MULTIPARAMETER IMMUNOFLUORESCENCE Carleton C. Stewart, Ph.D. & Sigrid J. Stewart R L L OSWE ARK P L Cancer Institute aboratory Flow of Cytometry Department of Health, State of New York Elm and Carlton Streets Buffalo, New York phone: fax:

ANTIBODY BINDING TO CELLS

KfKf KrKr Ab + Ep AbEp K f range is usually ~ 10 6 K r range is usually ~ K a = K f /K r = 10 6 /10 -3 = 10 9 K f = AbEp Ab Ep K r = AbEp Ab Ep THE LAW OF MASS ACTION

Specific Activity is the concentration of bindable antibody to its epitope divided by the protein concentration. 2 [F(ab')] SA = (protein)

Reasons Antibodies do not bind to cells: overconjugation not purified degradation of binding site aggregation

STORING OF ANTIBODIES : Proteases destroy antibodies in: ascitic fluid serum bacteria Use sodium azide Use highly purified albumin or gelatin as carrier Purify antibodies immediately

TITERING ANTIBODIES

SPECIFIC ANTIBODY NON-SPECIFIC ANTIBODY CONCENTRATION AMOUNT BOUND

Dilution Signal to Noise TITER

µg S/N Ab 278 IC 5.8 isotype control antibody cytokeratin.3 µg S/N Ab 100 IC µg S/N Ab 25.7 IC 2.6 number

Dilution Signal to Noise TITER

VERIFICATION OF ANTIBODY COMBINATIONS

CONTROL BATCH

BLOCKING IS IMPORTANT

INDIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING Primary Antibody:Second Antibody: murine monoclonalfluoresceinated antibodygoat anti-mouse IgG F(ab') 2 Fab epitope FcR AB CD Fc

ISOTYPE CONTROL- myeloma protein AUTOFLUORESCENCE CONTROL EFGH

BLOCKING WITH GOAT IgG goat IgG

add Mab Fab add fluoresceinated goat anti-mouse IgG F(ab') 2

VERIFICATION OF BLOCK 1. FcR and non-specific binding FL-MAB + PE-mIgG 2. gIgG + FL-MAB + PE-mIgG

EFFECT OF BLOCKING ON ANTIBODY BINDING TO MONONUCLEAR CELLS LOG FLUORESCENCE CELL VOLUME

number TOTAL A B C D channel number

variation in gamma 1 myeloma protein binding to macrophages mopc myeloma protein

Second Reagent Quality log fluorescence cell volume F(ab’) 2 of anti IgGanti IgG

DEAD CELLS CAN BE A PROBLEM They bind antibodies non-specifically They masquerade as specific subsets They cause data misinterpretation

ANTIBODIES BIND NON- SPECIFICALLY TO DEAD CELLS FL-KAPPA PE-LAMBDA dead cells ALL CELLSVIABLE CELLS AB

lysed, washed cells + 5 µg EMA 10 min. 18 cm. EMA PROCEDURE WASH, FIX, AND ANALYZE 1 3 2

Evaluating Viability with Ethidium Monoazide FSC SSC FSC SSC FSC FL3-EMA R9 % dead = 5% R2 % dead in gate = 1% R9

ONE COLOR IMMUNOPHENOTYPING RPCI LFC

1. IgG BlockMABwashFL-second antibody F(ab') 2 wash 2. IgG BlockB-MAB wash FL-Avidinwash 3. IgG BlockFL-MAB wash SINGLE COLOR IMMUNOPHENOTYPING

CORRELATED (LIST MODE) DATA ACQUISITION Entry No.ValueCell NumberParameter 1801FSC 21001SSC 3401Green 4201Red 5902FSC 61202SSC 71002Green 81102Red -50nFSC -75nSSC -110nGreen k120nRed

Analysis of List Mode Data CD4 FLUORESCENCE number of cells forward scatterCD4 fluorescence A B C region A region Bregion C

STRATEGIES IN MULTICOLOR FLOW CYTOMETRY RPCI LFC

CELLULAR ANTIGENS Adhesion Receptors Metabolic cytokines structure enzymes courtesy of Jim Bender

FLOW CYTOMETRY VS MICROSCOPIC IMAGING

TWO COLOR IMMUNOPHENOTYPING Antibodies labeled with fluorescein Antibodies labeled with phycoerythrin RPCI LFC

TWO COLOR IMMUNOPHENOTYPING 1. IgG Block + MAB wash FL- second antibody F(ab') 2 wash IgG Block + PE-MAB wash 2. IgG Block + B-MAB + FL-MAB wash PE-Avidinwash 3. IgG Block + FL-MAB + PE- MABwash

COMBINED INDIRECT AND DIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING Primary Antibody: mMABSecond Antibody: FGAMPE-mMAB

NO BLOCK CD8 + FGAM PE-CD4 21% 6% 49%

VERIFICATION OF BLOCK Second Reagent Block gIgG + MAB wash FL-GAM wash PE-mIgG gIgG + MAB wash FL-GAM wash mIgG + PE-mIgG

COMBINED INDIRECT AND DIRECT IMMUNOFLUORESCENCE STAINING BLOCKING Primary Antibody: mMABSecond Antibody: FGAMPE-mMAB

BLOCKED CD8 + FGAM PE-CD4 42% 12%

THREE COLOR IMMUNOPHENOTYPING Antibodies labeled with Fluorescein Antibodies labeled with Phycoerythrin Antibodies labeled with Tandem Complex to Avidin Tandem Complexes are Texas Red or CY 5 coupled to Phycoerythrin Per CP is a natural Tandem Complex of peridinin and chlorophyll a protein

SINGLE COLOR HISTOGRAMS

TWO COLOR PATTERN FL1-CD3 FL2-CD4 color CD3-CD4-black CD3+CD4-blue CD3-CD4+cyan CD3+CD4+green

THREE COLOR PATTERN CD3 CD4 CD3 CD4 CD8

FOUR COLOR SINGLE LASER IMMUNOPHENOTYPING Antibodies labeled with fluorescein Antibodies labeled with phycoerythrin (PE) Antibodies labeled with PE/Texas Red Antibodies labeled with PE/CY5 or PerCP

FOUR COLOR DUAL LASER IMMUNOPHENOTYPING Antibodies labeled with fluorescein Antibodies labeled with phycoerythrin (PE) Antibodies labeled with PE/CY5 or PerCP Antibodies labeled with APC, CY5 or CY7

CD3 CD56 CD8 FOUR COLOR PATTERN CD4CD8 CD56 CD8CD4

FL1FL3 FL2 FL1

FL2 FL3

VISUALIZING EACH POPULATION BIVARIATE DOT PLOTS CAN BE USED TO DISPLAY THE PATTERNS GENERATED BY MULTIPARAMETER DATA. FL1 FL4 FL2 FL4 FL3 FL4 FL1 FL3 FL2 FL3 FL1 FL2 4 COLOR 3 COLOR 2 COLOR

UNDERSTANDING BINARY LOGIC IS USEFUL Note that in binary logic the 1st parameter is sequenced as -/+, two parameters as --/++, 3 parameters as ----/++++, etc.. for as many parameters that are measured. The problem encountered after 3 parameters is...how does one visualize the multiple populations?

COLOR PATTERNS USED TO VIEW COEXPRESSION

Boolean Logic Table For Four Color Immunophenotyping

ABC DEF Ab1 Ab2 Types of Antigen Expression

THREE OR MORE COLOR IMMUNOPHENOTYPING 1. IgG Block + MAB wash Biotin- second antibody F(ab') 2 wash IgG Block + FL- MAB + PE-MAB + TC- Avidin wash 2. IgG Block + FL-MAB + PE-MAB + B-MABwash TC-Avidin wash 3. IgG Block + FL-MAB + PE-MAB + TC-MAB wash TC (third color) = PE/TR or PE/CY5 tandem or PerCP

STRATEGY FOR SELECTING FLUOROCHROME: EPITOPE DENSITYFLUOROCHROME Lowphycoerythrin, APC low-intermediatetandem, CY5 highfluorescein, PerCP

STAINING SOP 50 µl washed, and blocked* whole blood or bone marrow 15 min. on ice lyse, centrifuge, decant, blot, and resuspend pellet wash, fix, and analyse *add 10 µl mIg (10 mg/ml) to 1 ml washed whole blood. Add antibody combination

TANDEM COMPLEX PROPERTIES TO CONSIDER monocyte binding PE-CY5 light sensitivity PE-CY5 batch variation PE-TR and PE-CY5

Non-specific Binding to Monocytes A B C PerCP-CD4PE-CY5-CD4FSC SSC CD25

TC-CD45TC-CD3 Effect of Light Exposure on PE-CY5 Tandem Fluorescence PE-fluorescence

EFFECT OF BATCH VARIATION PE-fluorescence

FSC SSC CD16 CD32 CD64 CD16 CD64 Fc Receptor Expression on Blood Leukocytes

CD16CD64 All Cells CD16CD64 neutrophils T-cells eosinophilsbasophils monocytesNK cells B-cells CD32