The evaluation of the response of T-Cells to a prospective vaccine is a criti-cal aspect of any study of the effectiveness of vaccine candidates, such asprospective anti-cancer vaccines or vaccines against infectious diseases. TheT-Cell response is usually monitored by techniques such as the ELISPOTassay, or an intracellular cytokine-staining assay.

The ELISPOT assay involves the enumeration of the spots on a membraneafter incubating peripheral blood mononucleated cells (PBMCs) with specif-ic antigens in a 96-well plate. Each spot on the membrane represents a cellthat can produce an appropriate cytokine in response to the specific antigenstimulus. The response to the antigen is typically reported as Spot FormingCells per million peripheral blood mononucleated cells (SFC/PBMC). Thenumber of cells that are input for the assay is a vital parameter and the accu-racy and precision of the overall assay is clearly very dependent on the accu-racy and precision of the counting of the number of cells in the sample.

Several different technologies are commonly used to count the number ofviable cells, including staining with Trypan blue or with a fluorescence sub-strate, followed by visual counting methods. In our laboratories, it has beenfound that these techniques lead to a high variation when the counting isperformed at different times by the same operator, as well as when the count-ing is performed by different operators on the same sample. This does, ofcourse, lead to uncertainty in the number of cells in the sample, and therebyleads to an uncertainty in the overall evaluation of the efficacy of the vaccinecandidate.

Microcytometry provides an alternative approach for cell counting and thisarticle describes an evaluation of the ViaCount cell technology to determineif it is possible to obtain more reliable and accurate cell counts.

Methods Ten samples were collected from normal individuals and separated after stand-ing overnight by a standard Ficoll procedure. The remaining cells were cryopre-served and seven of these cell samples were used for counting after thawing (day1) and after standing overnight (day 2). Three additional samples were used assources of PBMCs. Cells in all the samples were counted using a Guava PersonalCell Analysis (PCA) system [Figure 1] in parallel with Trypan blue exclusion dye,Erythrosin B exclusion dye, and AOB fluorescent methods of staining andcounting in order to compare the accuracy, precision and linearity. A flow cyto-metric method (TruCOUNT, BD Biosciences) was also used for comparing theaccuracy of the counts. In order to compare the accuracy of the various meth-ods, ten different sample were run in triplicate. To determine the linearity of theresults, five different samples were run in a serial two fold dilution. In order to

compare the precision,three different sampleswere run ten times witheach procedure except forthe TruCOUNT method.

ResultsCounts that were obtainedwith the ViaCount were asaccurate as those obtainedwith the other procedures;no significant differenceswere observed (p>0.1). Itwas found that linearitywas lost from the 1:16dilution onwards for bothfresh and cryopreserved samples with initial count >2x106 PBMC/mL. For theGuava PCA method, the R2 value was >0.89; the range for the other methodswas >0.86. The precision using the three methods for cell counting describedabove is shown in Table 1.The ability of theViaCount system to pro-vide more precise valuesfor cell counts resulted inmore precise data with theELISPOT assay. The trendanalysis of SFC/106 PBMCdata obtained with theAMJ2L cell sample in theELISPOT assay was usedto illustrate this; the datashown in Figure 2 werecollected over a period of10 days. The yellow line represents results for each assay using the ViaCountmethod for cell counting, while the red line represents the results of the assayusing the Trypan blue method for determining the number of cells. Figure 3 rep-resents an analysis of the data obtained with ViaCount, with the Trypan bluemethod shown as a reference, to determine the mean +/- SD. It is clear that theViaCount assay technique generates results with considerably less variation overtime.

Conclusions

A new technique for enhanced cell counting precision with the ELISPOT assayby Dr. Guido Ferrari, Justin Pollara and Louise Saufley

The ELISPOT assay for the evaluation of the T-Cell response to vac-

cine candidates requires that the number of cells in the sample be

accurately determined. This article describes a study which demon-

strates that the use of the ViaCount cell counting technique from

Guava Technologies provides significantly more precise results than

manual procedures such as Trypan blue staining or the use of a flu-

orescent substrate prior to counting. The accuracy of the procedure

was found to be equivalent to the other techniques. An additional

benefit of this new technology is that an electronic record is gener-

ated that is easily transferable to other databases

Table 1. Data to show precision of the three methods ofcell counting.

Figure 1. The Guava personal cell analysis (PCA) system.

Figure 2. Plot of SFC/106 using the AMJ2L Cell Sample in an ELISPOT assay. Data aver-aged over 10 days (one sample/day). ViaCount method for cell counting = Yellow Line,Trypan blue method = Red Line.

C ell Counting as published in CLI November 2004

Cell counting using the Guava ViaCount technique clearly led to more precisecounts than were obtained using methods based on staining with Trypan blue orusing a fluorescence substrate prior to cell counting. The greater level of preci-sion lead to a significant improvement in the SFC/106 PBMC data for evalua-tion of vaccine candidates.

The authorsGuido Ferrari MD, andJustin Pollara Department of Surgery,Duke University,27710 NC, USA.Louise Saufley (corresponding author)Guava TechnologiesSan Francisco, CA, USA, Fax: +1 510 576 1500Email: lsaufley@guavatechnologies.com

Figure 3. Determination of the mean +/- SD of cell counts for Viacount and manualmethods.

C ell Counting as published in CLI November 2004