Aims Some types of monoclonal gammopathies are typified by a very limited availability of aberrant cells. Modern research use high throughput technologies and an integrated approach for detailed characterisation of abnormal cells. This strategy requires relatively high amounts of starting material which cannot be obtained from every diagnosis without causing inconvenience to the patient. The aim of this methodological paper is to reflect our long experience with laboratory work and describe the best protocols for sample collection, sorting and further preprocessing in terms of the available number of cells and intended downstream application in monoclonal gammopathies research. Potential pitfalls are also discussed.
Methods Comparison and optimisation of freezing and sorting protocols for plasma cells in monoclonal gammopathies, followed by testing of various nucleic acid isolation and amplification techniques to establish a guideline for sample processing in haemato-oncology research.
Results We show the average numbers of aberrant cells that can be obtained from various monoclonal gammopathies (monoclonal gammopathy of undetermined significance/light chain amyloidosis/multiple myeloma (MM)/MM circulating plasma cells/ minimal residual disease MM—10 123/22 846/305 501/68 641/4000 aberrant plasma cells of 48/30/10/16/37×106 bone marrow mononuclear cells) and the expected yield of nucleic acids provided from multiple isolation kits (DNA/RNA yield from 1 to 200×103 cells was 2.14–427/0.12–123 ng).
Conclusions Tested kits for parallel isolation deliver outputs comparable with kits specialised for just one type of molecule. We also present our positive experience with the whole genome amplification method, which can serve as a very powerful tool to gain complex information from a very small cell population.
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Abstract in Czech
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- Abstract in Czech - Online abstract
TŠ and KG contributed equally to this work.
Handling editor Mary Frances McMullin
Contributors TS, KG, ZKu, JF and PV performed the experiments. JF, FK and TJ performed the flow cytometry analyses. EK, TS and KG wrote the manuscript. ZKo, FK and RH designed the study and provided the clinical background. TS and KG contributed equally to this study.
Funding This study was supported by the Institutional Development Plan of the University of Ostrava (IRP201550) and the Ministry of Education, Youth and Sports (specific university research of the Faculty of Medicine, University of Ostrava) SGS03/LF/2015–2016 and SGS10/LF/2016–2017, University Hospital Ostrava DRO-FNOs/2016/21, DRO-FNOs/2014/17P and the Ministry of Health of the Czech Republic (15-29667A and 17-30089A).
Competing interests None declared.
Ethics approval The study was approved by the University Hospital Ostrava (66/2016).
Provenance and peer review Not commissioned; externally peer reviewed.
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