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Automated enumeration of lymphoid and plasma cells in bone marrow to establish normal reference ranges
  1. James Liang1,2,
  2. Jacques A J Malherbe1,3,
  3. Kathryn A Fuller1,4,
  4. Bob Mirzai1,4,
  5. Carly George1,5,
  6. Tina L Carter4,5,
  7. Catherine H Cole1,3,4,5,
  8. Belinda B Guo1,
  9. Katie Meehan1,
  10. Wendy N Erber1,3,4
  1. 1School of Biomedical Sciences, Faculty of Health and Medical Sciences, The University of Western Australia, Crawley, Western Australia, Australia
  2. 2Department of Haematology, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
  3. 3Medical School, Faculty of Health and Medical Sciences, The University of Western Australia, Crawley, Western Australia, Australia
  4. 4PathWest Laboratory Medicine, Nedlands, Western Australia, Australia
  5. 5Department of Haematology, Princess Margaret Hospital, Subiaco, Western Australia, Australia
  1. Correspondence to Professor Wendy N Erber, Faculty of Health and Medical Sciences (M500), The University of Western Australia, Crawley, Western Australia 6009, Australia; wendy.erber{at}uwa.edu.au

Abstract

Aims The number of precursor and mature lymphoid cells and plasma cells in normal bone marrow trephine (BMT) biopsies throughout the human lifespan is unknown. Reference ranges have been established from aspirated marrow, but due to haemodilution errors, these do not accurately reflect the native marrow milieu. We aimed to define age-specific, normal reference ranges for lymphoid and plasma cells in BMT biopsy specimens using a combined immunophenotyping and digital enumeration approach.

Methods Morphologically normal BMT biopsy specimens (n=483) were obtained from patients aged 1 month to 90 years of age. Immunohistochemistry was performed to identify lymphoid progenitors , T-lymphocytes (CD3), B-lymphocytes (CD20) and plasma cells (CD138 and MUM1). Positive cells were counted using digital enumeration software, and the percent positivity for each antigen was determined per case. Mean values were generated for specific age groups, and age-defined reference ranges were determined for each antigen using normalised data.

Results A mean of 16 609 cells (range: 7210–34 097) were counted per biopsy. Infant marrows showed a predominance of immature lymphoid progenitors and B cells. With increasing age, an increase in mean T cell and plasma cell numbers were observed. The results showed the same trends to flow cytometry references for aspirate material although the absolute values differed.

Conclusions Combined immunohistochemistry and automated enumeration gives an accurate, reproducible number of antigen-positive cells and has generated normal reference ranges for these cell types in BMT biopsies. The method and ranges we have established have the potential to be applied in routine clinical practice.

  • immunohistochemistry
  • bone marrow trephines
  • lymphocytes
  • digital pathology

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Footnotes

  • JL and JAJM contributed equally.

  • Handling editor Mary Frances McMullin.

  • Contributors JL, JAJM, CG, TLC, CHC and WNE selected suitable specimens for the study. JL and WNE reviewed bone marrow pathology. JL, JAJM, BM and KAF carried out the staining and digital enumeration. JL, JAJM, BBG, KAF and WNE extracted and interpreted the data. JAJM and KM conducted the statistical analyses. All authors were involved in writing the manuscript and had final approval of the submitted and published versions.

  • Funding JL received fellowships from the WA Cancer and Palliative Care Network Health and the WA Health Department. BBG received a Translational Cancer Pathology Postdoctoral Fellowship from Cancer Council Western Australia. KAF and WNE received a Sir Charles Gairdner Group grant for part of this study.

  • Competing interests None to declare.

  • Patient consent Not required.

  • Ethics approval The study had ethical approval from the Sir Charles Gairdner and Osborne Park Health Care Group (#2016-172), Child and Adolescent Health Service (2017004EP) and University of Western Australia (#RA/4/1/9099; #RA/4/1/9213) Human Research Ethics Committees.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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