A Questionnaire Survey of HLA Crossmatch Tests in Korea (2015)

BACKGROUND: We carried out a questionnaire survey for laboratories performing human leukocyte antigen-crossmatch (HLA-XM) to provide a basis for laboratory standardization of HLA-XM tests in Korea. METHODS: The questionnaires were distributed to 51 HLA laboratories participating in the HLA-XM part of the HLA proficiency survey program organized by the Korean Society for Laboratory Medicine and replies from 50 laboratories were analyzed. The questionnaires included following items: 1) HLA-XM methods performed and annual number of tests, 2) types of the specimen and lymphocyte separation methods, 3) test procedures and reagents for complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FCXM). RESULTS: The number of laboratories performing anti-human globulin (AHG) CDC-XM (47/49, 96%) and FCXM (30/50, 60%) was considerably increased compared to the 2005 survey (AHG CDC-XM, 35/43, 81%; FCXM, 7/44, 16%). As for the annual number of XM tests, more than 50% of the laboratories were low volume laboratories performing ≤50 tests, and only 10% of the laboratories were performing >500 tests. For cell isolation methods, negative selection was used by 43% (21/49) of laboratories performing CDC-XM. Number of cells reacted per 1 µL of serum varied among different laboratories in both CDC-XM (1,000–8,000) and FCXM tests (1,300-20,000). For the interpretation of FCXM, log fluorescence ratio (26/30, 87%) was more commonly used than channel shift values (5/30, 17%). CONCLUSIONS: Considerable variation is noted in both CDC-XM and FCXM methods performed by different laboratories. A continuous effort for laboratory standardization is needed to reduce inter-laboratory variation in the HLA-XM test results.

False-Positive T-Cell Cytotoxicity Crossmatch Results Due to Autoantibodies in Korean Network for Organ Sharing Crossmatch Tests / 대한이식학회지

BACKGROUND: Basic National Institute of Health (NIH) and sensitive antihuman globulin (AHG) methods are widely used for T-cell complement-dependent cytotoxicity crossmatch (XM) tests. Whereas NIH-negative, AHG-positive (NIH⁻/AHG⁺) results are caused by weak antibodies, NIH⁺/AHG⁻ results are usually due to autoantibodies. We found that solid organ transplantation candidates with NIH⁺/AHG⁻ XM results are repeatedly excluded from allocation of deceased donor organs by the Korean Network for Organ Sharing (KONOS) allocation system. Here, we attempted to demonstrate that these patients do not have donor-specific HLA antibodies (DSAs). METHODS: Sera showing NIH⁺/AHG⁻ results in the analysis of 1,668 KONOS T-cell XM tests were screened for panel reactive antibody (PRA) using a Luminex test. For screen-positive samples, antibody identification was conducted using a Luminex single antigen assay and the presence or absence of class I DSAs was determined. For positive controls, 42 KONOS XM tests showing probable true-positive (NIH⁻/AHG⁺ or NIH⁺/AHG⁺) results were reviewed for PRA results based on electronic medical records and the presence or absence of DSAs was determined. RESULTS: NIH⁺/AHG⁻ results were observed in 1.3% (21/1,668) of KONOS XM tests analyzed. Most of these (18/21, 85.7%) were negative for PRA or DSAs. All probable true-positive cases were either positive for DSAs (24/42, 57.1%) or had high PRA (mean, 92% [range; 42%~100%]), complicating accurate identification of antibody specificities. CONCLUSIONS: NIH⁺/AHG⁻ results are not rare (1.3%) in KONOS XM tests. Most of these results are not due to DSAs, and these patients should not be excluded from organ allocation.

A Family with Axenfeld-Rieger Syndrome: Report of the Clinical and Genetic Findings

PURPOSE: To describe clinical findings in a Korean family with Axenfeld-Rieger syndrome. METHODS: A retrospective review of clinical data about patients with diagnosed Axenfeld-Rieger syndrome. Five affected members of the family underwent a complete ophthalmologic examination. We screened the forkhead box C1 gene and the pituitary homeobox 2 gene in patients. Peripheral blood leukocytes and buccal mucosal epithelial cells were obtained from seven members of a family with Axenfeld-Rieger syndrome. DNA was extracted and amplified by polymerase chain reaction, followed by direct sequencing. RESULTS: The affected members showed iris hypoplasia, iridocorneal adhesions, posterior embryotoxon, and advanced glaucoma in three generation. None had systemic anomalies. Two mutations including c.1362_1364insCGG and c.1142_1144insGGC were identified in forkhead box C1 in four affected family members. CONCLUSIONS: This study may help to understand clinical findings and prognosis for patients with Axenfeld-Rieger syndrome.

Long-Term Outcomes and Predictive Factors for Success of Selective Laser Trabeculoplasty

PURPOSE: To investigate long-term outcomes and predictive success factors for selective laser trabeculoplasty (SLT). METHODS: This retrospective chart review included 66 eyes of 66 glaucoma or ocular hypertension patients who underwent SLT between 2004 and 2011. All patients were followed-up for at least 2 years after the procedure. The intraocular pressure (IOP) was measured before the treatment, at postoperative 1 week, 1 month and every 3 months thereafter. Success was defined as an IOP decrease > or = 3 mm Hg or > or = 20% of the pretreatment IOP. The success rate was determined based on the Kaplan-Meier survival analysis. The predictive success factors were analyzed using the Cox proportional hazard model. RESULTS: The mean follow-up period was 4.78 +/- 1.98 (2-8) years. The mean pretreatment IOP was 23.79 +/- 2.83 mm Hg. The mean IOP reduction was 6.64 +/- 3.21 mm Hg. The success rates were 80%, 75%, 69%, 63%, 50%, 45%, and 42% after 1, 2, 3, 4, 5, 6, and 7 years, respectively. Only high pretreatment IOP was significantly correlated with success (p = 0.044). CONCLUSIONS: SLT was an effective tool for lowering IOP in glaucoma patients. Higher pretreatment IOP was a predictive success factor.

Preoperative Selective Desensitization of Live Donor Liver Transplant Recipients Considering the Degree of T Lymphocyte Cross-Match Titer, Model for End-Stage Liver Disease Score, and Graft Liver Volume

Several studies have suggested that a positive lymphocyte cross-matching (XM) is associated with low graft survival rates and a high prevalence of acute rejection after adult living donor liver transplantations (ALDLTs) using a small-for-size graft. However, there is still no consensus on preoperative desensitization. We adopted the desensitization protocol from ABO-incompatible LDLT. We performed desensitization for the selected patients according to the degree of T lymphocyte cross-match titer, model for end-stage liver disease (MELD) score, and graft liver volume. We retrospectively evaluated 230 consecutive ALDLT recipients for 5 yr. Eleven recipients (4.8%) showed a positive XM. Among them, five patients with the high titer (> 1:16) by antihuman globulin-augmented method (T-AHG) and one with a low titer but a high MELD score of 36 were selected for desensitization: rituximab injection and plasmapheresis before the transplantation. There were no major side effects of desensitization. Four of the patients showed successful depletion of the T-AHG titer. There was no mortality and hyperacute rejection in lymphocyte XM-positive patients, showing no significant difference in survival outcome between two groups (P=1.000). In conclusion, this desensitization protocol for the selected recipients considering the degree of T lymphocyte cross-match titer, MELD score, and graft liver volume is feasible and safe.

Comparison of 3 Automated Immunoassays for Detection of Anti-Hepatitis A Virus Immunoglobulin M in a Tertiary Care Hospital

Three automated immunoassay kits for anti-Hepatitis A Virus (HAV) IgM-Architect, (Abbott Laboratories, USA), Elecsys (Roche Diagnostics, Germany), and ADVIA Centaur (Siemens Healthcare Diagnostics Inc., USA)-were compared. We included 178 consecutive samples, for which an anti-HAV IgM test was requested at Seoul National University Hospital from September 2009 to January 2010. Reviewing of medical records, reverse transcription (RT)-PCR for HAV RNA, or total anti-HAV assay were performed on 16 (9.0%) samples with discrepant results. The percent agreements (kappas) of the Architect and ADVIA Centaur, Architect and Elecsys, and ADVIA Centaur and Elecsys kits were 96.6% (0.91), 96.6% (0.92), and 97.8% (0.94), respectively. Eight out of 16 discrepant samples showed gray-zone values in Architect but were nonreactive in the others. Slightly earlier seroconversion was suspected in Elecsys. The 3 assays showed comparable performances with excellent agreements in a tertiary care hospital setting.

Comparison of Icare Rebound Tonometer with Goldmann Applanation Tonometry

PURPOSE: To compare Icare rebound tonometer (IRT) and Goldmann applanation tonometer (GAT) and investigate the clinical usefulness of IRT. METHODS: In a retrospective study of 131 eyes with glaucoma and glaucoma suspect, intraocular pressure (IOP) was measured with IRT and GAT. The correlation between IRT and GAT and the influence of refractive error (spherical equivalent; SE) and central corneal thickness (CCT) were analyzed. RESULTS: A distinct correlation between IRT and GAT was found. IOP measured with GAT was 2.23 +/- 3.16 mm Hg higher than with IRT (p or = -5 diopter) was measured relatively lower than in hyperopic eyes (SE < -5 diopter) with GAT (+1.50 +/- 0.68 mm Hg) and IRT (+1.88 +/- 0.75 mm Hg). CONCLUSIONS: IRT showed strong correlation with GAT, but IOP measured approximately 2 mm Hg lower. The IOP tends to measure lower at low CCT and in myopic eyes.

Analysis of Eye Drops Applying Behavior in Glaucoma Patients

PURPOSE: This study was conducted to understand the eye drop application method in patients with glaucoma. METHODS: A survey was performed in 5 hospitals on patients diagnosed with glaucoma using eye drops for more than 1 month. Variables associated with eye drop application such as pressing on a dacryocyst after application, time interval between multiple eye drop medications, hygienic management of an eye dropper and shaking a bottle of eye drops before use were evaluated. RESULTS: A total of 581 patients were surveyed in the present study. Regarding pressing on a dacryocyst after applying eye drops, 58 (10%) patients responded always, 103 patients (18%) sometimes, and 409 patients (70%) never. Three hundred fifteen patients (54%) did not touch the eye dropper to their eyes, 190 patients (33%) always shook the eye drop bottle before use and 106 patients (18%) shook the bottle sometimes. CONCLUSIONS: When using eye drops for glaucoma to reduce the intraocular pressure, most patients are committing errors, reducing the effect of treatment and likely causing side effects. When prescribing eye drops to glaucoma patients, the physician should educate patients on the proper method of application.

Analysis of Peripapillary Atrophy According to the Optic Disc Shape Using Spectral Domain OCT

PURPOSE: To analyze the structural changes in the beta-zone of peripapillary atrophy (PPA-beta) using cross-sectional image of the optic disc head from spectral-domain optical coherence tomography (SD-OCT) according to the optic disc shape. METHODS: One hundred thirty-seven eyes in 137 patients with glaucoma having PPA-beta and 31 normal eyes (control group) were evaluated retrospectively. Cross-sectional images of the optic disc were taken using the Cirrus HD-OCT. We classified optic disc patterns into normal, focal, myopic, generalized enlargement and senile sclerotic appearance types and analyzed the shape of Bruch's membrane (BM), composition of retinal layer and retinal slope according to the optic disc shape. RESULTS: Among the 137 eyes with glaucoma, 54 eyes were focal disc type, 34 eyes were myopic disc type, 28 eyes were generalized enlargement disc type and 21 eyes were senile sclerotic disc type. The myopic disc group showed a noticeable difference compared to the other groups in terms of a higher percentage of BM defect type, the lowest retinal slope (70.6 +/- 12.0degrees) and the earlier termination of retinal layers. The generalized enlargement disc group showed the highest percentage of curved BM type. Retinal slope angle increased with age and decreased with axial length. CONCLUSIONS: In the beta-zone of peripapillary atrophy, there were several differences in the shape of Bruch's membrane, composition of retinal layers and the retinal slope according to the optic disc shape.

Influence of Phacoemulsification on Progression of Glaucoma and Analysis of Related Factors in Glaucoma Patients

PURPOSE: To evaluate the factors affecting glaucoma progression after cataract surgery with phacoemulsification. METHODS: The medical charts of 109 eyes of 68 patients with glaucoma who had phacoemulsification were retrospectively reviewed. The course of glaucoma was followed up for at least 2 years. Age, sex, diabetes, hypertension, preoperative mean deviation, phacoemulsification time, cataract type, refractive error, preoperative and postoperative visual acuity, preoperative and postoperative intraocular pressure, axial length, and central corneal thickness between the progression group and the non-progression group were compared. RESULTS: Among 109 eyes with glaucoma, 19 (17.4%) eyes were classified into the glaucoma progression group. In the multivariate analysis, age (p = 0.026), preoperative mean deviation (p < 0.001), and phacoemulsification time (p < 0.001) were statistically associated with glaucoma progression. CONCLUSIONS: For patients with glaucoma, phacoemulsification may result in its progression. Special attention should be given to patients with the above risk factors.

A case of transfusion-related acute lung injury induced by anti-human leukocyte antigen antibodies in acute leukemia / 대한혈액학회지

Transfusion-related acute lung injury (TRALI) is a noncardiogenic pulmonary edema that occurs during or within 6 hours after transfusion. Risk factors for TRALI, which is relatively common in critically ill patients, include recent surgery, hematologic malignancy, and sepsis. Here, we report a case of TRALI induced by anti-human leukocyte antigen (anti-HLA) class II antibodies (HLA-DR) occurring after transfusion of platelet concentrates in a patient with acute leukemia. Although most patients with TRALI show improvement within 48-96 hours, our patient's condition rapidly worsened, and he did not respond to supportive treatment. TRALI is a relatively common and serious adverse transfusion reaction that requires prompt diagnosis and management.

Circulating Total Glutathione in Normal Tension Glaucoma Patients: Comparison with Normal Control Subjects

PURPOSE: Oxidative stress plays a critical role in the pathogenesis of glaucoma. Glutathione is a major antioxidant molecule present in intracellular or extracellular space. Herein, we aimed to examine circulating glutathione level in normal tension glaucoma (NTG), which comprises the largest proportion of glaucoma disease in the Korean population. METHODS: Nineteen NTG patients (NTG group) and 30 age- and gender-matched normal control subjects (control group) were included. Antecubital venous puncture was performed between 8 and 10 o'clock in the morning to obtain a 4 mL venous blood sample. Total glutathione level was measured by the spectrophotometric method at 412 nm. Correlation of total glutathione level with mean deviation and pattern standard deviation from the Humphrey visual field test was analyzed in the NTG group. RESULTS: Total glutathione level in circulating blood was 524.02 +/- 231.09 nmol and 586.06 +/- 156.08 nmol in the NTG group and the control group, respectively. The difference between these values was not statistically significant (p = 0.121, F = 2.212). Age had no significant effect on circulating total glutathione level in either the NTG group (p = 0.171, r = -0.328) or the control group (p = 0.380, r = -0.166). In the NTG group, circulating total glutathione level had no significant relationship with mean deviation (p = 0.226, F = 1.636) and pattern standard deviation (p = 0.200, F = 1.766) after correcting for age and gender. CONCLUSIONS: In NTG patients, circulating total glutathione levels were not different compared to those of normal subjects.

Evaluation of Flow Cytometric Crossmatch Results in Comparison with Donor-specific Antibodies Detected by Luminex-PRA Tests in Organ Transplantation Patients / 대한이식학회지

BACKGROUND: Two of the most sensitive methods for detecting donor-specific HLA antibodies (DSAs) are solid phase panel reactive antibody (PRA) assay using Luminex platform (Luminex-PRA), and a cell-based flow cytometric crossmatch (FCXM) test. We evaluated FCXM results in relation to DSAs detected by the Luminex-PRA method in solid organ transplantation candidates or post-transplant follow-up patients. METHODS: A total of 171 donor-recipient pairs were evaluated by Luminex-PRA (LIFECODES Class I and Class II ID kits; Gen-Probe, USA) and FCXM (T- and B-cells) tests. DSA levels were analyzed using a sum of median fluorescence intensity (MFI) values, and FCXM results were analyzed using MFI ratios. RESULTS: Class I and II DSAs were detected in 11.7% (20/171) and 11.1% (19/171) of tested sera, respectively. T-FCXM was negative in 97.4% (147/151) of Class I DSA negative sera, and B-FCXM was negative in 99.3% (137/138) of Class I and II DSA negative sera. T-FCXM was positive in 91.7% (11/12) of sera with moderate to strong Class I DSAs and B-FCXM was positive in 88.9% (16/18) of sera with moderate to strong Class II and/or Class I DSAs in the evaluation of sensitivities of FCXM in relation to DSA. There were significant correlations between FCXM ratios and DSA levels for both T-FCXM (P=0.008) and B-FCXM (P97%) and the sensitivities of T- and B-FCXM were satisfactory (>88%) in detecting moderate to strong DSAs.

Clinical Relevance of Pretransplant HLA Class II Donor-specific Antibodies in Renal Transplantation Patients with Negative T-cell Cytotoxicity Crossmatches

BACKGROUND: We evaluated the clinical relevance of pretransplant donor-specific HLA antibodies (DSA) in renal transplantation patients who had negative T-cell cytotoxicity crossmatches. METHODS: From 328 consecutive renal transplant recipients, we selected 28 patients who had positive pretransplant (historical or at the time of transplantation) flow cytometry crossmatches, but negative T-cell cytotoxicity crossmatches at the time of transplantation. The presence of DSA and its level at the time of transplantation were retrospectively tested using Luminex single antigen assays. RESULTS: DSA was present in 16 (57.1%) of 28 patients. Biopsy-proven acute rejection (9 patients) occurred more frequently in patients with DSA than in those without DSA (56.3% vs. 0.0%; P=0.003). The positivity rate of class II DSA was significantly higher in patients with antibody-mediated rejection (AMR) than in those without AMR (100% vs. 21.7%; P=0.003). However, the positivity rate of class I DSA was not different between the two groups (40% vs. 40.9%). Among patients with class II DSA, those with AMR tended to have higher antibody levels (median fluorescence intensity, MFI) than those without AMR (16,359 vs. 5,910; P=0.056). A cut-off MFI value of 4,487 for class II DSA predicted the occurrence of AMR with good sensitivity and specificity (100% and 87.0%). CONCLUSIONS: In patients with negative T-cell cytotoxicity crossmatches, the presence of class II DSA and its level at the time of transplantation were associated with the occurrence of AMR. Pretransplant DSA measurement with Luminex single antigen assay would be useful in renal transplantation.

The HLA-DRB1 Polymorphism is Associated With Atopic Dermatitis, but not Egg Allergy in Korean Children

PURPOSE: We investigated whether particular HLA-DRB1 polymorphisms contribute to egg allergy development in Korean children with atopic dermatitis (AD). METHODS: HLA-DRB1 alleles were determined by PCR-sequence-specific oligonucleotide (SSO) and PCR-single-strand conformation polymorphism (SSCP) methods in 185 patients with AD and 109 normal control (NC) subjects. AD patients were divided into two groups: 1) AD with egg allergy, consisting of 96 patients with egg allergies as determined by egg-specific immunoglobulin E (IgE) reactivity; and 2) AD without egg allergy, consisting of 89 patients without egg allergies. HLA-DRB1 alleles were classified into functional groups (A, De, Dr, E, Q, R, a). HLA-DRB1 phenotype and functional group frequencies in the AD, AD with egg allergy, and AD without egg allergy groups were compared with those in the NC group. RESULTS: The frequency of DRB1*08:02 was decreased in the AD with egg allergy group compared with the AD without egg allergy group (2.1% vs. 10.1%, P=0.021), and DRB1*15:01 was increased in the AD with egg allergy group compared with the AD without egg allergy group (22.9% vs. 11.2%, P=0.036). However, significance was lost after Bonferroni correction. HLA-DRB1*11:01 had a significantly higher frequency in AD patients compared with NCs (12.4% vs. 1.8%, corrected P=0.048) and was regarded as a susceptibility factor associated with AD. DRB1*08:03 was decreased in AD patients compared with NCs (10.8% vs. 19.3%, P=0.043). HLA-DRB1 functional group 'a', which includes DRB1*15:01, seemed to be associated with the development of egg allergy in AD (P=0.033), but this result was not significant after Bonferroni correction. CONCLUSIONS: HLA-DRB1 polymorphism is not associated with egg allergy, but HLA-DRB1*11:01 is associated with AD in Korean children.

Comparison of Measured Intraocular Pressure Change According to the Methods of Corneal Refractive Surgery

PURPOSE: To evaluate differences in intraocular pressure change after three different methods of corneal refractive surgery. METHODS: The medical records of 296 eyes of 150 patients who underwent corneal refractive surgery were reviewed. Spherical equivalent, central corneal thickness (CCT), and intraocular pressure before surgery, and one month, three months and six months after surgery were analyzed. RESULTS: The patients included those having undergone laser-assisted in situ keratomileusis (LASIK; 96 eyes), IntraLASIK (98 eyes), laser assisted sub-epithelial keratomileusis (LASEK; 102 eyes). Post operative intraocular pressure in ablated corneal depth and in CCT showed a meaningful correlation. Intraocular pressure decreased significantly after refractive surgery; however, there were no differences among the three groups. CONCLUSIONS: Post operative intraocular pressure after corneal refractive surgery is influenced by CCT. There were no differences in intraocular pressure change among the three groups.

False Homozygosity Results in HLA Genotyping due to Loss of Chromosome 6 in a Patient with Acute Lymphoblastic Leukemia / 대한진단검사의학회지

Loss of heterozygosity (LOH) in chromosome 6p has been reported in a number of tumors and some hematologic malignancies, including ALL. LOH in chromosome 6p, on which the HLA genes are located, can give rise to false homozygosity results in HLA genotyping of patients with hematologic malignancies. Here we report false homozygosity results in HLA genotyping due to the loss of whole chromosome 6 in the neoplastic cells of a patient with ALL. A 33-yr-old Korean female patient was admitted for the evaluation of leukocytosis detected during a workup for headache. Her initial white blood cell count was 336.9x109/L with 84% of blasts in the differential count. Precursor-B lymphoblastic leukemia was diagnosed from a subsequent bone marrow study. HLA high-resolution genotyping of the patient was requested at the time of diagnosis for possible hematopoietic stem cell transplantation. Homozygosity results (A*02:01, B*54:01, C*08:01, DQB1*04:01) were obtained, except for the DRB1 locus (DRB1*04:05, DRB1*11:01), in sequence-based typing. Conventional karyotyping of bone marrow metaphase cells revealed chromosomal abnormalities, with loss of multiple chromosomes including chromosome 6, and reduplication of the remaining chromosomes: 29,X,+X,+8,inv(9)(p11q13),+10,+14,+18,+21[15]/58,idemX2[3]/46,XX,inv(9)[2]. LOH at the HLA region was suspected and HLA genotyping was repeated with the peripheral blood in remission state after induction chemotherapy. All 5 HLA loci were typed as heterozygous (A*02:01, A*02:06, B*40:01, B*54:01, C*03:04, C*08:01, DRB1*04:05, DRB1*11:01, DQB1*03:01, DQB1*04:01). To avoid false HLA typing results in patients with hematologic malignancies, clinicians, as well as laboratory personnel, need to be aware of such problems and take appropriate precautions.

Erratum to "Assessment of Deceased Donor Kidneys Using a Donor Scoring System" by Bang K, et al. (Yonsei Med J 2010;51:870-876)

No abstract available.

Erratum to "Assessment of Deceased Donor Kidneys Using a Donor Scoring System" by Bang K, et al. (Yonsei Med J 2010;51:870-876)

No abstract available.

Glaucoma Filtering Surgery With Low Concentration of Cyclosporin A in Rabbits: A Pilot Study

PURPOSE: The effect of 0.2% cyclosporin A (CsA) as an adjuvant therapy after glaucoma-filtering surgery was the focus of this study. METHODS: A posterior lip sclerotomy was performed in 16 eyes of 8 rabbits, and 0.2% CsA was administered into the right eyes. The left eyes served as controls. The intraocular pressure (IOP) was measured 1, 3, 5, 7, 14, and 28 days after surgery. Hematoxylin-eosin (HE) and anti-bromodeoxyuridine (BrdU) immunocytochemical staining were performed at 1, 2, 4, and 8 weeks. RESULTS: The IOP at 7 and 14 days after surgery was lower in the 0.2% CsA group and statistically significant (P=0.047, P=0.48; respectively). HE staining did not show any difference between experimental and control eyes, but anti-BrdU staining showed a lower number of positive cells in the experimental eyes at 1 week. The fibroblast proliferation rate was significantly lower 1 week after surgery in the 0.2% CsA group (P=0.003). CONCLUSIONS: An effect of 0.2% CsA on early wound healing was observed. The data suggest that a low concentration of CsA can be useful when employed as adjuvant therapy in glaucoma filtering surgery.