Journal of Applied Laboratory Medicine最新文献

Background: Cancer immunotherapy research, immune microenvironment exploration, and biomarker discovery are key application areas of immune checkpoint analysis. PD-1 and CD28 are crucial receptors expressed on the surface of T cells, playing vital roles in regulating T cell activation and immune response. Accurate detection of these immune checkpoints, such as PD-1 and CD28 on lymphocytes, is essential for understanding immune responses, particularly in clinical contexts such as cancer immunotherapy. Flow cytometry offers a precise approach to detect these markers in whole blood samples.

Methods: This study developed and optimized a flow cytometry-based detection method utilizing the CYTEK NL-CLC flow cytometer to quantitatively assess the expression of PD-1 and CD28 on lymphocytes. A detailed protocol was established and validated, focusing on key performance parameters.

Results: Our method showed high sensitivity and specificity, providing a powerful tool for immune monitoring and treatment decision-making. Validation results, including precision, dilution linearity, fluorescence stability, reference interval, and accuracy, all met acceptable criteria and have been reviewed and approved for clinical testing.

Conclusions: The CYTEK NL-CLC flow cytometer is positioned as a reliable and effective platform for immune checkpoint analysis in both clinical and research settings, supporting its integration into cancer immunotherapy workflows and personalized medicine strategies.

Background: The QuidelOrtho TriageTrue® point-of-care (POC) high-sensitivity troponin I (hs-TnI) assay has been previously validated. We aimed to independently verify the assay's analytical performance and concordance with other assays and to verify secondary POC devices.

Methods: Intra-assay precision: 5 whole blood samples spanning the measuring interval were analyzed up to 20 times in succession. Inter-analyzer and inter-lot precision studies were also carried out and a precision curve generated. Hemolysis was assessed by spiking 8 samples with concentrations from 2 to 320 ng/L with hemolysate.Concordance between >200 TriageTrue samples and one other hs-TnI POC and 4 laboratory analyzers was assessed by Pearson correlation and kappa statistics at the limit of quantitation and upper reference limit.Paired measurements from 9 samples between a primary verified device and 8 secondary devices were compared against prespecified difference limits.

Results: Intra-assay precision CVs were 25% and 12.2% at mean concentrations of 2.4 ng/L and 8.5 ng, respectively, and ranged from 2.8% to 15.9% at higher concentrations >10 ng/L. There was minimal interference by hemolysis up to 10 g/L, which exceeded the manufacturer's claim of 1.96 g/L.Correlation coefficients were 0.89 to 0.99 with other assays with the exception of the Roche hs-TnT assay, where it was 0.79. These were similar to the laboratory assays. Of 116 comparisons with 8 secondary analyzers, all fell within acceptable limits.

Conclusions: The TriageTrue assay performed as reported in the package insert. The assay characteristics offer acceptable performance for use within the intended medical settings.

Background: Sepsis is a life-threatening complication of infection with high mortality. A high-throughput analysis of circulating blood proteins may provide mechanistic insight and potent therapeutic targets for the prevention of sepsis.

Methods: We used multivariable Cox regression analysis to examine the association of 4955 plasma proteins, measured by SomaScan, with the risk of incident sepsis among 11 065 participants of the Atherosclerosis Risk in Communities (ARIC) Study (visit 3 in 1993 to 1995; mean age, 60.1 years, 54.4% female, 21.0% Black). Proteins (false discovery rate [FDR] of P < 0.05) discovered at visit 3 were replicated using data at visit 5 (n = 4869 in 2011 to 2013: mean age, 75.5 years) and in the Cardiovascular Health Study (CHS) (n = 3512 in 1992 to 1993; mean age, 74.5 years). Canonical pathways were identified by enrichment analyses.

Results: At ARIC visit three, 669 proteins were associated with the risk of sepsis; 175 were replicated at visit 5. Of these, 90 were validated in the CHS. The top 20 proteins ranked by P value were relevant to acute inflammatory signaling in innate immunity. Pathway analyses implicated activation of pro-inflammatory pathways (e.g., cytokine storm signaling) as well as inhibition of anti-inflammatory pathways (e.g., liver X receptor/retinoid X receptor [LXR/RXR] activation), which also play relevant roles in lipid metabolism.

Conclusions: In this analysis, levels of acute inflammatory proteins measured during routine visits were associated with the subsequent incidence of sepsis. An increased risk of sepsis associated with the inhibition of anti-inflammatory pathways, such as LXR/RXR warrants further mechanistic investigation.

Background: Dipeptidyl peptidase 3 (DPP3) is a peptidase released from dying cells. It cleaves proteins in the renin-angiotensin pathway, which can result in hemodynamic instability. At elevated concentrations DPP3 is associated with worse outcomes, particularly in patients with shock. Herein we describe the assay performance of a DPP3 assay (4TEEN4 Pharmaceuticals GmbH) in human plasma.

Methods: DPP3 concentration was measured using the DPP3 immunoluminometric assay (4TEEN4 Pharmaceuticals GmbH) and the signal was read using a luminometer (Berthold Centro LB963). Analytical performance was established for precision, linearity, accuracy, detection limit, analytical specificity, reference interval, kit lot-to-lot comparison, specimen type, and sample stability.

Results: Limit of detection was verified at 1.6 ng/mL in EDTA plasma with a coefficient of variation (CV) of <10%. Precision studies revealed a CV ≤ 6% at 28.5 ng/mL and 59.4 ng/mL and comparability with a manufacturer performed assay was demonstrated between 7.7 and 195.2 ng/mL. An upper 97.5% limit of 22 ng/mL without age or sex associations was verified in healthy donors. The assay was not susceptible to interference from lipemia or bilirubin. However, measured DPP3 concentrations increased linearly with increasing hemolysis. DPP3 concentrations are stable in EDTA plasma for up to 24 h and at least 11 months when stored ambient or at -80°C, respectively.

Conclusions: DPP3 can be measured precisely in EDTA plasma using the immunoluminometric DPP3 assay. Given the potential clinical use of DPP3 in critical care patients, caution should be taken to avoid inducing pre-analytical hemolysis during sample collection.

Background: Circuit-induced hemolysis is relatively common in extracorporeal membrane oxygenation (ECMO) patients. Intravascular release of cell-free hemoglobin can lead to complications and requires timely recognition. Validation of plasma free hemoglobin (PFH) measurement using a direct spectrophotometric method is presented.

Methodology: We evaluated a method modified from Kahn et al. (Ann Clin Lab Sci 1981;11:126-31) on a stand-alone spectrophotometer (Cary 60) and compared its performance to the semiquantitative H-index on an Abbott Alinity c, including precision, linearity, recovery, reference interval verification, interference, and stability. Method comparison was performed relative to the H-index and the same method on a different spectrophotometer (Beckman DU 720). Lipemia interference was performed on the Cary 60, Cary 3500, and Beckman DU 720. Surrogate biomarkers for hemolysis detection were also investigated in ECMO patients.

Results: The PFH method on the Cary 60 demonstrated imprecision ranging from 1% (96.0 mg/dL) to 4% (3.0 mg/dL), linearity to 100 mg/dL, and recovery >80% for values >2 mg/dL hemoglobin-spiked plasma. Dilution expanded the reportable range to the maximum dilution tested (1000 mg/dL). Lipemia interfered with PFH measurement by the direct method, but the same method on the Cary 3500 was resistant to lipemia. Bilirubin did not cause significant interference. Direct and H-index methods were comparable with a mean difference of 5.03 mg/dL (95% CI -1.38, 11.44). Lactate dehydrogenase was the most reliable surrogate biomarker for hemolysis. with AUC of 0.921 (0.894, 0.949) at >50 mg/dL.

Conclusion: PFH measurement by a direct spectrophotometric method is more precise and sensitive compared to the H-index; however, PFH measurement is susceptible to lipemia unless performed on a high-end spectrophotometer.