Flow cytometric approach to the diagnosis and classification of mature B-cell lymphoid neoplasm 가천의대길병원진단검사의학과 안정열

Mature B-cell neoplasm 1. Mature B-cell : SIg(+) & lack of Ags of immaturity (TdT, CD34) or weak CD45 2. Diagnosis of mature B-cell lymphoma by FCM 1) identification of phenotypically abnormal cells 2) recognition of phenotypes characteristic of separate disease entities 3) provide prognostic information (CD38 in CLL) 4) evaluation of MRD (minimal residual disease) 5) identify targets for potential Ab-direct Tx

Identification of abnormal mature B-lymphoid cells I. Immunoglobulin light chain class restriction II. Aberrant antigen expression III. Significance of small populations of phenotypically abnormal B cells Flow cytometric classification of mature B-cell neoplasms

Identification of abnormal mature B-lymphoid cells I. Immunoglobulin light chain restriction 1. Neoplasm of mature B cells usually express only one class of Ig light chain (i.e., kappa or lambda), used as a surrogate marker of clonality 2. 고려할점 : (1) Ig light chain restriction = monoclonality? (2) Interpretation of staining for κ/λ

lambda kappa kappa lambda CD45 CD19 CD19

(1) Ig light chain restriction monoclonality 1) the presence of a clonal B-cell process by FCM : κ/λ ratios of more than 3.0 or less than 0.5 2) Rare monoclonal λ light chain restriction in reactive B-cell - monoclonal proliferations in tonsillar specimen in childhood. - multicentric Castleman disease - germinal center B cells in Hashimoto s thyroiditis => Ig light chain restriction is not synonymous with monoclonality or neoplasia 3) Light chain restricted populations that are truly monoclonal are not neoplastic : florid follicular hyperplasia in patients with HIV

(2) Interpretation of staining for κ/λ 1) Nonspecific (cytophilic ) binding - Fc receptor - adherence of Ab to sticky cells (damaged or dying cells) 2) Lack of staining for surface Ig - true weak or negative : lymphoblast (ALL), plasma cell (PCN), thymic B cell (mediastinal B-cell lymphoma), CLL -false negative ; soluble Ab interfere with the binding of detection Ab ( 해결 : initial wash step ) ; the epitope recognized has been deleted or altered (more frequently with MoAb than polyab)

CD45 CD19 lambda CD19 lambda kappa kappa

CD16 CD45 CD16 CD3 CD56 CD56

kappa lambda

poly lambda poly kappa

II. Aberrant antigen expression (1) the presence of Ags not normally expressed by B cells ( CD5 in CLL, CD13 or CD33 in LPL) (2) not typically present in a subset of B cells ( CD10+/bcl 2+ in follicular lymphoma) (3) alteration in intensity of staining for B-lineage associated Ags (dim CD19 and brighter CD10 in FL)

III. Significance of small populations of phenotypically abnormal B cells (1) Less than 5% of the total cells analyzed (2) In the staging of pts with previously characterized lymphoma => if the phenotype matches, can be used to determine the presence of involvement (3) do not have a previous diagnosis of lymphoid malignancy => significance is less clear ex) insignificant CLL-like populations in PB and BM from older patients =>it should not be used to establish a new diagnosis.

Flow cytometric classification of mature B-cell neoplasms (1) Useful to consider 4 broad groups as determined by their expression of CD5 and CD10 (2) CD5+ CD10- CD5- CD10+ CD5- CD10- CD5+ CD10+

Ab cocktails used in B-cell lymphoma (1) CD5/CD38/CD45/CD19 (2) Lambda/Kappa/CD45/CD19 (3) FMC7/CD23/CD45/CD22 (4) CD20/CD10/CD45/CD19

CD5/CD38/CD45/CD19 1) Aid in enumerating T and B cells 2) Assess CD5 expression on B cells 3) Assess CD38 on CD19+ cells to distinguish between activated mature B cells, hematogones, and plasma cells 4) Assess CD38 expression on CLL

CD5 CD5

CD19 CD5 CD38 CD38 CD45 CD19 CD5

Lambda/Kappa/CD45/CD19 1) Aid in enumerating B cells 2) Assess light chain reactivity on B cells ( monotypic or polytypic) 3) CD19 gate : aids in - characterizing light chain pattern - evaluation for surface Ig negative cells - small abnormal populations

CD19 CD19 Anti-kappa Anti-kappa Anti-lambda Anti-lambda

CD19 lambda kappa CD19

FMC7/CD23/CD45/CD22 1) Aid in characterizing mature CD5+ small B cell neoplasm 2) Typical CLL: CD23+FMC7-3) Typical Mantle cell lymphoma : CD23-FMC7+ 4) Assess for lymph node pattern: bimodal CD23/FMC7 - germinal center B cells : brighter FMC7 - mantle zone B cells : brighter CD23

CD5 CD5

CLL CD23 CD45 CD22 FMC7 CD22 FMC7 CD23

MCL CD23 FMC7 CD45 CD23 CD22 CD22 FMC7

CD20/CD10/CD45/CD19 1) Evaluation CD10 expression on B cells 2) Confirm normal circulating B cell phenotype (CD19+CD20+CD10-) 4) Confirm hematogones (CD19+CD20variableCD10+) 5) Assess CD19 expression on B cells (ex; aberrant dim is common in FL) 6) Assess for germinal center pattern ( minority of GC B cells with dim CD10+and slightly brighter CD20) 7) CD19+CD10+CD20-: D/Dx of negative for CD20 in Tx with rituximab anti-cd20

Chronic lymphocytic leukemia (CLL)

CD45 lambda CD19 lambda kappa kappa CD19

CD45 CD22 FMC7 CD22 FMC7 CD23 CD23

Mantle cell lymphoma

CD45 FMC7 CD23 CD23 CD22 CD22 FMC7

Follicular lymphoma

Anti-lambda CD19 Anti-lambda Anti-kappa Anti-kappa CD45 CD19

CD23 CD23 CD45 FMC7 CD22 CD22 FMC7

or CD45 FMC7 CD23 CD23 CD22 CD22 FMC7

Diffuse Large B-cell lymphoma (DLBCL)

Anti-lambda Anti-kappa Anti-kappa CD45 CD19 CD19 Anti-lambda

FMC7 CD45 CD22 CD22 FMC7 CD23 CD23

Hairy cell leukemia

CD45 CD19 Anti-lambda CD19 Anti-lambda Anti-kappa Anti-kappa

CD11c CD45 CD103 CD11c CD19 CD19 CD103

CD45 CD19 CD103 CD19 CD103 CD25 CD25

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