Implementation of Microfluidic Chip Electrophoresis for the Detection of B-cell Clonality

Abstract

Abstract Introduction: A clonal population of B-cells is defined as those cells arising from the mitotic division of a single somatic cell with the same rearrangement of immunoglobulin genes. This gives rise to DNA markers for each individual lymphoid cell and its progenies and enables us to study clonality in different B-cell malignancies using multiplex polymerase chain reaction - PCR. The BIOMED-2 protocol has been implemented for clonality detection in lymphoproliferative diseases and exploits multiplex PCR reaction, subsequently analyzed by heteroduplex analysis (HDA) using polyacrylamide gel electrophoresis (PAGE). With the advent of miniaturization and automation of molecular biology methods, lab-on-chip technologies were developed and replace partially the conventional approaches. We tested device for microfluidic chip, which is used for B-cells clonality analysis, using a PCR reaction for three subregions called frameworks (FR) of the immunoglobulin heavy locus (IGH) gene. Material and Methods: For the implementation and comparison of the two methods we analyzed three unknown B-cell chronic lymphocytic leukemia (B-CLL) samples. As positive control (PK) we used one formalin-fixed, paraffin-embedded (FFPE) sample from B-CLL lymph node. The DNA was extracted from FFPE sections and multiplex PCR was used to amplify IGH gene segments. After PCR, the HDA was performed, the DNA fragments were evaluated on the PAGE and the microfluidic chip electrophoresis as well, and the results were compared. Results: Using HDA with subsequent PAGE, we were able to confirm the clonality of the positive control and the tested samples. The same results were obtained by the Bioanalyzer 2100. The microfluidic chip electrophoresis was persuasive in all tested samples. Conclusion: The implementation of microfluidic chip electrophoresis for detection of B-cell clonality by BIOMED-2 protocol on the device Agilent 2100 Bioanalyzer was successful and yielded the same results as the HDA - PAGE. Moreover, chip electrophoresis system is faster for preparation and less laborious than the conventional HDA - PAGE method.