Journal: Current protocols in cytometry

Article Title: Monitoring of Measurable Residual Disease in Multiple Myeloma by Multiparametric Flow Cytometry

doi: 10.1002/cpcy.63

Figure Lengend Snippet: The strategy used to assess the performance of each mAb in the panel relies on defining negative (blue events) and positive (red events) populations for calculating stain indices. This is accomplished by first subtracting the median intensity of negative population from the median intensity of the positive population and dividing the number by 2 times the robust standard deviation of the negative population. A list of gating definitions can be found in Table 2. (A) For CD45, erythroid precursors are used as the negative population defined as SSClo, CD45- events (r1; blue dots) and T cells as the positive population defined as CD19-, CD56-, CD45br, SSClo events (r2 & r3; red dots). (B) For CD19, NK cells are used as the negative population defined as CD45br, CD56+ events (r4: blue dots) and B cells as the positive population defined as CD19+, CD45br events (r5; red dots). (C) For CD27, mast cells are used as the negative population defined as CD45dim, CD117br events (r6; blue dots) and T cells as the positive population defined as CD19-, CD56-, CD45br, SSClo events (r2 & r7; red dots). (D) For CD81, granulocytes are used as the negative population defined as CD45dim, SSChi events (r8; blue dots) and T cells as the positive population defined as CD19-, CD56-, CD45br, SSClo events (r9 & r10; red dots). (E) For CD56, B cells are used as the negative population defined as CD19+, CD56-, CD45br, SSClo events (r2 & r11; blue dots) and NK cells as the positive population defined as CD19-, CD56+, CD45br, SSClo events (r2 & r12; red dots). (F) For CD117, T cells are used as the negative population defined as CD19-, CD56-, CD45br, SSClo events (r2 & r13; blue dots) and mast cells as the positive population defined as CD27-, CD117br events (r14; red dots). (G) For CD138, B cells are used as the negative population defined as CD19+, CD56-, SSClo events (r2 & r15; blue dots) and CD-Chex CD103™ Plus cells as the positive population defined as CD81+, CD45dim events (r16; red dots). (H) For CD38, mature B cells are used as the negative population defined as CD19+, CD81dim events (r17; blue dots) and B progenitors as the positive population defined as CD19+, CD81br events (r18; red dots). (I) For cKappa, cLambda+ B cells are used as the negative population defined as CD19+, cLambda+, CD45br, SSClo events (r2 & r19; blue dots) and cLambda- B cells as the positive population defined as CD19+, cLambda-, CD45br, SSClo (r2 & r20; red dots). (J) For cLambda, cKappa+ B cells are used as the negative population defined as CD19+, cKappa+, CD45br, SSClo events (r2 & r21; blue dots) and cKappa- B cells as the positive population defined as CD19+, cKappa-, CD45br, SSClo (r2 & r22; red dots).

Article Snippet: Use the gating strategies detailed in Step 3 – 12 below to create a series of bivariate plots for identifying appropriate negative and positive populations used for calculating stain indices for each mAb. list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Stain index is a well-accepted and reliable measure that can be used to evaluate the performance of individual mAb employed in a multi-color tube; it can be calculated using the following formula: S t a i n I n d e x = M e d i a n F l u o r e s c e n c e I n t e n s i t i e s o f ( P o s i t i v e P o p u l a t i o n − N e g a t i v e P o p u l a t i o n ) 2 x R o b u s t S t a n d a r d D e v i a t i o n o f N e g a t i v e P o p u l a t i o n A summary of the gating definitions used to determine negative and positive populations for analysis of this equation in a one parameter histogram can be found in and the minimum recommended Stain Index for each fluorochrome-conjugated mAb can be found in . table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Tested Markers Negative Population Positive Population Phenotype Generic Name Phenotype Generic Name CD45 CD45-, SSClo Erythroid Precursors CD19-, CD56-, CD45br, SSClo T Cells CD19 CD45br, CD56+, SSClo NK Cells CD19+, CD45br, SSClo B Cells CD27 CD45+, CD117br Mast Cells CD19-, CD56-, CD45br, SSClo T Cells CD81 CD45dim,SSChi Granulocytes CD19-, CD56-, CD45br, SSClo T Cells CD56 CD19+, CD56-, CD45br, SSClo B Cells CD19-, CD56+, CD45br, SSClo NK Cells CD117 CD19-, CD56-, CD45br, SSClo T Cells CD27, CD117br Mast Cells CD138 CD19+, CD56-, CD45br, SSClo B Cells CD45dim, CD81+ Spiked Control a CD38 CD19+, CD81dim Mature B CD19+, CD81+ B-progenitors cKappa CD19+, CD45br, cLambda+, SSClo cLambda+ B Cells CD19+, CD45br, cLambda-, SSClo cLambda- B Cells cLambda CD19+, CD45br, cKappa+, SSClo cKappa+ B cells CD19+, CD45br, cKappa-, SSClo cKappa- B cells Open in a separate window a A procedural control for immunophenotyping, CD-Chex CD103 ™ Plus (Streck, Omaha, NE), which also expresses CD138 is spiked into the bone marrow sample and used as the positive population.

Techniques: Staining, Standard Deviation