How to do a perfect linearity measurement and test intermediate precision

Background

Many customers want to test the linearity and the precision of our instruments and check the CV values for different cell concentrations. Either they ask for our support in advance or they try it themselves and receive unsatisfying results. The correct sample preparation is very important and helps preventing much support from our site.

How tdo a perfect linearity measurement 

  1. Recommendations for sample handling and preparation: • Sample dilution should always be done in full media. We know that PBS will influence the viability depending on the cell type – dilution will decrease viability. Furthermore, PBS can influence the staining kinetic of Acridine Orange (effect known with T-cells and PBMCs). So, always recommend full media for sample dilution. • Sample mixing should be done carefully by pipetting. We do not recommend vortexing as the cells will stick to the plastic tube. Between the single measurements the sample can be inverted and always kept in movement. • Measure the samples at least in triplicates. • Avoid serial dilutions, the pipetting error will be carried to all samples. It is better to dilute every sample one by one and keep them between the measurements at 37°C.  • Always use an appropriate amount of cell sample for dilution, otherwise you will introduce errors. • It is possible to do technical replicates (measure the same sample several times) or biological replicates (dilute individual for each replicate). We recommend performing technical replicates otherwise more user errors will be introduced by sample preparation. 2. Use the attached excel sheet

We do have an excel sheet for performing linearity measurements. That sheet can be sent to the customer, all necessary information is already included, and the customer only needs to fill in the columns for total cell count and viability:

  • Sheet #1 (optional): The measured cell concentration can be entered in the column highlighted in green. The necessary amount of medium for dilution is will be automatically calculated. 
  • Sheet #2: The expected cell counts range from 4E6 to 7,5E4 cells/ml, this will avoid getting out of optimal range for the NC200. As the NC202 has an increased counting range, more cell concentrations can be added to the existing list, respectively. 3. Evaluation
  • The coefficient of variation (CV) % will be automatically calculated and can be compared for each cell concentration with the expected CV values for the instruments. You may send the performance documents for the NC200 or NC202 along with for the customers to compare.
  • The viability should always stay the same, if you see a drop in viability, ask the customer about sample preparation. Very often they´ve used PBS or mixed the samples very harsh.
  • Results which are out of the optimal range should be excluded and the measurement should be repeated.

Example of the linearity measurement for total cell count and viability.

How to test intermediate precision

  • Inter- instrument comparison: Test the same cell sample on several NC200 instruments one after the other by one user
  • Inter- person comparison: Test the same cell sample on the same NC200 instrument by different users. The instrument can be restarted in between to simulate different days.
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