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15-P: Solid Phase Antibody (SpA) Identification: Effect of Interfering Substances or Prozone Phenomenon
Abstracts
15-P
S29
SOLID PHASE ANTIBODY (SpA) IDENTIFICATION: EFFECT OF INTERFERING SUBSTANCES OR PROZONE PHENOMENON. Manish J. Gandhi, Nancy A. Ploeger, Deborah K. Falbo, Steven R. DeGoey. Division of Transfusion Medicine, Mayo Clinic, Rochester, MN, USA. Aim: HLA antibodies (HLA-Abs), especially donor specific antibodies (DSA) identified by SpA are used pre-transplant (Tx) to screen donors and for post Tx monitoring. However, there are questions regarding its technical limitation. Methods: Results from 11 recipients with low to high levels of HLA-Abs were compared after performing SpA by: 1.manufacturer protocol 2.after treatment at 56C for 30 minutes (inactivate complement) 3.after treatment at 63C for 13 minutes (inactivate IgM) 4.after 1:8 dilution. Results: Heat treatment resulted in increased mean flouresence intensity (MFI) of the negative control bead as shown below: Heat treatment resulted in increased number of allele specificities with MFI ⬎500: untreated⫽626, 56C⫽702, 63C⫽661, diluted⫽633. However, dilution demonstrated higher number of allele specificities with MFI ⬎6000 as shown below: Significantly, for BP and WK, untreated DSA MFI was ⬍4000. However, the diluted DSA MFI equivalent was ⬎50,000 and correlated with B-flow crossmatch channel shift ⬎500. Patient
Untreated
56C
63C
Dilution
AL BP WK WT RM BA HC HA SC TE VM Average
108 39 55 115 209 147 86 115 312 13 30 112
137 50 172 297 345 237 252 193 153 63 107 182
150 127 420 1029 1403 631 320 841 380 241 428 543
16 36 42 43 55 80 60 15 10 16 15 35
Patient
Untreated
56C
63C
Dilution
AL 23 28 21 47 BP 44 45 35 54 WK 35 41 36 49 WT 36 73 64 73 RM 39 45 34 51 BA 55 57 52 65 HC 0 0 0 0 HA 2 3 8 17 SC 0 0 0 0 TE 4 6 4 18 VM 0 1 0 1 Total 238 299 254 375 Conclusions: HLA-Abs detection by SpA can be effected by the steric interference from complement or IgM. Heat treating the sample helps however, there is increased background. Some cases with high level of antibodies also demonstrate a prozone phenomenon as demonstrated by significantly higher number of alleles with MFI ⬎6000.
15-P
S29
SOLID PHASE ANTIBODY (SpA) IDENTIFICATION: EFFECT OF INTERFERING SUBSTANCES OR PROZONE PHENOMENON. Manish J. Gandhi, Nancy A. Ploeger, Deborah K. Falbo, Steven R. DeGoey. Division of Transfusion Medicine, Mayo Clinic, Rochester, MN, USA. Aim: HLA antibodies (HLA-Abs), especially donor specific antibodies (DSA) identified by SpA are used pre-transplant (Tx) to screen donors and for post Tx monitoring. However, there are questions regarding its technical limitation. Methods: Results from 11 recipients with low to high levels of HLA-Abs were compared after performing SpA by: 1.manufacturer protocol 2.after treatment at 56C for 30 minutes (inactivate complement) 3.after treatment at 63C for 13 minutes (inactivate IgM) 4.after 1:8 dilution. Results: Heat treatment resulted in increased mean flouresence intensity (MFI) of the negative control bead as shown below: Heat treatment resulted in increased number of allele specificities with MFI ⬎500: untreated⫽626, 56C⫽702, 63C⫽661, diluted⫽633. However, dilution demonstrated higher number of allele specificities with MFI ⬎6000 as shown below: Significantly, for BP and WK, untreated DSA MFI was ⬍4000. However, the diluted DSA MFI equivalent was ⬎50,000 and correlated with B-flow crossmatch channel shift ⬎500. Patient
Untreated
56C
63C
Dilution
AL BP WK WT RM BA HC HA SC TE VM Average
108 39 55 115 209 147 86 115 312 13 30 112
137 50 172 297 345 237 252 193 153 63 107 182
150 127 420 1029 1403 631 320 841 380 241 428 543
16 36 42 43 55 80 60 15 10 16 15 35
Patient
Untreated
56C
63C
Dilution
AL 23 28 21 47 BP 44 45 35 54 WK 35 41 36 49 WT 36 73 64 73 RM 39 45 34 51 BA 55 57 52 65 HC 0 0 0 0 HA 2 3 8 17 SC 0 0 0 0 TE 4 6 4 18 VM 0 1 0 1 Total 238 299 254 375 Conclusions: HLA-Abs detection by SpA can be effected by the steric interference from complement or IgM. Heat treating the sample helps however, there is increased background. Some cases with high level of antibodies also demonstrate a prozone phenomenon as demonstrated by significantly higher number of alleles with MFI ⬎6000.