Current status of testing intervals for antiphospholipid syndrome testing: a 13-year retrospective national data analysis in South Korea.

OBJECTIVE: To assess the appropriateness of laboratory testing intervals and antiphospholipid syndrome (APS) incidence. METHODS: Between January 2010 and August 2022, insurance claims data of patients with disease codes for other thrombophilia (D68.6) and APS (V253) were retrieved in South Korea. Patients who received antiphospholipid antibody tests more than twice were classified as having suspected APS. The interval between the first 2 antiphospholipid antibody tests was evaluated in the patients with suspected APS. Patients with suspected APS who received anticoagulants for >180 days were classified as having APS. RESULTS: Overall, 8656 patients were classified as having suspected APS. The testing interval for the first 2 tests in patients with suspected APS was <6 and <12 weeks in 11.1% and 20.6% of cases, respectively, in 2010, gradually increasing to 21.0% and 35.4%, respectively, in 2021. Subsequently, 4344 patients were classified as having APS, with 65.0% being female. Only 330 patients were diagnosed with APS in 2021, down from 436 in 2020. CONCLUSION: This study showed a gradual increase in patients receiving antiphospholipid antibody testing with an inappropriate short-term interval, underscoring the need for laboratory stewardship to ensure an appropriate interval for APS testing.

Automated sepsis detection with vancomycin- and allantoin-polydopamine magnetic nanoparticles.

Rapid and accurate identification of the bacteria responsible for sepsis is paramount for effective patient care. Molecular diagnostic methods, such as polymerase chain reaction (PCR), encounter challenges in sepsis due to inhibitory compounds in the blood, necessitating their removal for precise analysis. In this study we present an innovative approach that utilizes vancomycin (Van) and allantoin (Al)-conjugated polydopamine (PDA)-coated magnetic nanoparticles (MNPs) for the rapid and automated enrichment of bacteria and their DNA extraction from blood without inducing clumping and aggregation of blood. Al/Van-PDA-MNPs, facilitated by IMS, eliminate the need for preliminary sample treatments, providing a swift and efficient method for bacterial concentration and DNA extraction within an hour. Employing Al/Van-PDA-MNPs within an automated framework has markedly improved our ability to pre-concentrate various Gram-negative and Gram-positive bacteria directly from blood samples. This advancement has effectively reduced the detection threshold to 102 colony-forming unit/mL by both PCR and quantitative PCR. The method's expedited processing time, combined with its precision, positions it as a feasible diagnostic tool for diverse healthcare settings, ranging from small clinics to large hospitals. Furthermore, the innovative application of nanoparticles for DNA extraction holds promising potential for advancing sepsis diagnostics, enabling earlier interventions and improving patient outcomes.

Increased PD-1 expression of bone marrow T-cells in acute myeloid leukaemia patients after stem cell transplantation, and its association with overall survival.

BACKGROUND: Immune checkpoints are involved in mechanisms by which tumours escape from the host immune system. Our aim was to evaluate acute myeloid leukaemia (AML) patients to determine expression levels of checkpoint molecules according to diagnosis and treatments, and to identify optimal candidates for checkpoint blockade. METHODS: Bone marrow (BM) samples were obtained from 279 AML patients at different disease status and from 23 controls. Flow cytometric analyses of PD-1 and PD-L1/PD-L2 expression were performed. RESULTS: Programmed death-1 (PD-1) expression levels on CD8+ T-cells at AML diagnosis were increased compared to controls. PD-L1 and PD-L2 expression levels on leukaemic cells at diagnosis were significantly higher in secondary AML than in de novo AML. PD-1 levels on CD8+ and CD4+ T-cells after allo-SCT were significantly higher than those at diagnosis and after CTx. PD-1 expression on CD8+ T-cells increased in the acute GVHD group than in the non-GVHD group. The overall survival of patients with high PD-1 expression on CD8+ T-cells was significantly shorter than that of patients with low PD-1 expression. CONCLUSIONS: In conclusion, patients who underwent allo-SCT exhibited high PD-1 expression, suggesting that allo-SCT increases PD-1 expression on T-cells, and the patients with high PD-1 expression on CD8+ T-cells after allo-SCT showed the poor prognosis. For these patients, PD-1 blockade could be an immunotherapeutic strategy.

Validation of High-sensitivity Flow Cytometry for Reliable Immune Cell Analysis in Real-world Laboratory Settings.

The adoption of high-sensitivity flow cytometry (HSFC) in routine laboratory settings has been slow owing to concerns regarding the reliability and reproducibility of results. Validation is an essential prerequisite for conducting assays, and implementing the CLSI guidelines has been confusing, primarily because many aspects are not yet established. We aimed to validate an HSFC protocol for detecting follicular helper T (Tfh) cells in a real-world laboratory environment. The analytical validity of the Tfh cell panel was ensured through rigorous testing, including evaluations of precision, stability, carryover, and sensitivity, following the CLSI H62 guidelines. We found that Tfh cells, present in very small numbers in the blood, could be sufficiently detected through HSFC, and concerns about the reliability and reproducibility of the results in real-world laboratories could be solved through systematic validation. Establishing the lower limit of quantification (LLOQ) is a critical step in HSFC evaluations. By selecting an appropriate sample, for example, collecting residual cells from CD4 isolation in our experiment and using them as low-level samples, the LLOQ could be accurately established. The strategic validation of flow cytometry panels can facilitate the adoption of HSFC in clinical laboratories, even with limited resources.

Effective and Practical Complete Blood Count Delta Check Method and Criteria for the Quality Control of Automated Hematology Analyzers.

Background: Delta checks increase patient safety by identifying automated hematology analyzer errors. International standards and guidelines for the complete blood count (CBC) delta check method have not been established. We established an effective, practical CBC delta check method and criteria. Methods: We assessed five delta check methods for nine CBC items (Hb, mean corpuscular volume, platelet count, white blood cell [WBC] count, and five-part WBC differential counts) using 219,804 blood samples from outpatients and inpatients collected over nine months. We adopted the best method and criteria and evaluated them using 42,652 CBC samples collected over two weeks with a new workflow algorithm for identifying test errors and corrections for Hb and platelet count. Results: The median delta check time interval was 1 and 21 days for inpatients and outpatients (range, 1-20 and 1-222 days), respectively. We used delta values at 99.5% as delta check criteria; the criteria varied among the five methods and between outpatients and inpatients. The delta percent change (DPC)/reference range (RR) rate performed best as the delta check for CBC items. Using the new DPC/RR rate method, 1.7% of total test results exceeded the delta check criteria; the retesting and resampling rates were 0.5% and 0.001%, respectively. Conclusions: We developed an effective, practical delta check method, including RRs and delta check time intervals, and delta check criteria for nine CBC items. The criteria differ between outpatients and inpatients. Using the new workflow algorithm, we can identify the causes of criterion exceedance and report correct test results.

Evaluation of laboratory diagnostic tests for light-chain clonality and bone marrow findings in AL amyloidosis.

Background: Light-chain amyloidosis (AL) is the most common form of systemic amyloidosis. This study aimed to evaluate the usefulness of laboratory tests for light-chain clonality and bone marrow (BM) findings in AL amyloidosis. Methods: We retrospectively enrolled patients newly diagnosed with AL amyloidosis on pathological examination who underwent a BM biopsy. Laboratory test data for light-chain clonality were collected and compared. Amyloid deposits were identified with H&E, Congo red, and PAS stains. Results: We reviewed 98 patients with AL amyloidosis. Light chain clonality (λ, 64 cases; κ, 34 cases) was detected by serum immunofixation electrophoresis (IFE) (63.3%), urine IFE (70.8%), serum protein electrophoresis (PEP) (44.9%), urine PEP (44.8%), serum free light chain (SFLC) ratio (79.5%), and BM immunohistochemistry (IHC) (85.7%). Flow cytometric (FCM) assay identified aberrant BM plasma cells in 92.9% of cases. BM amyloid deposits were identified in 35 of the 98 cases (35.7%); 71.4% (25/35) were Congo red-positive, and 100.0% (35/35) were PAS-positive. Conclusion: Laboratory tests for detecting light-chain clonality in AL amyloidosis in order of sensitivity include FCM assay for aberrant plasma cells, IHC for light chains on BM biopsy or clot section, SFLC ratio, and serum and urine IFE. Congo red staining of BM samples remains an important tool for identifying amyloid deposits in BM. Periodic acid-Schiff (PAS) staining can be useful in diagnosing some cases of Congo red-negative amyloidosis.

Natural Killer Cell Activity Test Helps to Suspect Aggressive Natural Killer Cell Leukemia - Diagnostic Challenge.

Aggressive natural killer cell leukemia (ANKL) is a rare disease with an aggressive clinical course. We aimed to assess the clinicopathological characteristics of the difficult to diagnose ANKL. During ten years, nine patients with ANKL were diagnosed. All the patients exhibited aggressive clinical course and underwent the BM study to rule out lymphoma and hemophagocytic lymphohistiocytosis (HLH). BM examination showed varying degrees of infiltration of neoplastic cells, which were mainly positive for CD2, CD56, cytoplasmic CD3 and EBV in situ hybridization. Five BM aspirates showed histiocytic proliferation with active heomphagocytosis. Normal or increased NK cell activity test results were obtained from 3 patients who were available for testing. Four had multiple BM studies until diagnosis. An aggressive clinical course and positive EBV in situ hybridization, often with associated secondary HLH, should raise the suspicion of an ANKL. Conducting additional supplementary tests such as NK cell activity and NK cell proportion would be helpful for the diagnosis of ANKL.

Lupus anticoagulants as a prospective independent predictor in COVID-19 patients.

OBJECTIVE: Lupus anticoagulant (LA) are commonly detected during SARS-CoV-2 infection. However, the relationship between LA and clinical significance is still unclear. METHODS: A retrospective chart analysis was performed on COVID-19 patients who were tested for LA at our hospital from March 2020 to November 2021. We analyzed the patient's characteristics based on the result of the LA test. In addition, subgroup analysis performed the LA-positive group who had undergone serial LA tests. RESULTS: A total of 219 COVID-19 patients were enrolled in the study, 148 patients (67.6%) were positive for LA test. The LA-positive group received more treatment of high flow nasal cannula (LA-positive 73.0%, LA-negative 57.7%, p = 0.024). The LA-positive group showed prolonged aPTT, higher levels of CRP and fibrinogen (all p's < 0.05). Among 148 LA-positive patients, 127 patients (86.5%) were found to be LA-positive within 10 days of SARS-CoV-2 positive, and LA-positive group confirmed a median time to LA loss of 10 days. However, there was a group that was negative for LA in the early stages of infection and became positive about 13 days later. A subgroup analysis showed that these patients had different characteristics due to their longer hospital stays and higher D-dimer levels. CONCLUSIONS: In COVID-19 patients, LA is expected to be associated to disease severity. Since the clinical significance of LA is different depending on the onset time of LA positivity, the LA test is suggested to be done at diagnosis of SARS-CoV-2 infection, even if LA is negative, follow-up test should be considered within 10 days.

Counting DNA molecules on a microchannel surface for quantitative analysis.

Microscopic visualization of DNA molecules is a simple, intuitive, and powerful method. Nonetheless, DNA-molecule quantification methods that employ microscopic visualization have not been reported so far. In this study, a new quantitative approach is presented that enables the counting of individual DNA molecules that have been rendered visible by fluorescence microscopy. Toward this, a microfluidic device was employed that directed DNA molecules into microchannels and deposited the molecules onto a positively charged surface. This microfluidic device had a vertically tapered channel inlet structure that prevented the accumulation of excess DNA molecules in the channel inlet while creating a tapering flow, thereby ensuring the even distribution of the DNA molecules in the microchannels. The channel heights and the density of positive charges on the surface were optimized for analysis. The linearity of this method with respect to the determination of the concentration of DNA in solutions was subsequently determined. The limit of detection was 0.48 fg/µL, which corresponds to 64 molecules of 7.25 kbp DNA in 1 µL of sample. This quantitative approach was finally used to count two types of plasmids co-transformed in an E. coli cell; a measurement that is typically considered challenging with gel electrophoresis.

Effect of changes in lymphocyte subsets at diagnosis in acute myeloid leukemia on prognosis: association with complete remission rates and relapse free survivals.

We prospectively investigated whether the characteristics of lymphocyte subsets at diagnosis in acute myeloid leukemia (AML) patients are different from healthy controls and affect treatment outcomes. A total of 91 AML patients classified into 3 genetic risk subgroups (favorable/intermediate/poor) according to 2022 NCCN guidelines were enrolled. We measured lymphocyte subsets by flow cytometry with peripheral blood samples at diagnosis and compared results with healthy controls. Influences of lymphocyte subsets on complete remission (CR) rates and survivals were also evaluated. AML patients had significantly lower numbers and proportions of CD56dimCD16+ natural killer (NK) cells, central memory T cells, and regulatory T cells than healthy controls. Higher proportion of helper/inducer T cells, CD4+CD31+ naïve T cells, and decreased proportion of NK cells significantly increased CR rates in 65 non-promyelocytic leukemia patients (P = 0.034, 0.027, and 0.019, respectively), and it was also significant in multivariable analysis with age/risk adjusted (P = 0.014, 0.016, and 0.045, respectively). NK cells < 4.8% of lymphocytes demonstrated significantly shorter relapse free survivals (RFS) in both univariate and multivariate analyses with risk adjusted (P = 0.006 and 0.037, respectively). AML patients showed significant lower numbers of CD56dimCD16+ NK cells, central memory T cells, and regulatory T cells than healthy controls at diagnosis. Higher proportion of helper/inducer T cells and CD4+CD31+ naïve T cells and decreased proportion of NK cells at diagnosis were independent factor of increasing probability of CR, and proportion of NK cells < 4.8% at diagnosis had adverse impact in RFS.

Remedying Airbnb COVID-19 disruption through tourism clusters and community resilience.

Peer-to-peer (P2P) accommodation markets have been disrupted by the COVID-19 pandemic. However, little attention is paid to how to remedy the disruption in terms of P2P accommodation performance. This study empirically investigates the spatially heterogeneous COVID-19 disruptions in the Airbnb business and offers place-based remedying strategies through local resources, including tourism clusters and community resilience. Using real data on Airbnb operating performance and local resources in Florida, we employ spatial econometric models and visualization techniques to estimate the pandemic-disrupted Airbnb performance model. The results show that leisure and hospitality clusters and three resilience resources-social, community capital, and environmental-had spatially heterogeneous effects on Airbnb revenue and booking performance across Floridian counties during the pandemic. Furthermore, community resilience moderated the effect of tourism clusters on Airbnb performance across individual and subclustered counties. These findings enable P2P accommodation hosts and policymakers to adopt destination-specific remedying strategies to cope with the pandemic.

Establishing Reference Range and Assessing Precision Performance of the TEG6s System; Verification of TEG6s to Introduce to Clinical Laboratory.

BACKGROUND: Thromboelastography (TEG) provides assessment of global coagulation. The TEG6s (Haemonetics Corporation) is a newly developed cartridge-based system, fully automated and the first true point-of-care TEG. In this study, we evaluated the precision and established the reference intervals (RIs) for TEG6s. METHODS: TEG assays were performed on the blood of healthy donors to determine RIs and on the QC materials for precision testing. The study design was developed in accordance with Clinical and Laboratory Standards Institute Guidelines. RESULTS: TEG6s precision testing yielded low variability except R, due to a low value for the mean. The newly established RIs of R, MA, and LY30 were similar to the manufacturer's RIs. Some were different, showing short K and increased α. CONCLUSIONS: This study has shown that TEG6s has high precision and each institution should verify the manufacturer's RIs before adopting TEG6s and establish RIs if necessary.

Clinical implications and genetic features of clonal cytopenia of undetermined significance compared to lower-risk myelodysplastic syndrome.

Clonal cytopenia of undetermined significance (CCUS) is characterized by persistent cytopenias with genetic aberrations, which do not meet the diagnostic criteria for myelodysplastic syndrome (MDS). We aimed to compare the clinical and genetic characteristics of CCUS with lower-risk MDS and identify patients with CCUS with a high risk of progression. We performed targeted sequencing of bone marrow (BM) samples from patients with idiopathic cytopenia of undetermined significance (ICUS) (n = 139) and MDS (n = 226). Overall survival (OS) of patients with CCUS (n = 78) was worse than non-clonal ICUS (n = 61) and superior to lower-risk MDS (n = 99). Patients with CCUS showed similar characteristics to those with lower-risk MDS, except for higher haemoglobin, lower BM cellularity, and less frequent SF3B1 mutations. Lower haemoglobin, DDX41 (biallelic germline and somatic), ETV6, and RUNX1 mutations were independent prognostic factors for worse OS. Lower haemoglobin and DDX41 mutations were also associated with lower progression-free survival. Patients with CCUS with high-risk features showed similar or worse OS than patients with lower-risk MDS. Our findings suggest that patients with CCUS having certain clinical or genetic features should be regarded and treated as lower-risk MDS despite lacking significant dysplasia or MDS-associated chromosomal abnormalities.

Clinical Application for Diagnosis of Myelodysplatic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis.

BACKGROUND: Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/ MPN-RS-T) was newly introduced as a full entity in the 2016 revision of the WHO classification. In this study, we investigated the morphologic, laboratory, and clinical features of MDS/MPN-RS-T. METHODS: We reviewed the bone marrow and genetic studies of patients whose diagnoses were coded as "refractory anemia with ring sideroblasts (RARS)" or "MDS/MPN, unclassifiable" between January 2008 and April 2018. RESULTS: A total of 8 cases fulfilled the criteria for a diagnosis of MDS/MPN-RS-T. All of them had no specific symptoms. Half of the cases had less than 450 × 109/L platelet counts by an automated hematology analyzer; however, all platelet counts exceeded 450 × 109/L when performed manually. JAK2 mutation tests were performed in 7 cases, and a heterozygous mutation was detected in 1 case. SF3B1 mutations were present in 3 of the 4 cases tested. CONCLUSIONS: When RARS is suspected in patients without thrombocytopenia, manual platelet counts should be performed. For patients with suspected essential thrombocythemia, RS evaluation through careful observation of an iron-stained slide is crucial. Since the independent evaluation of RS was reflected in the revised classification, the ambiguous disease classification becomes clearer and more consistent.