Background: Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing’s syndrome (CS). We compared UFC determination by both direct and extraction immunoassays using Abbott Architect, Siemens Atellica Solution, and Beckman DxI800 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we evaluated the value of 24-hr UFC measured by six methods for diagnosing CS. Methods: Residual 24-hr urine samples of 94 CS and 246 non-CS patients were collected.A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by direct assays (D) using Abbott, Siemens, and Beckman platforms and by extraction assays (E) using Siemens and Beckman platforms. Method was compared using Passing–Bablok regression and Bland–Altman plot analyses. Cut-off values for the six assays and corresponding sensitivities and specificities were calculated by ROC analysis. Results: Abbott-D, Beckman-E, Siemens-E, and Siemens-D showed strong correlations with LC-MS/MS (Spearman coefficient r = 0.965, 0.922, 0.922, and 0.897, respectively), while Beckman-D showed weaker correlation (r = 0.755). All immunoassays showed proportionally positive bias. The areas under the curve were 0.975 for Abbott-D, 0.972 for LCMS/MS, 0.966 for Siemens-E, 0.948 for Siemens-D, 0.955 for Beckman-E, and 0.877 for Beckman-D. The cut-off values varied significantly (154.8–1,321.5 nmol/24 hrs). Assay sensitivity and specificity ranged from 76.1% to 93.2% and from 93.0% to 97.1%, respectively. Conclusions Commercially available immunoassays for measuring UFC show different levels of analytical consistency compared to LC-MS/MS. Abbott-D, Siemens-E, and Beckman-E have high diagnostic accuracy for CS.

Objective:To assess the applicability of fully automatic pipeline automated testing for internal quality control (automated quality control).Methods:Stability, assay efficiency and implementation costs of 18 biochemical tests, 5 immunoturbidimetric tests and 11 chemical illuminescent tests in the Department of Laboratory Medicine of Peking Union Hospital from January 2019 to July 2022 were evaluated using automated quality control implementation methods. The detailed method is as follows: quality control materials for biochemical, immunoturbidimetric and chemiluminescent tests were stored in the refrigerator in the pipeline which was controlled by the intermediate software, and were automatically retrieved and tested as pre-set followed by documenting and storing. The quality control setup for the biochemical tests included refreshing quality control materials daily and weekly,both of which were paralleled for 3 months. The on-line storage stability of quality control materials in the pipeline was evaluated by comparing the coefficients of variation ( CV) of the quality control results between the two patterns. Effect of automated quality control application was evaluated using 6 indicators, including the results′ variation of automatically performed and manually performed quality controls, the out-of-controlled rate, the consumption of quality control materials, the change of staff workload, the impact on the testing time of the first sample, and the failure rate of automated quality control. Results:(1) Storage stability of quality control materials in the pipeline: under the pattern of weekly refresh of the biochemical quality control materials, except for total carbon dioxide (TCO 2) (the CVs of low and high level quality control were respectively 20.24% and 21.82%) and sodium (the CV of low level quality control was 1.51%) that were greater than the allowable variation set by the laboratory, the CVs of the rest tests meet the lab requirements on the allowable variations. (2) The results′ variation of quality control in automatically performed and manually performed control patterns: in the patterns of daily refresh of biochemical quality control materials and weekly refresh of immunoturbidimetric and chemiluminescent quality control materials, the CVs of both low and high levels of quality control were lower in the automatically performed control pattern than that in manually performed pattern for 8 chemiluminescent items of dehydroepiandrosterone sulfate, estradiol, follicle stimulating hormone, luteinizing hormone, serum ferritin, serum folic acid, vitamin B12 and testosterone, 3 immunologic items of complement 3, C reactive protein and immunoglobulin G, and 10 biochemical items of alkaline phosphatase, glucose, calcium, chloride, potassium, lactate dehydrogenase, sodium, urea, low density lipoprotein cholesterol, and adenosine deaminase. The out-of-control rates of biochemistry, immunoturbidimetric and chemiluminescence tests in both quality control patterns conformed with the clinical routine work requirements. (3) Comparison of quality control materials′ consumption: compared with manually performed quality control, weekly consumption of automatically performed chemiluminescent quality control materials decreased 37.5% (from 8 ml to 5 ml); weekly consumption of automatically performed immunoturbidimetric quality control materials decreased 33.3% (from 3 ml to 2 ml). (4)Comparison of staff workload and first sample testing time: compared with manually performed quality control, automatical quality control reduced manual work by about 156 steps per week, and the daily initial testing time was earlier by 15 min on average. The failure rate was 54.5% (37/64) during the early-stage application of the automated quality control which dropped to 10.2% (13/128) in the late-stage. Conclusion:The results of automated quality control detected in the pipeline system meet the quality indicators′ requirements of the laboratory, and the application of automated quality control can improve the quality control, save costs, reduce workload, and improve work efficiency.

Clinical application of mass spectrometry technology has attracted the attention of clinical laboratory experts due to its high sensitivity, high specificity, and capacities of simultaneous detection of multiple compounds. In recent years, mass spectrometry technology has made significant achievements in the fields of identification of pathogenic microorganism, detection of trace elements and heavy metals, small molecule hormones, vitamins, amino acids, peptides and proteins, as well as therapeutic drug monitoring (TDM) and poisoning drugs screening. In order to further clarify the opportunities and challenges brought by this complex mass spectrometry technology in the field of clinical laboratory, the Chinese Journal of Laboratory Medicine invited experts and scholars of laboratory medicine to share their experience and opinions on related items focusing on the positioning of mass spectrometry technology in the clinical laboratory, the development and improvement of the clinical laboratory by mass spectrometry technology, the challenges of interpreting mass spectrometry test results, the challenges of operating and managing clinical mass spectrometry laboratories, and ways of improving the application of clinical mass spectrometry laboratories with this technology. Agreement was achieved in that the introduction of mass spectrometry technology into the clinical laboratory could bring new directions and opportunities for clinical testing and research, and also is associated with a series of challenges such as the difficulty of sample pretreatment, the high cost and complexity of mass spectrometry technology, the complexity of data processing and interpretation, the lack of standards and norms, and the issue of determining the price of mass spectrometry examinations.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology has the characteristics of high specificity and high throughput, making it rapidly applied and developed in the field of clinical testing. Its application in the monitoring of therapeutic drugs can effectively improve the quantitative accuracy and sensitivity, and formulate a personalized and optimal dosing plan for patients. However, this technology still faces some challenges, and automation, quality control, and quantitative traceability will be the future development direction.

Objective:To investigate the efficacy and safety of protein A immunoadsorption (PAIA) combined with rituximab (RTX) in highly sensitized patients who underwent haplo-hematopoietic stem cell transplantation (haplo-HSCT) .Methods:The clinical data of 56 highly sensitized patients treated with PAIA and RTX before haplo-HSCT at the First Affiliated Hospital of Soochow University and Soochow Hopes Hematonosis Hospital between March 2021 and June 2023 were retrospectively analyzed. The number of human leukocyte antigen (HLA) antibody types and the mean fluorescence intensity (MFI), humoral immunity, adverse reactions during adsorption, and survival within 100 days before and after adsorption were measured.Results:After receiving the PAIA treatment, the median MFI of patients containing only HLA Ⅰ antibodies decreased from 7 859 (3 209-12 444) to 3 719 (0-8 275) ( P<0.001), and the median MFI of HLA Ⅰ+Ⅱ antibodies decreased from 5 476 (1 977-12 382) to 3 714 (0-11 074) ( P=0.035). The median MFI of patients with positive anti-donor-specific antibodies decreased from 8 779 (2 697-18 659) to 4 524 (0–15 989) ( P<0.001). The number of HLA-A, B, C, DR, and DQ antibodies in all patients decreased after the PAIA treatment, and the differences were statistically significant (A, B, C, DR: P<0.001, DQ: P<0.01). The humoral immune monitoring before and after the PAIA treatment showed a significant decrease in the number of IgG and complement C3 ( P<0.001 and P=0.002, respectively). Forty-four patients underwent HLA antibody monitoring after transplantation, and the overall MFI and number of antibody types decreased. However, five patients developed new antibodies with low MFI, and nine patients continued to have high MFI. The overall survival, disease-free survival, non-recurrent mortality, and cumulative recurrence rates at 100 days post-transplantation were 83.8%, 80.2%, 16.1%, and 4.5%, respectively. Conclusions:The combination of PAIA and RTX has a certain therapeutic effect and good safety in the desensitization treatment of highly sensitive patients before haplo-HSCT.

A young female patient with acne and elevated testosterone level underwent plasma steroid hormones testing and found a significant increase in aldosterone. We excluded testing interference and verified the absence of hypertension, hypokalemia, and adrenal occupancy, as well as primary and secondary hyperaldosteronism. During follow-up, a temporal correlation was found between aldosterone levels and the use of drospirenone and ethinylestradiol tablets. It was observed that the combination of drospirenone and ethinylestradiol could lead to the increase of aldosterone level and the concentration ratio of aldosterone to direct renin through different mechanisms. Drospirenone exerts an antagonistic effect on mineralocorticoid receptor to prevent the development of hypertension or hypokalemia. In clinical practice, it is necessary to pay attention to the effect of this drug on screening markers for primary aldosteronism. In the laboratory examination, when female patients with no symptoms of hypertension and hypokalemia but with elevated aldosterone levels are encountered, it can be verified whether they have a history of use of compound estrogen-progestin such as drospirenone and ethinylestradiol tablets, and appropriate tips are provided in the report.

OBJECTIVE: To explore the genetic etiology for a child with profound intellectual disabilities and obvious behavioral abnormalities. METHODS: A male child who had presented at the Zhongnan Hospital of Wuhan University on December 2, 2020 was selected as the study subject. Peripheral blood samples of the child and his parents were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. Short tandem repeat (STR) analysis was carried out to determine its parental origin. The splicing variant was also validated in vitro with a minigene assay. RESULTS: WES results revealed that the child had harbored a novel splicing variant of c.176-2A>G in the PAK3 gene, which was inherited from his mother. The results of minigene assay have confirmed aberrant splicing of exon 2. According to the guidelines from the American College of Medical Genetics and Genomics, it was classified as a pathogenic variant (PVS1+PM2_Supporting+PP3). CONCLUSION The novel splicing variant c.176-2A>G of the PAK3 gene probably underlay the disorder in this child. Above finding has expanded the variation spectrum of the PAK3 gene and provided a basis for genetic counseling and prenatal diagnosis for this family.
Child ; Female ; Humans ; Male ; Pregnancy ; Exons ; Intellectual Disability/genetics* ; Mothers ; Mutation ; p21-Activated Kinases/genetics* ; Parents ; RNA Splicing

Objective: To understand the current status of diagnosis and treatment of chronic lymphocytic leukemia (CLL) /small lymphocytic lymphoma (SLL) among hematologists, oncologists, and lymphoma physicians from hospitals of different levels in China. Methods: This multicenter questionnaire survey was conducted from March 2021 to July 2021 and included 1,000 eligible physicians. A combination of face-to-face interviews and online questionnaire surveys was used. A standardized questionnaire regarding the composition of patients treated for CLL/SLL, disease diagnosis and prognosis evaluation, concomitant diseases, organ function evaluation, treatment selection, and Bruton tyrosine kinase (BTK) inhibitor was used. Results: ①The interviewed physicians stated that the proportion of male patients treated for CLL/SLL is higher than that of females, and the age is mainly concentrated in 61-70 years old. ②Most of the interviewed physicians conducted tests, such as bone marrow biopsies and immunohistochemistry, for patient diagnosis, in addition to the blood test. ③Only 13.7% of the interviewed physicians fully grasped the initial treatment indications recommended by the existing guidelines. ④In terms of cognition of high-risk prognostic factors, physicians' knowledge of unmutated immunoglobulin heavy-chain variable and 11q- is far inferior to that of TP53 mutation and complex karyotype, which are two high-risk prognostic factors, and only 17.1% of the interviewed physicians fully mastered CLL International Prognostic Index scoring system. ⑤Among the first-line treatment strategy, BTK inhibitors are used for different types of patients, and physicians have formed a certain understanding that BTK inhibitors should be preferentially used in patients with high-risk factors and elderly patients, but the actual use of BTK inhibitors in different types of patients is not high (31.6%-46.0%). ⑥BTK inhibitors at a reduced dose in actual clinical treatment were used by 69.0% of the physicians, and 66.8% of the physicians had interrupted the BTK inhibitor for >12 days in actual clinical treatment. The use of BTK inhibitors is reduced or interrupted mainly because of adverse reactions, such as atrial fibrillation, severe bone marrow suppression, hemorrhage, and pulmonary infection, as well as patients' payment capacity and effective disease progression control. ⑦Some differences were found in the perceptions and behaviors of hematologists and oncologists regarding the prognostic assessment of CLL/SLL, the choice of treatment options, the clinical use of BTK inhibitors, etc. Conclusion: At present, a gap remains between the diagnosis and treatment of CLL/SLL among Chinese physicians compared with the recommendations in the guidelines regarding the diagnostic criteria, treatment indications, prognosis assessment, accompanying disease assessment, treatment strategy selection, and rational BTK inhibitor use, especially the proportion of dose reduction or BTK inhibitor discontinuation due to high adverse events.
Female ; Humans ; Male ; Aged ; Middle Aged ; Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy* ; Prognosis ; Lymphoma, B-Cell ; Immunohistochemistry ; Immunoglobulin Heavy Chains/therapeutic use*

Objective: Retrospective analysis of the efficacy and influencing factors of bladder preservation integrated therapy for unresectable invasive bladder cancer confined to the pelvis was done, also including the bladder function preservation and adverse effects analysis. Methods: Sixty-nine patients with unresectable locally invasive bladder cancer who received radiotherapy-based combination therapy from March 1999 to December 2021 at our hospital were selected. Among them, 42 patients received concurrent chemoradiotherapy, 32 underwent neoadjuvant chemotherapyand 43 with transurethral resection of bladder tumors (TURBT) prior to radiotherapy. The late adverse effect of radiotherapy, preservation of bladder function, replase and metastasis and survival were followed-up. Cox proportional hazards models were applied for the multifactorial analysis. Results: The median age was 69 years. There were 63 cases (91.3%) of uroepithelial carcinoma, 64 of stage Ⅲ and 4 of stage Ⅳ. The median duration of follow-up was 76 months. There were 7 grade 2 late genito urinary toxicities, 2 grade 2 gastrointestinal toxicities, no grade 3 or higher adverse events occurred. All patients maintained normal bladder function, except for 8 cases who lost bladder function due to uncontrolled tumor in the bladder. Seventeen cases recurred locally. There were 11 cases in the concurrent chemoradiotherapy group with a local recurrence rate of 26.2% (11/42) and 6 cases in the non-concurrent chemoradiotherapy group with a local recurrence rate of 22.2% (6/27), and the difference in local recurrence rate between the two groups was not statistically significant (P=0.709). There were 23 cases of distant metastasis (including 2 cases of local recurrence with distant metastasis), including 10 cases in the concurrent chemoradiotherapy group with a distant metastasis rate of 23.8% (10/42) and 13 cases in the non-concurrent chemoradiotherapy group with a distant metastasis rate of 48.1% (13/27), and the distant metastasis rate in the non-concurrent chemoradiotherapy group was higher than that in the concurrent chemoradiotherapy group (P=0.036). The median 5-year overall survival (OS) time was 59 months and the OS rate was 47.8%. The 5-year progression-free survival (PFS) time was 20 months and the PFS rate was 34.4%. The 5-year OS rates of concurrent and non-concurrent chemoradiotherapy group were 62.9% and 27.6% (P<0.001), and 5-year PFS rates were 45.4% and 20.0%, respectively (P=0.022). The 5-year OS rates of with or without neoadjuvant chemotherapy were 78.4% and 30.1% (P=0.002), and the 5-year PFS rates were 49.1% and 25.1% (P=0.087), respectively. The 5-year OS rates with or without TURBT before radiotherapy were 45.5% and 51.9% (P=0.233) and the 5-year PFS rates were 30.8% and 39.9% (P=0.198), respectively. Multivariate Cox regression analysis results showed that the clinical stage (HR=0.422, 95% CI: 0.205-0.869) was independent prognostic factor for PFS of invasive bladder cancer. The multivariate analysis showed that clinical stages (HR=0.278, 95% CI: 0.114-0.678), concurrent chemoradiotherapy (HR=0.391, 95% CI: 0.165-0.930), neoadjuvant chemotherapy (HR=0.188, 95% CI: 0.058-0.611), and recurrences (HR=10.855, 95% CI: 3.655-32.638) were independent prognostic factors for OS of invasive bladder cancer. Conclusion: Unresectable localized invasive bladder cancer can achieve satisfactory long-term outcomes with bladder-preserving combination therapy based on radiotherapy, most patients can retain normal bladder function with acceptable late adverse effects and improved survival particularly evident in patients with early, concurrent chemoradiotherapy and neoadjuvant chemotherapy.
Humans ; Aged ; Treatment Outcome ; Retrospective Studies ; Combined Modality Therapy ; Chemoradiotherapy/methods* ; Urinary Bladder Neoplasms/radiotherapy* ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use* ; Neoplasm Staging

Objective:To establish and validate an LC-MS/MS method for simultaneous determination of Aβ 1-42, Aβ 1-40, and Aβ 1-38 in cerebrospinal fluid. Additionally, the consistency between this method and three mainstream detection methods was evaluated.Methods:This study involved method establishment, validation, and consistency evaluation. The N15 labeled β-amyloid protein was used as the internal standard. Extraction was performed using Waters MCX 96-wells solid phase extraction plate, and the eluent was collected to QuanRecovery MaxPeak 700 μl plate. At the positive ion mode, the multi-reaction ion monitoring mode based on electric spray ionization is chosen for the determination of CSF Aβ 1-42, Aβ 1-40, and Aβ 1-38. Referring to the CLSI C62-A and EP-15A3 guidelines, the method is evaluated and verified, including quantitation of limit (LOQ), linearity, recovery, precision, and accuracy. In addition, a total of 57 clinical residual CSF samples were collected and the concentrations of Aβ 1-42 and Aβ 1-40 were determined based on manual INNOTEST ELISA assay and Lumipulse G and Roche Elecsys fully automated biochemical analyzers. The comparison analysis and deviation evaluation were conducted by passing-bablok and Bland Altman methods.Results:The analysis time of this method is 8 min, and the LOQ of Aβ 1-42, Aβ1-40 and Aβ1-38 is 0.1 ng/ml, 0.5 ng/ml, and 0.1 ng/ml, respectively, and the linear range can meet the needs of clinical detection. Respectively, the recovery is 86.2%-93.8%, 100.9%-103.9% and 103.3%-107.1%; the total imprecision is 4.7%-7.4%, 3.5%-4.6% and 5.2%-10.9%. The measured values of Aβ 1-42 certified reference materials are all within the allowable uncertainty requirements. Moreover, the carryover rate of three analytes was all≤0.11%. In addition, the correlations of Aβ 1-42 and Aβ1-40 in CSF between this LC-MS/MS method and the INNOTEST ELISA method, Lumipulse G and Roche Elecsys fully automated biochemical analyzers were all deemed good, with correlation coefficient (r) ranging from 0.920 to 0.970. However, the measured values between the four methods were remarkably different.Conclusion:We established and validated a robust method based on LC-MS/MS technology for simultaneous determination of Aβ 1-42, Aβ 1-40, and Aβ 1-38 in CSF. The method is accurate, simple, and suitable for clinical measurements. However, despite good correlations, there were substantial differences in the measurement results of Aβ 1-42 and Aβ 1-40 among different analytical platforms, indicating the need for further promotion of harmonization and standardization processes for AD classic biomarkers.