Analysis of human chromosomes by imaging flow cytometry

Abstract

Chromosomal analysis is traditionally performed by karyotyping on metaphase spreads, or by fluorescent in situ hybridization (FISH) on interphase cells or metaphase spreads. Flow cytometry was introduced as a new method to analyze chromosomes number (ploidy) and structure (telomere length) in the 1970s with data interpretation largely based on fluorescence intensity. This technology has had little uptake for human cytogenetic applications primarily due to analytical challenges. The introduction of imaging flow cytometry, with the addition of digital images to standard multi‐parametric flow cytometry quantitative tools, has added a new dimension. The ability to visualize the chromosomes and FISH signals overcomes the inherent difficulties when the data is restricted to fluorescence intensity. This field is now moving forward with methods being developed to assess chromosome number and structure in whole cells (normal and malignant) in suspension. A recent advance has been the inclusion of immunophenotyping such that antigen expression can be used to identify specific cells of interest for specific chromosomes and their abnormalities. This capability has been illustrated in blood cancers, such as chronic lymphocytic leukemia and plasma cell myeloma. The high sensitivity and specificity achievable highlights the potential imaging flow cytometry has for cytogenomic applications (i.e., diagnosis and disease monitoring). This review introduces and describes the development, current status, and applications of imaging flow cytometry for chromosomal analysis of human chromosomes.

Imaging flow cytometry shows monosomy 17 in circulating plasma cells in myeloma

“…8 To improve stability of the antibody-antigen complex on the plasma cell membrane, in order to withstand the acidic and high temperatures for FISH, bis (sulfosuccinimidyl) suberate was added. 8,9 The cells were fixed with 4% formaldehyde postimmunophenotyping to preserve cellular integrity. Double stranded DNA was then denatured (78 o C for 5 min), FISH probes added and hybridised at 37 C for 24 h. For this assessment, probes to the centromere of chromosome 17 (CEP17-SG) and the 17p region (17p12-OR) were assessed to determine whether there was a secondary abnormality leading to the progressive disease.…”

Imaging flow cytometry-based multiplex FISH for three IGH translocations in multiple myeloma

“…Various attempts have been made to use flow cytometry technology to evaluate chromosome status since the development of flow karyotyping in the mid-1970s [ 24 , 25 ]. Then, the combination assay of the FISH technique and the immunophenotyping of cells of interest using flow cytometric procedure, the so-called immune-S-FISH, has been introduced as a successor modality [ 25 – 29 ].…”

“…Various attempts have been made to use flow cytometry technology to evaluate chromosome status since the development of flow karyotyping in the mid-1970s [ 24 , 25 ]. Then, the combination assay of the FISH technique and the immunophenotyping of cells of interest using flow cytometric procedure, the so-called immune-S-FISH, has been introduced as a successor modality [ 25 – 29 ]. Unlike the standard FISH for cells attached to the glass slide, the immuno-S-FISH enabled the high-throughput evaluation of the chromosome status of many immunophenotyped cells in suspension.…”