Application of Blood Lymphocyte Immunophenotype and TCR Gene Rearrangement in the Diagnosis of CTCL.

BACKGROUND: The immunophenotype of peripheral blood lymphocytes and T-cell receptor (TCR) gene rearrangement of cutaneous T cell lymphoma (CTCL) patients were retrospectively analyzed to explore their value in the diagnosis of CTCL. METHODS: A total of fifty patients' results were enrolled from 2013 to 2021, including 29 malignant skin disorders and 21 benign skin disorders. The immunophenotype of peripheral blood lymphocytes were analyzed by flow cytometry and TCR gene rearrangement was detected by capillary electrophoresis. Lymphocyte subsets, CD4/CD8 ratio, the percentage of CD3+CD4+CD7- cells and CD45RA/CD45RO ratio was calculated between malignant and benign skin disorders. Peripheral blood lymphocyte immunophenotype and TCR gene rearrangement was compared with skin biopsy to evaluate their sensitivity and specificity. RESULTS: Lymphocyte subsets between malignant and benign groups have no significant difference in percentage of T cell (p > 0.05). The CD4/CD8 ratio is higher in patients with malignant lymphoma than the healthy range. The percentage of CD3+CD4+CD7- cells in malignant groups is higher than that in benign groups and CD45RA/ CD45RO ratio has significant difference between malignant and benign groups (p < 0.05). The sensitivity and specificity of TCR rearrangement for CTCL were 51.7% and 42.9%. The sensitivity and specificity of peripheral blood lymphocyte immunophenotype for CTCL were 44.8% and 33.3%. Combining the two methods, the sensitivity and specificity reached 69.0% and 38.1%, respectively. CONCLUSIONS: CD4/CD8 ratio of lymphocyte subsets, the proportion of CD4+CD7-T cells and CD45RA/CD45RO ratio can effectively distinguish benign and malignant dermatosis. TCR rearrangement method combined with lymphocyte immunophenotype can improve the sensitivity and specificity of CTCL diagnosis.

A Single-Tube 21-Color Multiparameter Flow Cytometry Improve the Sensitivity of B-ALL MRD Analysis.

BACKGROUND: Detection of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) is a well-established risk stratification factor and therapeutic modification strategy in B acute lymphoblastic leukemia (B-ALL). However, current 8 color (8c)-MFC for MRD detection had the sensitivity of 0.01% with false negative or positive. Hence, a more sensitive and applicable MFC-MRD method is urgently needed. The aim of this study is to establish a single-tube 21c-MFC method to detect B-ALL MRD, evaluate its performance, and to investigate its preliminary clinical application. METHODS: We selected 21 markers to establish a single-tube 21c-MFC method. The repeatability and sensitivity of this method was validated by adding Nalm-6 cells to normal bone marrow. Samples from control group (n = 6), B-ALL group (n = 7) and complete remission (CR) group (n = 26) were detected by 21c- and 8c-MFC separately. The expression characteristics of these markers was analyzed in control and B-ALL group, and the consistency of 21c- and 8c-MFC in detecting MRD was compared. RESULTS: Repeatability of this method was 1.91% of CV and sensitivity was up to 0.005%. In control group, the expression of CD81, CD97, and CD200 gradually decreased and CD44, HLA-DR, CD73, and CD72 gradually increased with the maturation of normal B cells. In B-ALL group, CD73stro, CD81low, CD44stro, CD123stro, and CD58stro showed high-frequency expression. The consistency rate of 21c- and 8c-MFC in detecting MRD was 96%. CONCLUSIONS: A single-tube 21c-MFC method was established for MRD detection in B-ALL and had higher sensitivity than the 8c-MFC method.

IGH rod-like tracer: An AlphaFold2 structural similarity extraction-based predictive biomarker for MRD monitoring in pre-B-ALL.

Sequence variation resulting from the evolution of IGH clones and immunophenotypic drift makes it difficult to track abnormal B cells in children with precursor B cell acute lymphoblastic leukemia (pre-B-ALL) by flow cytometry, qPCR, or next-generation sequencing (NGS). The V-(D)-J regions of immunoglobulin and T cell receptor of 47 pre-B-ALL samples were sequenced using the Illumina NovaSeq platform. The IGH rod-like tracer consensus sequence was extracted based on its rod-like alpha-helices structural similarity predicted by AlphaFold2. Additional data from published 203 pre-B-ALL samples were applied for validation. NGS-IGH (+) patients with pre-B-ALL had a poor prognosis. Consistent CDR3-coded protein structures in NGS-IGH (+) samples could be extracted as a potential follow-up marker for pre-B-ALL children during treatment. IGH rod-like tracer from quantitative immune repertoire sequencing may serve as a class of biomarker with significant predictive values for the dynamic monitoring of MRD in pre-B-ALL children.

Clinical Evaluation of the Pre-Analytical Capabilities of Hemostasis Instrument.

Objective: Evaluate the technical performance of the pre-analytical hemolysis-icterus-lipemia (HIL) check module on the ACL-TOP-750. Methods: 8433 routine coagulation samples were evaluated for HIL, the presence of clotting and low sample volume by both visual inspection and the pre-analytical HIL check module on the ACL-TOP-750. Results: 7726 samples were in agreement with both methods and 707 were not consistent. 356 samples with low volume were identified by visual inspection and 920 by the instrument (2.7 mL threshold). Visual inspection identified 56 lipemic samples while 13 of those with moderate or high lipemia were identified by the instrument. Visual inspection identified 47 hemolyzed samples while 7 with moderate or high hemolysis were identified by the instrument. Both visual inspection and the instrument identified 36 icteric samples. For triglyceride concentration and bilirubin concentration, there was good correlation between the ACL-TOP-750 and the DXC800 biochemistry analyzer. Among 30 samples with varying amounts of clotting, 27 were discovered by visual inspection and 3 were discovered by the instrument. Conclusion: The pre-analytical check module on the ACL-TOP-750 improved the detection rate of samples below the target 2.7 mL volume, and the accuracy in detection of HIL. However, the automated method could not replace visual assessment of clotting in samples.

Establishment of the reference intervals of lymphocyte subsets for healthy Chinese Han adults and its influencing factors.

BACKGROUND: Cellular immune monitoring is becoming more critical in the clinic, but its application has not yet become sufficiently widespread. One reason may be the different reference intervals among clinical laboratories due to several factors. Percentage and number of lymphocyte subsets are standard indicators of cellular immune detection. The present study aimed to establish standardized reference intervals of lymphocyte subsets in the healthy Chinese Han adult population and examine such influencing factors as age, gender, region, and measurement instruments. METHODS: A total of 496 healthy Chinese Han people aged 18-59 years from 3 China Mainland regions (north, east, and south) were enrolled. The sample of each center was simultaneously examined by three flow cytometers (FACSCantoTMII, FACSLyricTM, and FACSCaliburTM). A single-platform flow cytometry-based absolute count technique was used to quantify the percentage and number of each lymphocyte subset. The flow cytometry results were analyzed by variance analysis and Z test to determine the influence of age, gender, and instruments on lymphocyte subsets. RESULTS: Multi-center, age-specific, and gender-specific reference intervals of healthy Chinese Han adults' lymphocyte subsets were established. There was no statistical difference in the results from the three flow cytometers. Gender affected the results of CD4+ (%) and the absolute count of CD3-CD16+CD56+, where CD4+ (%) was higher in women, and the absolute count of CD3-CD16+CD56+ was higher in men. Age mainly affected the CD4+/CD8+ ratio, which was statistically higher in groups aged over 40 years; the percentage and number of CD3-CD19+ were more elevated in age groups below 30 years; however, the difference was not statistically significant. CONCLUSIONS: This study established the reference intervals of lymphocyte subsets for healthy Chinese Han adult populations under the standardized methods. This study was the first nationwide study in China to use a flow cytometry-based single-platform method to establish the reference intervals of lymphocyte subsets of the healthy Chinese Han adult population. Gender and age were shown to influence the results of lymphocyte subsets.

Contradictory Phenomenon Between Serum Separator Tube and Plasma Tube: A Case Report.

Separator gels in blood collection tubes are used to separate serum from clotted whole blood or plasma from cells. Here we present a case of a patient with a contradictory phenomenon between the serum separator tube and the plasma tube. The serum separator tube showed mixed serum and separator gel and distinctly less serum. However, the plasma tube showed fewer cells. Laboratory study revealed an IgG level of 78.9 g/L. Serum immunofixation electrophoresis analysis identified the abnormal pattern as a dense IgG band with a corresponding dense light chain band of λ. Bone marrow smear showed 53% proplasmacytes. The patient was diagnosed with multiple myeloma. The marked hyperproteinemia, especially hyperimmunoglobulinemia, may have resulted in the density alteration of serum that was mixed or located above the separator gel. This phenomenon is also seen in patients injected with iodinated radiologic contrast media such as iohexol and in patients on hemodialysis with a concentrated sodium citrate solution.

The platelet surface glycosylation caused by glycosidase has a strong impact on platelet function.

: Platelet surface glycosylation defects has been reported to be significantly associated with many diseases. Our previous study found that platelet surface glycosylation is altered in coronary heart disease. In this study, we further investigated whether altered glycosylation affects platelet function. Platelets were obtained from ten healthy volunteers. The platelet surface terminal sialic acid was removed by neuraminidase A, and N-linked oligosaccharides was removed by PNGase F. The function of the enzyme-treated platelet was measured. The activation and platelet adhesion to von Willebrand factor (vWF) was measured by flow cytometry. Platelet aggregation induced by ADP, arachidonic acid and collagen was detected through light transmission aggregometry, and platelet-leukocyte aggregates (PLAs) was detected by flow cytometry. Neuraminidase A treatment caused sialic acid level decrease and ß-galactose level increase significantly on platelet surface. Activation marker CD62P did not change. Platelet adhesion to vWF was increased significantly (P < 0.05). ADP-induced platelet aggregation was significantly reduced (P < 0.05). Platelet-granulocytes aggregates and platelet-monocytes aggregates increased (P < 0.05). Platelet surface sialic acid was increased after PNGase F treatment. Platelet aggregation by all agonists were significantly reduced (P < 0.05). There is no difference in the binding of vWF and PLAs for PNGase F treated platelet. We demonstrated that asialoglycosylation enhances platelet binding to vWF and forming PLAs, suggest that it may be associated with high platelet reactivity and the increased risk of thrombosis.