Selection of a Suitable Assay

Summary

This article aims to:

• provide a broad overview of the three stage process of introducing a new or modified method into the clinical diagnostic laboratory

• outline general principles and strategies that can be utilised in the process of selecting a suitable method

• give a practical example of how these principles and strategies can be applied.

Introduction

The introduction of a new or modified method (assay) is a core value adding activity of the clinical diagnostic laboratory. Historically, the main drivers of these activities have been clinical demand and the desire to improve the quality of an existing assay. More recently, with the ever-increasing demand on limited healthcare resources, the need to reduce assay-associated costs may now also be a factor driving the change process.

The selection of a suitable assay is an important step in the process of introducing a new or modified method into the clinical diagnostic laboratory.

The aim of this paper is to provide a broad overview of the process of introducing a new or modified method into the clinical diagnostic laboratory, primarily to define where in the overall process selection of a suitable assay fits. Following this, the paper will then examine in more detail the specific factors to be considered in the selection of a suitable assay.

Process Overview

The process of introducing a method into routine use in a clinical diagnostic laboratory is shown in Figure 1.^1–3 This process may be initiated either by the introduction of a new test or because an improvement is required in the quality or cost effectiveness of an existing test.

Figure 1.

High-level overview of the process. Adapted from Westgard J. Method Validation. Selecting a method to validate.¹ Westgard QC Inc. copyright 2008.

Introduction of New Tests

New putative markers are typically the result of research undertaken in public sector institutes; hospitals; established In Vitro Diagnostic (IVD) companies; pharmaceutical companies; and, more recently, from a new breed of diagnostic companies that have emerged as a result of studies on the human genome and proteome.

The caveats to be considered when introducing a new assay are i) does the result provide information of significant clinical utility; and ii) does the assay facilitate the re-capture of resources, resulting in cost savings or reinvestment of freed up resources.³

Existing Assay

A new method for an existing assay may provide the laboratory with opportunities to improve quality by expanding the linear range, reducing imprecision, removing interferences, improving turnaround time, or allowing a new scenario, e.g. POCT.

New methods for existing assays may also provide opportunities to reduce costs, for example, by transferring the test to the core laboratory’s automated platform.

It is important that the clinical requirement (in the case of a new test) or the improvement required (in the case of an existing assay) is well defined before any assays are selected.

Once selected, the process moves into the second stage, method validation. ISO 15189 states that “method validations shall be as extensive as necessary to meet the needs of the given application”.⁴ It is also a requirement of the standard that only validated procedures be used to confirm that the method is suitable for the intended use.

The validation phase may identify opportunities to improve method performance. Improvements could be as simple as introducing a multipoint calibration curve, or changing a secondary wavelength.

The next step in this stage is implementation of the assay and would include activities such as updating Standard Operating Procedures and incorporating them into the laboratory manual, training staff and educating clinicians.

The third and final stage is performing the test and, through the laboratory’s quality management system, the continuous monitoring of the performance in order to verify the quality of patient results.

The remainder of this paper will focus on the steps involved in the selection of a suitable assay.

Selecting a Suitable Assay

Selecting a suitable assay for the clinical laboratory is a three-step process.

Step 1: Defining the clinical need and/or improvement required

The clinical need or improvement required could be well established as a result of:

1. Clinical outcome trials

   For example, the UK Prospective Diabetes study and the Diabetes Control and Complications trial that established HbA1_c as the standard measure of long-term glycaemic control.

2. Regulatory requirements that set analytical performance criteria

   For example, the Centers for Disease Control and Prevention (CDC) in the USA has established, through the Cholesterol Reference Method Laboratory Network, a process whereby manufacturers obtain certification for Total, HDL and LDL Cholesterol methods as having met National Cholesterol Education Programme (NCEP) bias and coefficient of variation (CV) specifications. Certified methods are publicly available on the CDC website.⁵

3. Recommendations of relevant professional bodies

   Contemporary examples of professional bodies making recommendations with respect to the performance criteria of various assays include -

   1. National Kidney Disease Education Programme - plasma creatinine.

   2. US CDC and the American Heart Association - CRP

   3. The American College of Cardiology and European Society of Cardiology - Troponin

4. Published Literature

5. Discussions with Clinicians

Step 2: Review available methods

Information on a range of methods is available from:

1. Published Literature

2. IVD companies

   1. Reagent package inserts.

   2. A number of IVD companies provide users with access to web based libraries.

   3. User groups.

3. Conferences - e.g. AACB annual scientific meeting

4. Professional Organisations – e.g. IFCC, AACB & AACC

5. External Quality Assessment (EQA) Program - An EQA end of cycle report is a valuable independent guide to the performance of a range of methods in common use.

Step 3: Select methods with characteristics that best suit the laboratory’s service requirements

Method characteristics can be categorised as¹:

1. Application Characteristics: These are factors that determine if a method can be implemented into a particular laboratory and would typically include things such as:

   • Where is the test to be run: Core Lab, Satellite Lab or at the Point of Care

   • Cost per test

   • Suitable sample types

   • Expected test volume

   • Equipment required

   • Turn around time required

   • Occupation, Health and Safety Issues

     • Operator, Patient, Environment, and Third party risk assessment

     • Product supply and support

     • Personnel requirements and training

2. Methodology Characteristics: These are factors that contribute to best performance and include:

   • Method: Is there a

     • IFCC recommended method; or

     • Reference method available.

   • Standardisation and Calibration

     • Metrological Traceability to certified primary reference material

   • Regulatory Requirements

     • Laboratory accreditation requirements

     • Australian Register of Therapeutic Goods (ARTG) listing

   • Other

     • Minimum number of steps

     • Ease of use

     • Robustness

     • Kit configuration

3. Performance Characteristics: Demonstrate how well a method performs and will include:

   • Precision (imprecision)

   • Interference (analytical specificity)

   • Trueness (bias)

   • Measuring range

   • Clinical validation

   • Reference interval validation

Combined, the method characteristics form assay boundary conditions (Figure 2).

Figure 2.

Assay boundary conditions determined by Method Characteristics.

Result

Based on the above analysis, Candidate Method 1 meets or exceeds the required boundary conditions and therefore would be the most suitable method to proceed to Stage 2 of the process.

Conclusion

The validation and implementation of a new or modified method is a resource intensive process, requiring adherence to well defined procedures in order to meet increasingly stringent laboratory accreditation requirements. Therefore, it is important that the most suitable assay is selected at the outset of the change process.

However, before a suitable method can be selected, it is essential that the clinical need or desired improvement be well defined. Only then is the clinical laboratory in a position to determine the context in which a new or modified assay is to be evaluated.

One approach to determining the context in which a new or modified method is to be assessed, which is outlined in this paper, is to identify applicable boundary conditions in terms of application, methodology and performance characteristics, required to be met in order to achieve the desired outcome. A suitable assay will be one that meets or exceeds all boundary conditions, and in so doing, becomes the ideal candidate to proceed to the second stage of the process of introducing a new method, validation and implementation.

Table.

The Table shows a worked example of how the process could be utilised to select a new Amylase method for the Core Laboratory.

Desired Outcome: Replace Saccharogenic amylase method with a Chromogenic amylase method (candidate methods 1 and 2).

Application Characteristics	Boundary Condition	Candidate Method 1	Candidate Method 2	Comments
Availability	Core Laboratory	Suitable	Suitable
Cost per Test	<8 cents	5.0 cents	5.5 cents
Sample Type	Serum, Plasma & Urine	Yes	Yes	All acceptable. EDTA plasma not recommended (10% decrease in values)
Equipment Required	Validated Instrument application available	Yes	Yes	In house
				verification required as per laboratory procedures
Turn Around Time	Total Analysis Time less than 10 minutes (theoretical)	8 minutes	3 minutes
OH&S	Acceptable level of risk to Operator, Patient, Environment and Third Party	Yes	Yes	Based on clinical laboratory’s risk assessment procedure
Product Supply and Support	Available directly from manufacturer or fully owned subsidiary	Yes	Yes
Personnel requirements	No additional headcount requirements	Yes	Yes
Methodology Characteristics	Boundary Condition	Candidate Method 1	Candidate Method 2	Comments
Method	Chromogenic, utilising a soluble p-nitrophenol (PNP) Oligosaccharide Substrate	5 Ethylidene–G7- pNP (EPS)	2-Chloro-4- nitrophenyl–α-D-Maltotrioside (CNP-G3)
Metrological Traceability	Traceable to certified primary reference material CRM456 and IFCC reference measurement	Yes	No	Refer to traceability tree
Regulatory requirements	Conformité Européene marked	Yes	Yes
Ease of use	Supplied ready to use	Liquid stable	Liquid stable
Robustness	>18 months shelf life	20 months from Date of Manufacture	36 months from Date of Manufacture
Kit Configuration	400 –500 tests per kit	425	450	Based on supplied recommended procedure
Performance Characteristics	Boundary Condition	Candidate Method 1	Candidate Method 2	Comments
Repeatability	CV< 3% or SD <3 U/L	CV<1.5% SD<2 U/L	CV<2.5% SD <2.5 U/L	Package insert data
Intermediate Precision	CV<5% or SD<5 U/L	CV<2.5% SD<2.5 U/L	CV<3% SD<3 U/L	Package insert data
Analytical Specificity	Hb–No interference up to 10 g/L	10 g/L	10 g/L	Package insert data
	Lipid - No interference up to 22.6 mmol/L	22.6 mmol/L	11.3 mmol/L	Package insert data
	Glucose–No interference up to 120 mmol/L	120 mmol/L	120 mmol/L	Package insert data
	Bilirubin- No interference up to 900 μmol/L	>900 μmol/L	900 μmol/L	Package insert data
	Ascorbic Acid–No interference up to 2 g/L	>2 g/L	>2 g/L	Package insert data
Measuring Range	Up to 2000 U/L	4–2000 U/L	4–1800 U/L	Package insert data
Lot to Lot Variability	<3 %	<3%	<3%	Information supplied by manufacturer
On Board Reagent Stability	>21 days	30 days	45 days	Package insert data