The effect of preformed antibodies targeting human leukocyte antigens (HLAs) on the outcome of organ transplantation has been demonstrated in kidney, heart, and lung transplantation, and a lymphocyte crossmatch test (LCT) is considered mandatory. On the other hand, such effect is controversial in liver transplantation, even in living donor liver transplantation (LDLT) [
In the present study, we retrospectively analyzed the targeted HLA and the amount of DSA in the preserved serum of patients with a positive LCT prior to LDLT to clarify the relationship between the amount of DSA and the clinical outcome.
Between January 2000 and March 2008, 616 patients underwent blood type identical or compatible LDLT. Among them, 21 recipients (3.4%) were LCT positive preoperatively. Pretransplant sera from 18 of these 21 recipients were preserved and available for examination in the present study. These 18 patients were enrolled in this study and their characteristics are shown in Table
Profiles of crossmatch-positive recipients.
Patient no. | Primary disease | Age (years) | Sex | Donor | Blood type compatibility | Graft type | GRWR (%)* | History of pregnancy | History of blood transfusion | History of abdominal surgery |
---|---|---|---|---|---|---|---|---|---|---|
1 | PBC† | 67 | F | Son | Identical | Left lobe | 1.43 | Yes | Yes | Cholecystectomy, Hassab operation |
2 | PBC | 47 | F | Son | Compatible | Right lobe | 1.29 | Yes | Yes | Cholecystectomy, choledocojejunostomy, hepaticojejunostomy |
3 | Biliary atresia | 19 | F | Mother | Identical | Right lobe | 1.16 | No | Yes | Kasai operation |
4 | HCV-LC‡ | 49 | F | Husband | Identical | Right lobe | 1.41 | Yes | Yes | Appendectomy |
5 | PBC | 53 | F | Daughter | Identical | Right lobe | 0.98 | Yes | Unknown | No |
6 | HBV-FHF$ | 62 | F | Daughter | Identical | Left lobe | 0.77 | Yes | No | No |
7 | Metastatic neuroendocrine tumor of pancreas | 47 | F | Husband | Compatible | Left lobe | 1.00 | Yes | Yes | Distal pancreatectomy and splenectomy and repair of portal vein injury |
8 | HCV-LC | 53 | F | Father | Identical | Right lobe | 0.75 | Yes | Unknown | Hysterectomy |
9 | Graft failure due to portal vein thrombosis after LDLT⋀ | 25 | F | Sister | Identical | Right lobe | 1.66 | No | Yes | Kasai operation, splenectomy, distal splenorenal shunt, 1st LDLT |
10 | Congenital liver fibrosis | 44 | F | Husband | Identical | Right lobe | 1.26 | Yes | Yes | Splenectomy, esophageal transection, cholecystectomy, subtotal gastrectomy |
11 | LC | 55 | F | Son | Identical | Right lobe | 1.16 | Yes | No | No |
12 | PBC | 50 | F | Daughter | Identical | Right lobe | 0.96 | Yes | Yes | No |
13 | Cryptogenic LC (AIH#) | 51 | F | Sister | Identical | Left lobe | 0.72 | No | Yes | No |
14 | Biliary atresia | 0 (6 M) | F | Mother | Identical | Lateral segment | 3.19 | No | No | Kasai operation, revision |
15 | Biliary atresia | 30 | F | Mother | Identical | Left lobe | 0.91 | No | No | Kasai operation |
16 | HBV-LC, HCC** | 50 | M | Wife | Compatible | Right lobe | 0.82 | No | No | No |
17 | Wilson's disease | 8 | M | Mother | Identical | Lateral segment | 1.34 | No | Yes | No |
18 | Graft failure due to outflow block after LDLT | 0 (11 M) | F | Mother | Compatible | Lateral segment | 2.94 | No | Yes | Kasai operation, 1st LDLT |
*Graft recipient weight ratio; †primary biliary cirrhosis; ‡hepatitis C virus-related liver cirrhosis; $hepatitis B virus-related fulminant hepatic failure; ⋀living donor liver transplantation; #autoimmune hepatitis; **hepatocellular carcinoma.
This study was approved by the Ethics Committee of Kyoto University Hospital according to the Declaration of Helsinki of 1975 as revised in 2008.
All recipients underwent LDLT employing our standard methods. In our protocol, the target trough level of tacrolimus was 10 to 15 ng/mL for the first 2 weeks, and then it was tapered and adjusted individually depending on each patient’s condition [
Liver specimens were fixed in 10% buffered formalin, processed routinely, and cut into 3
Polyclonal antibody against C4d complement (BI-RC4D; Biomedia, Vienna, Austria; dilution 1 : 50) was used for immunostaining with an automated immunostainer (BENCHMARK XT, Ventana Medical Systems, Tucson, AZ). For antigen retrieval, deparaffinized and rehydrated sections were treated with protease I (Ventana Medical Systems; 0.5 U/mL) at 37°C for 20 minutes [
Biopsy specimens in which only the vascular endothelium was stained were evaluated as endothelial positive. Biopsy specimens in which both the vascular endothelium and the stroma were stained were evaluated as endothelial and stromal positive (E&S positive). Any C4d staining on elastic fibers within the arteries and stroma was regarded as a nonspecific finding without clinical significance [
Pretransplant LCT was performed using both direct complement-dependent cytotoxicity (CDC) and CDC with added anti-human globulin (AHG-CDC) tests. Incubation was conducted using 1 milliliter of donor lymphocyte suspension and 5 milliliters of recipient serum in a Terasaki plate (Nunc, Roskilde, Denmark) at room temperature for 30 min. In the AHG-CDC test, AHG (Goat IgG k and l light chains) was added and incubated at room temperature for 3 min. Five microliters of rabbit complement were added to each well and the mixture was incubated at room temperature for 60 min. Two microliters of 5% eosin solution were added and the mixture was examined using phase-contrast microscopy (IMT-2; Olympus, Tokyo, Japan). The results were considered positive when more than 20% of the donor lymphocytes were killed by the recipient’s serum in either test. Dithiothreitol was not used for the inactivation of IgM antibodies.
Tissue typing was performed in patients and donors for HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ for class I and II loci using WAKFlow (Wakunaga Corp., Hiroshima, Japan) and Luminex xMAP technology (Luminex Corp., Austin, TX) [
Pretransplant sera were retrospectively analyzed for HLA antibodies employing a multiplexed microsphere-based suspension array from Luminex xMAP technology (Luminex Corp.). In brief, 5 microliters of LABScreen Mixed (One Lambda, Canoga Park, CA) color-coded microbeads coated with purified HLA were incubated in the dark for 30 min at 20°C to 25°C with 20 microliters of test serum. Any HLA antibodies present in the sera were bound to the LABScreen Mixed surface antigens coating the microbeads and were subsequently labeled with R-phycoerythrin-conjugated goat anti-human IgG. The microbead fluorescent emission of R-phycoerythrin was then detected and quantified using the LABScan 100 flow analyzer (One Lambda).
The determination of positive and negative sera was performed with One Lambda software (LABScreen PRA software, One Lambda) according to the manufacturer’s protocol. Sera reactivity was assessed based on the fluorescent signal for each HLA-coated microbead following correction for nonspecific binding to the negative control microbead. In the LABScreen Mixed assay, the normalized fluorescent signal is equal to the value of the antigen-coated microbead minus the value of the negative control microbead. If any one microbead in the mixed assay is positive, the result is considered positive.
The Single Antigen bead assay is essentially the same as the assay outlined earlier according to manufacturer’s protocol. In brief, 20 microliters of test serum were incubated with 5 microliters of the selected single beads and 5 microliters of LABScreen Singles control beads. Samples were read on the LABScan 100 flow analyzer (One Lambda). Raw trimmed MFIs were obtained from the output file generated by the flow analyzer and normalized.
A
The patient survival rate was 72% on postoperative day (POD) 60, 67% on POD 90, and 61% on POD 180 until 10 years (Figure
Patient survival curve.
Results of the CDC and AHG-CDC tests, the LABScreen Mixed assay (One Lambda), and LABScreen Single Antigen assay (One Lambda) are shown in Table
Results of lymphocyte crossmatch test, Mixed assay, and Single Antigen assay and histological findings and clinical outcomes in crossmatch-positive recipients.
Patient no. | LCT* | Mixed assay |
MFI$ of anti-Class I |
Mixed assay |
MFI$ of anti-Class II |
Histological findings of the first liver biopsy (POD) | C4d deposition | Outcome | Causes of death | Histological findings of the last liver biopsy in alive patients (POD) | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CDC† | AHG-CDC‡ | HLA-Ab class I | DSA⋀ | Non-DSA | HLA-Ab class II | DSA⋀ | Non-DSA | Alive/dead (POD) | |||||
1 |
|
|
|
High | High | − | Low | High | Cholangitis (40) | E# | Dead (118) | Sepsis | |
2 |
|
|
|
High | High |
|
High | High | Cholangitis (183) | E & S** | Alive (3479) | Chronic cholangitis, possible PBC‡‡ recurrence (2360) | |
3 |
|
|
|
High | High |
|
Negative | Negative | No biopsy |
|
Dead (28) | Hepatic artery rupture due to biliary leakage | |
4 |
|
|
|
High | High |
|
High | High | Cholestasis (18) | E & S | Dead (69) | Sepsis after repeated steroid pulse therapy for refractory ACR | |
5 |
|
|
|
High | High |
|
Low | Low | No biopsy | Dead (26) | Sepsis and fungal infection after steroid pulse therapy for refractory ACR | ||
6 |
|
|
|
High | High |
|
Negative | Negative | No biopsy |
|
Alive (1286) | No biopsy | |
7 |
|
|
|
High | High |
|
Negative | Negative | Lobular inflammation (3) | E & S | Dead (49) | Intra-abdominal hemorrhage, sepsis after steroid pulse therapy for severe ACR | |
8 |
|
|
|
High | High |
|
High | Low | Acute cholangitis (18) | E | Alive (1160) | Chronic cholangitis, HCV$$ recurrence (339) | |
9 |
|
|
|
High | High |
|
Negative | Negative | No biopsy |
|
Dead (72) | Sepsis of intestinal perforation | |
10 |
|
|
|
High | High |
|
Low | Low | No biopsy |
|
Dead (12) | Intra-abdominal hemorrhage and hepatic failure | |
11 |
|
|
|
High | High |
|
Negative | Negative | Steatosis (32) | E | Alive (1407) | Mild portal inflammation (1377) | |
12 |
|
|
|
Low | High |
|
Negative | Negative | Cholangitis (183) | E & S | Alive (2660) | Resolving cholangitis (2286) | |
13 |
|
|
|
Low | Low |
|
Negative | Negative | Steatosis (61) | E | Alive (777) | Steatohepatitis (613) | |
14 |
|
|
|
Negative | Low |
|
Negative | Negative | Cholangitis (28) | N | Alive (1885) | Mild portal inflammation (913) | |
15 |
|
|
|
Negative | Negative |
|
Negative | Negative | Cholestasis (20) | N | Alive (1490) | ACR (468) | |
16 |
|
|
|
Negative | Negative |
|
Negative | Negative | No biopsy |
|
Alive (1095) | No biopsy | |
17 |
|
|
|
Negative | Negative |
|
Negative | Negative | Severe ACR†† (8) | N | Alive (3318) | No remarkable findings (720) | |
18 |
|
|
|
Negative | Low |
|
Negative | Negative | Centrilobular hemorrhage and congestion (228) | N | Alive (2765) | Mild portal inflammation and fibrosis (2273) |
*Lymphocyte crossmatch test; †direct complement-dependent cytotoxicity; ‡anti-human globulin with added CDC; $mean fluorescence intensity (high, >10,000; low, <10,000); ⋀donor-specific antibody; #endothelial deposition; **endothelial and stromal deposition; ††acute cellular rejection; ‡‡primary biliary cirrhosis; $$hepatitis C.
Four patients showed both positive CDC and AHG-CDC tests, 10 patients showed negative CDC and positive AHG-CDC tests, and 4 patients showed positive CDC and negative AHG-CDC tests. Based on the Mixed assay, 15 patients had anti-HLA Class I antibodies. Based on the Single Antigen assay, DSA was detected in 13 out of the 18 patients and non-DSA was detected in 15 patients. When a patient showed positive DSA or non-DSA against more than 2 HLAs of donor or non-donor, the highest value was selected as “the peak value.” The MFI peak value in each patient ranged from 571 to 20,259 (median, 15,864) in 13 DSA-positive patients and 901 to 20,576 (median, 14,399) in 15 non-DSA-positive patients. Based on receiver operating characteristic (ROC) curve, the point with the best sensitivity and specificity for mortality was 12,211 in patient 4. The area under the curve was 0.792. The next small value was 8,272 in patient 12. The 18 patients were divided into 3 groups: high (MFI > 10,000;
Among 7 patients who had anti-HLA Class II antibodies based on the Mixed assay, DSA was positive in 6 patients based on the Single Antigen assay. All 6 patients presented positive Class I DSA. The MFI peak value in each patient ranged from 2,793 to 18,760 (median, 8,776) in 6 DSA-positive patients.
Regarding the relationship between the LCT and the Single Antigen assay, all 4 patients with positive CDC and AHG-CDC tests had DSA with high MFI. Ten patients with negative CDC and positive AHG-CDC tests consisted of patients from the 3 groups (7 high MFI, 2 low MFI, and 1 negative DSA). Four patients with positive CDC and negative AHG-CDC tests showed negative DSA on the Single Antigen assay.
Regarding the relationship between high DSA or high non-DSA and possible backgrounds, there was no significant relationship between history of blood transfusion and high DSA (
The MFI of DSA, the histological findings of the first liver biopsy after transplantation, and the clinical outcomes of the 18 patients are shown in Table
Eight of 12 initial biopsy specimens showed positive C4d staining: stromal and endothelial deposition in 4 patients and endothelial deposition in 4 patients. All 4 cases with endothelial C4d staining only showed focal staining (portal C4d immunolabeling of fewer than 50% of portal tracts). All 4 cases with endothelial and stromal C4d staining showed diffuse staining pattern (C4d deposition in the hepatic artery, portal vein, or capillary endothelium of more than 50% of portal tracts). There were cases showing sinusoidal C4d staining. Three of the 4 patients with stromal and endothelial deposition (75%) and 3 of the 4 patients with endothelial deposition (75%) showed positive DSA with high MFI. All C4d-negative patients showed negative DSA on the Single Antigen assay. A significant correlation between MFI strength and C4d deposition was found on univariate analysis (
Seven patients died within 4 months after transplantation. The causes of death were sepsis in 5 and vascular complications in 2.
All of the 7 patients who died early had DSA with high MFI prior to LDLT. The risk factors for mortality were analyzed and a high level of Class-I DSA was found to be a significant risk factor (Fisher exact test,
Risk factors for mortality.
Characteristics | Number | Mortality | |
---|---|---|---|
% |
|
||
Age | 0.245 | ||
<18 years old | 3 | 0 | |
≥18 years old | 15 | 47 | |
History of pregnancy | 0.367 | ||
No | 8 | 25 | |
Yes | 10 | 50 | |
History of blood transfusion | 0.093 | ||
No | 5 | 0 | |
Yes | 11 | 55 | |
Unknown | 2 | — | |
History of upper abdominal surgery | 0.335 | ||
No | 9 | 22 | |
Yes | 9 | 56 | |
Donor selection | 0.335 | ||
Husband-son-daughter | 9 | 56 | |
Others | 9 | 22 | |
Graft type | 0.358 | ||
Right lobe | 10 | 50 | |
Left lobe | 5 | 40 | |
Lateral segment | 3 | 0 | |
GRWR† | 0.245 | ||
>0.8 | 15 | 47 | |
<0.8 | 3 | 0 | |
CDC‡ | 1.000 | ||
Negative | 10 | 40 | |
Positive | 8 | 38 | |
AHG-CDC$ | 0.119 | ||
Negative | 4 | 0 | |
Positive | 14 | 50 | |
Mix assay Class I | 0.245 | ||
Negative | 3 | 0 | |
Positive | 15 | 47 | |
FI of anti-Class I-DSA⋀ | 0.015 | ||
Negative | 5 | 0 | |
Low | 2 | 0 | |
High | 11 | 64 | |
FI of anti-Class I-non-DSA | 0.110 | ||
Negative | 3 | 0 | |
Low | 3 | 0 | |
High | 12 | 58 | |
Mix assay Class I | 0.205 | ||
Negative | 11 | 27 | |
Positive | 7 | 39 | |
FI of anti-Class I-DSA⋀ | 0.057 | ||
Negative | 12 | 25 | |
Low | 3 | 100 | |
High | 3 | 33 | |
FI of anti-Class I-non-DSA | |||
Negative | |||
Low | |||
High | |||
C4d staining | 0.709 | ||
Negative | 4 | 0 | |
Endothelial | 4 | 25 | |
Endothelial and stromal | 4 | 50 |
*Fisher exact test; †graft recipient weight ratio; ‡compliment-dependent cytotoxicity; $anti-human globulin with added compliment-dependent cytotoxicity; ⋀fluorescence intensity of single antigen assay for anti-Class I donor-specific antibody (high, >10,000; low, <10,000).
Eleven patients are alive and the follow-up period ranged from 777 to 3,479 days. All but one showed normal hepatic chemistries and their performance status was 0. Case 2 showed an AST of 21 U/L, an ALP of 2,140 U/L, and a total bilirubin level of 4.4 mg/dL, and her performance status was 2 at 3,479 days after transplantation. Histological findings from the last liver biopsy specimens on postoperative days (PODs) 339 to 2,360 of 9 out of 11 alive patients are shown in Table
To the best of our knowledge, this is the first study to clarify the relationship between LCT and Single Antigen assay in liver transplantation with the following important findings. First, both the positive CDC and AHG-CDC tests indicated a high level of DSA against HLA. Second, the positive AHG-CDC test indicated the presence of DSA against HLA. Third, the positive CDC test with negative AHG-CDC test indicated contribution of IgM-DSA. Based on these findings, we deduced that the AHG-CDC test is not sufficiently sensitive to predict postoperative mortality but could indicate the presence of a high level of DSA against Class I.
Castillo-Rama reported the significance of the Mixed assay in liver transplantation [
The incidence of preformed DSA is approximately 10% in deceased donor liver transplantation in Western counties [
All DSA-positive patients showed positive non-DSA. It can be considered that positive DSA is part of the phenomena of sensitization against HLA including donor-specific antigens. However, in this study, high DSA positivity was found to be a significant risk factor for mortality. Blood transfusion is theoretically the most important contributing factor for sensitization. However, blood transfusion was found to be independent of the high positivity of DSA and non-DSA. Therefore, we analyzed the combined effect of high DSA or high non-DSA and history of blood transfusion. A significant difference in the incidence of mortality between positive history of blood transfusion and high DSA (
Based on these results, we changed our policy of donor selection. A donor candidate to whom a recipient is highly sensitized with MFI > 10,000 is rejected. A donor to whom a recipient is sensitized with MFI < 10,000 is not considered when other donors are available; however, such a donor can be accepted after B cell desensitization using our protocol for ABO incompatible transplantation, which involves administration of rituximab, plasma exchange, and intravenous immunoglobulin. During 1 year from December 2009, 100 patients were evaluated regarding their LDLT and 12 patients were found to be positive for anti-class I antibodies. Five of them had DSA against donor candidates. Only 1 patient was highly sensitized and another family member to whom the patient had no DSA donated the graft. Another patient could change the donor, but the remaining 3 could not. All 5 patients survived after the transplantation.
A limitation of this study is that it did not reveal the relationship between the specific HLA and the clinical outcome in recipients with preformed DSA. Although the significance of MFI generated by the Luminex analyzer for the DSA assay has not yet been established, this study showed that DSA-MFI > 10,000 had a significant effect on the clinical outcome and a significant relationship with LCT. Further studies to clarify the meaning of low MFI and postoperative changes in DSA using the Single Antigen assay are required.
Donor-specific antibody
Living donor liver transplantation
Human leukocyte antigens
Lymphocyte cross-match test
Direct complement-dependent cytotoxicity
Anti-human globulin
Mean fluorescence intensity.
Atsushi Yoshizawa and Hiroto Egawa wrote the paper. Hiroto Egawa organized the study. Aya Miyagawa-Hayashino and Hironori Haga performed the pathological analysis. Kimiko Yurugi, Rie Hishida, Hiroaki Tsuji, Eishi Ashihara, and Taira Maekawa conducted the HLA analysis, LCT, and Luminex assays. Satoshi Teramukai carried out the statistical analysis. Shinji Uemoto supervised the study.
All authors of this paper reported no biomedical financial interests or potential conflict of interests.