Check units, dilutions and replicates

Keep numerical values and units consistent

Declare the concentration/dose unit and response unit supported by the chosen family. The application retains unit labels; changing ng/mL to pg/mL is not an automatic numerical conversion. Ensure source values, standards and procedure use consistent units before importing or changing a label.

Relative activity is a dimensionless test-to-reference ratio; percentage relative potency is 100 times that ratio. A ratio of 0.8 is 80%. Absolute potency multiplies the ratio by the assigned reference potency once. It is not the fitted horizontal shift itself. Check the reference assignment and potency unit in the saved configuration and report. The current manual potency setup initializes the reference assignment to 100 and uses the concentration unit for potency.

Distinguish dose-series construction from sample correction

In relative potency, Initial concentration, Dilution factor and the mapped level determine the positive dose for each observation. For example, starting at 128 with factor 2 gives 128, 64, 32 and so on. Check these doses against your sample preparation records.

In concentration determination, a sample dilution factor is a denominator of at least one. The interpolated concentration on the curve is multiplied by that factor to obtain the undiluted concentration. A curve estimate of 6 ng/mL with factor 10 represents 60 ng/mL in the original sample. Do not apply that correction twice after downloading the report.

The reportable interval applies to the diluted concentration on the calibration curve, before sample correction. An undiluted result can therefore exceed the highest standard concentration without being an extrapolation.

Distinguish standards, anchors, unknowns, QCs and blanks

The reportable endpoints are derived from the lowest and highest included reportable standard levels. Excluding every observation at an endpoint changes that range; inspect it again. The editor does not provide independent manual range endpoints.

Declare what repeated observations represent

A different well number does not prove an independent experimental unit. For concentration work, choose the declared replicate treatment from the actual preparation and measurement procedure. Technical pseudoreplicates are repeated readings of one underlying unit; independent replicates represent independent units under the procedure.

Eligible dilution-corrected concentrations are combined as an equal-weight geometric mean, not an arithmetic mean of responses. Unknowns and QCs use the same aggregation rule. Inspect which observations contributed: observations outside the reportable range are omitted, not treated as zero.

All sample estimates share uncertainty in their fitted calibration curve. The calculation for combined observations also treats readings within a technical group as fully correlated for their response-error contribution. This conservative rule does not claim that physical correlation is always exactly one. Choose technical or independent replicates according to how the samples were prepared and measured.

Relative-potency precision instead uses variation within replicated preparation–dose cells. Retain the required balanced design and justify the experimental replication; the software does not infer or separately estimate every biological or preparation variance component.

Check before running and after recovery

1. In Define the dilution series, inspect units, level concentrations, sample dilution factors, replicate treatment and expected QC concentrations where present.
2. Check Mapped observations against the physical plate and original preparation record. Keep missing measurements distinct from measured zero.
3. After a map, role or exclusion change, inspect the derived concentrations and reportable range again. Confirm the saved configuration before Run analysis.