Fatima Abbasi

Overview of monoclonal B‐cell lymphocytosis

Monoclonal B‐cell lymphocytosis (MBL) has been the subject of more intensive investigation for the last 10 years. The increased presence of MBL in unaffected, first‐degree relatives with familial chronic lymphocytic leukaemia (CLL) suggest that it is surrogate marker for early disease. In normal population studies, MBL is found to be increased in ageing subjects. Consensus criteria for the diagnosis of MBL have been proposed. The differential diagnosis has been further clarified and the prevalence of MBL is most prominent in the elderly. The aetiology of MBL is unknown but probably involves immune mechanism of senescence or altered response. Environmental health studies suggest that exposure to certain toxins may lead to MBL but further work is needed. MBL is a precursor to CLL but may also regress, remain stable or progress to clinical CLL.

Prevalence and natural history of monoclonal and polyclonal B-cell lymphocytosis in a residential adult population.

Monoclonal B‐cells can be detected in the peripheral blood of some adults without B‐cell malignancies, a condition recently termed monoclonal B‐cell lymphocytosis (MBL). The risk of individuals with MBL progressing to a B‐cell malignancy is unknown. Polyclonal B‐cell lymphocytosis (PCBL) has not been systematically studied in the general population.

Monoclonal B‐cell lymphocytosis in blood donors

Monoclonal B‐cell populations have been detected in the peripheral blood of apparently healthy individuals by flow cytometry. In 2005, the term monoclonal B‐cell lymphocytosis (MBL) was proposed to describe these findings. MBL may be immunophenotypically similar to chronic lymphocytic leukaemia (CLL) and, like CLL, the prevalence is higher in males and older individuals. We studied the prevalence of MBL in blood donors from the Midwestern United States. Samples from 5141 donors were examined and seven (0·14%) were found to have immunophenotypic characteristics of MBL or CLL. Immunoglobulin heavy chain analysis yielded monoclonality or oligoclonality. Prior and subsequent to the study, an additional undetermined number of blood donors were screened and seven of these expressed immunophenotypic characteristics of MBL or CLL. We thus found a total of 14 healthy blood donors with monoclonal expansions of B‐lymphocyte populations. Of these, 12 were presumptively classified as MBL and two as CLL. All but two of the donors were male; the mean age was 59 years. The clinical importance of these findings with regard to transfusion medicine has not been established.

Toward quantitative fluorescence measurements with multicolor flow cytometry

A procedure is presented for calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure involves two steps. First, each of the fluorescence channels of the flow cytometer is calibrated using Ultra Rainbow beads with assigned values of equivalent number of reference fluorophores (ERF). The objective of this step is to establish a linear relation between the fluorescence signal in a given fluorescence channel of multicolor flow cytometers and the value of ERF. The second step involves a biological standard such as a lymphocyte with a known number of antibody binding sites (e.g., CD4 binding sites). The biological standard is incubated with antibodies labeled with one type of fluorophores for a particular fluorescence channel and serves to translate the ERF scale to an ABC scale. A significant part of the two‐step calibration procedure involves the assignment of ERF values to the different populations of Ultra Rainbow beads. The assignment of ERF values quantifies the relative amount of embedded fluorophore mixture in each bead population. It is crucial to insure that the fluorescence signal in a given range of fluorescence emission wavelengths is related linearly to the assigned values of ERF. The biological standard has to posses a known number of binding sites for a given antibody. In addition, this antibody has to be amenable to labeling with different types of fluorophores associated with various fluorescence channels. The present work suggests that all of the requirements for a successful calibration of a multicolor flow cytometer in terms of ABC values can be fulfilled. The calibration procedure is based on firm scientific foundations so that it is easy to envision future improvements in accuracy and ease of implementation. Published 2007 Wiley‐Liss, Inc.

CD69 expression as an index of T-cell function: assay standardization, validation and use in monitoring immune recovery

BACKGROUND CD69 is a surrogate marker of T-cell responsiveness to mitogen and Ag stimulus and can be used as a measure of T-lymphocyte activation. Quantitative flow cytometric determination of CD69 expression on T lymphocytes has several advantages over traditional lymphocyte proliferation assays, but this method has not yet been standardized for clinical applications. METHODS We qualified a commercially available assay using the manufacturers procedures for measurement of T-cell response to a mitogen (PHA), superantigen (Staphylococcus endotoxin B; SEB) and Ca(2+) ionophore (phorbyl myristate acetate; PMA) with peripheral blood from healthy volunteers. Following this, we tested the usefulness of the assay in determining T-cell responses to PHA and SEB for six immunocompromised patients. RESULTS Healthy volunteers showed 17-fold increases in T-cell CD69 Ab bound per cell (ABC) with PHA stimulation compared with the baseline. SEB was also an effective T-cell activating agent, increasing CD69 ABC by 5-fold, comparable with results obtained with PMA stimulation. PHA- and SEB-stimulated T-cell CD69 ABC for patients 100 days post-BM transplant were generally below 1 SD of that from healthy volunteers. SEB-stimulated T-cell CD69 expression was significantly depressed for CD8(+) T cells while CD4(+) T-cell responses to SEB were generally within 1 SD of the mean for healthy volunteers. DISCUSSION These results suggest that quantitative measurement of CD69 surface expression by flow cytometry is a useful diagnostic tool for detailed assessment of T-lymphocyte and subset activation.

Prevalence of monoclonal B-cell lymphocytosis: A systematic review†‡§

Individuals with monoclonal B‐cell lymphocytosis (MBL) have been identified in clinic outpatients, in unaffected relatives of patients with chronic lymphocytic leukemia (CLL), and in general populations. MBL and its relationship with CLL have been actively investigated over the last decade. This report systematically reviews the prevalence of MBL in the context of the populations studied and the evolution of laboratory methods used to define MBL.

Monoclonal B-cell lymphocytosis in healthy blood donors: an unexpectedly common finding

Circulating monoclonal B cells may be detected in healthy adults, a condition called monoclonal B-cell lymphocytosis (MBL). MBL has also been identified in donated blood, but no systematic study of blood donors has been reported. Using sensitive and specific laboratory methods, we detected MBL in 149 (7.1%; 95% confidence interval, 6.0% to 8.3%) of 2098 unique donors ages 45 years or older in a Midwestern US regional blood center between 2010 and 2011. Most of the 149 donors had low-count MBL, including 99 chronic lymphocytic leukemia-like (66.4%), 22 atypical (14.8%), and 19 CD5(-) (12.8%) immunophenotypes. However, 5 donors (3.4%) had B-cell clonal counts above 500 cells per µL, including 3 with 1693 to 2887 cells per µL; the clone accounted for nearly all their circulating B cells. Four donors (2.7%) had 2 distinct MBL clones. Of 51 MBL samples in which immunoglobulin heavy chain (IGH)V-D-J genotypes could be determined, 71% and 29% used IGHV3- and IGHV4-family genes, respectively. Sequencing revealed 82% with somatic hypermutation, whereas 18% had >98% germ-line identity, including 5 with entirely germ-line sequences. In conclusion, MBL prevalence is much higher in blood donors than previously reported, and although uncommon, the presence of high-count MBL warrants further investigations to define the biological fate of the transfused cells in recipients.

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes as potential disease biomarkers. CD20 expression in B‐cell chronic lymphocytic leukemia (B‐CLL) is one of the best examples of such a biomarker, but results from the use of different QFC methods vary considerably.

Discrepancy in Measuring CD4 Expression on T-lymphocytes using Fluorescein Conjugates in Comparison with Unimolar CD4-phycoerythrin Conjugates

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO‐TROL™ Control Cells and T‐lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate.

Improved ZAP-70 assay using two clones, multiple methods of analysis and clinical correlation.

In a companion methodological study, we compared two anti‐ZAP‐70 clones (1E7.2 AF 488 and SBZAP PE) and four selected methods of analysis. Clinical correlations are required for validation.