NRICHDX SCIENTIFIC FRAMEWORK

The Sensitivity
Ladder™

How much sample input does your assay really need?

The Sensitivity Ladder™ is an nRichDX-developed framework for empirically evaluating how sample input affects a defined assay endpoint, identifying where meaningful performance plateaus, and translating that learning into a reproducible workflow.

The framework does not assume that more sample is always better, does not claim that recovery alone determines sensitivity or specificity, and does not replace analytical or clinical validation.

THE INPUT-RESPONSE QUESTION Find the performance region.
DECISION REGION Increasing sample input Selected assay endpoint
THE OBJECTIVE Identify the lowest input that reaches the desired performance region.
THE DEVELOPMENT PRINCIPLE Sample input should be discovered through performance and proven through workflow.
01 WHY INPUT MATTERS

Low-abundance signals make the upstream workflow matter.

In early-stage cancer and MRD, disease-associated molecular signal may be scarce before analysis even begins.

For teams developing MCED, single-cancer early detection, MRD, and other liquid-biopsy assays, downstream technologies can only analyze the molecular material that ultimately reaches the analytical workflow.

That makes the quantity and quality of molecular input important upstream variables. Sample volume, extraction recovery, analyte integrity, transfer into the assay, downstream chemistry, background, sequencing, and interpretation can all influence the final result.

THE DEVELOPMENT QUESTION Was the input selected because the assay demonstrated that it was appropriate, or because the existing workflow dictated what was practical?
02 FOLLOW THE MOLECULE

Sample volume is only the beginning.

Molecular information moves through a series of biological, preanalytical, recovery, transfer, and analytical steps before becoming a measurable result.

01
Biologically Present The disease-associated signal must exist before it can be collected.
02
Collected Sampling determines what molecular material enters the specimen.
03
Recovered Sample preparation determines what material is recovered for analysis.
04
Transferred Only part of the recovered material may enter the analytical reaction.
05
Measured Chemistry, detection, sequencing, and interpretation convert molecules into evidence.
A milliliter is not a molecule.

Specimen volume, recovered analyte, assay input, analytical performance, and clinical performance are connected quantities, but they are not interchangeable.

03 THE INPUT-RESPONSE RELATIONSHIP

More sample is not the strategy.

More sample may create additional molecular opportunity. It does not guarantee improved assay performance.

Increasing sample input may improve a selected endpoint, produce diminishing returns, have little meaningful effect, or introduce additional background, variability, inhibition, cost, sequencing demand, or workflow burden.

The purpose of an input study is therefore not to prove that larger volume is better. It is to measure the relationship that actually exists for the assay and workflow being developed.

THE OBJECTIVE Performance optimization, not volume maximization.
04 THE PLATEAU

The plateau is the decision.

As input increases, a selected performance endpoint may improve and then begin to level as another factor becomes limiting.

The scientifically useful region is where additional sample no longer produces a prespecified meaningful improvement in the endpoint being evaluated.

The candidate input can then be evaluated against specificity, background, reproducibility, sample quality, collection requirements, cost, and workflow practicality.

Assay performance Workflow burden
DECISION REGION Increasing sample input
OPERATING PRINCIPLE Once meaningful performance has plateaued, additional volume becomes a workflow decision rather than an assumed sensitivity strategy.
05 THE FRAMEWORK

Evaluate. Measure. Optimize.
Standardize. Automate.

Turn sample input from an inherited workflow constraint into a controlled development variable.

01
COMPARE INPUTS

Evaluate

Test a scientifically justified range of sample inputs under controlled and comparable conditions.

02
TRACK THE RESPONSE

Measure

Follow recovered molecular material, the selected assay endpoint, background, reproducibility, invalid results, and operational behavior.

03
FIND THE REGION

Optimize

Determine where additional input delivers meaningful value and identify the lowest input that enters the desired performance region.

DECISION REGION
04
LOCK THE CONDITION

Standardize

Define specimen input, processing requirements, elution conditions, quality controls, and relevant acceptance criteria.

05
SCALE EXECUTION

Automate

Translate the selected workflow into repeatable execution appropriate for the intended laboratory environment and throughput.

THE DECISION IS MULTIVARIABLE

Sensitivity cannot be optimized in isolation.

A useful input decision considers the entire controlled system rather than extraction yield or volume alone.

PERFORMANCE Selected Performance Endpoint

Determine whether increasing input produces a meaningful improvement in the endpoint defined for the study.

ASSAY BEHAVIOR Specificity & Background

Evaluate whether additional input changes background, false-positive behavior, or other assay characteristics.

MOLECULAR INPUT Quantity & Quality

Consider how much usable molecular material reaches downstream analysis and whether it remains suitable for the intended assay.

EXECUTION Reproducibility & Workflow Burden

Balance performance with collection, processing, throughput, cost, consistency, and operational practicality.

FROM DECISION TO EXECUTION

Prove the input.
Standardize the workflow.
Scale what works.

A scientific decision is only useful if the surrounding sample-preparation workflow can reproduce it.

The Revolution Platform provides a path from flexible sample-preparation development into standardized and automated execution across supported liquid-biopsy workflows.

cfDNA cfRNA cfTNA CTC
nRichDX Revolution Pro nRichDX nRicher Cartridge
THE EXECUTION LAYER Automation should preserve the workflow selected through measurement.
SCIENTIFIC BOUNDARY

Measurement, not assumption.

The framework is intentionally bounded by what the data demonstrate.

Not “more is always better.”

The framework is designed to reveal the input-response relationship that actually exists.

Not “recovery determines everything.”

Biology, preanalytics, recovery, molecular quality, downstream chemistry, background, sequencing, and interpretation can all contribute.

Not a replacement for validation.

The framework helps evaluate an upstream development variable before analytical or clinical validation locks the workflow around an untested assumption.

THE SENSITIVITY LADDER™

Build the ladder.
Find the plateau.

Let’s evaluate where your assay reaches its optimal molecular input and how that workflow can be standardized.