How Does Immunoturbidimetric HbA1c Testing Work?

How Does Immunoturbidimetric HbA1c Testing Work?

Hemoglobin A1c (HbA1c) is one of the most established biomarkers used for the assessment of long-term glycemic status. Unlike a single glucose measurement, which reflects glucose concentration at a particular point in time, HbA1c provides information related to glycemic exposure over the preceding months.

A variety of analytical principles are available for HbA1c measurement, including ion-exchange high-performance liquid chromatography (HPLC), affinity chromatography, capillary electrophoresis, enzymatic methods and immunoassay-based techniques.

Among these approaches, immunoturbidimetric HbA1c assays enable HbA1c measurement on automated clinical chemistry analyzers by combining an antigen-antibody reaction with photometric detection.

Understanding how this analytical principle works—and how factors such as calibration, standardization, precision and interference influence assay performance—is important when evaluating an HbA1c method for routine laboratory use.

What Is HbA1c?

HbA1c is formed when glucose becomes irreversibly attached to hemoglobin.

The process begins with the formation of an unstable Schiff base, sometimes referred to as pre-HbA1c or labile HbA1c. This intermediate can dissociate or undergo an Amadori rearrangement, resulting in the formation of a stable ketoamine structure.

Because erythrocytes circulate for approximately 120 days, HbA1c reflects glycemic exposure over a considerably longer period than a single plasma glucose measurement. The contribution is weighted toward more recent glycemia rather than representing a simple 120-day average.

Why Is HbA1c Measured?

HbA1c is widely used in the diagnosis and monitoring of diabetes mellitus. In contrast to a single blood glucose measurement, HbA1c provides information about longer-term glycemic exposure.

An important analytical consideration is that HbA1c interpretation depends on the relationship between glycation and the normal lifespan of erythrocytes. Conditions that alter red blood cell survival, certain hemoglobin variants and other clinical factors may influence the relationship between an HbA1c result and the patient's actual glycemic status.

For this reason, HbA1c results should always be interpreted within the appropriate clinical and analytical context.

What Methods Are Used to Measure HbA1c?

HbA1c can be measured using several analytical principles. Some methods distinguish glycated from non-glycated hemoglobin based on charge differences, while others rely on structural differences.

Immunoturbidimetric methods are particularly relevant to laboratories seeking to perform HbA1c testing within an automated clinical chemistry workflow.

What Is Immunoturbidimetric HbA1c Testing?

Immunoturbidimetry is an analytical approach in which an antigen-antibody reaction produces changes in turbidity that can be measured photometrically.

In an HbA1c immunoturbidimetric assay, antibodies specifically recognize glycated hemoglobin. The resulting reaction generates agglutination, producing a change in turbidity. The analyzer measures this change in absorbance and uses the assay's calibration system to determine the HbA1c result.

How Does the Immunoturbidimetric HbA1c Method Work?

Although exact reaction designs vary among manufacturers, a particle-enhanced immunoturbidimetric HbA1c assay can be understood through several analytical stages.

1. Preparation and Hemolysis of the Whole Blood Sample

HbA1c is measured in whole blood because the analyte is contained within erythrocytes. The erythrocytes therefore need to be lysed to release hemoglobin before the analytical reaction can take place.

For Archem HbA1c II DIRECT, anticoagulated human venous whole blood is the recommended specimen, with K2-EDTA or K3-EDTA specified as recommended anticoagulants.

2. Interaction with Latex Particles

In the Archem assay principle, total hemoglobin and HbA1c interact with latex particles. These particles provide a surface on which the subsequent immunochemical reaction can occur.

3. HbA1c-Specific Antibody Reaction

An antibody directed against HbA1c is introduced into the reaction. A particularly important analytical consideration is the distinction between stable HbA1c and labile HbA1c.

In Archem HbA1c II DIRECT, the antibody is designed not to cross-react with labile HbA1c and is specific for the stable ketoamine form of HbA1c.

4. Agglutination

The antibody reaction causes latex-HbA1c complexes to agglutinate. As the complexes form, the optical properties of the reaction mixture change. The amount of agglutination is related to the amount of HbA1c associated with the latex particle surface.

5. Photometric Measurement

The turbidity generated by agglutination is measured as a change in absorbance.

For Archem HbA1c II DIRECT, the reaction is measured at 660 nm, with 800 nm specified for dual-wavelength applications.

The analytical system ultimately expresses HbA1c relative to total hemoglobin.

How Are HbA1c Results Standardized and Reported?

Reliable HbA1c measurement requires more than an appropriate analytical reaction. Results produced by different laboratory systems also need to be linked to established reference systems.

Two systems are particularly important: NGSP and IFCC.

NGSP Reporting

NGSP expresses HbA1c results as a percentage. The NGSP standardization system links HbA1c results to the evidence base established through the Diabetes Control and Complications Trial (DCCT).

IFCC Reporting

The IFCC reference system provides a metrologically based reference measurement framework for HbA1c. IFCC results are expressed in mmol/mol, whereas NGSP results are commonly expressed as % HbA1c.

For example, an HbA1c value of approximately 7.0% NGSP corresponds to 53 mmol/mol IFCC.

What Should Laboratories Consider When Evaluating an Immunoturbidimetric HbA1c Assay?

Selecting an HbA1c assay involves more than considering the measurement principle alone. Laboratories should evaluate analytical and operational characteristics relevant to their own analyzer configuration and workflow.

Immunoturbidimetric HbA1c Testing on Automated Clinical Chemistry Analyzers

One of the practical characteristics of immunoturbidimetric HbA1c methodology is its ability to be implemented on compatible automated clinical chemistry platforms.

This allows laboratories to incorporate HbA1c testing into existing chemistry workflows rather than necessarily relying on a separate dedicated HbA1c analyzer.

For an analyzer-specific application, performance should be appropriately validated and the manufacturer's specified application parameters, calibration procedures and quality-control requirements should be followed.

Archem HbA1c II DIRECT is intended for the quantitative in vitro determination of HbA1c in human whole blood using autoanalyzers in clinical laboratory settings.

Archem HbA1c II DIRECT

HbA1c II DIRECT is Archem Diagnostics' liquid, dual-reagent HbA1c assay designed for use on compatible automated clinical chemistry analyzers.

The assay uses an immunoturbidimetric measurement principle based on latex particles and an HbA1c-specific monoclonal antibody.

Method	Immunoturbidimetric
Sample	Human anticoagulated venous whole blood
Reagent Format	Liquid, dual reagent
Preparation	Ready to use
Measuring Interval	4.0–15.5% HbA1c / 20–140 mmol/mol
Limit of Detection	2.7%
Limit of Quantitation	4.0%
Open-Vial Stability	30 days at 2–8°C under optimum conditions
Calibration Stability	30 days*
Reporting	NGSP (%) / IFCC (mmol/mol)

*Calibration stability depends on the application characteristics and cooling capacity of the autoanalyzer. On-board reagent stability is also analyzer-dependent.

NGSP Traceability Across Multiple Chemistry Platforms

Archem has successfully completed NGSP Manufacturer Certification for HbA1c systems on multiple automated clinical chemistry platforms for the certification period May 1, 2026 – May 1, 2027.

The certified systems are traceable to the Diabetes Control and Complications Trial (DCCT) Reference Method, with comparisons performed through European Reference Laboratory ESRL #13.

Manufacturer	Certified Platform
Abbott	Alinity C
Abbott	ARCHITECT c4000
Abbott	ARCHITECT c8000
Beckman Coulter	DxC 700 AU
Olympus	AU480
Olympus	AU5800
Mindray	BS2000M
Mindray	BS800 / BS800M

NGSP manufacturer certification applies to the specific analytical system, reagent/calibrator configuration and instrumentation evaluated during certification.

Abbott Alinity C

ARCHITECT c4000

ARCHITECT c8000

DxC 700 AU

Olympus AU480

Olympus AU5800

Mindray BS2000M

Mindray BS800 / BS800M

Frequently Asked Questions

Conclusion

Immunoturbidimetric HbA1c testing combines the specificity of an antigen-antibody reaction with photometric detection, enabling HbA1c measurement on compatible automated clinical chemistry analyzers.

However, the measurement principle is only one part of assay performance. Standardization, traceability, precision, measuring interval, interference, calibration, reagent stability and analyzer-specific performance all contribute to the reliability of an HbA1c testing system.

With HbA1c II DIRECT, Archem Diagnostics provides an immunoturbidimetric HbA1c solution for automated clinical chemistry platforms, supported by defined analytical performance characteristics and NGSP manufacturer certification across multiple analyzer systems.