#li{i:il:fff 'll,ii',il'iu"'li'ii'i'. ol, oittltt p'nJt#il::Ht'"??:'i;3 nm The estimation "'n';J'illlltg fl ';;il.;i;i;ffi;s;;;;THil,lililfi THlrflh:,il1:: ji jT
CONTENTS
'l x30mL

ffi!r%'#i:%1:,j;$'fi'l',

-tq,+:L:^\grgr.shly and bt it mrnutes.

lo,Tn a

stand at room ten This hemorysate wiil be used in steps 6 rrtl.r1rt" preparation (Notes 2 and 3)

ror

10-15

[r,ffi*

5.

the upper cap of the column and then snap tre bp ofi

:e

,rff:nn*,*

Using the flat end of rhe upper disc oown ro he resin i care not to compiess it. drain comptetetv

flT^g:l*l rritl!.jrin

o
ix30mL
1 x240 nL 4x450mL 1x100

Separation and Reading of HbAr. fraction uarefully pipette on

the upper filter:

1x50mL
'I

x 450 mL
1

x20

In order to Or.'n

rny

rrr#

left

bove

tnt'Pp" oi",

p,prn.-

1.
2.

coMPOSfTfoN
soorum azide 0.95 g/1. Xeagent. phosphate

Reagent. potassium p htalate 50 mmol/l, detergent 5 g/1, pH 5.0,

bu1

with phosphate b Use on,y

I i*::l#;:tmr: ii;fili ;; t*j,Tff;ff yi

amount

of resin equitibrated q'!su ri"iilili?;,-etghted microc;il ;) ;;l[]l,i;ii,,,lji1iilJ::Tj;: ;qrv'

,l::,Hifl:l;,
Reading of Hbrour.
1

;.tiri,o

'",t.,

ri,i]l;T;: $f_,lli.:,,f

5ifi:,fl?

Store at .15-300C.

STORAGE

'1. Pipette into a test tube:

Reagents are stable until thr tightly ctosed aniliffiffie^,e,:ltrydate shown on the labet when srored is prevented during their use. fndications

of particulate material, turbidity. Microcolumns (4): Absence of buffer over th. orin irO.

Reagents: presence

"t

o.r.nr*""all0n

,,

hour

l,5,H,y,llnnrrl distilled watei(R"nr.,^, _..-. ,nururAL,,.

\,

,,,oEJi. J*[** "':s oulurodllce

CALCULATIONS

i-}i absorbance is rrte

(A) ar 415 nm against

stable

lor at least one

ADDITIONAL EQUIPMENT
Spectrophotometer or photomeier with a 415 nm filter (405425)

The HbArc relative concentr:ation in the sample is calculated rollowing general using the formula:

Arur.

X Vgunrc

,fi fooo Hemoglobin

useo as

ore

cotlectedo,

r-.olf

anticoagulants.

Arc is stable frrr 7 days

I::r::,
at
2-8oc. Heparin or EDTA may be

AHbtorAL X Vscrc;,.r

x'100 = o/oHbAtc

ii'dfl;[;'Hi#['HH,; il :.T;]:,T: ilJ: jrTLi,#r,I:

Hemorysatepreparation

jT:r:-tt'.,ff
Erimination

1.
2.

Bring the columns anrJ reagents to room temperature (21-26c) (Note

1).

Pinptlo i^+^ a +^^r r. , Pipette into ^ test tube:

The resurts obtained

\,lational

il,13ti',iy':5T:;:[":i*,#[i.'Jd;,*:ffi f :l1i#; $3,1?J:1m.1i.,.:llll:*:ir,,yf il;.J"fi [#*:: TJ;J,li8.itff J?x ly,.r ;;;;r;;l ;:t d;eXTi,:.r.r1 'fj;'.fl.;,?J::ffi il:l:ffi,yJ:{l?i,ifi
in te m a tio
n a

Gtycoh.r*i"ui, "it"*T:I^1.]hod are

*nnts

equivarenr

to a

US

HbArrlFcc (mmol/mol) =

10.g3 x HbArrNGSp-DC

cT

(o/o\

_23

REFERENCE VALUES
The following cut-off points have been established by the Diabetes Control and Complications Trial Research Group (DCCT) and have been adopted by many countries for a reference population (Non diabetic) and for the evaluation of the degree blood glucose control in diabetic patientsa,s.

HbAt. levels are a valuable adiunct to glucose determinations in the assessment and follow up of individuals with diabetes mellitus, providing

much more reliable information for glycemia monitoring than

do

determinations of glucose. Numerous studies have shown that diabetes related complications may be reduced by the long term monitoring and
tight control of blood glucose levels.

NGSP-DCCT

fk)

(mmol/mol)
20-48
42-53 53-64
>64

IFCC

Reference values / Degree of conkol Non Diabetic Goal Good Control

The HbAr. concentration may also be a useful tool in the diagnosis of

diabeteslo.

4,0 - 6.5 6.0 - 7.0 7.0 - 8.0 > 8.0

Clinical diagnosis should not be made on the findings of

result, but should integrate both clinical and laboratory data.

a single

test

Action suggested
1.

NOTES
The obtained values are temperature-independent when working in the recommended interval (21-26"C).lf working temperature is out of
range, multiply the obtained value by the corresponding factor showed in the following table: Working

QUALITY CONTROL
It is
recommended to use the Hemoglobin Ar. Controls, Normal (cod. '18001) and Elevated (cod. '18002), to verify the performance of the

measurement orocedure.

Each laboratory should establish its own internal Quality Control scheme and procedures for conective action if controls do not recover within the
acceotable tolerances. M

temperature 1B-200C 27-300C

Factor
1,15 0.90

The storage of the columns may lead to an excessive packing of the

resin, diminishing the flow rate and lengthening the elution. To avoid it, invert the column, do a gentle spin movement, let it stand upside down for 10 minutes, then place it back to its upright position and let the resin settle for a few minutes before of opening the column.
3.

ETROLOGICAL CHARACTERISTICS
4.0%=20 mmol/mol.
o/o

Detection limit Lowerthan

Linearity limit: At least

17 .0

= 162 mmol/mol.

Repeatibility (within run):

Some air bubbles may occasionally appear inside the resin bed. Their presence does not alter the test performance.

Concentration CV mmol/mol | 5.4 "/" | 6.3 o/o | 9.9 % = 85 mmol/mol I

l\4ean
7

.2 ok = 55

25 25

BIBLIOGRAPHY
Abraham EC. New less temperature-sensitive Biss6 microchromatographic method for the separation and quantitation of
glycosylated hemoglobins using
Chromatog 1985; 344: 81'91.

E,

Reproducibility (run to run):

Mean Concentration

non-cyanide buffer system. J

mmol/mol | 7.3o/o | 9.9%=85mmolimol I 5.9% |

7.2ok = 55

25 25

Hoelzel W,

et al. IFCC reference

system for measurement of

standardization a method-

hemoglobin A1c

in human blood and the national

schemes in the United States, Japan, and Sweden:

comparison study. C/ln Chem2004,50: 166-174.

Trueness: Results obtained with this method did not show systematic differences when compared with reference methods. Details of the
comparison experiments are available on request. Interferences: Bilirubin (20 mg/dL)and llpemia (triglycerides 10 g/L)do not interfere, Some drugs and other substances may interfereo.

et al. 2010 Consensus statement on the worldwide standardization of the hemoglobin Ar. measurement C/tn Chem Lab Med 2010: 48.775-776.
Hanas R,
4.

Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th

ed. Burtis CA, Ashwood ER, Bruns DE. WB Saunders Co, 2005.

In the ionic exchange chromatographic methods, the presence of hemoglobin C or S in the sample may slightly alter results, but
differences are not clinically significantT. Other hemoglobin variants like HbE, HbF, carbamyl-Hb and acetyl-Hb can interfereT'8. The incubation with Reagent (1)eliminates the interference due to HbArc-labile

The Diabetes Control and Compltcations Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long term complications in insulin-dependent diabetes mellitus. N EnglJ Med 1993; 329: 977-986.
Young DS. Effects of drugs on clinical laboratory tests, 5th ed. Press, 2000.
7.

ln hemolytic anemia, iron deficiency anemia and transfusion, the average age of erythrocytes is altered. Caution should be used when
interpreting the HbArc results from patients with these conditions.

MCC
eight

Roberts WL

et al. Effects of hemoglobin C and S traits on

DIAGNOSTIC CHARACTERISTICS
HbArc is the product of the ineversible condensation of glucose with the Nterminal residue of the B-chain of hemoglobin A.

glycohemoglobin methods. Clin Chem 20Q2. 48: 383-385.

Bry L, Chen PC, Sacks DB. Effects of hemoglobin vanants anc chemically modified derivatives on assays for glycohemoglobin. C/in
Chem 2001 ,47: 1 53-1 63. Nathan DM, et al. Translating the A1C assay into estimated average glucose values. Diabetes Care 2008, 31 . I 47 3-1 47 8.
10.

The HbArc concentration in blood is directly proportional to the mean

concentration of glucose prevailing in the previous 6-8 weeks, equivalent to the lifetime of the erythrocytes4, and the estimated average glucose (eAG) during this period can be calculated with the formulas belowe. eAG (mg/dl)=28.7 x HbATTNGSP-DCCI (o/o)-46'7 eAG (mmol/L) = 1.59 x HbAr'NGSP-DCCT (%)

Nathan DM, et al. International Expert Committee report on the role of

the HbAIC assay in the diagnosis of diabetes. Diabetes care 2009:

32:1327-1334.

2 59

eAG (mg/dL) = 2.64 x HbAr.lFCC (mmol/mol) + 15'0 eAG (mmol/L) = 0.146 x HbAr-IFCC (mmol/mol)+ 0.843