Biomedical Engineering Reference
In-Depth Information
help in getting the instrument settings and compensation for a new assay especially for
multifluorochrome analysis.
Staining of samples can be performed in test tubes that fit onto the cytometer
platform or in multiwell plates. The use of multiwell microtiter plates is ideal for
staining a large number of samples; however, wash volumes are limited by well
volume. Nonetheless, if plates are used and the flow cytometer is equipped with a
multiwell plate sampler, the samples can be directly acquired on the flow cytometer.
Alternatively, if tubes are preferred, additional automated solutions are available.
Samples manually stained in test tubes can be lysed, washed, and fixed using
automated instruments such as Lyse Wash Assistant (BD Biosciences), then directly
loaded onto a FACSLoader for automated acquisition.
Optimization of temperature, time, and antibody concentration is important for
establishing a good quality flow cytometry assay. Staining should be performed at
colder temperatures, ideally 4 C, and with buffers that contain sodium azide to
prevent antibody capping. Antibody titrations using these time(s)/temperature(s) are
appropriate for determining the saturating concentration of antibody during assay
development.When a laboratory initiates awork stream to study a new antigen (or one
they are unfamiliar with), it is strongly suggested that the impact of receptor shedding/
soluble receptors on antibody titrations are considered. Soluble receptors impact the
saturating concentration of an antibody reagent, and the concentration of a soluble
receptor can vary in a population of normal versus diseased individuals. Measures that
ensure that saturating concentrations of antibodies are used will ensure good signal-
to-noise ratio and overall assay performance. In cases where whole blood is the
desired specimen and the level of saturating receptor is high and/or variable, a simple
wash with PBS immediately prior to staining safeguards against erroneous titrations
and subsequent staining conditions when the assay moves to the production stage.
12.3.1.4 Sample Fixation It is not always possible to analyze samples on a flow
cytometer immediately after staining, therefore cells may have to withstand delays in
storage or shipment (hours to days). It is critical to stabilize labeled cells by fixation in
a low concentration of formaldehyde. Most laboratories store fixed samples at 4 C,
and all should store the samples protected from light. Fixation also alleviates potential
biohazard concerns regarding the samples and is always recommended for clinical
sample preparation. Protein markers are affected differently by fixation techniques;
therefore, it is important to evaluate stability of all markers over a range of fixation
time. Because of these effects, some laboratories opt to fix all samples even if they are
analyzed soon after processing. Once stability in fixative is established, these assay
conditions can be used for subsequent validation.
12.3.1.5 Sample Collection, Processing, and Stability It is critical that all
relevant cellular and antigenic properties are maintained on cell lines or primary
cells to be used for method development. For cell lines, particularly adherent cell
lines, it is important to generate suspension cultures using reagents that preserve the
expression of cell surface markers. For peripheral blood samples, anticoagulants must
be evaluated and selected on the basis of antigenic integrity and stability.
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