Appendix 1: List of materials Balanced salt solution (BSS), pH 7.5, Ampliqon, Batch nr.: 150774AN23, Varenr.: AMPQ40040.1000 RPMI Medium 1640 (1X), gibco by life technologies, Ref. nr.: 31870-025, Lot nr.: 1530384 Lymphoprep, Axis-Shield, Ref. nr.: 1114547, Lot nr.: 12H6505 7-AAD, eBioscience, Lot nr.: E00031-1638 CD3 FITC, Beutan Dickinson, clone SK7, Lot nr.: 4199773 CD19 PE, Beutan Dickinson, clone SJ25C1, Lot nr.: 4148551 CD45 APC, Beutan Dickinson, clone 2D1, Lot nr.: 4136641 Heparin unconserved, Amgros I/S, varenr.: 741827 Vi-CELL Reagent Pak, Beckman Coulter, Lot nr.: 3199276
I
Appendix 2: Lymphocyte defrost OBS Der må max. optøs 10-12 cryorør ad gangen. Der må max. komme lymfocytter fra 2 cryorør i 1 spidsglas. Optøede lymfocytter bør ikke genfryses. 1. Tænd for 37 °C vandbadet. 2. Fyld 5 mL BSS (Balanced Salt Solution) i et spidsglas. Brug en 20 mL sprøjte. Anbring spidsglasset i 4 °C vandbad. 3. Klargør et stativ med centrifugeglas og Pasteur-pipetter. 4. Tag cryorørene op af cryobeholderen, og placer dem i stålkassen. OBS Umiddelbart derefter, SKAL optøningen foregå. 5. Placer cryorørene i stativ. 6. Ryst stativet i 37 °C vandbadet. VIGTIGT: Når der er en lille isklump på størrelse med en ært tilbage i cryorøret, tages spidsglasset fra 4 °C vandbadet og ind i Laf-bænken. 7. Overfør cellerne med pasteur-pipette til spidsglasset - husk max. 2 cryorør pr. spidsglas. Skyl cryorøret med ca. 1 mL BSS fra spidsglassene. 8. Centrifuger spidsglassene 10 min. ved 180G. 9. Sug supernatanten fra. Tilsæt 5 mL BSS til hvert glas, og resuspendér cellerne. 10. Centrifugér igen 5 min. ved 180G. 11. Cellesuspensioner til panel og HLA typning afleveres i Serologien. 12. Sug supernatanten fra og tilsæt et kendt volumen 20 % S-RPMI og resuspendér. 13. Tæl og indstil lymfocytterne (i suspensionen) - se instruktion: Lymfocytter, tælling i mikroskop 14. Ved lymfocytter, der skal i kultur, foretag celleviabilitetstest: 100 µL cellesuspension + 25 µL trypanblåt. Tælles i Bürker-Turk tællekammer. Krav: 80 % viabilitet. 15. Ved beregning af cellekoncentrationen skal der lægges 20 % celler til tælletallet pga. fortynding med trypanblåt.
II
Appendix 3: Isolation of PBMC 1. Bland 30 mL BSS + heparin (6 dråber) 2. Fortynd blodet 1:1 i BSS + heparin (eller 0,9 % NaCl) 3. Tag det ønskede 50 mL rør og hæld 10 mL lymphoprep i hvert glasrør 4. Læg op til 18 mL fortyndet blod oven på lymphoprepen 5. Centrifuger (program 1) ved 1070 x g, RT, 20 min. 6. Hvis der ikke er mere BSS + heparin, laves mere nu og der overføres 7 mL til et passende antal rør 7. Efter centrifugering overføres interfasen m. pipette til ca. 7 mL BSS + heparin 8. Centrifuger (program 2) ved 500 x g, RT, 10 min. 9. Hæld supernatanten fra og resuspender pellet i 1 mL 20 % S-RPMI (RPMI med 20 % serum) 10. Tæl cellerne i trypanblåt (100 µL celler)
III
Appendix 4: Magnetic separation of lymphocytes 2. Protocol 2.1 Sample preparation When working with anticoagulated peripheral blood or buffy coat, peripheral blood mononuclear cells (PBMCs) should be isolated by density gradient centrifugation (e.g. Ficoll-Paque™, see "General Protocols" in the User Manuals or visit www.miltenyibiotec.com/protocols). ▲ Note: Remove platelets after density gradient separation: resuspend cell pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully remove supernatant. Repeat washing step and carefully remove supernatant. When working with tissues, prepare a single-cell suspension by a standard
preparation
method
(see
"General
Protocols"
in
the
User
Manuals
or
visit
www.miltenyibiotec.com/protocols). ▲ Note: Dead cells may bind non-specifically to MACS MicroBeads. To remove dead cells, we recommend using density gradient centrifugation or the Dead Cell Removal Kit (# 130-090-101). 2.2 Magnetic labeling ▲ Work fast, keep the cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling. ▲ Volumes for magnetic labeling given below are for up to 107 total cells. When working with fewer than 107 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×107 total cells, use twice the volume of all indicated reagent volumes and total volumes). ▲ For optimal performance it is important to obtain a singlecell suspension before magnetic separation. Pass cells through 30 µm nylon mesh (Pre-Separation Filters # 130-041-407) to remove cell clumps which may clog the column. 1. Determine cell number. 2. Centrifuge cell suspension at 300×g for 10 minutes. Pipette off supernatant completely. 3. Resuspend cell pellet in 80 µL of buffer per 107 total cells. 4. Add 20 µL of CD3 MicroBeads per 107 total cells. 5. Mix well and incubate for 15 minutes at 4−8 °C. ▲ Note: Working on icemay require increased incubation times. Higher temperatures and/or longer incubation times lead to non-specific cell labeling. 6. (Optional) Add staining antibodies, e.g. add 10 µL of CD3-FITC (# 130-080-401), and incubate for 5 minutes at 4−8 °C.
IV
7. Wash cells by adding 1−2 mL of buffer per 107 cells and centrifuge at 300×g for 10 minutes. Pipette off supernatant completely. 8. Resuspend up to 108 cells in 500 µL of buffer. ▲ Note: For higher cell numbers, scale up buffer volume accordingly. ▲ Note: For depletion with LD Columns, resuspend up to 1.25×108 cells in 500 µL of buffer. 9. Proceed to magnetic separation (2.3). 2.3 Magnetic separation ▲ Choose an appropriate MACS Column and MACS Separator according to the number of total cells and the number of CD3+ cells (see table in section 1.3). Magnetic separation with MS or LS Columns 1. Place column in the magnetic field of a suitable MACS Separator (see "Column data sheets"). 2. Prepare column by rinsing with appropriate amount of buffer: MS: 500 µL LS: 3 mL 3. Apply cell suspension onto the column. 4. Collect unlabeled cells which pass through and wash column with appropriate amount of buffer. Perform washing steps by adding buffer three times, each time once the column reservoir is empty. MS: 3×500 µL
LS: 3×3 mL
Collect total effluent. This is the unlabeled cell fraction. 5. Remove column from the separator and place it on a suitable collection tube. 6. Pipette appropriate amount of buffer onto the column. Immediately flush out fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column. MS: 1 mL LS: 5 mL ▲ Note: To increase the purity of the magnetically labeled fraction, it can be passed over a new, freshly prepared column.
V
Appendix 5: Clinical data ID Age at TX 1 51 2 27 3 68 4 49 5 16 6 61 7 46 8 44 9 46 10 56 11 61 12 45 13 57 14 54 15 54 16 52 17 66 18 66 19 52 20 61 21 30 22 51 23 47 24 45 25 50 26 53 27 69 28 51 29 56 30 63 31 62 32 53 33 72 34 61 35 42 36 43 37 29 38 42 39 67 40 52
Sex F F M M M M M F F M F M F M M F F F M F M M M M M M M M M M M F M F M M M F M M
Prev. TX No Yes No No No No No Yes (2) No No No No No Yes (3) No No No Yes No No No No No No Yes No No No No No No No No Yes No No No No Yes (2) No
DM at TX No No No No No No No Yes No Yes No No Yes No No No No No No No No No Yes No Yes No No No No Yes No No Yes No No Yes No No No No
Basis disease Unknown Glomerulonephritis Unknown Unknown Glomerulonephritis Nephrectomy Polycystic kidney disease Diabetic nephropathy Glomerulonephritis Chronic kidney failure Chronic kidney failure Polycystic kidney disease Diabetic nephropathy Unknown Polycystic kidney disease Unknown Medullary cystic kidney Terminal kidney failure Polycystic kidney disease Anti-GBM-disease Interstitial nephritis Chronic kidney failure Nephropathy Unknown Kidney failure Polycystic kidney disease Polycystic kidney disease Congenital contracted kidney Congenital contracted kidney Diabetic nephropathy Glomerulosclerosis Polycystic kidney disease Kidney failure Not given Glomerulonephritis Diabetic nephropathy IgA nephritis Unknown Glomerulonephritis Glomerulosclerosis
TX = Transplantation Prev. TX = Previous transplantation; Yes indicates one previous kidney transplantation, number in brackets indicates multiple previous kidney transplantations DM = Diabetes mellitus F = Female M = Male
VI
ID 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
Age at TX 53 65 27 62 58 63 67 58 62 67 69 58 70 54 61 68 68 69 42 35 45 64 51 49 35 59 74 68 42 64 58 29 37 56 29 61 61 50 27 22
Sex F M F F M F F M M F M M F F M M M M M F M M M F F M M M F F M F F M M F F F F M
Prev. TX No No No No No No No No No No No No No No No No No No No No No No No Yes No Yes No No No No No No Yes (3) No No No No No No No
DM at TX No No No No No Yes No No No No Yes No No No No No No Yes No No No No No No No No No No No No No No No Yes No No Yes No No No
Basis disease Glomerulosclerosis Polycystic kidney disease Glomerulonephritis Nephrectomy and kidney failure IgA nephritis Diabetic nephropathy Unknown Chronic kidney failure Unknown Unknown Diabetic nephropathy Polycystic kidney disease Hypertension Polycystic kidney disease Unknown Unknown Obstructive nephropathy Diabetic nephropathy Unknown Kidney infections Unknown Hypertension Polycystic kidney disease Unknown Hypertension Polycystic kidney disease Kidney failure Hypertension Polycystic kidney disease Congenital contracted kidney Glomerulonephritis Acute kidney failure Small kidney IgA nephritis IgA nephritis Lithium nephropathy Diabetic nephropathy Hypertension Congenital myelomeningocele Congenital vulvulae urethra
TX = Transplantation Prev. TX = Previous transplantation; Yes indicates one previous kidney transplantation, number in brackets indicates multiple previous kidney transplantations DM = Diabetes mellitus F = Female M = Male
VII
ID Age at TX 81 60 82 72 83 64 84 56 85 62 86 48 87 46 88 26 89 38 90 61 91 53 92 41 93 61 94 44 95 47 96 34 97 66 98 52 99 64 100 53 101 26 102 60 103 58 104 43 105 40 106 58 107 64 108 52 109 71 110 74 111 62 112 49 113 39 114 47 115 58 116 26 117 67 118 59 119 69 120 53
Sex F F F F F F M M F M M F M M M M M M M F M F F M M M F F M F F M M M F M F M M F
Prev. TX No No No Yes Yes No No Yes Yes No No No No No No Yes No No No Yes No No No No Heart No No No No Yes (2) Yes (3) Nej Yes (3) No No No No No
DM at TX No No No No No No No No No Yes No No No Yes Yes No No No No No No Yes No Yes No No No No No Yes No No No Yes Yes No No Yes No No
Basis disease Polycystic kidney disease Unknown Hypertension Glomerulosclerosis Polycystic kidney disease Polycystic kidney disease Bilateral kidney stones Glomerusclerosis Vesicouteral reflux Diabetic nephropathy IgA nephropathy Terminal kidney failure IgA nephritis Diabetic nephropathy Diabetic nephropathy Immunoglobulin E nefropathy Glomerulonephritis Nephritis Kidney failure Polycystic kidney disease AA-amyloidosis Polycystic kidney disease Kidney cysts Diabetic nephropathy Polycystic kidney disease Kidney failure from calcineurin inhibitor Lithium nephropathy Polycystic kidney disease Polycystic kidney disease Diabetic nephropathy Nephrotic syndrome IgA nephritis Polycystic kidney disease Diabetic nephropathy Diabetic nephropathy Unknown Hypertension Diabetic nephropathy Nephropathy Unknown
TX = Transplantation Prev. TX = Previous transplantation; Yes indicates one previous kidney transplantation, number in brackets indicates multiple previous kidney transplantations; Heart = Previous heart transplantation DM = Diabetes mellitus F = Female M = Male
VIII
ID Age at TX 121 49 122 41 123 50 124 58 125 60 126 70 127 62 128 54 129 36 130 30 131 45 132 64 133 59 134 49 135 37 136 42 137 58 138 54 139 43 140 49 141 22 142 48 143 49 144 64 145 68 146 36 147 60
Sex M M M M F M M F M F M M F M M M M F F M F M M M M M M
Prev. TX Yes No No No No No No No No No Yes No No No No Yes Yes No Liver No Yes Yes No No No No No
DM at TX No No Yes No No No No No No No No No No No No No Yes Nej Yes No No No Yes No No No Yes
Basis disease Unknown Glomerulonephritis Diabetic nephropathy Glomerulosclerosis and hypertension Polycystic kidney disease Sepsis Kidney failure Polycystic kidney disease Glomerulosclerosis Reflux nephropathy Kidney cirrhosis Polycystic kidney disease IgA nephritis Polycystic kidney disease Unknown Unknown Diabetic nephropathy Polycystic kidney disease Acute tubule-interstitial nephropathy Reflux nephropathy Schönlein-Henochs purpura Nephrotic syndrome Diabetes Hypertension Hypertension Hypertension Diabetic nephropathy
TX = Transplantation Prev. TX = Previous transplantation; Yes indicates one previous kidney transplantation; Liver = Previous liver transplantation DM = Diabetes mellitus F = Female M = Male
IX
ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
CDC-XM Mismatch degree T-, B3:0 T-, B1:2 T-, B3:1 T-, B2:0 T-, B2:1 T-, B2:0 T-, B1:1 T-, B3:1 T-, B4:2 T-, B4:1 T-, B3:1 T-, B4:2 T-, B3:1 T-, B- * 2:0 T-, B- * 1:1 T-, B3:1 T-, B2:1 T-, B2:1 T-, B4:1 T-, B3:2 T-, B2:1 T-, B1:1 T-, B- * 1:2 T-, B3:1 T-, B3:1 T-, B2:1 T-, B3:1 T-, B- * 3:2 T-, B- * 3:2 T-, B4:1 T-, B4:1 T-, B2:2 T-,B2:1 T-,B2:1 T-, B- * 2:1 T-,B3:0 T-,BT-,B+ 3:1 T-,B2:1 T-,B3:1
HLA-antibodies None HLA-II None None HLA-I and HLA-II None None HLA-I and HLA-II HLA-I HLA-I HLA-II HLA-I and HLA-II Present HLA-II None None None
Flow-XM DSA T-, B+
DQ4, DQ2
T-, BT-, B+
Dr13, DQ6
T+, B+ T+,B-
None HLA-I and HLA-II HLA-I and HLA-II None None None None None None None
None None None HLA-I None None HLA-I and HLA-II T+, None None
DQ7, B7 None A68 DQ7 A23, B49, B62, DQ7 None Cw5, DQ2
B18, DR7 B60
A2
B7
CDC-XM = Complement dependent cytotoxicity crossmatch HLA = Human leucocyte antigen Flow-XM = Flow cytometry crossmatch DSA = Donor specific antibodies T- = Negative T-cell crossmatch; T+ = Positive T-cell crossmatch B- = Negative B-cell crossmatch; B+ = Positive B-cell crossmatch Mismatch degree is given in HLA-A and B:HLA-DR number of mismatches * = Results are obtained from laboratory working papers X
ID 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
CDC-XM Mismatch degree T-,B2:1 T-,B4:1 T-,B2:1 T-,B2:1 T-,B2:1 T-,BT-,BT-,B2:0 T-,B3:1 T-,BT-,B2:1 T-,B2:0 T-, B- * 2:1 T-,B2:1 T-, B- * T-, B- * T-,B2:1 T-,BT-,B3:1 T-, B- * T-,BT-,B0:0 T-,B3:1 T-,B2:1 T-,B2:0 T-,BT-,B3:0 T-,BT-,BT-,BT-,B3:1 T-,B3:1 T-,B1:1 T-, B- * 2:1 T-, B- * 2:1 T-,B2:1 T-,B4:1 T-,B4:0 T-,B2:1 T-,B2:1
HLA-antibodies
Flow-XM DSA
None None HLA-I and HLA-II T+,B+ None None T-,BNone None None None None None None HLA-I and HLA-II T+,B+
None None None None
A2, B60, DR4
CW17
T-,B-
None
HLA-I HLA-I None None HLA-II
T+,B+
B57
HLA-II
DR17, DQ2
HLA-I None HLA-I HLA-I
A25
None None
CDC-XM = Complement dependent cytotoxicity crossmatch HLA = Human leucocyte antigen Flow-XM = Flow cytometry crossmatch DSA = Donor specific antibodies T- = Negative T-cell crossmatch; T+ = Positive T-cell crossmatch B- = Negative B-cell crossmatch; B+ = Positive B-cell crossmatch Mismatch degree is given in HLA-A and B:HLA-DR number of mismatches * = Results are obtained from laboratory working papers XI
ID 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120
CDC-XM Mismatch degree T-, B2:1 T-, B2:1 T-, B3:1 T-, BT-, B2:1 T-, B2:1 T-, B3:1 T-, B- * 3:1 T-, B3:0 T-, B2:1 T-, B2:1 T-, BT-, BT-, B1:1 T-, B4:0 T-, BT-, B2:0 T-, BT-, BT-, BT-, BT-, BT-, B- * 0:0 T-, B2:2 T-, B2:1 T-, B1:1 T-, BT-, BT-, BT-, BT-, B2:0 T-, B0:0 T-, BT-, B_ 1:1 T-, B- * 2:1 T-, B2:1 T-, BT-, BT-, BT-, B2:1
HLA-antibodies
Flow-XM DSA
HLA-II HLA-I
DQ6 B8
HLA-I and HLA-II
A23
A24, DR13 None DQ5 None None HLA-I None None None None HLA-I and -II None HLA-II None HLA-I and -II None
T+, B+
A1, B51, B44, Q7
T-, B+
DQ2, DQ7
None HLA-I HLA-I and -II
B60
HLA-I HLA-I and -II None
B51, D11
None HLA-I
CDC-XM = Complement dependent cytotoxicity crossmatch HLA = Human leucocyte antigen Flow-XM = Flow cytometry crossmatch DSA = Donor specific antibodies T- = Negative T-cell crossmatch; T+ = Positive T-cell crossmatch B- = Negative B-cell crossmatch; B+ = Positive B-cell crossmatch Mismatch degree is given in HLA-A and B:HLA-DR number of mismatches * = Results are obtained from laboratory working papers XII
ID 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147
CDC-XM Mismatch degree T-, B2:0 T-, B- * 1:0 T-, BT-, B3:2 T-, B3:1 T-, B3:1 T-, BT- B? * 2:0 T-, B3:1 T-, B3:1 T-, BT-, B3:1 T-, B2:1 T-, B2:1 T-, B- * 1:2 T-, B- * 2:1 T-, B2:1 T-, B4:1 T-, B2:1 T-, B1:1 T-, B2:1 T-, B1:1 T-, B0:0 T-, B2:1 T-, B1:1 T-, B2:1 T-, B2:1
HLA-antibodies Flow-XM DSA None None None None None None None None None None HLA-I None None HLA-I and -II None None None HLA-II None None
B39, B38, B76 T-, B-
None None
CDC-XM = Complement dependent cytotoxicity crossmatch HLA = Human leucocyte antigen Flow-XM = Flow cytometry crossmatch DSA = Donor specific antibodies T- = Negative T-cell crossmatch B- = Negative B-cell crossmatch B? = Inconclusive B-cell crossmatch result Mismatch degree is given in HLA-A and B:HLA-DR number of mismatches * = Results are obtained from laboratory working papers
XIII
ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Basis biopsy Normal Fibrosis, sclerosis Normal Normal Normal Fibrosis, arteria sclerosis Fibrosis, arteria sclerosis Arteria sclerosis Normal Normal Arteria sclerosis Arteria sclerosis Arteria sclerosis Normal Normal Sclerosis, fibrosis Normal Normal Sclerosis, fibrosis, tubule atrophy Fibrosis, tubule atrophy Arteria sclerosis Thickening of the intima Arteria sclerosis Arteria sclerosis, thickening of the intima Arteria sclerosis Normal Arteria sclerosis Fibrosis, thickening of the intima Intima fibrosis Normal Fibrosis Normal Arteria sclerosis Fibrosis, arteria sclerosis Normal Normal Normal Arteria sclerosis Normal
IT eGFR 3M eGFR 1Y Biopsy w/o rejection (time post-TX) T 10D 56 53 T 3D, 7D, 14D, 40D, 48D, 55D, 63D 5 5 T 76D, 6M + 1D 38 36 T 1D, 6D, 15D, 18D 17 15 T T 27D 36 39 T 62 T 36 45 T 8D, 15D, 3M 36 22 T 67 74 T 25 35 T 2D, 17D, 5M 16 20 T 13D 30 33 T 16D 29 27 T 7D, 1M + 20D 30 36 T 4D 31 29 T 61 76 T 20D 38 48 T 7D, 21D 37 32 T 17D 44 54 T 5D, 10D, 30D, 2M + 9D, 5M 16 12 T 5D 39 46 T > 90 > 90 T 20D, 29D, 1M + 14D, 2M 22 23 T 7D 50 T 13D 37 38 T 7D, 1M, 1M + 15D, 2M 20 25 T 13D 59 42 T 13D 22 29 T 71 66 T 56 T 47 45 T 56 61 T 5D, 12D, 8M 48 48 T 2M, 1Y + 1M 48 54 T 68 T 3M, 6M 22 T 3D, 9D 41 T 7D, 1M 19 25 T 6D 45 47
IT = Immunosuppressive treatment; T = Tacrolimus eGFR 3M = eGFR obtained three months after transplantation eGFR 1Y = eGFR obtained one year after transplantation TX = Transplantation Biopsy w/o rejection is given in years (Y) + months (M) + days (D), and the biopsies are divided with comma
XIV
ID 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
Basis biopsy Fibrosis, edema Normal Normal Profileration of the intima Normal Normal Fibrosis, profileration of the intima Fibrosis, tubule atrophy Normal Normal Normal Profileration of the intima Normal Arteria sclerosis Normal Normal Mesangioprofileration Acute humoral rejection Was not representative Normal Fibrosis, tubule atrophy, sclerosis Normal Normal Normal Arteria sclerosis Fibrosis, arteria sclerosis Normal Was not representative Normal Normal Normal Sclerosis, thickening of the intima Normal Normal Was not representative Arteria sclerosis Normal Normal Mesangioprofileration
IT eGFR 3M eGFR 1Y Biopsy w/o rejection (time post-TX) T 65 50 T 56 63 6D T 69 64 T T 63 69 9D, 4M + 9D T 15 13 6D, 12D, 21D, 3M + 7D, 4M + 7D, 5 M T 17 17 14D, 1M + 14D T 43 43 2M + 14D, 3M + 14D T 19 - 1M + 14D, 2M + 7D, 3M, 4M T 40 40 3M T 42 53 1M T 50 56 5D T 60 59 T 84 83 1D T 73 54 2M T > 90 > 90 6D, 2Y T 64 55 2M + 14D T 34 56 3M T 6 4 T 63 78 T 57 65 7D, 2M + 7 D, 2M + 10D T 15 5 2D, 9D, 17D, 24D, 1M, 1M + 9D, 4M T 65 T 25 13 15D, 2M, 2M + 14D, 3M + 14D, 1Y + 2M, 1,5Y T 64 60 4D, 3M + 14D T 49 46 5D T 30 28 6D, 15D, 3M + 14D T 33 27 5D, 1Y + 5M T 6 5 22D, 1M + 15D, 2M + 8D T 41 43 7D, 1M, 1M + 14 D, 2M T 38 41 6M T 28 - 1M + 3D, 2M + 3D T 35 41 2M + 23D T 50 56 T 55 50 4M + 7D T > 90 - 21D, 1M T 29 10 6D, 14D, 9M + 14D T 21 18 8D, 1M, 5M, 6M + 14D T T 35 43 4D, 2M, 2M + 7D
IT = Immunosuppressive treatment; T = Tacrolimus eGFR 3M = eGFR obtained three months after transplantation eGFR 1Y = eGFR obtained one year after transplantation TX = Transplantation Biopsy w/o rejection is given in years (Y) + months (M) + days (D), and the biopsies are divided with comma
XV
ID
T T T T T T T T T T T T T T T T T T
eGFR 3M 28 35 10 24 57 36 53 46 60 62 39 42 > 90 45 50 46 57
eGFR 1Y 20 30 19 26 33 35 63 29 59 30 62 82 59 31 53
T
12
12
Fibrosis, arteria sclerosis Changes in the tubules Fibrosis
T T
33 10
41 17
T
37
47
Was not representative Fibrosis Sclerosis Mesangioproliferation Changes in the tubules, sclerosis, mesangioproliferation Normal Normal Fibrosis Fibrosis Arteria sclerosis Arteria sclerosis, fibrosis, thickening of the intima Arteria sclerosis, fibrosis, thickening of the intima Normal Normal Normal Normal Normal
T T T T
58 59 46 27
53 59 44 25
T
89
-
T T T T T T
50 47 62 45 36
55 -
T
31
40
T
17
-
T T T T T
83 43 31 36 72
108 54 19 41 74
Basis biopsy
IT
Normal Fibrosis, sclerosis Normal Sclerosis, arteria sclerosis Glomerulosclerosis Normal Proliferation of the intima Normal Proliferation of the intima Normal Changes in the tubules Changes in the tubules Normal Proliferation of the intima Fibrosis Normal Fibrosis
100 101 102 103 104 105 106 107
81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
108 109 110 111 112 113 114 115 116 117 118 119 120
63 37 49
Biopsy w/o rejection (time post-TX) 4D, 3M + 24D, 4M + 14D 5D 1M + 14D, 2M, 2Y 8D 10D, 4M + 14D, 9M + 14D, 10M, 1Y + 2M + 14D 9D 3D, 14D, 22D
6D, 16D, 2M + 14D 13D 7D 13D 8D 5D, 24D 5D, 14D, 1M, 1M + 2D, 1M + 14D 6D, 9D, 23D, 1M + 10D, 1M + 19D, 10M + 21D, 11M, 1Y 6D, 25D, 2M, 1.5 Y 2D, 1M + 14D, 6M 1 M + 14D, 3 M + 14D, 3M + 21D, 4M, 1Y + 6M, 1Y + 6M, 1Y + 7M 4D, 21D, 4M + 21D 4D, 4M + 14D 2D, 8D, 17D, 30D 4D, 22D, 5M
2D, 4M + 10D, 5M 5M + 19D 5D, 12D, 8M + 14D 4D, 1M + 1D 6D, 15D, 3Y + 3M 5D 6D, 1M, 1M + 7D, 6M + 7D, 7M + 7D, 8M 21D, 1M + 3D, 2M + 2D
IT = Immunosuppressive treatment; T = Tacrolimus eGFR 3M = eGFR obtained three months after transplantation eGFR 1Y = eGFR obtained one year after transplantation TX = Transplantation Biopsy w/o rejection is given in years (Y) + months (M) + days (D), and the biopsies are divided with comma XVI
ID
Basis biopsy
IT
eGFR 3M
eGFR 1Y
121
Arteria sclerosis
T
36
37
3D, 26D, 2M + 20D
122
Normal
T
59
61
3D
123
Fibrosis, sclerosis
T
43
41
5D, 9D
124
T
25
40
5D, 16D, 1M + 4D
T
22
15
6D, 3M + 14D
126
Normal Fibrosis, sclerosis, thickening of the intima Sclerosis, tubule atrophy, fibrosis
T
27
8
6D
127
Normal
T
59
64
6D
128
Normal
T
56
66
5D
129
Normal
T
36
50
15D, 2M + 18D, 6M + 10D, 3Y + 3M + 7D, 3Y + 8M
130
Not representative
T
34
65
2M + 14D
131
Normal
T
37
29
17D, 1M + 3D,
132
Normal
T
50
52
133
Normal
T
27
60
134
Normal
T
54
-
135
Fibrosis
T
29
30
136
Fibrosis, arteria sclerosis
T
-
-
137
Normal
T
36
47
138
Normal
T
56
57
139
Normal
T
43
73
140
Normal
T
54
66
141
Normal
T
97
95
142
Normal
T
19
16
143
Normal
T
62
66
144
Normal
T
32
-
5D, 12D
145
Normal
T
31
36
3D, 12D,
146
Normal
T
48
40
5D, 2M
147
Normal
T
61
57
4M + 14D, 1Y + 4M
125
Biopsy w/o rejection (time post-TX)
3M + 2D, 5M 9D, 1M 1D 1M + 14D, 4M 1M + 21D, 3M
4D, 1M+2D, 10M + 6D
IT = Immunosuppressive treatment; T = Tacrolimus eGFR 3M = eGFR obtained three months after transplantation eGFR 1Y = eGFR obtained one year after transplantation TX = Transplantation Biopsy w/o rejection is given in years (Y) + months (M) + days (D), and the biopsies are divided with comma
XVII
ID 2 11 12 18 21 24 26 27 29 44 46 47 50 55 59 62 64 67 68 69 74 77 78 85 86 92 93 95 97 101 104 107 114 115 116 124 126 129 135 136 142 147
R1 Bord. 1A Hum. 2A Bord. 2A 2A Bord. 2A Hum. 2A Bord 1B Bord. Acute 2A Bord. 2A 2A 2A 2A 2A Bord. Bord. Chro. Bord. 1A 2A 2A 2A 1A Bord. 2B Bord. 1B 2A Bord. 2A 1A Necr. 1A 2A
T1 None MP, Solu. Rit. None MP MP MP None MP Gr.ectomy None None MP MP Gr.ectomy Gr.ectomy MP MP MP Rit. Solu. MP None MF MF None MP MP None None MP MP None None MP None None MP None None None MP
R2 2A
T2 Gr.ectomy
R3
T3
R4
Hum.
None
Necr.
None
Bord.
Bord.
None
2A
None
2A
None
Chro.
Gr.ectomy
2A
MP
Bord.
Bord. 2A 2A 2A 2A
MP None Solu. MP MP
Bord.
Solu.
2A Bord Bord.
None None None
B 1A
T4
R5
T5
R6
T6
2A
TG
Chro.
Gr.ectomy
Bord. Hum.
MMF ↑ Plasmaferese
2A
TG
1A
None
Bord.
None
Bord.
None
TG
Bord. 1A
MP
Bord. 1A
MP
Bord. 1B
None Solu.
Bord.
Gr.ectomy ↑P
Bord.
None
2B
None
Bord.
None
1B
Solu.
1A
P
RX and TX = Number of rejection and corresponding treatment Bord. = Borderline rejection Acute = Acute rejection Hum. = Humoral 1-2A + 1-2B = BANFF classification of rejection Chro. = Chronic Necr. = Necrotic XVIII
MF = Myfenax P = Prednisolon MP = Methylprednisolon Solu. = Solumedrol TG = Thymoglobulin Gr.ectomy = Graftectomy Rit. = Rituximab MMF = Mycophenolate mofetil
XIX
Appendix 6 – Experimental data Test sample Pre-sep B-pos B-neg T-pos T-neg Lymphocyte source Spleen Spleen Spleen Spleen Spleen V (μL) 50 100 100 100 100 CFlowcytometry (T-cells/μL) 1,274 64 63.2 40.7 160 CFlowcytometry (B-cells/μL) 180 420 19.7 39.5 1.68 T-cell-viabilityFlowcytometry (%) 61.71 75.94 73.48 33.97 80.74 B-cell-viabilityFlowcytometry (%) 42.69 28.57 56.4 60.76 56.4
The results are obtained from the same sample and divided in B- and T-cell fractions Pre-sep = Sample values before separation Neg = Sample values from the negative sample fraction Pos = Sample values in the positive sample fraction V = Volume extracted for flow cytometry analysis C = Concentration Test sample Pre-sep B-pos B-neg T-pos T-neg Lymphocyte source PB PB PB PB PB V (μL) 100 100 100 100 100 CFlowcytometry (T-cells/μL) 976 3.3 318 257 7.5 CFlowcytometry (B-cells/μL) 75.8 21.6 1.55 1.5 14.5 T-cell-viabilityFlowcytometry (%) 98.14 30.3 99.41 97.83 93.84 B-cell-viabilityFlowcytometry (%) 88.41 96.52 100 71.88 97.53
The results are obtained from the same sample and divided in B- and T-cell fractions Pre-sep = Sample values before separation Neg = Sample values from the negative sample fraction Pos = Sample values in the positive sample fraction PB = Peripheral blood V = Volume extracted for flow cytometry analysis C = Concentration Test sample B-Pre-sep T-Pre-sep B-pos1 B-neg T-pos1 T-neg B-pos2 T-pos2 Lymphocyte source PB PB PB PB PB PB PB PB V (μL) 50 50 50 50 50 50 50 50 CFlowcytometry (T-cells/μL) 2,234 451 23.6 425 187 2.84 1.28 1.92 CFlowcytometry (B-cells/μL) 86.8 101 2.84 2.44 1.16 23.6 0 0 T-cell-viabilityFlowcytometry (%) 96.02 99.51 19.72 96.89 96.12 83.5 71.88 84.78 B-cell-viabilityFlowcytometry (%) 97.76 96.23 95.93 98.33 72.41 99.16 -----
The results are obtained from the same sample and divided in B- and T-cell fractions Pre-sep = Sample values before separation Neg = Sample values from the negative sample fraction Pos = Sample values in the positive sample fraction, which was performed twice (1 and 2) PB = Peripheral blood V = Volume extracted for flow cytometry analysis C = Concentration
XX
Test sample B-Pre-sep T-Pre-sep B-pos B-neg T-pos T-neg Lymphocyte source PB PB PB PB PB PB V (μL) 30 35 50 50 50 50 CFlowcytometry (T-cells/μL) 2,422 2,417 0 590 403 7.92 CFlowcytometry (B-cells/μL) 103 148 16.5 7.52 0.72 17.9 T-cell-viabilityFlowcytometry (%) --96 92 71 B-cell-viabilityFlowcytometry (%) 62 79 0 66
The results are obtained from the same sample and divided in B- and T-cell fractions Pre-sep = Sample values before separation Neg = Sample values from the negative sample fraction Pos = Sample values in the positive sample fraction PB = Peripheral blood V = Volume extracted for flow cytometry analysis C = Concentration Test sample ① ② ③ ④ Lymphocyte source PB PB PB PB CFlowcytometry (Lymphocytes/μL) 946 1,781 1,437 1,228 CFlowcytometry (T-cells/μL) 782 1,489 990 720 CFlowcytometry (B-cells/μL) 85.8 112 104 91.5 T-cell-viabilityFlowcytometry (%) 96.3 98.76 96.75 96.62 B-cell-viabilityFlowcytometry (%) 98.81 98.87 99.76 99.73 Vi-CELL setting CHO HumLymph HumLymph HumLymph CVi-CELL (Lymphocytes/μL) 2,720 1,300 1,870 2,100 Lymphocyte-viabilityVi-CELL (%) 97.8 98.7 96.7 97.1
The results are obtained from three different samples, where the third sample was divided into two (3 and 4) Pre-sep = Sample values before separation Neg = Sample values from the negative sample fraction Pos = Sample values in the positive sample fraction PB = Peripheral blood C = Concentration Test sample B-neg B-pos T-neg T-pos Lymphocyte source PB PB PB PB V (μL) 50 50 50 50 CFlowcytometry (Lymphocytes/μL) 315 21 88,4 301 CFlowcytometry (T-cells/μL) 239 0 5.57 298 CFlowcytometry (B-cells/μL) 4.66 20.8 23.2 2.25 T-cell-viabilityFlowcytometry (%) 99.58 --95.45 99.08 B-cell-viabilityFlowcytometry (%) 100 99.6 100 100 CVi-CELL (Lymphocytes/μL) 350 13 * 250 230 Lymphocyte-viabilityVi-CELL (%) 97.6 100 97.6 100
Vi-CELL setting: HumLymph * = Vi-CELL Analyzer reported too low cell concentration to measure accurately The results are obtained from the same sample and divided in B- and T-cell fractions Neg = Sample values from the negative sample fraction Pos = Sample values in the positive sample fraction PB = Peripheral blood XXI
V = Volume extracted for flow cytometry analysis C = Concentration Test sample 100 % 75 % 50 % 25 % 0% Lymphocyte source Spleen PB/Spleen PB/Spleen PB/Spleen PB V (μL) 100 100 100 100 100 CFlowcytometry (Lymphocytes/μL) 1,803 1,512 1,380 1,238 1,154 Lymphocyte-viabilityFlowcytometry (%) 62.67 72.22 83.58 90.39 98.68 CVi-CELL (Lymphocytes/μL) 1,070 950 1,050 1,130 1,370 Lymphocyte-viabilityVi-CELL (%) 65.8 76.4 84.8 93.4 98.5
Test sample indicates percentage of spleen cells in the sample Vi-CELL setting: HumLymph PB = Peripheral blood V = Volume extracted for flow cytometry analysis C = Concentration Test sample Lymphocyte source V (μL) CFlowcytometry (Lymphocytes/μL) Lymphocyte-viabilityFlowcytometry (%) CVi-CELL (Lymphocytes/μL) Lymphocyte-viabilityVi-CELL (%)
100 % CH2O PB 100 713
80 % CH2O PB 100 777
60 % CH2O PB 100 862
40 % CH2O PB 100 897
20 % CH2O PB 100 975
0 % CH2O PB 100 1,044
98
98
99
99
99
99
1,100 96.1
1,060 97.1
1,100 96.4
1,000 95.1
1,000 95.9
1,120 98
Test sample indicates percentage of CH2O-treated lymphocytes mixed with non-CH2O-treated lymphocytes Vi-CELL setting: HumLymph PB = Peripheral blood V = Volume extracted for flow cytometry analysis C = Concentration Test sample Lymphocyte source V (μL) CFlowcytometry (Lymphocytes/μL) Lymphocyte-viabilityFlowcytometry (%) CVi-CELL (Lymphocytes/μL) Lymphocyte-viabilityVi-CELL (%)
100 % CH2O PB 50 523
80 % CH2O PB 50 528
60 % CH2O PB 50 521
40 % CH2O PB 50 452
20 % CH2O PB 50 449
0 % CH2O PB 50 601
99
99
98.8
99
98
98.5
470 89.3
300 93.5
340 95.7
380 93.6
580 96.7
510 97.7
Test sample indicates percentage of CH2O-treated lymphocytes mixed with non-CH2O-treated lymphocytes Vi-CELL setting: HumLymph PB = Peripheral blood V = Volume extracted for flow cytometry analysis C = Concentration
XXII
Test sample Lymphocyte source V (μL) CFlowcytometry (Lymphocytes/μL) CFlowcytometry (T-cells/μL) CFlowcytometry (B-cells/μL) T-cell-viabilityFlowcytometry (%) B-cell-viabilityFlowcytometry (%) Lymphocyte-viabilityFlowcytometry (%) 1 CVi-CELL (Lymphocytes/μL) 1 Lymphocyte-viabilityVi-CELL (%) 2 CVi-CELL (Lymphocytes/μL) 2 Lymphocyte-viabilityVi-CELL (%) Mean CVi-CELL (Lymphocytes/μL) Mean lymphocyte-viabilityVi-CELL (%)
1.1 1.2 2.1 2.2 PB PB PB PB 100 100 100 100 1,827 1,696 1,330 1,121 1,171 1,078 1,098 924 150 135 131 111 98.4 99 100 99.7 99.6 99.7 99.7 99.8 99 99.35 99.85 99.75 1,990 1,560 1,690 99.1 98.9 98.6 1,830 1,820 1,600 1,670 98.4 97.7 99.1 98.8 1,830 1,905 1,580 1,680 98.4 98.4 99.0 98.7
3.1 PB 100 1,706 1,408 182 99.5 99.7 99.6 2,770 99 2,730 98.6 2,750 98.8
3.2 PB 100 1,763 1,456 185 99.4 99.6 99.5 2,800 98.2 2,850 98.5 2,825 98.4
Lymphocytes from three blood samples each divided into two samples (x.1 and x.2). The samples were run in duplicates on the Vi-CELL Analyzer (1 and 2). Vi-CELL setting: HumLymph Lymphocyte-viabilityFlowcytometry = Mean viability of B- and T-cell viability Mean CVi-CELL = Mean concentration of lymphocyte concentrations from duplicates Mean lymphocyte-viabilityVi-CELL = Mean viability of lymphocyte viabilities from duplicates PB = Peripheral blood V = Volume extracted for flow cytometry analysis C = Concentration
XXIII
