[Challenges and solutions in current human leukocyte antigen confirmatory typing of hematopoietic stem cell transplantation].

The impact of human leukocyte antigen (HLA) on hematopoietic stem cell transplantation (HSCT) necessitates high precision in HLA genotyping. Confirmatory typing for patients and their related or unrelated donors before HSCT is critical. This study seeks to standardize HLA confirmatory typing in laboratories by examining the current state of HLA genotyping in the country, building upon the National Standards and Industrial Standards for HLA, and highlighting the significance of confirmatory typing for patients and potential donors prior to HSCT. A retrospective analysis over a decade reveals initial typing errors, indicating potential issues and critical considerations in pre-analytical, analytical, and post-analytical stages. Problems are attributed to three main causes: (1) random human errors, including technical mistakes, sample mix-up, and transcription inaccuracies; (2) limitations of technical methods, such as the varied sequence ranges between confirmatory and initial typing; (3) patient factors, involving high tumor burden, the influence of certain drugs on HLA genotyping results, and the second transplantation. Solutions are proposed for these problems, along with recommendations to standardize HLA confirmatory typing.

[Clinical significance of HLA-A, -B, -C, -DRB1, -DQB1 haplotype gene frequencies].

Objective: To analyze family-based haplotype frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 genes and their clinical significance. Methods: The data of HLA genotyping in 3568 families undergoing related haploidentical transplantation between 2012 and 2017 at the First Affiliated Hospital of Soochow University were retrospectively evaluated. The HLA genotyping was performed by PCR amplification with sequence-based typing (PCR-SBT) and sequence-specific oligonucleotide probe (PCR-SSOP) methods. The family genetic analysis and haplotype frequencies were also investigated. Results: All the families were divided into 3 groups, including group1 of 1 422 entire families; group2 of 1 310 patients and either of their parents or one of their children; group3 of 836 patients and their HLA≥5/10 matched sibling donors. In the haplotypes with frequencies greater than 0.1% in group1+ group2, the frequency of A*11∶01-B*40∶01-C*03∶04-DRB1*11∶01-DQB1*03∶01, A*02∶07-B*51∶01-C*14∶02-DRB1*09:01-DQB1*03∶03 were significantly different between group1 and group2 (P=0.029, 0.033) . The frequency of A*11∶01-B*46∶01-C*01∶02∶01G-DRB1*09∶01-DQB1*03∶03 was significantly different between group1 and group3 (P=0.035) . The frequency of A*02∶01-B*40∶01-C*07∶02-DRB1*09∶01-DQB1*03∶03 was significantly different between group1 and group2 (P=0.034) , or group1 and group3 (P=0.034) . The frequency of A*24∶02-B*13∶01-C*03∶04-DRB1*12∶02-DQB1*03:01 was significantly different between group2 and group3 (P=0.046) . Conclusion: In this study, we summarize the prevalence of haplotype frequencies in terms of HLA-A, -B, -C, -DRB1 and-DQB1. Based on the database of family haplotype analysis, patients and donor candidates are sorted with matched HLA genotype while unmatched HLA haplotype. Even in patients without entire family information, HLA haplotype analysis assists in choosing the optimal related or unrelated donors.

[Immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation].

Objective: To investigate the immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation (allo-HSCT) with KIR-AA genotype. Method: 75 donor-recipient pairs were performed by KIR genotying using PCR-SSP, and all donors were identified with KIR-AA genotype. Dynamic detections (including unrelated-donor on the day of transplantation and the recipient each month post allo-HSCT) of the expression of KIR2DL1/3DL1 on NK cell and mRNA level were performed in 291 cases using flow cytometry (FCM) and real-time fluorescent quantitation PCR (RT-qPCR) . Result: ①The median expression of KIR2DL1 in unrelated-donor on transplant's day was 21.60%, the median expression of KIR2DL1 in recipient 1M, 2M, 3M and 3-6M after transplantation were 7.40%, 12.00%, 16.92%, 17.64% respectively. The median expression of KIR2DL1 in unrelated-donor on transplant's day was 265.14 copies/10 000abl copies, the median expression of KIR2DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 332.17, 438.31, 723.25, 414.17, 180.76 and 234.67 copies/10 000abl copies respectively. The median expression of KIR2DL1 on NK cells and mRNA level gradually increased at all time points after transplantation, and reached the highest expression at 3 months after transplantation. But mRNA expression levels increased earlier than NK cell membrane proteins. ②The median expression of KIR3DL1 in unrelated-donors on transplant's day was 18.56%, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M after transplantation were 23.83%, 22.57%, 23.02%, 21.60% respectively. The median expression of KIR3DL1 in unrelated-donor on transplant's day was 572.29 copies/10 000abl copies, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 1 233.74, 1 140.42, 876.73, 1 057.07, 739.02 and 514.43 copies/10 000abl copies respectively. The median expression of KIR3DL1 on NK cells and mRNA level were higher than donors at 1 month after transplantation, and stable expression at all time points after transplantation, so mRNA and NK cell membrane proteins expression increased at the same time. Conclusion: The immune reconstruct regularity of KIR2DL1 and KIR3DL1 gene were different, which provided an experimental basis for selecting the best time to detect the expressions of KIR2DL1 and 3DL1 after transplantation.

[Distribution of donor-specific aKIR after unrelated allogeneic hematopoietic stem cell transplantation].

Objective: To analyze the distribution and proportion of donor-specific activated killer cell immunoglobulin like receptor (aKIR) genes and their clinical application values in unrelated allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: Retrospective analyses of KIR genotyping using polymerase chain reaction with sequence specific primers (PCR-SSP) were performed in 216 pairs of donors and recipients. Results: The frequency of donor-specific KIR genes was 53.7% (116/216) in 216 patients receiving unrelated allo-HSCT, with the frequency of 78.3% (112/143) in the KIR genes mismatched group and 5.5% (4/73) in matched group. Of the 116 patients with detectable donor-specific KIR genes, 99.1% (115/116) patients had various donor-specific aKIR genes. Among 55 pairs of donors' KIR-Bx genotype and patients' KIR-AA genotype group, the most commonly observed genotypes were Bx1, Bx2, Bx3, Bx4, in which the donor-specific KIR genes were respectively KIR 3DS1, 2DL5A, 2DS5, 2DS1; KIR 3DS1, 2DL5A, 2DS3, 2DS1; KIR 2DS2, 2DL2; KIR 2DS2, 2DL2, 3DS1, 2DL5A, 2DS5, 2DS1. Of 44 pairs of donors' KIR-AA genotype and patients' KIR-Bx (AB) genotype group, 36.4% (16/44) recipients had donor-specific KIR2DS4 (FUL) gene. In 143 pairs of KIR mismatched group, the frequencies of donor-specific KIR genes were KIR2DS1 (35.7%) , KIR3DS1 (32.9%) , KIR2DS5 (29.4%) , KIR2DS4 (FUL) (25.9%) , KIR2DL2 (25.2%) , KIR2DS2 (24.5%) , KIR2DS3 (21.7%) and KIR3DL1 (8.4%) , respectively. Conclusion: The donor-specific aKIR genes mainly existed in KIR mismatched group after unrelated allo-HSCT, and the different pairs of donors' and patients' KIR genotypes led to the diverse donor-specific aKIR. But there were higher specific aKIR genes in higher frequency of KIR AA, Bx1, Bx2, Bx3, Bx4 genotypes. All these can provide the experimental basis for studying the role of the donor-specific aKIR genes on the prognosis of HSCT.

[The impact of HLA haplotype and alleles mismatches of donor-recipient pairs on outcome of haploidentical hematopoietic stem cell transplantation with a third part cord blood unit].

OBJECTIVE: To analyze allele mismatches of HLA- A, - B, - C, - DRB1, - DQB1 and haplotype mismatch of donor- recipient pairs on the outcome of haploidentical transplantation combined with a third part cord blood unit. METHODS: 230 pairs of donor-recipient were performed HLA-A, B, C, DRB1, DQB1 typing using SBT and SSOP methods from January 2012 to December 2014. RESULTS: Pairs were divided into HLA- 5/10、6/10、7/10 and ≥8/10 groups according to HLA- A, B, C and DRB1 highresolution typing and matched degrees, the 3-year probability of overall survival (OS) for each group were 48.7%, 59.3%, 71.1%, 38.3% (P=0.068) respectively. HLA-6/10 matched group associated with significant favorable effect on OS compared with HLA- 5/10 matched one (P=0.041).When the HLA class I antigen matched on the recipient and donor, improved OS and event free survival (EFS) in HLA- 6/10 matched group than in HLA-5/10 matched one (P=0.017,P=0.088), especially in single HLA-A loci allele matched one (P=0.013,P=0.013), were observed. As to the third part cord blood unit, sharing the same haplotype with the recipient-donor pairs produced better platelet recovery than the misfit one (95.3%vs 86.2%,P= 0.007), similar result was found in terms of neutrophil recovery (98.8%vs 96.1% ,P=0.022). CONCLUSIONS: HLA locus mismatch and haplotype mismatch of the donor and recipient should be useful for selection of the most optimum donor. Co- infused of an unrelated cord blood unit sharing the same haplotype with the recipient-donor pairs could improve hematopoietic recovery.

[Use early morning serum cortisollevel to evaluate the effect of Cushing's disease after transsphenoidal surgery].

OBJECTIVE: To predict the therapeutic effect of Cushing's disease after transsphenoidal surgery by using morning serum cortisol level. METHODS: The clinical data of 275 cases that had transsphenoidal surgery in Peking Union Medical College Hospital from 2010 to 2014 were analyzed retrospectively.Early morning serum cortisol level less than 140 nmol/L 3 days postoperation was usedto predict endocrinological remission. And long-term efficacy was evaluated by follow-up. RESULTS: Of the 275 patients, there were 49 males and 226 females; average age was 36.5 years old.Remission wasconfirmed in 201 cases, the remission rate was 73.1%, and 8 cases recurrent duringfollow-up.There were 17 macroadenomas, theremission rate was 47.1%; 258 microadenomas and MRI negative adenomas, the remission rate was 74.8%.And 43 recurrent cases had reoperations; the remission rate was 46.5%. CONCLUSION: Early morningserum cortisol 3 days post operation can evaluate the effectof transsphenoidal surgery, but even if the level of cortisol is less than 140 nmol/L, there is still tumor recurrence.Patients should be follow-up for a lifetime.

[Analysis of outcomes and learning curve of endoscopic transsphenoidal surgeries for 124 patients with pituitary adenomas].

OBJECTIVE: To investigate the outcomes of the Endoscopic transsphenoidal surgery for patients with pituitary adenomas, analyze the learning curve and provide reference for future surgeries. METHODS: Retrospective analysis was carried out on 124 patients by endoscopic transsphenoidal surgery with a single neurosurgeon over a period spanning from January 2010 to January 2014 at Peking Union Medical College Hospital.The changes of endocrine and tumor imaging before and after surgery were analysed. Operative time and complication rates of one surgeon in the early period of learning curve were compared with that in later period. RESULTS: There were significant differences in Gross total resection (GTR) rate of pituitary adenomas with different sizes and different Knosp classifications (P<0.01, P<0.01). GTR rate of huge adenomas was significantly lower than that of macroadenoma and adenomas (P<0.05). GTR rate of Knosp 4 grade adenoma was significantly lower than that of Knosp 0-3 level (P<0.05). No significant difference in GTR among all types of functional pituitary adenomas and hormone levels after surgery was observed (P>0.05). In addition, no significant difference (P>0.05) in complications among different sizes, Knosp grade and type of pituitary adenomas was observed.GTR of Knosp 4 adenoma in later period of the learning curve was significantly higher than that in early period (P<0.05). Meanwhile the operative time was significantly lower than early period (P<0.05). CONCLUSIONS: Endoscopic transsphenoidal pituitary adenoma resection has the advantages of wider surgical field, higher GTR rate, less trauma, fewer complications and better life quality of patients.Through standardized learning, the GTR rate of the invasive pituitary adenomas can be improved.

The impact of KIR2DS4 alleles and the expression of KIR in the development of acute GVHD after unrelated allogeneic hematopoietic SCT.

The role of killer Ig-like receptors (KIR) in SCT was analyzed. A total of 75 Chinese patients were transplanted with T-depleted hematopoietic stem cells from unrelated donors. Among the 75 donor-recipient pairs, 60 were HLA 10/10 matched and 15 had some mismatches at HLA-C. Transplants from KIR haplotype B/x group donors showed significantly higher overall survival rates compared with those from KIR haplotype A/A donors (relative risk (RR) 3.1 (95% confidence interval (CI) 1.1-8.6), P=0.007). In the haplotype A/A group, a higher risk of acute GVHD (aGVHD) (RR 9.0 (95% CI 1.2-66.9), P=0.01), especially grade III-IV aGVHD (P=0.006), was observed when the donor was homozygous for the full-length expressed KIR2DS4*00101 allele. Real-time PCR showed that a high expression of inhibitory KIR (2DL1 and 3DL1) in the early stages (<90 days) after transplantation correlated with the development of aGVHD (z=2.558, P=0.011). Our findings indicated a significant association of full-length KIR2DS4 or KIR2DL1/3DL1 expression with the occurrence of aGVHD. In aggregate these results suggested that combining KIR and HLA genotyping could help in the selection of transplant donors and improve the outcome of transplantation. Dynamic detection of KIR2DL1/3DL1 expression would be beneficial for prediction of aGVHD after transplantation.

[Factors influencing the prognosis of gastric cancer patients: a study using international gastric cancer staging classification].

In the 18-year period from 1973 to 1991, 502 cases with gastric cancer were treated surgically at the Peking Union Medical College Hospital. A unified international classification for staging of gastric cancer had been applied to these patients in this study. The pathologic staging classification is based on the extent of the disease at the time of surgical exploration of the abdomen and/or histopathologic study of the excised surgical specimens. According to the new TNM staging classification, the 5-year cumulative survival rates of all surgically treated patients Ia, Ib, II, IIIa, IIIb and IV were 96.25%, 87.34%, 66.11%, 43.70%, 30.33% and 9.36% respectively. It was showed that there is a decreasing tendency in the 5-year survival rates for the depth of invasion of the gastric cancer. The histological types of the gastric cancer had the same prognostic significance as depth of invasion. The 5-year cumulative survival rate of patients with curative surgery was better in the group with chemotherapy (55.6%) than in that without chemotherapy (43%).

[The common causes and differential diagnosis of malignant jaundice].

To investigate the common causes and differential diagnosis of malignant jaundice, we reviewed 903 cases with obstructive jaundice in PUMC hospital in recent 16 years. 383 of them were malignant jaundice (42.4%). The most common origin of malignant jaundice was carcinoma of the pancreatic head with 198 patients (51.7%), and carcinoma of the ampulla Vater with 94 cases (24.5%) and carcinoma of the extrahepatic bile duct with 71 cases (5.2%). The clinical symptoms and signs were not much helpful to the differentiation of malignant jaundice. No specific early signs were found to the malignant jaundice, but most of the patients felt epigastric distension and distress, anorexia, loss of body weight and fatigue before jaundice appeared. More than one third patients had discontinuous fever. The imaging investigation had decisive roles in the diagnosis and differential diagnosis of the malignant jaundice. The positive rate of diagnosis in sonography was 95.5%, but the correct rate only 85.0% (P < 0.05). We regard that sonography might be the first imaging examination for the malignant jaundice and clue for further investigation. ERCP can clearly reveal the papilla, biliary and pancreatic ducts with high positive rate (97.7%) and correct rate (95.1%). PTC was only used in those patients who had the contraindications to ERCP or the cannulation of ERCP was not successful. The positive rate of PTC was 95.8% in the cases with extrahepatic cholangiocarcinoma. The combination of ERCP and PTC could determine the position and extent of extrahepatic cholangiocarcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)