CellPrint has developed and patented an analytical platform (CellPrint™) that surpasses other available technologies in assessing molecular expression in human cells. CellPrint™ is based on flow cytometry, an established tool widely used in clinical and research laboratories, that allows for single-cell analysis such that assessments of molecular expression can be assigned to specific cell types. CellPrint™ conserves this important and distinguishing feature of standard flow cytometry but transforms it with a level of precision and a dynamic range of detection that are superior by orders of magnitude.

Single Cell Resolution. Like standard flow cytometry, CellPrint™ allows assignment of assessments to specific cell types. Other analytical platforms such as microarrays, genomic analysis, mutational studies, and immunoblotting, either assess expression levels across mixtures of cell types in a specimen or require preparatory subpopulation enrichment. Reporting expression across multiple cell types degrades analytical performance (accuracy and precision) since expression levels in important cell types are averaged with levels in irrelevant cell types. Subpopulation enrichment is complicated, expensive, time-consuming and error prone. Since the procedure is rarely complete, it also carries unacceptable risks of flawed measurements and misleading findings.

Sensitivity and Precision. Despite standard flow cytometry’s powerful features, it suffers from poor sensitivity. CellPrint scientists have solved this problem by amplifying the fluorescent signal 10-100 fold by binding an enzyme to the targeted molecule that catalyzes the deposition of fluorescent markers, in contrast to regular flow cytometry where a fluorescent marker is bound to the targeted molecule. This innovative catalyzed reporter deposition technology allows detection of clinically relevant molecular expression when it may be undetectable using standard flow cytometry.

Example of Enhanced Sensitivity with CellPrint™ versus Standard Flow CytometrySensitivity4small

• Results are from a CellPrint study of immune molecules.
• Left panel shows results using standard flow cytometry. Right panel shows results using CellPrint™.
• The y-axes show the count of events at each level of fluorescence intensity (x-axes).
• Gray histograms reflect controls. Blue histograms reflect expression of the molecule.
• Example shows a 59-fold enhancement of the fluorescent signal with CellPrint™ compared to standard flow cytometry.

An additional benefit of this enhanced sensitivity is an expansion of the dynamic range of detection from a fraction of a log, typical with standard flow cytometry, to 1-2 logs with CellPrint™. This expanded range of detection enables powerful quantitative analysis of the level of molecular expression in cell donor populations (e.g., treated and untreated patients) or cell types (e.g. monocytes, B cells, T cells, or platelets) that is impossible with standard flow cytometry.

Example of Expanded Dynamic Range of Detection with CellPrint™ versus Standard Flow CytometryDR6

• Results are from a CellPrint study of PTEN (a phosphatase important in signaling pathways and oncogenesis) expression in peripheral blood mononuclear cells from 25 donors.
• The y-axis reflects PTEN expression as measured by median fluorescence ratio.
• Orange columns show study results with standard flow cytometry. The range of values (from 1 to 3) provides limited information about differences in expression across donors.
• Blue columns show study results with CellPrint™. The range of values (from 3 to 60) allows powerful quantitative analysis.
• Results from the two platform were correlated with r = 0.7.

The enhanced sensitivity and expanded dynamic range afforded by CellPrint™ allows precise quantification of molecular relationships that is not feasible using other technologies. In the example below, protein co-expression characterizes biological differences between healthy and diseased persons. Some correlations are maintained between populations, but others are lost with the disease. With findings such as this, CellPrint scientists are developing important insights about pathogenesis and advancing the discovery and development of novel therapies.

Example of CellPrint™ Revealing Molecular Organization in Healthy and Diseased Patientsprecision3

• Selected results are from a study using CellPrint™ to assess molecular organization in peripheral blood mononuclear cells.
• Each axis reflects protein expression as measured by median fluorescence ratio.
• Relationship between expression of Protein 1 and expression of Protein 2 in healthy persons is maintained in diseased persons. However, relationship between expression of Protein 1 and expression of Protein 3 in healthy persons is lost in diseased persons.
• For two-tailed t-tests of r values, p ≤ 5.0×10-6, except for the lower-right panel where p ≤ 0.29.
• With the high precision afforded by CellPrint™, important findings were obtained in this study using clinical samples from only 22 healthy and 9 diseased persons. The ability to leverage small samples allows acceleration of preclinical and clinical trials.

Protein Analysis. CellPrint™ is particularly powerful in assessing protein expression. Some technologies, such as microarray, attempt to explain disease or drug effects by measuring messenger RNA (mRNA) expression. Yet protein, not mRNA, is directly responsible for cellular function and organismal phenotype. Many studies have demonstrated a poor correlation between mRNA expression and protein expression, including one showing that less than one-quarter of the variability of protein expression can be explained by mRNA expression (Zhang et al. (2014) Nature. Sep 18; 513(7518):382-70). The capability of CellPrint™ to precisely assess protein expression is a key advantage over platforms assessing mRNA expression.

Clinical Utility. With its roots in flow cytometry, CellPrint™ maintains the benefits of a proven clinical platform including rapid data generation, detection of cellular lineage, and the ability to probe intact cells. CellPrint™ is a validated technology and has demonstrated the high reproducibility essential for research investigations and human clinical trials.

Example of High Reproducibility of Results with CellPrint™reproducibility3

• This example shows selected results using CellPrint™ in two separate studies of bivariate relationships of protein expression in monocytes cells from healthy donors.
• Each axis reflects protein expression as measured by median fluorescence ratio.
• The results demonstrated high reproducibility with high Pearson’s r values for the same five protein pairs in both studies.
• There was no attempt to match the two studies. Antibody lots were distinct and the mix of donors was different with respect to gender and age.