4813: Problem-based Learning Workshop Advanced Laboratory studies for Primary Immunodeficiency Disorders Moderator: Richard Wasserman, MD, PhD Discussion Leader: Roshini Abraham, PhD ● Understand B cell flow analysis in CVID (PBL case)
HUMAN PERIPHERAL B CELL DIFFERENTIATION Marginal zone B cells Bone marrow Immature B cells Transitional B cells Memory B cells Plasmablasts Naïve B cells Periphery
MARKERS FOR PERIPHERAL B CELL SUBSETS - CURRENT Total B cells: CD19 and/or CD20 Transitional B cells: CD19+CD38+IgM+ Total IgM+ B cells: CD19+IgM+ (includes naïve B cells) Memory B cells: CD19+CD27+ switched memory B cells: CD19+CD27+IgM-IgD- marginal zone B cells: CD19+CD27+IgM+IgD+ IgM-only memory B cells: CD19+CD27+IgM+IgD- Plasmablasts: CD19+CD38+IgM- CD21+ B cells: CD19+CD21+ CD21- B cells: CD19+CD21- ● With newer information, more suitable cellular markers are available for accurate identification of transitional B cells and plasmablasts, in particular, but also for naïve B cells
NEWER B CELL MARKERS FOR B CELL SUBSET QUANTITATION Total B cells and B cell subsets can be quantitated in blood using multicolor flow cytometry: Total B cells: CD45+CD19+20+ For B cell subset analysis, the gating strategy uses CD45+19+20+/- (depending on the subset being studied), thus these markers are not specifically repeated in the panel below:- Transitional B cells: CD24 hi 38 hi 10+27-21 low IgM+++ T1: T2: CD24 hi 38 hi 10+27-21 int IgM+++ Naïve B cells: IgM+IgD+27-38-21+++ Memory B cells: Marginal zone B cells: CD27+IgM+IgD+ IgM-only memory: CD27+IgM+IgD- IgD-only memory: CD27+IgM-IgD+ Class-switched memory: CD27+IgM-IgD- (IgG+ or IgA+ or IgE+)
Plasmablasts: CD19+20-IgD-IgM-CD38++27+138+/- Assessment of CD21 expression on B cells: CD21+: CD19+20+21+ CD21-: CD19+20+21- CD21 low : CD19+20+21 low
EXAMPLES OF B CELL SUBSET ANALYSIS Healthy Donor CD19+CD20+ total B cells CD19+CD27+ IgM+IgD+ (marginal zone B cells) CD19+CD27+ (memory B cells) CD19+CD27+ IgM-IgD- CD19+CD27+ IgM+IgD- (switched memory B cells) (IgM-only memory B cells)
Healthy Donor CD19+CD38++IgM+ CD19+CD38++IgM- defined as transitional B cells in EUROclass defined as plasmablasts and Freiburg schemes in EUROclass and Freiburg schemes ● Other laboratories may define the entire upper right quadrant as transitional B cells and the entire upper left quadrant as plasmablasts without differentiating between intensity of expression of CD38 on B cells in conjunction with presence or absence of IgM ● This could result in quantitative variability for these B cell subsets between clinical laboratories depending on gating strategy Healthy Donor CVID Patient
PBL CASE: B CELL SUBSET ANALYSIS CD19+CD21 dim/low CD19+CD21- CD19+CD21+ ● Decreased total memory and switched memory B cells, expansion of CD21 dim/low B cells
OTHER EXAMPLES OF ABNORMAL B CELL SUBSETS IN CVID PATIENTS Patient A ● Decreased total memory and switched memory B cells, expansion of CD21 dim/low B cells (pediatric CVID patient) Healthy Donor Patient B A B C D ● Increased total IgM+ B cells with a population having bright IgM expression (in red circle, panel A), decreased total memory and switched memory B cells, unusual and large increase in IgM-only memory B cells (panel C, in red circle), all CD21+ B cells are of the CD21 dim/low phenotype (panel D- see comparison with healthy donor example for CD21+ B cells (pediatric CVID patient)
SUMMARY OF PARIS AND FREIBURG B CELL SUBSET CLASSIFICATION FOR CVID PATIENTS AND CLINICAL CORRELATION MB0 48% MB1 43% MB2 9% >11% CD27+ Not MB0 or MB1 <11% CD27+ Memory B cells Paris scheme Memory B cells <8% switched memory (2003) B cells (CD27+IgM-IgD-) N = 57 Paris/ Freiburg 23%/19% 47%/ 58% * splenomegaly 34%/41% 17%/17% CVID patients lymphadenopathy 28%/32% 18%/22% granulomas 17%/20% * 0%/4% 8%/14% 14%/11% autoimmune 17%/26% 12%/15% cytopenias II Ia Ib Freiburg scheme 23% 56% (2002) 21% Switched memory B Switched memory B Switched memory B cells (CD27+IgM-IgD-) cells (CD27+IgM-IgD-) cells (CD27+IgM-IgD-) N = 30 >0.4% of PBLs (lymphocytes) <0.4% of PBLs (lymphocytes) <0.4% of PBLs (lymphocytes) % CD19+CD21 low/dim % CD19+CD21 low/dim B cells B cells >20% <20% Piqueras B et al, J Clin Immunol, 2003, 23: 385-400 Warnatz K et al, Blood, 2002, 99: 1544-1551 * Associations with clinical phenotypes were found to be statistically significant by the authors’ of both classifications
SUMMARY OF EUROclass (2008) CLASSIFICATION FOR CVID PATIENTS AND CLINICAL CORRELATION <2% switched memory >1% B cells B cells (grp. B+) (CD27+IgM-IgD-) smB- >=9% Transitional B cells (CD19+CD38 hi IgM hi ) smB-Tr hi > =2% switched memory B cells <9% Transitional (CD27+IgM-IgD-) smB+ B cells (CD19+CD38 hi IgM hi ) smB-Tr normal 42% of total patients 58% of total 19% of smB- 81% of smB- CVID patients N = 303 54% 51% 24% 52%* (after removal 41% of Grp. B-) splenomegaly 57%* 22% 22% 24% 26% lymphadenopathy 24% 18% granulomas 12% 4% 17%* autoimmune 19% 21% 20% cytopenias 19% 26% <=1% B cells (grp. B-) Wehr C et al, Blood, 2008, 111: 77-85 * Associations with clinical phenotypes were found to be statistically significant
EUROclass (2008) CLASSIFICATION (CONTD.) > =2% switched memory <2% switched memory B cells B cells (CD27+IgM-IgD-) smB+ (CD27+IgM-IgD-) smB- <10% CD21dim/low B cells >=10% CD21dim/low B cells <10% CD21dim/low B cells >=10% CD21dim/low B cells smB-21 low smB+21 low smB+21 low smB-21 low 49% 51% 33% 67% 42% 50%* 14% 60%* splenomegaly >1% B cells 38% (grp. B+) 25% lymphadenopathy 17% 20% granulomas 14%* 2% 15% 20%* autoimmune 15% 10% 27% cytopenias 24% Summary of clinical correlation and B cell subsets: 1. Significant correlation between splenomegaly and decreased total % B cells, marginal zone B cells and switched memory B cells 2. Very significant correlation between splenomegaly and increased CD21 low/dim B cells 3. Significant correlation between increased transitional B cells (Tr B cells) and lymphadenopathy 4. Significant correlation between granulomatous disease and severe reduction (<2% of B cells) in switched memory B cells; 5. Lesser, but still significant, correlation between granulomatous disease and reduced marginal zone B cells 6. Significant correlation between autoimmune cytopenias and close to absent absolute count of plasmablasts (but excluded from EUROclass scheme due low numbers in both groups) Key B cell subsets in EUROclass scheme: class-switched memory B cells, transitional B cells and CD21 low/dim B cells Wehr C et al, Blood, 2008, 111: 77-85 * Associations with clinical phenotypes were found to be statistically significant
OTHER STUDIES USING B CELL SUBSET ANALYSIS FOR CLASSIFICATION OR CLINICAL “BINNING” OF CVID PATIENTS 1. Detkova D et al (Chest, 2007, 131: 1883-1889): (n =41) Association between memory B cell reduction and lung disease as well as intestinal involvement (uses the Paris classification) :– statistically significant correlation between CVID pts with chronic lung disease (bronchiectasis and reduced FVC+/-FEV1) and reduced total memory B cells and low class-switched memory B cells compared to those with normal memory B cell count - Malabsorption syndrome/chronic non-infectious diarrhea in CVID pts with low total memory B cells and decreased class-switched memory B cells compared to those with normal memory B cell count 2. Vodjgani M et al (J Investig Allergol Clin Immunol, 2007, 17: 321-328): (n = 29) Decreased class-switched memory B cells in CVID patients compared to controls. Correlation observed between low switched memory B cells and recurrent respiratory infections, development of bronchiectasis 3. Sanchez-Ramon S et al (Clin Immunol, 2008, 128: 314-321): (n = 105) Decreased class-switched memory B cells identified as an independent risk factor for granulomas, autoimmunity and splenomegaly in CVID. Gender association documented with fewer switched memory B cells in males compared to female CVID pts. Lower baseline IgG was independent predictor of pneumonia and severe infections in this group 4. Kalina T et al (Cytometry Part A, 2009, 75A: 902-909): (n = 48) Mathematical modeling of B cell subset patterns in CVID patients revealed clusters :– smB-CD21 low , smB-CD21 normal , smB+CD21 low and smB+CD21 normal . No clinical correlations were performed in this study
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