Ultimate Guide to Validating an LDT for COVID/Flu/RSV qPCR

Today, we're going to explore how to conduct a Laboratory Developed Test (LDT) for the COVID/Flu/RSV qPCR Assay. This guide will serve as a comprehensive resource, detailing each step and explaining the reasoning behind it.

Table of Contents

1. Creating a Validation Plan 📝
2. Materials and Methods 🛠
3. Sensitivity Study 🎯
4. Precision Study 📊
5. Specificity Study 🎯
6. Accuracy Study ✅
7. Final Steps 📋

1️⃣ Creating a Validation Plan 📝

Steps:

• Create a validation plan that includes:
  • Sensitivity study
  • Specificity study
  • Accuracy study
  • Precision study
  • Reportable Range
  • Reference Range (Normal Values)

Why?

A validation plan is crucial because it serves as a roadmap for your LDT. It outlines the key studies you'll conduct to ensure the assay's reliability and accuracy. Plus, you'll need your lab director's approval for the validation plan to ensure compliance. Per 42 CFR Part 493, an LDT requires a validation providing the performance characteristics of sensitivity, specificity, accuracy, precision, reportable range, and reference values. 

Consequences of Not Doing This:

Without a validation plan, you risk having a non-compliant validation. Validations should be planned prior to beginning, with the specific studies and acceptance criteria approved by the lab director. If the studies you perform are not approved, you may end up doing work that is not useful.

2️⃣ Materials and Methods 🛠

Steps:

• Gather all necessary materials:
  • Validation plan
  • Plastics
  • Kit
  • Standards and reagents
  • Clinical samples

Why?

Having all materials ready ensures a smooth testing process. Each component, from plastics to reagents, plays a role in the assay's performance.

Consequences of Not Doing This:

Lack of preparation can lead to delays and potential errors.

3️⃣ Sensitivity Study 🎯

Steps:

• Determine the Limit of Detection (LOD) using standards (We recommend purchasing from ATCC).
• Perform dilutions and replicates to confirm LOD.
• Dilute a high concentration standard 10-fold so that you have 4-5 enumerated standards decreasing in concentration.
• Run each standard in triplicate - choose the lowest concentration which gives you amplification for 3/3 replicates as your LOD.
• Run the LOD with 20 replicates - 19/20 (95%) must pass to be confirmed as the LOD. 

Why?

The LOD is the lowest concentration of the target that the assay can reliably detect. Knowing this ensures that your test is sensitive enough to detect low levels of the pathogen.

❌ Common Mistakes to Avoid ❌

1. Not Keeping Everything Cold

Why?

Keeping reagents and samples cold prevents degradation and ensures that the chemical reactions occur as intended. Elevated temperatures can accelerate reactions, leading to unreliable results.

Consequences:

If the reagents or samples get warm, you risk inaccurate results, including false negatives or positives.

2. Using the Wrong Plastics

Why?

Using low DNA-binding dilution tubes ensures that your sample doesn't stick to the container, which could lead to lower sensitivity. 

Consequences:

Using the wrong plastics can result in nucleic acid loss, leading to an artificially high LOD.

3. Diluting with Water

Why?

While water seems like an obvious choice for dilution, using TE buffer keeps RNA more stable, leading to more reliable results.

Consequences:

Using water for dilution can result in RNA degradation, affecting the test's sensitivity and leading to high LOD's.

4. Going for the Lowest LOD Possible

Why?

While it might seem advantageous to detect the lowest possible concentration, aiming for an LOD that's too low can make the sensitivity and precision studies less reliable and harder to reproduce. 

Consequences:

Choosing an LOD that's too low can result in wasted time, energy, and money spent during sensitivity and precision studies.

5. Testing Each Standard One at a Time

Why?

Testing all the standards together speeds up the process without significantly affecting sensitivity.

Consequences:

Doing them one at a time is time-consuming and offers no significant advantage in terms of sensitivity.

4️⃣ Precision Study 📊

Steps:

• Determine intraday and between-day precision.
• For intraday precision, use the LOD confirmation PCR run data. Calculate the Coefficient of Variation of each target and confirm it is less than 10%. 
• For Between-day precision, run 4-6 replicates of 20xLOD, 5xLOD, and negative controls.
• You can arrange the plate in a "checkerboard" pattern to test for carryover as well. 
• Ensure the Coefficient of Variation (CV) is within 10%.

Why?

Precision studies validate the reproducibility of your test results. A low CV indicates that your test is reliable.

Consequences of Not Doing This:

A high CV indicates inconsistent results, which can lead to incorrect diagnoses and treatments.

5️⃣ Specificity Study 🎯

Steps:

• Conduct interfering substance and cross-reactivity studies.
• For interfering substances, spike your samples with endogenous and exogenous materials prior to extraction. Run them and see if you get the expected results.
• For Cross-reactivity, run samples which are positive for respiratory viruses unrelated to your PCR kit, such as rhinovirus or adenovirus. 
• Ensure no false positives or negatives.

Why?

Specificity ensures that your test is targeting the correct pathogen and is not compromised due to unrelated substances or pathogens. 

Consequences of Not Doing This:

Poor specificity can result in false positives, leading to incorrect treatment and unnecessary anxiety.

6️⃣ Accuracy Study ✅

Steps:

• Use 20 clinical samples (10 positive and 10 negative) for testing.
• Achieve at least a 90% positive and negative correlation for a pass.

Why?

Accuracy ensures that your test provides reliable results. A high correlation between the test and a reference method is crucial for its credibility.

Consequences of Not Doing This:

Inaccurate results can lead to incorrect diagnoses and treatments, affecting patient care.

8️⃣ Final Steps 📋

• Compile all data into a validation report.
• Include sections for Reportable range and Reference Range
• Get it signed off by your lab director.
• Create a Standard Operating Procedure (SOP).
• Train all personnel.

Why?

These final steps ensure that your test is ready for implementation in a clinical setting. Mentioning reportable range and reference range is necessary in any LDT validation report. Since PCR is qualitative, the reportable range is simply "Detected" or "Not detected" for each pathogen, and reference range is simply "Not detected" for each pathogen in a healthy population.

Consequences of Not Doing This:

Failure to complete these steps can result in the test not being approved for clinical use, wasting all the effort put into the validation process.

Conclusion 🎉

Congratulations, you've successfully conducted an LDT for the COVID/Flu/RSV qPCR Assay! Remember, the validation is ultimately up to your lab director - ensure all validations are approved before starting clinical testing.