FLOW CROSSMATCH TESTING PROTOCOLS

I. Purpose

These protocols describe the method for performing a flow cytometric crossmatch using three colors to simultaneously detect alloantibodies reacting with T cells and B cells. II. Principle

The complement dependent cytotoxicity (CDC) crossmatch is adequate for detecting most antibodies that can cause hyperacute graft rejection, but it does not detect very low concentrations of antibodies that may be the cause of accelerated graft rejection in some patients. The flow cytometric crossmatch (FCXM) is not dependent upon complement fixation and can detect much lower levels of anti-HLA antibodies than the CDC assay. Patients with a negative CDC crossmatch but a positive FCXM have been shown to be at risk for an increased number of rejection episodes and possibly early graft failure. The FCXM may be important in identifying a subgroup of patients at increased risk for graft failure. The FCXM is performed by incubating donor cells with potential recipients sera followed by addition of a fluoresceinated (FITC) goat anti-human polyclonal antibody reagent. A phycoerythrin (PE) labeled monoclonal antibody that detects B cells (anti-CD19) and a peridinin chlorophyll protein (PerCP)-conjugated monoclonal antibody that detects T cells (anti-CD3) are added. This three-color combination allows for the simultaneous detection of alloantibodies reacting with T cells and B cells and eliminates background binding due to NK cells and monocytes. Results are analyzed by flow cytometry and expressed as positive or negative based on a shift in median channel fluorescence intensity of the test serum with respect to negative control or autologous serum. III. Testing Policy Flow crossmatch testing will be performed on all new Renal Transplant Candidates with a potential living donor. Renal Patients on the UNOS Waitlist who have been transplanted with a deceased donor organ will be flow crossmatched retrospectively. Waitlist patients who have had an unexpected positive crossmatch with a deceased donor will also be crossmatched on the next business day. Both a recipient auto T and B cell flow crossmatch and Donor-Recipient T and B cell flow crossmatch will be performed. For new Renal and LRD samples, extract lymphocytes from two yellow top ACD tubes of blood ~8mL each by the RosetteSep method. For deceased donors, lymphocytes will be isolated from either lymph nodes or spleen following the cell isolation protocol.

IV.

Protocols A. Protocol for Isolation of Lymphocytes from Deceased Donor Lymph Nodes or Spleen B. Protocol for Lymphocytes Isolation from Peripheral Blood C. Protocol for Three Color Flow Cytometric Crossmatch D. Protocol for Converserion of Anti-human IgG-FITC Fluorescence into MESF values

A. Protocol for Isolation of Lymphocytes from Deceased Donor Lymph Nodes or Spleen
I. Purpose This protocol describes a method to isolate deceased lymph nodes and splenocytes for use in the flow cytometry crossmatch. II. Principle In order to perform flow crossmatch, lymphocytes need to be isolated from deceased lymph node or splenic tissue. The use of a sterile cell strainer will allow the tissue to be ground into a single cell suspension and free of debris that may clog the flow cytometer. III. Materials A. Specimen Requirements Lymph nodes or spleen collected in McCoys medium store in the 4rC fridge B. Reagents 1. McCoys Medium (Life Technologies, cat# 16600-082) 2. Red Blood Cell Lysing Solution (1x) (5 Prime, cat# 2900030) C. Supplies 1. 2. 3. 4. 5. 6. 7. 8. 9. 15 mL and 50 mL Falcon plastic conical tubes with caps 70 uM cell strainer P-200 and P-1000 Pipettes with tips Fisher tubes Fisher tabletop Centrifuge model 59 1mL 25G 5/8 syringe 60 mm petri dish scissors/forceps glass pipettes

IV. Method for lymph nodes 1. Carefully take out the donor lymph node from the 15 mL conical tube in medium using forceps and place into a 60 mm petri dish 2. Using a 1 mL syringe filled with McCoys medium poke holes into the lymph node while injecting medium to release the lymphocytes into suspension.

3. Once you have about 5 mL of cell suspension, add this to four Fisher tubes and spin down at 1500 g for 1 min in a Fisher tabletop centrifuge. 4. Aspirate supernatant and resuspend cells in 1.5 mL of RBC lysis buffer in two tubes. Mix and incubate at room temperature for 5 min and then spin down. 5. Wash the cell pellet 2x using 1 mL of McCoys Medium 6. If cell debris is present in the resuspend cell prep, add the cells in solution dropwise over a 70 uM strainer on top of a 50 mL conical tube to ensure debris is removed. Add a few drops of McCoys Medium to ensure all cells get through the strainer. 7. Count cells on the hemocytometer. Use 500,000 cells/ tube for flow crossmatch application ** Very Important. Debris can clog the flow cytometer and should be avoided at all times.

V. Method for spleen 1. Carefully take out the donor spleen from the 50 mL conical tube in medium using scissors and forceps and place into a 60 mm petri dish 2. Cut a small piece of spleen (~1 cm) and place it into the 70 uM strainer sitting on top of a 50 mL conical tube. 3. Using the reverse side of a syringe plunger, make circular motions to grind up the spleen into a single cell suspension. Add McCoys medium to help filter cells through the strainer. (Use about 2 mL of medium) 4. Add the cell suspension to two fisher tubes and spin down at 1500 g for 1 min in a Fisher tabletop centrifuge. 5. Aspirate supernatant and resuspend cells in 1.5 mL of RBC lysis buffer. Mix and incubate at RT for 5 min and then spin down. 6. Wash the cell pellet 2 times using 1 mL of McCoys Medium 7. If cell debris are present in the resuspend cell prep, add the cells in solution dropwise over a new 70 uM strainer on top of a 50 mL conical tube to ensure debris are removed. Add a few drops of McCoys Medium to ensure all cells get through the strainer. 8. Count cells on the hemocytometer. Use 500,000 cells/ tube for flow crossmatch application

** Very Important. Debris can clog the flow cytometer and should be avoided at all times.

B. Protocol for Lymphocyte Cell Isolation from Peripheral Blood

RosetteSep Total Lymphocyte Enrichment Cocktail I. Purpose This protocol describes a method to isolate and enrich lymphocytes for use in the Flow cytometry crossmatch. II. Principle RosetteSep Total lymphocyte Enrichment is a rapid and easy cell separation procedure for the isolation of highly purified total lymphocytes directly from whole blood or buffy coat preparations. This procedure is used to isolate lymphcytes for use in flow cytometry cross-match. It combines the specificity of antibody-mediated cell separation with the ease of density gradient centrifugation. RosetteSep Total lymphocytes Enrichment cocktail links unwanted granulocytes, platelets, and monocytes to red blood cells using tetrameric antibody complexes containing monoclonal anti-CD66b specific for granulocytes, CD36 for platelets, CD16 for monocytes, and anti-Glycophorin A found on the red blood cell surface. Once binding of the target cells occurs, antibody complexes form upon which rosettes are created. When the diluted blood sample is centrifuged over a Lymphoprep density layer, unwanted cells (embedded in rosettes) will pellet along with free red blood cells and the desired cells (total lymphocytes) are then easily collected as a purified population from the upper interface of the lymphoprep. III. Materials A. Specimen Requirements 1. ACD is the preferred anticoagulant for the HLA laboratory. 2. Two 8mL tubes of ACD anticoagulated blood are required for the isolation of total lymphocytes for flow crossmatch. 3. Specimens should be kept at room temperature until isolation is performed 4. Lymphocytes must isolated within 48 hrs from the time of collection. For older samples it is mandatory to test the cell viability by 7AAD staining according to Flow Cytometry protocols. Only samples with >80% of viable cells will be tested for Flow Cytometry crossmatch. B. Reagents 1. McCoys Medium (Life Technologies, cat# 16600-082) 2. RosetteSep Total Lymphocyte Enrichment Cocktail (Store at 2-8rC) (2mL X 5 vials, 18 month shelf-life) (StemCell Technologies, cat# 15263) 3. Lymphocyte Separation Medium (MediaTech, cat# 25-072-CL) 4. Red Blood Cell lysing solution (1x) (5 Prime, cat# 2900030)

5. HBSS (w/o Ca2+/Mg2+/Phenol Red) (MediaTech, cat# 21-022CV)

C. Supplies 10. 15mL Falcon plastic conical tubes with caps (Fisher, cat# 12-959-11B) 11. Disposable glass transfer pipets 12. P-200 and P-1000 Pipettes with tips 13. Fisher tubes 14. Fisher tabletop Centrifuge model 59 15. IEC Centra-7 Centrifuge 16. Vacuum aspirator

IV. Method 1. For each donor and/or recipient centrifuge two 8 mL ACD yellow top tubes at 3000 rpm for 7 minutes. (LOW BRAKE, Level 3) 2. Using a glass pipet transfer the buffy coat from the yellow tops into the 15 mL tube labeled with sample name. 3. For each 1 mL of buffy coat add 50 uL of RosetteSepTotal Lymphocyte Enrichment cocktail directly into the 15 mL tube. ACD Buffy 1 mL 1.5 mL 2 mL 2.5 mL RosetteSep 50 uL 75 uL 100 uL 150 uL

4. Mix well by gently vortexing the tube and incubate 20 min at room temp on the rotator. 5. After incubation, dilute sample to 7 mL with room temp HBSS w/o Ca2+/Mg2+/Phenol Red and mix. 6. Add 4 mL of Lymphoprep into a 15 mL glass tube then overlay the diluted sample using a glass pipette. 7. Centrifuge for 20 min at 2200 rpm at room temperature (LOW BRAKE, Level 3). 8. Collect all the buffy coat layer and transfer to 15 mL tube fill to the top with HBSS then mix by inversion. (NOTE: The Lymphoprep is not toxic for the cells unless the exposure to it is prolonged) 9. Centrifuge at 2000 rpm for 5 min with the brake on HIGH. Decant the supernatant

10. If red blood cells are present add 0.5 mL of 1x red blood cell lysing solution. Mix well and transfer to a Fisher tube. Incubate at room temp for 5 mins. 11. Add 0.5 mL of McCoys medium and centrifuge at 2000 rpm for 1 min on tabletop centrifuge. Decant the supernatant and resuspend the cell pellet in 1 mL of McCoys medium and centrifuge. Wash one more time with 1 mL McCoys medium for a total of two washes. 12. After 2 washes add 1 mL McCoys and count cells on the hemocytometer. The cell count in five 0.04 mm squares X 50 gives you how many cells/uL. 13. Each tube for flow crossmatch will require 500,000 cells. Use the equation below to divided 500,000 cells by the calculated cell count in cells/uL to obtain the correct volume of cell suspension that equates to 500,000 cells 500,000 cells uL [cell count X 50] = uL volume of cell suspension equivalent to 500,000 cells

14. Pipette the volume calculated in step 13 that to equal 500,000 cells into each Fisher tube used for flow cytometry crossmatch.

C. Protocol for Three Color Flow Cytometric Crossmatch

I. Purpose This protocol describes the method for performing a flow cytometric crossmatch using three colors to simultaneously detect alloantibodies reacting with T cells and B cells. II. Principle The complement dependent cytotoxicity (CDC) crossmatch is adequate for detecting most antibodies that can cause hyperacute graft rejection, but it does not detect very low concentrations of antibodies that may be the cause of accelerated graft rejection in some patients. The flow cytometric crossmatch (FCXM) is not dependent upon complement fixation and can detect much lower levels of anti-HLA antibodies than the CDC assay. Patients with a negative CDC crossmatch but a positive FCXM have been shown to be at risk for an increased number of rejection episodes and possibly early graft failure. The FCXM may be important in identifying a subgroup of patients at increased risk for graft failure. The FCXM is performed by incubating donor cells with potential recipients sera followed by addition of a fluoresceinated (FITC) goat anti-human polyclonal antibody reagent. A phycoerythrin (PE) labeled monoclonal antibody that detects B cells (anti-CD19) and a peridinin chlorophyll protein (PerCP)-conjugated monoclonal antibody that detects T cells (anti-CD3) are added. This three-color combination allows for the simultaneous detection of alloantibodies reacting with T cells and B cells and eliminates background binding due to NK cells and monocytes. Results are analyzed by flow cytometry and expressed as positive or negative based on a shift in median channel fluorescence intensity of the test serum with respect to negative control or autologous serum. III. Materials A. Specimen Requirements Lymphocytes or mononuclear cells with viability >80%, separated from peripheral blood, lymph node, or spleen: 500,000 cells per tube. Note: Separation using Rosette-Sep is recommended. Do not use a cell prep that has been treated with Percoll or Lympho-Kwik (which contains Percoll). Both of these products increase the negative background binding, which reduces the sensitivity of the assay and may result in a false negative result. If it is necessary to use cells that have been treated with either of these products, incubate the prep for a minimum of 1 hour at 37C in RPMI with 10%FCS. Wash 3x with flow wash buffer. This will remove the Percoll. Serum samples from potential recipients. Note: All fresh (unfrozen) serum should be flash-frozen then hard spun in the Beckman microcentrifuge for 5 minutes to remove aggregated immunoglobulin and immune complexes which could cause a false positive crossmatch.

B. Reagents, Supplies and Equipment Negative control (normal human serum or pooled normal human sera) Positive control (pooled positive sera) titered for flow cytometry FITC-conjugated goat anti-human IgG [F(ab)2, Fc specific] (Jackson Labs, cat# 109-016-098) Anti-human CD3, PerCP (BD, cat# 347344) Anti-human CD19, PE (BD, cat# 555413) Flow wash buffer (PBS) (Thermo, cat# SH30256.01) Pipetman with tips Fisher tubes 5mL polystyrene falcon tubes Fisher Scientific tabletop centrifuge Vacuum aspirator FACScan Flow cytometer capable of 3 color analysis C. Reagent notes: 1. Although using monoclonal antibodies at the manufacturers recommended concentration is acceptable, some may be titered prior to use. 2. After reconstituting the FITC reagent, hard spin the entire stock and aliquot into 0.5 mL eppendorf microtubes and store at -80C for up to 1 year. When ready to use just thaw and dilute to the established working dilution. Place any unused portion in 4C refrigerator for up to 7 days. 3. CD3 PerCP and CD19 PE may be combined using equal volumes of the appropriate dilution of each reagent. The combination may be stored at 4C for up to one month. 4. Keep reagents cold and in the dark. 5. Due to the high sensitivity of flow cytometry, all cell concentrations, serum and antibody dilutions, and volumes must be exact and accurate. Small fluctuations will result in erroneous and inconsistent results.

IV. Method A. Staining 1. Flash freeze all fresh (unfrozen) serum, then hard spin all serum samples and positive and negative controls in Beckman microfuge. 2. Place 500,000 cells in each Fisher tube labeled to indicate cell sample. Mix the cell prep well before putting cells in tubes to ensure consistency from tube to tube. The number of tubes needed will depend upon the number of serum samples to be tested. Fisher Tubes should be labeled and set up as follows for one patient with one donor: Tube # 1 2 3 4 5 6 7 Primary Antibody/Serum Negative (NHS) Donor Pooled Positive (1:10) Donor Patient sample #1 Donor XM 7AD working stock Donor Negative (NHS) Recipient Patient sample #1 Auto XM 7AD working stock Recipient Secondary Antibody anti-human IgG FITC CD3 PerCP & CD19 PE anti-human IgG FITC CD3 PerCP & CD19 PE anti-human IgG FITC CD3 PerCP & CD19 PE anti-human IgG FITC CD3 PerCP & CD19 PE anti-human IgG FITC CD3 PerCP & CD19 PE -

3. Wash cells 2 times with 1 mL of cold PBS. Centrifuge cells to a pellet in the Fisher Scientific tabletop centrifuge for 1 minute at 700 x g. 4. Aspirate supernatant, being careful not to aspirate cell pellet. Residual fluid volume should be <20 l. 5. Add 30 L of the appropriate serum directly to the pellet in each corresponding tube, being careful not to allow the serum to run down the side of the tube. Vortex to ensure proper mixing of serum and cells. 6. Incubate tubes for 30 minutes at 4rC in the dark 7. During this incubate step several tasks can be performed: y Add 250 ul of cold PBS to each 7AAD tube and place in the fridge. 10 min before analysis add 2.5 uL of 7AAD. See step 14. y Turn on the FACS machine then computer and run FACS comp procedure. y Label 5 mL FACS tubes similar to Fisher tubes. y Dilute the anti-human IgG-FITC according to the set dilution for the present lot.

8. Wash cells with 1mL of cold PBS. Centrifuge cells to a pellet in Fisher tabletop for 1 minute at 700 x g. 9. Aspirate supernatant and repeat wash three more times for a total of FOUR washes. Note: be sure to aspirate positive control LAST to decrease the chance of carryover. Rinse vacuum apparatus tip after aspirating positive control. 10. Add 20 L of diluted anti-human IgG-FITC to each tube and incubate at 4rC in the dark for 10 minutes. Note: Check titer and proper dilution of ALL reagents prior to use. 11. Create a Master Mix of CD3 PerCP and CD19 PE. Both antibodies have a diluted working solution that has been titrated for optimal staining. To determine how much of each antibody to add calculate the number of samples plus one extra and multiply by 10 (Example: (5 samples + 1)*10 = 60uL of CD3 PerCP + 60uL of CD19 PE from each working solution vial 12. After 10 minutes, add 20 L of CD3 PerCP/CD19 PE master mix to each tube, vortex gently and incubate at 4rC in the dark for an additional 20 minutes. 13. Wash cells TWICE with 1 mL cold PBS wash buffer and centrifuge. 14. In order to check cell viability resuspend an unlabelled cell pellet with 250 uL of wash buffer and add 2.5 uL of 7AAD working dilution. Wait 10 min and run on FACScan along with XM samples 15. Since the stained cells will be analyzed immediately, resuspend cell pellet in 200 l of wash buffer, transfer to flow tubes and analyze on the FACScan flow cytometer. The final volume for analysis should always be 200 l B. Acquisition 1. Turn on the FACScan cytometer FIRST then the computer. Perform FACSComp procedure each time the instrument is turned on. (See FLOW CYTOMETER INITIAL INSPECTION, START-UP, BASIC CALIBRATION AND SHUT-DOWN document) 2. Double click on the Flow Crossmatch template icon on the desktop. 3. Click on ACQUIRE in the menu bar, select CONNECT TO CYTOMETER (or Apple B). Drag the ACQUISITION CONTROL window to the lower right side of the desktop. 4. Open the four window instrument settings for the FACScan (Hold Apple 1,2,3,4) This includes general instrument settings, compensation, theshold and machine diagnostics 5. Click on ACQUIRE in the menu bar, select COUNTERS. Drag the COUNTERS WINDOW to the bottom of the desktop 6. Expand the COUNTERS window by clicking the green + button. Under SOURCE change accept to COLLECT for the first two. Under MODE change the top one to RATE and ensure the second collect mode is ACCUMULATE. 7. Click on the PARAMETER DESCRIPTION box to the lower right of the desktop.

8. Locate Directory: and click on CHANGE button. In the file path scroll to the left and click on FLOW CYTOMETRY folder which should then be highlighted. Click NEW FOLDER and label the new FOLDER with todays date. (This is where data will be stored) Then click CREATE. Ensure the new folder with date is highlighted and click CHOOSE. 9. Locate FILE: and click on CHANGE button. Correct the FILE COUNT to 1 at the beginning of the run. Click OK 10. Complete the Sample ID field that is specific for each condition tested. 11. In the general instrument settings, adjust the SSC setting to 318 and in compensation settings adjust the FL3%FL2 setting to 20. 12. Change the fluidics control from STANDBY to RUN in the LO position. 13. Click Acquire and view all gated cell populations. If cells are not within the gate adjust the gate or compensation values. (eg. If B cells are too low decrease the FL3 %FL2 number to raise the population higher) 14. Ensure the SETUP box is unchecked in the ACQUISITION CONTROL window and click ACQUIRE. 15. The FACScan will acquire gated events until 2500 lymphocyte B cells have been counted. If the sample has low cell count, fewer events may be acquired by clicking PAUSE and then SAVE in the acquisition menu. 16. The file count automatically advances from 001 to 002 as each sample is acquired. After each sample is acquired, type in the name of the next sample in the SAMPLE ID field 17. Once all samples have been acquired print all sample files in the folder on hp deskjet 9300 using (Hold apple and P). You can go to the next sample (Hold apple and [ or ] ) You can scroll through all files acquire. 18. Once printed record the T and B cell mean and median on the Excel worksheet template and calculate the change in mean channel fluorescence (T cells) or median channel fluorescence (B cells) by subtracting the corresponding NHS background. Also note the Date, Serum, Cells, NHS lot, POS control lot and % of live cells (7AAD sample data) for each worksheet. 19. Refer to the MESF protocol if this is the first run in two weeks. If the MESF procedure was previously performed during the week or prior week and all the settings have not changed during the two week period then open MESF template.xls and start the MESF protocol at step 8. 20. In order to clean the FACScan, place a 5 mL flow tube containing 1 mL FACS Clean on the sample probe and arm to the side for 1 min. Then replace the arm under the tube and allow the FACScan to run for 5 min. Remove tube and discard 21. Place a 5 mL flow tube containing 1 mL FACS Rinse on the sample probe and arm to the side for 1 min. Then replace the arm under the tube and allow the FACScan to run for 5 min. Remove tube and discard 22. Place a 5 mL flow tube containing 1mL DiH20 on the sample probe and arm to the side for 1 min. Then replace the arm under the tube and allow the FACScan to run for 5 min.

23. Check the SETUP button, then ACQUIRE for 1 min. After a min, record the number of events in the FACScan logbook. If >30 events are present the cleaning procedure (steps 15-17) need to be repeated. 24. Then switch the fluidics control to STANDBY. Keep the machine in standby for at least 5 min to allow the laser to cool down. 25. Flip the vent valve to the down position to release pressure in the tubing. Empty the waste container and refill the reservoir with shealth fluid. C. Interpretation The change in Mean (T cell)/Median (B cell) channel fluorescence calculated for each sample in the FXM template excel worksheet needs to be compared to the established positive cutoff value. T cells and B cells will have their own defined positive cutoff value. The cutoff values for interpretation of results will change with each new lot of NHS or anti-human IgG FITC. Please consult the Reagent Data Notebook for the current T cell and B cell positive cutoff values. These values are only to be used as guidelines for interpretation and troubleshooting. If a sample should fall outside of these ranges it does not necessarily invalidate the test but should indicate the need for closer evaluation of the results and/or review by the director. When channel shift values exceed the cutpoints, results must be reviewed by director or manager before reporting the results as positive. There are situations where the cutpoints are exceeded but the interpretation of the crossmatch will still be negative. For positive and negative serum MCF values outside expected range consult director or manager. If the positive serum values are significantly greater than the expected values it is not necessarily a cause for concern or indication to repeat the test. However, if the values are significantly below the expected ranges, repeating the test may be indicated if the crossmatch is negative.

V. Policy Flow cytometry crossmatches will be used for living donor renal transplants where the recipients have had prior sensitizing events (transfusions, pregnancies or prior transplants) as well as recipients of cadaver donor kidneys. All results will be reviewed by the director or manager before reporting to transplant surgeons. VI. References 1. Garovoy MR, et al. Flow cytometry analysis: A high technology crossmatch technique facilitating transplantation. Transp. Proc. 15:1939; 1983. 2. Chapman JR, et al. Analysis of flow cytometry and cytotoxicity crossmatches in renal transplantation. Transp. Proc. 17:2480; 1985.

3. Terasaki PI, et al. Overview. In: Terasaki PI, ed. Clinical Transplantation 1986. Los Angeles: UCLA Tissue Typing Laboratory, CA. pg 367-392. 4. Raftery MJ, et al. Successful renal transplantation despite a positive fluorescenceactivated cell sorter crossmatch following plasma exchange of donor-specific anti-HLA antibodies. Transplantation. 41:131; 1986. 5. Rodey G, et al. Extra reactivities in flow-cytometry-Positive, CDC-Negative crossmatches are definable HLA specificities. Transp. Proc. 19:778; 1987. 6. Thistlethwaite JR, et al. T cell immunofluorescence flow cytometry cross-match results in cadaver donor renal transplantation. Transp. Proc. 19:722; 1987. 7. Cook DJ, et al. An approach to reducing early kidney transplant failure by flow cytometry crossmatching. Clin. Transp. 1:253; 1987. 8. Lazda VA, et al. The relationship between flow cytometer crossmatch results and subsequent rejection episodes in cadaver renal allograft recipients. Transplantation. 45:562; 1988. 9. Talbot D, et al. Rapid detection of low levels of donor specific IgG by flow cytometry with single and dual color fluorescence in renal transplantation. J. Immun. Meth. 112:279; 1988. 10. Scornik JC, et al. Posttransplant antidonor antibodies and graft rejection. Transplantation. 47:287; 1989. 11. Bray RA, Lebeck LL, and Gebel HM. The flow cytometric crossmatch: Dualcolor analysis of T and B cells. Transplantation 48:834; 1989. 12. Mahoney RJ, et al. The flow cytometric crossmatch and early renal transplant loss. Transplantation 49:527; 1990. 13. Karuppan SS, Lindholm A, and Moller E. Fewer acute rejection episodes and improved outcome in kidney patients transplanted with selection criteria based on crossmatching. Transplantation 53:666; 1992. 14. Bray RA. Flow cytometry in the transplant laboratory. Annls. N.Y. Acad. Sci. 677:138; 1993. 15. Flow crossmatch procedure, Emory University Hospital Medical Laboratories.

Director Review:__________________________________ Date:_______________ Manager Review:__________________________________Date:_______________ Technologist Review:_______________________________Date:_______________ Created: 09/17/10 by PTJ In use:

D. Protocol for Conversion of Anti-human IgG-FITC Fluorescence into MESF Values

I. Purpose Direct quantitation of anti-human IgG-FITC fluorescence intensity in a sample by conversion to MESF units. II. Principle Molecules of Equivalent Soluble Fluorochrome (MESF) microspheres (7-9um) are labeled with a known quantity of fluorescence. A standard curve of fluorescence intensity is generated by the MESF beads, which includes unstained MESF beads as a baseline and five additional MESF beads with increasing amount of fluorochrome. By plotting each bead populations fluorescence intensity versus its known MESF value, a standard MESF curve is generated and the MESF value of anti-human IgG-FITC bound to T or B cells may be easily determined. A unit of MESF is equal to the fluorescence intensity of a given number of pure fluorochrome molecules in solution. For example a MESF-FITC bead with a MESF value of 10,000 has the same fluorescence intensity as a T or B cell containing 10,000 FITC molecules. The use of MESF values provides a tool to validate the flow cytometers linearity in the FITC channel (FL-1) and compare quantitative measurements over time and across flow cytometric platforms.

III. Materials Quantum FITC-5 MESF kit (Premix, 5 labeled, 1 blank) (Bangs Labs, cat# 555) Quick Cal Template Excel Worksheet FXM template Worksheet PBS (Thermo, cat# SH30256.01) IV. Method 1. Vigorously shake the two bottles to ensure uniform suspensions of microspheres. Do not sonicate. 2. Pipette 0.5 mL of PBS into two 5 mL flow tubes and add one drop of blank microspheres to one tube and add one drop of premixed FITC MESF beads to the other tube and mix. 3. At the computer, open the cellquest mesf template on the desktop. Change the directory to the file date just run and create a new folder titled MESF then CHOOSE. Change the file to 1 and label unstained beads 4. With Setup checked, run the unstained MESF beads on the flow cytometer. Adjust the SSC from 318 to 267 and ensure the bead population is within the R1 gate. Then

uncheck setup and then click acquire. No further adjustments to the fluorescence settings or FSC can be made (eg. amplifier gains, PMT voltages, compensation) 5. Acquire 10,000 total events for both the unstained and premixed FITC MESF beads on the flow cytometer 6. Once finished acquiring the unstained and premixed FITC MESF beads, adjust the M1-M6 gates to confirm the whole population of the peak is included. 7. Create a calibration standard curve using the QuickCal quantitative software. Open Excel document: MESF template.xls and enter the median channel values for the Blank thru Bead #5 in red. (entering these values will create a standard curve). 8. Use the calibration plot to determine the MESF value that corresponds to each samples peak channel by copying over the T cell mean channel from the FXM template worksheet and paste it into the channel column in the MESF template worksheet. This will create the MESF values in the next column based on the calibration standard curve. Copy and paste these values back into the worksheet to calculate the T cell mean channel shift in MESF values. Repeat this step for the median B cell channel.

V. Policy Run the MESF protocol approximately every two weeks. If the MESF procedure was previously performed during the week or prior week and none of the settings have changed during the two week period there is no need to run the MESF beads. In this case, open the MESF template.xls and start the MESF protocol at step 8 to convert T and B cell Mean/ Median channel fluorescence respectively into MESF values.

VI. References 1. Vogt, R.F., G.D. Cross, L.O. Henderson, D.L. Phillips. 1989. Model system evaluating fluorescein-labeled microbeads as internal standards to calibrate fluorescence intensity on flow cytometers. Cytometry, 10(3):294-302. 2. Sisken, J.E. 1989. Fluorescent standards. In: Taylor, D.L., Wang, Y., Eds. Methods in cell biology, Vol. 30. San Diego, CA: Academic Press Inc., pp. 30. 3. Schwartz, A. 1989. Instrument compensation and calibration methods: reducing theory to practice. Progress in Cytometry. Belgium: Reports from the Third European Cytometry Users Meeting; June 5-7, 1989. 4. Longobardi Givan, A. 1992. Flow cytometry: first principles. New York: Wiley-Liss, pp. 88-90. 5. Horan, P.K., K.A. Muirhead, S.E. Slezak. 1990. Standards and controls in flow cytometry. In: Melamed, M.R., T. Lindmo, M.L. Mendelsohn, Eds. Flow cytometry and sorting, 2nd ed. New York, NY: Wiley-Liss.

6. National Committee for Clinical Laboratory Standards. 1993. Clinical applications of flow cytometry: immunophenotyping of leukemic cells. Proposed Guideline, NCCLS document H43-P (ISBN 1-56238-219-5). Villanova, PA: NCCLS.