Automated enumeration of lymphoid and plasma cells in bone marrow to establish normal reference ranges

James Liang, Jacques A.J. Malherbe, Kathryn A. Fuller, Bob Mirzai, Carly George, Tina L. Carter, Catherine H. Cole, Belinda B. Guo, Katie Meehan, Wendy N. Erber

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)916-925
Number of pages10
JournalJournal of Clinical Pathology
Volume71
Issue number10
DOIs
Publication statusPublished - 1 Oct 2018

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Plasma Cells
Reference Values
Bone Marrow
Lymphocytes
Biopsy
Antigens
Lymphoid Progenitor Cells
Immunohistochemistry
T-Lymphocytes
Immunophenotyping
Hemodilution
B-Lymphoid Precursor Cells
Bone Marrow Cells
Flow Cytometry
B-Lymphocytes
Software
Age Groups
Cell Count

Cite this

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title = "Automated enumeration of lymphoid and plasma cells in bone marrow to establish normal reference ranges",
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.",
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Automated enumeration of lymphoid and plasma cells in bone marrow to establish normal reference ranges. / Liang, James; Malherbe, Jacques A.J.; Fuller, Kathryn A.; Mirzai, Bob; George, Carly; Carter, Tina L.; Cole, Catherine H.; Guo, Belinda B.; Meehan, Katie; Erber, Wendy N.

In: Journal of Clinical Pathology, Vol. 71, No. 10, 01.10.2018, p. 916-925.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Automated enumeration of lymphoid and plasma cells in bone marrow to establish normal reference ranges

AU - Liang, James

AU - Malherbe, Jacques A.J.

AU - Fuller, Kathryn A.

AU - Mirzai, Bob

AU - George, Carly

AU - Carter, Tina L.

AU - Cole, Catherine H.

AU - Guo, Belinda B.

AU - Meehan, Katie

AU - Erber, Wendy N.

PY - 2018/10/1

Y1 - 2018/10/1

N2 - 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.

AB - 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.

KW - bone marrow trephines

KW - digital pathology

KW - immunohistochemistry

KW - lymphocytes

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U2 - 10.1136/jclinpath-2018-205168

DO - 10.1136/jclinpath-2018-205168

M3 - Article

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JO - Journal of Clinical Pathology

JF - Journal of Clinical Pathology

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