Piston Pumps for Dilution, Spiking and Proportioning

0120 μL sample, 60 μL diluent, then a 3 μL aliquot

The decreased-sample setting in Roche GLUC3 V7.0 for cobas c 303, c 503 and c 703 predilutes 20 μL of sample with 60 μL of the specified NaCl diluent, then uses 3 μL of the diluted sample. The normal sample setting is 1.5 μL.^[1]

Assuming additive volumes, complete mixing and no analyte loss, the dilution vessel contains one-quarter of the original concentration. Its 3 μL aliquot therefore contains the equivalent of 0.75 μL of original sample—half the 1.5 μL normal sample input. This explains the method sheet's 1:2 rerun dilution and automatic result multiplication by two. The fourfold concentration dilution in the vessel and the method's twofold rerun correction describe different stages; do not multiply the correction factors again. The analyzer handles the reported concentration according to the method.^[1]

The 50 μL and 100 μL capacities are EA standard configurations. The calculations use nominal capacity divided by full-stroke steps; they are not delivery-accuracy results at these working points. EA-100-PEEK's full-stroke accuracy and repeatability specifications of ≤0.5%, under the stated test conditions, cannot be transferred directly to partial strokes or mixture concentration. This design example does not imply that the Roche analyzer uses FOREACH products.^[2][3]

Budget any isolation segments, retraction and retained volume in the same stroke. If sample-side auxiliary actions exceed the usable 50 μL stroke, compare 100 μL; the 3 μL aliquot would then correspond to 60 full steps, and the control program must change accordingly. If aspiration/dispensing mixing requires a larger liquid movement, arrange an appropriate mixing action or separate channel instead of assuming that the small-dose metering capacity must also handle the whole mixture.

02Specify diluent, reaction water and calibration matrix separately

Dilution changes salt concentration, protein matrix and reaction conditions as well as analyte concentration. Establish the assay-approved diluent and any required stabilization interval before designing the metering sequence. The method determines whether water, saline or a dedicated matrix is suitable.

High-protein or abnormal immunoglobulin specimens may also precipitate or produce matrix interference during processing, so assay suitability must be assessed separately. Once the liquids are known, list what actually passes through the pump, valves, probe and tubing—including maintenance cleaners—and evaluate material compatibility against composition, concentration, temperature and contact time.^[5][6]

03Choose separate channels, sequential switching or system-fluid displacement

Separate metering addresses capacity and timing; system-fluid displacement addresses where the sample contacts the instrument. They can be combined. Liquid-handling systems such as Hamilton Microlab 600 provide examples of fluid-path organization, but a FOREACH assembly must still be designed around the selected pumps, valves and connections.^[7]

If a single-pump program aspirates both liquids as one segmented column, it needs space beyond 20 μL plus 60 μL for isolation and other movements. If it aspirates and dispenses them separately, budget the switching displacement instead. Eliminating a pump does not necessarily reduce reagent use or shorten the cycle; shared-path recovery to the required composition can consume both liquid and time.

System fluid is not part of the formulation while it remains outside the useful mixture. If it enters the mixture, it becomes additional dilution. A system-fluid-driven channel must therefore deliver the complete useful sample segment, not merely execute an equivalent piston displacement.

Where installation space is limited, compare SM or TM using the already-defined working points, then recheck that model's step volume, connections and performance. Where chamber gas is difficult to clear, compare EAS with a suitable priming program. Continuing air aspiration, inlet leakage or inadequate liquid level must still be corrected at their source; extra steps cannot reliably compensate a changing gas-compression volume.^[8]

04Confirm mixing and usable volume before taking the next aliquot

Correct sample and diluent volumes do not guarantee a correct local concentration. Addition position, vessel geometry and liquid level affect mixing; premature aspiration may collect a sample-rich or diluent-rich region. Establish the mixing sequence in the actual vessel and make mixing completion a prerequisite for resampling.

Mixing may use an appropriate mechanical stirrer or aspiration/dispensing movements. For the latter, the mixing volume must remain below the volume safely accessible at that moment, with the probe at the specified immersion. Mechanical mixing also needs control of bubbles and vessel-to-vessel carryover. More mixing cycles add time and can add tip retention or bubbles; evaluate adequacy through concentration consistency at different sampling positions and times.

An 80 μL nominal total cannot simply be divided by 3 μL to declare the number of available tests. Inaccessible bottom volume, the minimum level for mixing, wall retention and repeat-test reserve reduce usable liquid. Evaluate the last aliquot separately from the first. If several assays share one predilution, schedule them within each assay's allowed holding time.

Resampling is its own small-dose action. Acceptance of the original sample dose does not validate aspiration, tip retention and delivery of the diluted aliquot. Wash the probe according to its actual contact path before it changes tasks.

05Separate volume error, dilution-ratio error and subsequent transfer error

For analyte-free diluent, additive volumes, complete mixing and no analyte loss, the concentration ratio is Vsample ÷ (Vsample + Vdiluent). The target for 20 μL plus 60 μL is 0.25. The directions of the two volume errors jointly determine the actual ratio.

Evaluate each channel, the mixed concentration and the later aliquot separately. Allocate verification requirements for volume, mixing, interface loss and resampling from the customer's allowable final concentration or analytical-result error. One pump specification cannot serve as the error allowance for every stage.

Liquid-handling verification combines volume measurement with sample-integrity evaluation. If only particular specimens fail after dilution, also investigate method suitability and matrix changes. Locate the failing stage before changing both pump calibrations and the software dilution factor together.^[9][10]

06Convert a spike addition into its final concentration

In reagent development, sample preparation or method evaluation, a piston pump can meter a known-concentration standard into sample or blank matrix for spike recovery or concentration-series preparation. Define the standard, stock concentration, matrix, added volume and final volume together. Routine calibrators and QC materials still follow their preparation instructions; this is not permission to reformulate them.

For that example, compare an EA 100 μL full stroke for the standard and 90% of an EA 1 mL stroke for the matrix. A full 100 μL stroke leaves no same-stroke space for auxiliary liquid; adjust capacity or sequencing if predispense, isolation or reserve is required. Protein matrices, special solvents and low-concentration analytes also require checks of mixture stability, wetted compatibility and adsorption. Repeatable volume alone does not demonstrate spike recovery.^[2]

A concentration series may be prepared independently from the same stock or serially from the preceding level. Independent preparations reduce dependence on the previous vessel's metering and mixing error, although all levels still share stock-concentration uncertainty. Serial dilution may conserve stock and replace impractically small stock aliquots with more manageable intermediate volumes, but earlier ratio and mixing errors propagate. List stock volume, diluent volume and final volume at every level, then compare the lowest working volume, mixing burden and allowable error.

A standard channel used infrequently also needs first-dose-after-idle testing. Whether an additional dilution stage is permitted, which matrix to use and how to calculate the result are method decisions when the required range exceeds one preparation step.

07Connect selection requirements to the instrument sequence and recovery logic

Pumps and valves execute aspiration, dispensing and switching; the instrument coordinates the dilution vessel, mixer, probe motion and method calculations. The full program must establish priming and gas clearance, correct liquid sources and valve states, and adequate mixing before resampling. Include cleaning and low-level handling; completed motion is not proof that the liquid state is correct.

After air aspiration, gas entry, incorrect valve switching or an interrupted preparation, isolate the affected mixture and let the instrument method determine whether to remake it or apply another approved response. Restore supply and the liquid column, then recheck the first dose and critical working points. A mixture with uncertain composition should not be returned to reportable testing simply by adding a theoretical missing volume.

For selection, provide the sample and diluent identities, every metered volume, target ratio, mixing method, resampling volume, available specimen volume, allowed cycle time and cleaning conditions. FOREACH can then match capacities, wetted materials, valves and drive configuration and define validation in the actual vessel and fluid path.

References

Related guides

• Piston-pump capacity and configuration
• Sample aspiration and transfer
• Reagent dispensing and first-dose recovery
• Piston-pump models and configurations