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Published on: December 6, 2014
Evaluation of a method allowing preservation of fresh lymph nodes for flow cytometric immunophenotyping
Patricia Brincat1, James Degaetano, Craig Donaldson
1Haematology Laboratory, Department of Pathology, Mater Dei Hospital, Malta. patricia.farrugia@gov.mt
Insights
A new cryopreservation method allows for delayed flow cytometric immunophenotyping (FCI) of lymph nodes (LN). This technique preserves diagnostic accuracy for hematolymphoid neoplasms (HLN) and reactive processes, enabling morphology-based analysis.
Area of Science:
- Hematopathology
- Immunophenotyping
- Cryopreservation
Background:
- Flow cytometric immunophenotyping (FCI) of lymph nodes (LN) traditionally requires immediate analysis of fresh tissue.
- Delays between surgery and analysis can compromise sample integrity and diagnostic accuracy.
Purpose of the Study:
- To evaluate a novel cryopreservation method for preserving fresh lymph node biopsies.
- To enable histomorphology-based FCI for improved diagnostic capabilities.
Main Methods:
- Prospective study on 30 lymph nodes (LN) suspected of hematolymphoid neoplasms (HLN).
- FCI performed on both fresh and cryopreserved LN cell suspensions.
- Analysis of percentage positivities (PP) and mean fluorescent intensities (MFI) for lymphoid antigens and viability.
Main Results:
- Cryopreservation showed minimal impact on PP and MFI for most tested antigens.
- A slight increase in apoptotic cells was observed but was not diagnostically significant.
- Both reactive processes and HLN were accurately identified and differentiated in cryopreserved samples.
- Viability remained above 75% for cryopreserved LN.
Conclusions:
- The developed cryopreservation method allows for delayed FCI of LN biopsies.
- This approach supports morphology-based immunophenotypic analysis, enhancing sensitivity, specificity, and cost-effectiveness.
- The method maintains the benefits of FCI for LN specimen management.
Background:
Flow cytometric immunophenotyping (FCI) of lymph nodes (LN) requires fresh unfixed tissue, with analysis being carried out within few hours post surgery. This study evaluated a novel method for fresh LN preservation, in order to allow histomorphology-based FCI.
Methods:
This study was carried out prospectively on 30 LN with suspected involvement by haematolymphoid neoplasms (HLN). FCI was performed on each fresh and post cryopreserved LN cell suspension. Percentage positivities (PP) and mean fluorescent intensities (MFI) were calculated on both preparations for a combination of T and B-lymphoid antigens together with viability.
Results:
The cryopreservation method applied in this study did not affect significantly PP and had minor impact on MFI of the tested antigens. Overall, there was minimal decrease in PP and MFI on the cryopreserved cells when compared with fresh cells, for most antigens with only a mild increase in apoptotic cells. However, these changes were not diagnostically significant, since both reactive processes and HLN present within analyzed LN could be identified and differentiated. Viability was more than 75% for all cryopreserved LN composed of haematolymphoid cells.
Conclusion:
The method presented in this study confers the possibility of storing fresh LN biopsies for later FCI, thus allowing a morphology-based immunophenotypic approach. This would allow a more sensitive, specific, and cost-effective management of LN specimens, whilst maintaining the important benefits provided by FCI.
