BD

Total Spread Matrices for the BD Leukocyte Panel Blocks

What is a Total Spread Matrix (TSM)?

While designing multicolor flow cytometry panels, it is important to assess fluorescence spillover to prevent or minimize loss of resolution due to spreading. The Spillover Spread Matrix (SSM) was developed as a tool to monitor and compare instrument performance over time, especially when experiments are standardized or calibrated across different
instruments.⁽¹⁾

The SSM is independent of fluorochrome brightness. While this feature is important for the comparison of instruments, it may lead to inaccurate spread prediction and sub-optimal panel design. The Total Spread Matrix (TSM) is the Spillover Spreading Matrix without normalization to the fluorescent intensity of the probe. This makes it possible to evaluate the true spreading in a given panel, based on the signal intensity of the different marker/fluorochrome combinations in each channel.

Please note for all tables shown below, the spread displayed is only found on the subpopulations that are positive for a given marker.

To view the instrument configuration on which these TSMs were collected, please scroll down.

Watch the webinar to learn more about TSM

Lymphocyte Backbone Panel Block

Total Spread Matrix	BUV615	BUV661	BUV737	BV421	BV480	BV605	BV650	BV711	BV750	BV786	BB515	BB630	BB660	BB700	RB744	RB780	PE	PE-CF594	PE-Cy5	APC
BUV395 CD4	11	21	21	0	0	60	24	13	40	26	0	13	21	17	22	29	29	26	18	22
BUV496 CD8	15	56	43	0	78	94	36	25	57	40	18	10	24	29	22	39	55	55	0	48
BUV563 CD19	18	53	35	0	0	72	0	8	23	0	0	38	35	17	22	10	169	120	48	45
BUV805 CD45	13	36	62	31	17	65	29	20	121	118	21	23	22	22	48	113	38	38	15	31
PE-Cy7 CD56	12	22	14	17	0	0	10	18	82	41	0	0	16	28	148	284	28	40	16	18
R718 HLA-DR	0	22	230	0	0	22	22	44	218	92	0	1	37	34	56	116	15	0	46	128
APC-H7 CD3	9	26	36	0	16	57	25	22	140	123	24	22	25	22	48	140	24	32	29	44

Table 1. A representative TSM showing the impact of the backbone markers on unused channels. Whole blood was single stained with the markers from the Backbone Panel Block and TSM calculated in Flowjo version 10.8.1. Highlighted cells in the table show the highest spread for reference.

Figure 1. Representative data images from highlighted cells in Table 1. Human whole blood was stained with the Lymphocytes Backbone Panel Block (see above). The effect of R718 HLA-DR on BUV737 (A) and of PE-Cy7 CD56 on RB780 (B) are shown.

T cell Differentiation Panel Block

Total Spread Into Empty Channels
Total spread from T Cell Differentiation Panel Block		BUV615	BUV661	BUV737	BV421	BV480	BV711	BV750	BB700	RB780	PE	PE-Cy5

	BV605 CD127	16	54	44	0	0	32	77	20	29	67	36

	BV650 CD27	15	214	123	0	0	99	229	34	36	43	86
	BV786 CD28	9	28	54	0	0	30	325	16	52	25	23
	BB515 CD45RA	13	35	27	0	31	23	61	37	47	48	26
	PE-CF594 CD25	9	24	17	0	9	17	33	30	48	76	82
	APC CCR7	6	38	38	0	0	20	42	27	33	0	89

Table 2. A representative TSM showing the impact of the markers in the T cell Differentiation Panel on unused channels. Cells were single stained with the markers from the T cell Differentiation Panel and TSM calculated in Flowjo version10.8.1. Highlighted cells in the table show the highest spread in the panel block.

Figure 2. Representative data images from highlighted cells in Table 1. Human blood was stained with the T cell Differentiation Panel Block (see above). The spread from BV650 CD27 into BUV661 (A) and BV750 (B) and from BV786 CD28 into BV750 (C) are shown.

B cell and B cell Subset Panel Blocks.

Total Spread Into Empty Channels
Total spread from B cell Maturation and Subsets Panel Blocks		BUV615	BV421	BV605	BV711	BV750	BB515	BB700	RB780	PE	PE-Cy5	APC

	BUV661 IgG	6	15	23	39	75	0	54	38	0	166	366

	BUV737 CD20	0	31	0	42	267	0	174	252	0	17	67
	BV480 IgD	0	0	77	19	53	12	0	0	21	17	0
	BV650 CD27	9	0	56	78	180	5	29	27	30	76	140
	BV786 CD38	8	55	0	26	395	9	0	56	0	24	0
	PE-CF594	11	0	42	22	40	0	70	77	152	189	65

Table 3. A representative TSM showing the impact of the markers in the B cell and B cell Subset Panels on unused channels. Cells were single stained with markers from the B cell and B cell Subset Panel and TSM calculated in Flowjo v10.8.1. Highlighted cells in the table show the highest spread in the panel block.

Figure 3. Representative data images from highlighted cells in Table 1. Human blood was stained with the B cell and B cell subset Panel Block (see above). The spread of BUV737 CD20 into RB780 (A) and BV750 (B) and BUV661 IgG into APC (C) and BV786 CD38 into BV750 are shown.

Unconventional T cells and NK cells Panel Block

Total Spread Into Empty Channels
Total Spread From Unconventional T cells and NK cell Panel Block		BUV661	BV421	BV480	BV605	BV650	BV786	BB515	RB780	PE	PE-CF594	PE-Cy5	APC

	BUV615 TCRgd	178	0	12	64	27	0	0	30	104	291	102	116

	BUV737 CD16	19	0	0	28	22	30	0	44	44	16	26	28
	BV711 CD161	32	12	0	58	22	52	0	27	30	0	24	41
	BV750 CD7	31	38	14	76	32	189	22	58	29	29	26	18
	BB700 TCR Va7.2	40	0	8	42	25	25	8	46	0	0	0	63

Table 4. A representative TSM showing the impact of the markers in the Unconventional T cells and NK cells panel blocks on unused channels. Cells were single stained with the markers from the Unconventional T cells and NK cells panel blocks and TSM calculated in Flowjo version10.8.1. Highlighted cells in the table show the highest spread in the panel block.

Figure 4. Representative data images from highlighted cells in Table 1. Human blood was stained with the Unconventional T cells and NK cells panel blocks (see above). The spread from BV750 into BV786 (A) and BUV615 TCRgd into PE-CF594 are shown.

Myeloid Backbone Panel Block

Total Spread Into channels
Total Spread from Myeloid Backbone markers		BUV395	BUV563	BUV615	BUV661	BUV737	BV421	BV480	BV605	BV650	BV711	BV750	BB515	BB700	RB780	PE	PE-CF594	PE-Cy5	PE-Cy7	APC

	BUV496 CD16	429	48	23	101	50	42	197	67	30	23	51	38	22	0	31	71	0	9	46

	BUV805 CD45	278	20	8	27	54	32	17	39	22	16	133	21	22	98	25	32	15	63	24
	BV786 CD15	143	14	6	34	192	655	14	48	41	74	1136	8	28	179	22	18	22	96	35
	BB515 CD33	52	17	9	15	17	0	18	73	26	16	39	0	45	44	23	26	11	0	54
	Alexa 700 HLA-DR	35	59	13	33	48	195	0	44	9	17	75	0	25	41	23	31	30	50	40
	APC-H7 CD14	26	0	10	24	37	76	0	42	10	11	144	0	18	120	26	28	26	185	42

Table 5. A representative TSM showing the impact of the markers in the myeloid backbone panel blocks on unused channels. Cells were single stained with the markers from the myeloid backbone panel block and TSM calculated from total cells gate (indlucing lymphocytes, monocytes, and granulocytes) in Flowjo version 10.8.1. Highlighted cells in the table show the highest spread in the panel block. Red highlight is showing spread over 1000. Assigning a marker to BV750 on a CD15 expressing cell should be avoided.

Figure 5. Representative data images from highlighted cells in Table 1. Human blood was stained with the myeloid backbone panel block (see above). The spread from BUV805 CD45 into BUV395 (A) BUV496 CD16 into BUV395 (B), BV786 CD15 into BV421 (C) and BV786 CD15 into BV750 (D) are shown.

The Dendritic Cell (DC) Subset Panel Block

Total Spread Into channels
Total Spread from Myeloid Backbone markers		BUV395	BUV563	BUV661	BUV737	BV421	BV605	BV650	BV711	BB515	BB700	RB780	PE	PE-CF594	PE-Cy5	APC

	BUV615 CD141	95	39	59	45	57	69	20	16	0	24	35	71	140	64	40

	BV480 CD11c	111	38	41	34	70	139	32	21	0	20	18	55	55	32	34
	BV750 CD1c	11	22	1	54	46	20	0	25	0	9	36	16	16	0	10
	PE-Cy7 CD123	28	0	18	40	37	14	0	11	0	66	793	52	47	22	18

Table 6. A representative TSM showing the impact of the markers in the dendritic cell (DC) subset panel block on unused channels. Cells were single stained with the markers from the dendritic cell (DC) subset panel block and TSM calculated from total cells gate (indlucing lymphocytes, monocytes, and granulocytes) in Flowjo version 10.8.1. Highlighted cells in the table show the highest spread in the panel block.

Figure 6. Representative data images from highlighted cells in Table 1. Human blood was stained with the the dendritic cell (DC) subset panel block (see above). The spread from PE-Cy7 CD123 into RB780 channel is shown.

The Granulocyte Panel Block

Total Spread Into channels
Total Spread from Myeloid Backbone markers		BUV395	BUV563	BUV615	BUV661	BUV737	BV421	BV480	BV650	BV750	BB515	BB700	RB780	PE	PE-CF594	PE-Cy5	PE-Cy7

	BV605 FcER1a	91	39	141	199	142	98	69	200	338	0	48	70	359	413	205	92

	BV711 Siglec 8	42	12	5	81	333	83	0	88	654	0	80	105	22	22	22	63
	APC CD66b	0	8	0	27	32	27	1	18	21	8	21	43	13	18	101	48

Table 7. A representative TSM showing the impact of the markers in the granulocyte panel block on unused channels. Cells were single stained with the markers from the granulocyte panel block and TSM calculated from total cells gate (indlucing lymphocytes, monocytes, and granulocytes) in Flowjo version 10.8.1. Highlighted cells in the table show the highest spread in the panel block.

Figure 7. Representative data images from highlighted cells in Table 1. Human blood was stained with the granulocyte panel block (see above). The spread from BV711 Siglec 8 into BUV737 (A) BV605 FcER1a into BV750 (B), BV605 FcER1a intoPE-CF594 (C) and BV711 Siglec 8 into BV750 (D) are shown.

Myeloid-derived Suppressor Cell (MDSC) Panel Block

Total Spread Into channels
Total Spread from Myeloid Backbone markers		BUV563	BUV615	BUV661	BUV737	BV421	BV480	BV605	BV650	BV711	BV750	BB515	BB700	RB780	PE	PE-CF594	PE-Cy5	PE-Cy7

	BUV395 CD11b	0	7	0	0	0	0	0	0	0	0	0	0	0	0	21	0	0

	BV786 CD15	14	6	34	192	655	14	48	41	74	1136	8	28	179	22	18	22	96
	APC CD66b	8	0	27	32	27	1	35	18	12	21	8	21	43	13	18	101	48

Table 8. A representative TSM showing the impact of the markers in the myeloid-derived suppressor cell (MDSC) panel block on unused channels. Cells were single stained with the markers from the myeloid-derived suppressor cell (MDSC) panel block and TSM calculated from total cells gate (indlucing lymphocytes, monocytes, and granulocytes) in Flowjo version 10.8.1. Highlighted cells in the table show the highest spread in the panel block. Red highlight is showing spread over 1000. Assigning a marker to BV750 on a CD15 expressing cell should be avoided.

Figure 8. Representative data images from highlighted cells in Table 1. Human blood was stained with the myeloid-derived suppressor cell (MDSC) panel block (see above). The spread from BV786 into BV421 (A) and BV750 (B) are shown.

BD FACSymphony™ A3 Instrument Configuration

Lasers	Filter	LP Mirror	Fluorochrome
UV 355 nm 100 mW	379/28	370	BUV395

	515/30	490	BUV496
	580/20	550	BUV563
	610/20	600	BUV615
	670/20	630	BUV661
	735/30	690	BUV737
	810/40	770	BUV805
	Violet 405 nm 100 mW	431/28	410	BV421

525/50		505	BV480/BV510
586/15		550	BV570
605/40		595	BV605
677/20		635	BV650
710/50		685	BV711
750/30		735	BV750
810/40		770	BV786
Blue 488 nm 100 mW		488/10	N.A.	SSC

	530/30	505	BB515
	610/20	600	BB630-P2
	670/30	635	BB660-P2
	710/50	685	BB700
	750/30	735	BB755-P
	810/40	770	BB790-P
	Yellow-Green 561 nm 100 mW	586/15	570	PE/BYG584-P

610/20		600	PE-CF594
670/30		635	PE-Cy5

780/60		750	PE-Cy7
Red 637 nm 140 mW		670/30	655	APC

		730/45	690	AF700/R718
		780/60	750	APC-Cy7

Table 9. All TSM matrices were collected on the BD FACSymphony™ A3 configuration as shown.