HEMOGLOBINA

GLICOSILADA
Mtodo Inmunoturbidimtrico

REACTIVOS
Fabricante: RAL TECNICA PARA EL LABORATORIO, S.A. Componentes y concentraciones
Av. Mare de Du de Montserrat 51 R1: tampn 20 mmol/L ltex 0.14 %
08970 Sant Joan Desp (Barcelona) - Espaa R2: tampn 10 mmol/L, anticuerpo monoclonal HbA1c antihumano de ratn (5.5
mg/dL), anticuerpo policlonal IgG anti-ratn de cabra (67 mg/dL), estabilizantes
IVD PARA USO DIAGNSTICO IN VITRO R3: hemolizante
A una temperatura de 2 - 8 C, los reactivos despus de abiertos se conservan al
menos durante un mes siempre que se evite la contaminacin y la evaporacin.
CONSERVAR A 2 - 8 C. Los reactivos estn listos para su uso.
Advertencias y medidas de precaucin
(S25) Evitar el contacto del reactivo con los ojos.
LEER LAS INSTRUCCIONES ANTES DEL USO
En casos excepcionales, las muestras de pacientes con gammapatas pueden dar
resultados incorrectos.
Observar todas las medidas de precaucin necesarias para la manipulacin de
REF Cont. reactivos de laboratorio.
Deben seguirse las disposiciones locales para la gestin de residuos.
Referencia: Presentacin:
G N 86900: 1 x 30 mL Reactiv o 1 MUESTRAS
1 x 10 mL Reactiv o 2 Sangre entera EDTA.
1 x 125 mL Reactiv o 3 hemolizante Desechar las muestras contaminadas.
Preparacin de muestras
1000 L de solucin hemolizante y 20 L de muestra (o calibrador o control).
USO PREVISTO Mezclar y dejar reposar 5 minutos o hasta que se haya completado la hemlisis.
El reactivo de Hemoglobina glicosilada de Gernon es un test inmunoturbidimtrico
para la determinacin de HbA1c en muestras de sangre entera. Las muestras de sangre entera son estables 1 semana a 2 8 C.
Las muestras de hemolizado son estables 10 horas a 15 25 C 10 das a 2 8
RESUMEN C
La hemoglobina A1c (HbA1c) es una hemoglobina glicosilada que se origina a
partir de una reaccin no enzimtica de glucosa con hemoglobina nativa. Este ESQUEMA DE LA PRUEBA
proceso se produce constantemente mientras el eritrocito se encuentra en la Hay disponibles, a peticin, aplicaciones para sistemas automticos.
circulacin sangunea (vida media de los eritrocitos : 100-120 das). El grado de Longitud de onda: 660 nm
glicosilacin es directamente proporcional a la concentracin de glucosa en Paso ptico: 1 cm
sangre. La proporcin de HbA1c en la hemoglobina total representa el nivel medio Temperatura: 37 C
de glucosa en sangre de las ltimas 6-8 semanas de modo que la HbA1c sirve Medida: Respecto aire
como parmetro de glucemia para el seguimiento retrospectivo en casos de
diabetes mellitus. Los estudios clnicos han demostrado que un ajuste adecuado Muestra / Std Blanco
del valor HbA1c puede evitar o retrasar las consecuencias de la diabetes a largo Muestra / Std 8 uL ---
plazo. Reactivo 1 300 uL 300 uL
Debido a que la cantidad de HbA1c tambin depende de la cantidad total de Mezclar e incubar 5 minutos
hemoglobina, se indica el porcentaje de HbA1c en la hemoglobina total. Reactivo 2 100 uL 100 uL
En enfermedades relacionadas con una vida media reducida de los eritrocitos y en Mezclar y leer la absorbancia exactamente a los 5 minutos
prdidas de sangre considerables durante las semanas anteriores (proporcin
mayor de eritrocitos jvenes), se pueden presentar valores bajos falsos. Se han CALIBRADORES Y CONTROLES
observado falsos valores elevados en anemias por dficit de hierro (mayor Para la calibracin se recomienda utilizar el kit de calibracin (GN 86930) con 4
proporcin de eritrocitos viejos).
concentraciones diferentes adems de una solucin de NaCl (9 g/L) para
determinar el punto cero.
PRINCIPIO Para el control de calidad interno se recomienda utilizar el control Hemoglobin A1c
La hemoglobina total y la HbA1c se unen con la misma afinidad a las partculas de
(GN86910) con cada serie de muestras.
ltex del reactivo 1 en sangre hemolizada. El anticuerpo monoclonal HbA1c anti-
humano (ratn) se une a la HbA1c ligada a las partculas de ltex. El anticuerpo
CARACTERSTICAS
policlonal IgG anti-ratn (cabra) reacciona con el anticuerpo monoclonal HbA1c
Rango de medida
anti-humano, producindose una aglutinacin. La absorcin medida es El test tiene un rango de medida de 3 16 % segn DCCT/NGSP (15 150
proporcional a la HbA1c unida a las partculas que, a su vez, es proporcional al mmol/mol de HbA1c segn IFCC).
porcentaje de HbA1c en la muestra.
El test est indicado para una concentracin total de hemoglobina de 6 26 g/dL.
ESTANDARIZACIN Especificidad/Interferencias
La prueba ha sido estandarizada a partir del mtodo de referencia aprobado por la Debido a los anticuerpos que contiene, Hemoglobin A1c es un inmunoensayo
IFCC. Tambin es posible calibrar segn DCCT/NGSP. Los valores correspondientes especfico para HbA1c humana. No se presentan interferencias con cido ascrbico
de calibracin se indican en la tcnica del kit de calibracin. hasta 50 mg/dL, con bilirrubina conjugada y no conjugada hasta 50 mg/dL, con
Los valores DCCT/NGSP muestran una relacin linear a los del IFCC. Por lo tanto lipemia hasta 2000 mg/dL de triglicridos, con FR hasta 250 lU/mL, con
hemoglobina carbamilada hasta 7.5 mmol/L, y con hemoglobina acetilada hasta
se pueden calcular mediante la siguiente frmula:
5.0 mmol/L.
IFCCb = (NGSPa 2.15) / 0.915
NGSPa = 0.915 x IFCC b + 2.15 No se presentan interferencias por uremia, intermedios lbiles (base de Schiff) y
las variantes de hemoglobina HbS, HbC y HbA2. Elevados valores de HbF pueden
a: Valores NGSP (%) producir falsos resultados negativos de HbA1c.
b: Valores IFCC (mmol/mol) El alcoholismo y la ingestin de elevadas dosis de Aspirina pueden influir en los
Conversin de valores IFCC (%) en mmol/mol: [mmol/mol IFCC] = 10 * [% IFCC] valores de HbA1c.
IFCC: International Federation of Clinical Chemistry
DCCT: Diabetes Control and Complications Tria l Sensibilidad del test/Lmite de prueba
NGSP: National Glycohemoglobin Standardization Program
El lmite inferior de prueba es de 1% (10 mmol/mol) de HbA1c.
HbA1c Y CONCENTRACIONES MEDIAS DE GLUCOSA Imprecisin (Hitachi 917, sistema de 3 componentes):
Existe una correlacin lineal entre la HbA1c y las concentraciones medias de Valores segn IFCC
glucosa, de modo que se pueden convertir en valores estimados de
concentraciones medias de glucosa por medio de las siguientes ecuaciones: En la serie Valor
DS [%] (CV)[%]
n = 20 medio[%]
Normalizacin segn IFCC: Muestra 1 5.97 0.138 2.31
[Glucosa media en mg/dL] = 2.63* (HbA1c en mmol/mol IFCC) +15.01 Muestra 2 8.49 0.072 0.85
[Glucosa media en mmol/L] = 0.146 * (HbA1c en mmol/mol IFCC) +0.829 Muestra 3 12.21 0.152 1.24

Normalizacin segn NGSP: Da a da Valor

[Glucosa media en mg/dL] = 28.7 * (HbA1c en % NGSP) 46.7 DS [%] (CV)[%]
n = 20 medio[%]
[Glucosa media en mmol/L] = 1.59 * (HbA1c en % NGSP) 2.59 Muestra 1 5.95 0.190 3.2
No se observaron diferencias significativas en la ecuacin de regresin para ambos Muestra 2 8.33 0.093 1.12
sexos, presencia o ausencia de diabetes, tipo de diabetes, franja de edad o Muestra 3 12.15 0.179 1.47
procedencia.
Aunque esta ecuacin puede emplearse en la mayora de individuos, cada
laboratorio debe verificar su validez para cada grupo de pacientes.

T el: (+3 4 ) 9 3 4 8 0 8 0 4 7 Fax: (+3 4 ) 9 3 3 7 3 0 0 9 2 e - mail: ral@ ral- s a.c om web: www.ral- s a.c om
 HEMOGLOBINA
GLICOSILADA
Mtodo Inmunoturbidimtrico

Comparacin de mtodos
En la comparacin de Hemoglobin A1c (y) con otro test inmunoturbidimtrico (x)
se obtuvieron los siguientes resultados para 40 muestras:
y = 0.98 x + 0.14 %; r = 0.987.

VALORES DE REFERENCIA
Cada laboratorio debe comprobar y determinar los rangos de referencia con
pacientes no diabticos.
Valores de referencia recomendados para HbA1c:
% NGSP mmol/mol IFCC
Pacientes no diabticos 46 20-42
Objetivo de terapia <7 < 53
Modificacin de terapia >8 > 64

BIBLIOGRAFA
Thomas L. Clinical Laboratory Diagnostics. 1st ed. Frankfurt:
TH-Books Verlagsgesellschaft; 1998. p. 142-48.
Sacks DB. Carbohydrates. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of
Clinical Chemistry. 3rd ed. Philadelphia W.B. Saunders Company; 1999. p. 790-6.
Jeppsson JO, Kobold U, Barr J, Finke A et al. Approved IFCC reference method for
the measurement of HbA1c in human blood. Clin Chem Lab Med 2002; 40:78-89.
Hoelzel W, Weykamp C et al. IFCC Reference System for Measurement of
Hemoglobin A1c in Human Blood and the National Standardization Schemes in the
United States, Japan, and Sweden: A Method-Comparison Study. Clin Chem 2004;
50:1:166-74.
The Diabetes Control and Complications Trial Research Group. The effect of
intensive treatment of diabetes in the development and progression of long-term
complications in insulin-dependent diabetes mellitus. N Engl J Med.1993; 329:977-
86.
Little RR, Rohlfing CL, Wiedmeyer HM, Myers GL et al. The National
Glycohemoglobin Standardization Program: A Five- Years Progress Report. Clin
Chem 2001; 47:1985-92.
Miedema K. Standardization of HbA1c and Optimal Range o Monitoring. Scand J
Clin Lab Invest 2005;65 (Suppl 240):61-72.
Pantheghini M, John WG on behalf of the IFCC Scientific Division. Implementation
of HbA1c results traceable to the IFCC reference system: the way forward. Clin
Chem Lab med 2007; 45 (8): 942-4
Nordin G, Dybkaer R. Recommendation for term and measurement unit for HbA1c.
Clin Chem Lab med 2007; 45 (8): 1081-2.
Sacks DB. Translating Hemoglobin A1c into Average Blood Glucose: Implications
for Clinical Chemistry. Clinical Chemistry 2008; 54: 1756-80

IU/GN86900/R4/1214

T el: (+3 4 ) 9 3 4 8 0 8 0 4 7 Fax: (+3 4 ) 9 3 3 7 3 0 0 9 2 e - mail: ral@ ral- s a.c om web: www.ral- s a.c om
 GLYCATED HEMOGLOBIN
Immunoturbidimetric Method

Fabricante: RAL TECNICA PARA EL LABORATORIO, S.A. REAGENTS

Av. Mare de Du de Montserrat 51 Components and concentrations
08970 Sant Joan Desp (Barcelona) - Spain R1: buffer 20 mmol/l, Latex 0.14 %
R2: buffer 10 mmol/l, Mouse anti-human HbA1c monoclonal antibody (5.5 mg/ml),
IVD FOR IN VITRO DIAGNOSTIC goat anti-mouse IgG polyclonal antibody (67 mg/dl), stabilizers
R3: Hemolysing solution

STORE AT 2 - 8 C. After opening, the reagents are stable at least for one month when at 2-8C and
contamination and evaporation are avoided.
The reagents are ready to use.
READ INSTRUCTIONS Warnings and precautions
(S25) Avoid contact with eyes.
In very rare cases, samples of patients with gammapathy might give false results.
Take the necessary precautions for the use of laboratory reagents.
REF Cont.
Please refer to local legal requirements for the waste management .
Reference: Package:
G N 86900: 1 x 30 mL Reagent 1 SAMPLE
1 x 10 mL Reagent 2 Whole blood collected with EDTA.
1 x 125 ml Ly sing Reagent 3 Discard contaminated specimens.
Sample preparation
INTENDED USE Hemolyzing solution 1000 l
Gernon Glycated hemoglobin reagent is an immunoturbidimetry assay to quantify the Sample / calibrator / control 20 l
percentage of HbA1c in samples of whole blood. Mix and allow to stand for 5 minutes or until complete lysis is apparent.
Whole blood simples are stable for 1 week at 2 8 C.
SUMMARY
Hemoglobin A1c (HbA1) is a glycated hemoglobin which is formed by the non- Hemolisate samples are stable for 10 hours at 15 25 C or 10 days at 2 8 C
enzymatic reaction of glucose with native hemoglobin. This process runs continuously
PROCEDURE
throughout the circulation life of the red cell (average life time 100-120 days). The
rate of glycation is directly proportional to the concentration of glucose in the blood. Application sheets for automated systems are available on request.
Wavelength: 660 nm
The blood level of HbA1c represents the average blood glucose level over the
Optical path: 1 cm
preceding 6 to 8 weeks (due to the kinetics of erythrocyte turnover this period is
more affected by the blood glucose level than the preceding weeks). Therefore, Temperature: 37 C
Measurement: Read absorbance against air
HbA1c is suitable for retrospective long-term monitoring of blood glucose
concentration in individuals with diabetes mellitus. Clinical studies have shown that
lowering of HbA1c level can help to prevent or delay the incidence of late diabetic Sample / Std Blank
complications. Sample / Std 8 ul ---
As the amount of HbA1c also depends on the total quantity of haemoglobin the Reagent 1 300 ul 300 ul
reported HbA1c value is indicated as a percentage of the total haemoglobin Mix, incubate for 5 minutes, then add
concentration. Reagent 2 100 ul 100 ul
Falsely low values (low HbA1c despite high blood glucose) may occur in people with Mix, read absorbance after exactly 5 minutes
conditions with shortened red blood cell survival (haemolytic diseases) or significant
recent blood loss (higher fraction of young erythrocytes). Falsely high values (high CALIBRATORS AND CONTROLS
HbA1c despite normal blood glucose) have been reported in iron deficiency anaemia For calibration use it is recommended to use the calibration kit (GN86930) with 4
(high proportion of old erythrocytes). These circumstances have to be considered in different concentrations.
clinical interpretation of HbA1c values. For internal quality control use the Hemoglobin A1c control (GN86910) which should
be assayed with each batch of samples.
PRINCIPLE
Total Hb and HbA1c in hemolyzed blood bind with the same affinity to particles in R1. PERFORMANCE CHARACTERISTICS
The amount of binding is proportional to the relative concentration of both Measuring Range
substances in the blood. The test has been developed to determine concentrations of HbA1c within a
Mouse anti-human HbA1c monoclonal antibody binds to particle bound HbA1c. Goat measuring range from 3 16 % according to DCCT/NGSP (1.5 15 mmol/mol
anti-mouse IgG polyclonal antibody interacts with the monoclonal mouse anti-human according to IFCC).
HbA1c antibody and agglutination takes place. The measured absorbance is The assay is applicable for haemoglobin concentrations in blood from 6 26 g/dl.
proportional to the HbA1c bound to particles, which in turn is proportional to the
percentage of HbA1c in the sample. Specificity / Interferences
Due to its antibodies, HbA1c kit is a specific immunoassay for human HbA1c. No
STANDARDIZATION interference was observed by ascorbic acid up to 60 mg/dl, conjugated and
The assay is standardized according to the approved IFCC reference method. unconjugated bilirubin up to 60 mg/dl, lipemia up to 2000 mg/dl triglycerides, RF up
Calibration according to DCCT/NGSP is also possible. Corresponding calibrator values to 250 IU/ml, carbamylated Hb up to 7.5 mmol/l, and acetylated Hb up to 5.0
are listed in the package insert of the calibrator set. mmol/l.
DCCT/NGSP and IFCC values show a linear relationship and can therefore be No interference is observed by uremia, labile intermediates (Schiff bases), and
calculated from each other using the following equation: Hemoglobin variants HbS and HbA2. Elevated levels of HbF may lead to falsely low
IFCCb = (NGSPa 2.15) / 0.915 HbA1c values. Alcoholism and ingestion of large doses of aspirin may lead to
NGSPa = 0.915 x IFCC b + 2.15 inconsistent results.
a: Valores NGSP (%)
b: Valores IFCC (mmol/mol) Sensitivity / Limit of detection
The limit of detection is 1 % HbA1c.
Conversin de valores IFCC (%) en mmol/mol: [mmol/mol IFCC] = 10 * [% IFCC]
IFCC: International Federation of Clinical Chemistry Imprecision (Hitachi 917, 3-components system)
DCCT: Diabetes Control and Complications Trial Values according to IFCC
NGSP: National Glycohemoglobin Standardization Program

Within-run
HbA1c AND AVERAGE GLUCOSE CONCENTRATIONS Valor
precision DS [%] (CV)[%]
Due to a linear correlation between hemoglobin A1c and average glucose medio[%]
n = 20
concentrations HbA1c values can be converted in estimated average glucose values Sample 1 5.97 0.138 2.31
by means of the following equations: Sample 2 8.49 0.072 0.85
Standardization according to IFCC: Sample 3 12.21 0.152 1.24
[Average Glucose in mg/dL] = 2.63* (HbA1c in mmol/mol IFCC) +15.01 Between day
[Average Glucose in mmol/L] = 0.146 * (HbA1c in mmol/mol IFCC) +0.829 Valor
precision DS [%] (CV)[%]
medio[%]
Standardization according to NGSP: n = 20
[Average Glucose in mg/dL] = 28.7 * (HbA1c in % NGSP) 46.7 Sample 1 5.95 0.190 3.2
[Average Glucose in mmol/L] = 1.59 * (HbA1c in % NGSP) 2.59 Sample 2 8.33 0.093 1.12
Sample 3 12.15 0.179 1.47
No significant differences in the regression equation were observed for variations in
individuals tested, including sex, presence or absence of diabetes, type of diabetes,
Method comparison
age, race, and ethnicity. Although this equation can be used for the majority of
A comparison between Gernon HbA1c (y) and another immunoturbidimetric assay (x)
individuals each laboratory has to reassure itself if the regression equations
using 40 samples gave following results: y = 0.98 x + 0.14; r = 0.987.
mentioned are applicable for the patient group to be examined.

T el: (+3 4 ) 9 3 4 8 0 8 0 4 7 Fax: (+3 4 ) 9 3 3 7 3 0 0 9 2 e - mail: ral@ ral- s a.c om web: www.ral- s a.c om
 GLYCATED HEMOGLOBIN
Immunoturbidimetric Method

REFERENCE RANGE
Reference intervals should be established or verified by the laboratory based on an
appropriate non-diabetic patient population.

Suggested target values for HbA1c:

% NGSP mmol/mol IFCC
Non-diabetics 46 20 - 42
Target of therapy <7 < 53
Change of therapy >8 > 64

LITERATURE
Thomas L. Clinical Laboratory Diagnostics. 1st ed. Frankfurt: TH-Books
Verlagsgesellschaft; 1998. p. 142-48.
Sacks DB. Carbohydrates. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of
Clinical Chemistry. 3rd ed. Philadelphia W.B. Saunders Company; 1999. p. 790-6.
Jeppsson JO, Kobold U, Barr J, Finke A et al. Approved IFCC reference method for
the measurement of HbA1c in human blood. Clin Chem Lab Med 2002; 40:78-89.
Hoelzel W, Weykamp C et al. IFCC Reference System for Measurement of
Hemoglobin A1c in Human Blood and the National Standardization Schemes in the
United States, Japan, and Sweden: A Method-Comparison Study. Clin Chem 2004;
50:1:166-74.
The Diabetes Control and Complications Trial Research Group. The effect of intensive
treatment of diabetes in the development and progression of long-term complications
in insulin-dependent diabetes mellitus. N Engl J Med.1993; 329:977-86.
Little RR, Rohlfing CL, Wiedmeyer HM, Myers GL et al. The National Glycohemoglobin
Standardization Program: A Five- Years Progress Report. Clin Chem 2001; 47:1985-
92.
Miedema K. Standardization of HbA1c and Optimal Range o Monitoring. Scand J Clin
Lab Invest 2005;65 (Suppl 240):61-72.
Pantheghini M, John WG on behalf of the IFCC Scientific Division. Implementation of
HbA1c results traceable to the IFCC reference system: the way forward. Clin Chem
Lab med 2007; 45 (8): 942-4
Nordin G, Dybkaer R. Recommendation for term and measurement unit for HbA1c.
Clin Chem Lab med 2007; 45 (8): 1081-2.
Sacks DB. Translating Hemoglobin A1c into Average Blood Glucose: Implications for
Clinical Chemistry. Clinical Chemistry 2008; 54: 1756-8

IU/GN86900/R4/1214/EA

T el: (+3 4 ) 9 3 4 8 0 8 0 4 7 Fax: (+3 4 ) 9 3 3 7 3 0 0 9 2 e - mail: ral@ ral- s a.c om web: www.ral- s a.c om