Performance evaluation of the new Sysmex XR-Series haematology analyser.

Background: The new XR-Series haematology analyser from Sysmex provides increased throughput and automation, along with a new reagent in WDF channel for optimised WBC differential. Methods: An analytical performance study for the XR analyser was conducted to evaluate the WDF channel parameters in comparison to the instrument specifications. Additionally, 7460 samples were measured on XR and XN analysers to compare selected parameters and flags, and 930 randomly selected samples were further evaluated with microscopy. Results: All investigated aspects of the analytical performance study for the XR fell within the manufacturer specifications. The correlation coefficients between the two systems for the parameters tested were greater than 0.983 for the main CBC and DIFF parameters, greater than 0.909 for the Extended Inflammation Parameters, and greater than 0.932 for the parameters used in the workflow rulesets of the Extended IPU. Similarly high sensitivities for the detection of abnormal cells were observed for the 'Blasts/Abn Lympho?' flag (XN: 100%, XR: 99.0%) and WPC abnormal flags ('Blasts?' or 'Abn Lympho?') (XN: 97.0%, XR: 96.0%). XN with WPC channel had a 26% reduction of false positive smears compared to XR with 22% reduction, a statistically non-significant difference. Conclusion: The XR analyser had very good analytical performance, and highly comparable results to the predecessor XN analyser in all investigated parameters, flags and workflow aspects.

Automated measurement of malaria parasitaemia among asymptomatic blood donors in Malawi using the Sysmex XN-31 analyser: could such data be used to complement national malaria surveillance in real time?

BACKGROUND: The recent worldwide increase in malaria cases highlights the need for renewed efforts to eliminate malaria. The World Health Organization advocates that malaria surveillance becomes a core intervention. Current methods to estimate the malaria burden rely on clinical malaria case reports and surveys of asymptomatic parasite infection mainly from children < 5 years. In this study the hypothesis was that screening blood donors for malaria parasites would provide real-time information on the asymptomatic reservoir of parasites in the adult population and mirror other surveillance data. METHODS: This study was conducted in Malawi, a high malaria burden country, at the Malawi Blood Transfusion Service, which collects blood units at donation sites countrywide. A secondary analysis was conducted on data obtained from a prior Sysmex XN-31 analyser malaria diagnostic evaluation study utilizing residual donor blood samples. XN-31 malaria results, donor age, sex, geographical location, and collection date, were analysed using standard statistical methods. RESULTS: The malaria parasite prevalence in blood donors was 11.6% (614/5281 samples) increasing seasonally from December (8.6%) to April (18.3%). The median age was 21 years and 45.9% of donors were from urban areas, which showed a lower prevalence compared to non-urban regions. The Central administrative region had the highest and the Northern region the lowest malaria parasite prevalence. The donors were predominantly male (80.2%), 13.1% of whom had malaria parasites, which was significantly higher (p < 0.0001) than for female donors (7.4%). Multivariable logistic regression analysis showed that age, location, and collection month were significant predictors of malaria positivity in males, whereas in females only location was significant. There was no gender difference in parasite density nor gametocyte carriage. CONCLUSIONS: This study demonstrates the powerful utility of screening blood donors for malaria parasites using the XN-31, which not only improves the safety of blood transfusion, but provides valuable complementary surveillance data for malaria control, especially targeting males, who are generally excluded from periodic household surveys. Blood donations are sourced countrywide, year-round, and thus provide dynamic, real-time information on the malaria burden. Furthermore, the XN-31 identifies the asymptomatic human reservoir of infectious gametocytes, which must be targeted to eliminate malaria.

Residual red cells in blood components: A multisite study of fully automated enumeration using a hematology analyzer.

BACKGROUND: Manufacture of platelet concentrates (PCs) and plasma may fail to remove all residual red blood cells (rRBCs). Measuring rRBCs for compliance to guidelines has proven challenging, leading to an absence of a consensus methodology. Sysmex hematology analyzers with the Blood Bank mode (BB mode) analysis option offer the potential for automated rRBC counting. We therefore performed a two-site appraisal of the system. STUDY DESIGN AND METHODS: Performance characteristics were determined using platelet and plasma samples spiked with RBCs. Sample stability (n = 47) and the impact of sample type were also assessed. Components (platelets, n = 1474; plasma, n = 77) prepared using different routine manufacturing methods were tested to assess variation in rRBC concentration. RESULTS: Linearity studies up to 19 000 RBCs/µL demonstrated good correlation between expected and observed results (R2 ≥ 0.9731), and flow cytometric results also correlated well with BB mode (R2 = 0.9400). Precision analysis gave a limit of quantitation of 6 to 7 RBCs/µL, and carryover was 0.03%. Ethylenediaminetetraacetic acid and plain tube results were not significantly different (P ≥ 0.10), and samples were stable up to 24 hours. Apheresis PCs produced at two sites had lower rRBC concentrations (medians, 17 and 13 RBCs/µL) than those produced with the buffy coat method either manually (median, 681 RBCs/µL) or with the automated Terumo Automated Centrifuge and Separator Integration process (median, 81 RBCs/µL). All PCs failing visual inspection as having RBCs ≥4000 RBCs/µL were also detected by the BB mode. CONCLUSION: The BB mode had acceptable performance characteristics and has the potential for integration into a fully automated process control system for rRBC enumeration in plasma and PCs.

A novel haemocytometric COVID-19 prognostic score developed and validated in an observational multicentre European hospital-based study.

COVID-19 induces haemocytometric changes. Complete blood count changes, including new cell activation parameters, from 982 confirmed COVID-19 adult patients from 11 European hospitals were retrospectively analysed for distinctive patterns based on age, gender, clinical severity, symptom duration, and hospital days. The observed haemocytometric patterns formed the basis to develop a multi-haemocytometric-parameter prognostic score to predict, during the first three days after presentation, which patients will recover without ventilation or deteriorate within a two-week timeframe, needing intensive care or with fatal outcome. The prognostic score, with ROC curve AUC at baseline of 0.753 (95% CI 0.723-0.781) increasing to 0.875 (95% CI 0.806-0.926) on day 3, was superior to any individual parameter at distinguishing between clinical severity. Findings were confirmed in a validation cohort. Aim is that the score and haemocytometry results are simultaneously provided by analyser software, enabling wide applicability of the score as haemocytometry is commonly requested in COVID-19 patients.

The use of a hematology analyzer with a new generation of software as an alternative to flow cytometry for enumerating residual white blood cells in blood components.

BACKGROUND: Leukoreduction of blood components was implemented to reduce transfusion-associated risks. The detection level for residual white blood cells (rWBCs) required to demonstrate leukoreduction was originally considered too low for hematology analyzers. Developments enabling cell counts in body fluids have, however, renewed interest in rWBC counting. An assessment of Sysmex XN hematology analyzers with software offering automated rWBC enumeration intended for use on blood components was performed. STUDY DESIGN AND METHODS: Performance characteristics were determined using platelet, red blood cell (RBC), and plasma samples spiked with WBCs. Subsequently, components (platelets, n = 1367; and plasma, n = 80) were tested and results compared with flow cytometry, to monitor leukoreduction efficiency to a level of less than 1 × 106 /unit. Components identified by flow cytometry as having poor leukoreduction, exceeding this limit, were also tested (platelets, n = 3; and RBCs, n = 10). RESULTS: Linearity studies up to 32 WBCs/µL showed good correlation between observed and expected results (R2 > 0.9996). Precision analysis gave an average limit of quantitation of 2 WBCs/µL with coefficients of variation less than 20%. Average carryover was 0.1%. Plain sample tubes were a source of aberrant results with routine components. Using ethylenediaminetetraacetic acid tubes the analyzer gave results greater than 1 × 106 /unit in 2.7% of cases compared with 1.4% by flow cytometry, but overall results were within specification, with more than 90% of components having rWBC values below the limit. All incidences of poor leukoreduction, with flow cytometry results greater than 13 rWBCs/µL were correctly identified, with an excellent correlation between results (R2 = 0.9818). CONCLUSION: The analyzer demonstrated acceptable performance characteristics for enumeration of rWBCs; consequently, additional multisite evaluations are warranted.

Therapeutic combination of radiolabeled CLR1404 with external beam radiation in head and neck cancer model systems.

BACKGROUND AND PURPOSE: CLR1404 is a phospholipid ether that exhibits selective uptake and retention in malignant tissues. Radiolabeled CLR1404 enables tumor-specific positron-emission tomography (PET) imaging ((124)I) and targeted delivery of ionizing radiation ((131)I). Here we describe the first preclinical studies of this diapeutic molecule in head and neck cancer (HNC) models. MATERIAL AND METHODS: Tumor-selective distribution of (124)I-CLR1404 and therapeutic efficacy of (131)I-CLR1404 were tested in HNC cell lines and patient-derived xenograft tumor models. Monte Carlo dose calculations and (124)I-CLR1404 PET/CT imaging were used to examine (131)I-CLR1404 dosimetry in preclinical HNC tumor models. RESULTS: HNC tumor xenograft studies including patient-derived xenografts demonstrate tumor-selective uptake and retention of (124)I-CLR1404 resulting in a model of highly conformal dose distribution for (131)I-CLR1404. We observe dose-dependent response to (131)I-CLR1404 with respect to HNC tumor xenograft growth inhibition and this effect is maintained together with external beam radiation. CONCLUSIONS: We confirm the utility of CLR1404 for tumor imaging and treatment of HNC. This promising agent warrants further investigation in a developing phase I trial combining (131)I-CLR1404 with reduced-dose external beam radiation in patients with loco-regionally recurrent HNC.

"Aged sample" software on automated routine hematology analyzer enables differentiation between pathological and non-pathological WBC flagging in aging samples.

BACKGROUND: Storing K(x)EDTA-conjugated blood samples at room temperature or under insufficient cooling conditions results in various morphological changes such as swelling of the blood cells. These changes are reproducible and have already been described well. However, they can lead to incorrect flagging when using automated hematology analyzers for complete blood counts and white blood cell differentials. The aim of this study was to determine if those changes can be detected automatically and used to prevent false positive flagging. METHODS: 150 blood samples were aged under controlled conditions and the impact on the "Aged sample" software was checked retrospectively. The results were verified in a second retrospective study including 6288 routine samples. RESULTS: When tested in a routine laboratory, the "Aged sample" software was able to reduce overall flagging by 23% without increasing false negative flagging. CONCLUSIONS: The "Aged sample" software of XN-Series analyzers does not only detect and flag samples that are aging or were stored under suboptimal conditions but also prevents false positive flagging.

Sym004, a novel anti-EGFR antibody mixture, augments radiation response in human lung and head and neck cancers.

Sym004 represents a novel EGF receptor (EGFR)-targeting approach comprising a mixture of two anti-EGFR antibodies directed against distinct epitopes of EGFR. In contrast with single anti-EGFR antibodies, Sym004 induces rapid and highly efficient degradation of EGFR. In the current study, we examine the capacity of Sym004 to augment radiation response in lung cancer and head and neck cancer model systems. We first examined the antiproliferative effect of Sym004 and confirmed 40% to 60% growth inhibition by Sym004. Using clonogenic survival analysis, we identified that Sym004 potently increased cell kill by up to 10-fold following radiation exposure. A significant increase of γH2AX foci resulting from DNA double-strand breaks was observed in Sym004-treated cells following exposure to radiation. Mechanistic studies further showed that Sym004 enhanced radiation response via induction of cell-cycle arrest followed by induction of apoptosis and cell death, reflecting inhibitory effects on DNA damage repair. The expression of several critical molecules involved in radiation-induced DNA damage repair was significantly inhibited by Sym004, including DNAPK, NBS1, RAD50, and BRCA1. Using single and fractionated radiation in human tumor xenograft models, we confirmed that the combination of Sym004 and radiation resulted in significant tumor regrowth delay and superior antitumor effects compared with treatment with Sym004 or radiation alone. Taken together, these data reveal the strong capacity of Sym004 to augment radiation response in lung and head and neck cancers. The unique action mechanism of Sym004 warrants further investigation as a promising EGFR targeting agent combined with radiotherapy in cancer therapy.

Inactivation of HNSCC cells by 90Y-labeled cetuximab strictly depends on the number of induced DNA double-strand breaks.

UNLABELLED: Radioimmunotherapy is considered to have great potential for efficient and highly specific treatment of tumors. The aim of this study was to determine the efficacy of radioimmunotherapy when using (90)Y-labeled cetuximab and to determine to what degree induction and repair of DNA double-strand breaks (DSBs) are decisive for this approach. METHODS: This study was performed with 9 cell lines of squamous cell carcinoma of the head and neck (HNSCC) differing strongly in epidermal growth factor receptor (EGFR) expression. The radionuclide (90)Y was coupled by the chelator trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA)/linker construct to the EGFR-directed antibody cetuximab to yield (90)Y-Y-CHX-A″-DTPA-cetuximab with a specific activity of approximately 1.2 GBq/mg. EGFR expression was determined by immunofluorescence and Western blotting, cetuximab binding by fluorescence-activated cell sorter analysis, the number of DSBs by immunofluorescence staining γH2AX/53BP1-positive repair foci, and cell survival by colony formation. RESULTS: For the 9 HNSCC cell lines, cetuximab binding correlated with the amount of EGFR present in the cell membrane (r(2) = 0.967, P < 0.001). When cells were exposed to (90)Y-Y-CHX-A″-DTPA-cetuximab, the number of induced DSBs increased linearly with time (r(2) = 0.968, P = 0.016). This number was found to correlate with the amount of membranous EGFR (r(2) = 0.877, P = 0.006). Most DSBs were repaired during incubation at 37°C, but the small number of remaining DSBs still correlated with the amount of membranous EGFR (24 h: r(2) = 0.977, P < 0.001; 48 h: r(2) = 0.947, P < 0.001). Exposure to (90)Y-Y-CHX-A″-DTPA-cetuximab also resulted in efficient cell killing, whereby the extent of cell killing correlated strongly with the respective number of remaining DSBs (r(2) = 0.989, P < 0.001) and with the amount of membranous EGFR (r(2) = 0.967, P < 0.001). No cell killing was observed for UTSCC15 cells with low EGFR expression, in contrast to the strong reduction of 86% measured for UTSCC14 cells showing a strong overexpression of EGFR. CONCLUSION: (90)Y-Y-CHX-A″-DTPA-cetuximab affected cell survival through the induction of DSBs. This treatment was especially efficient for HNSCC cells strongly overexpressing EGFR, whereas no effect was seen for cells with low levels of EGFR expression. Therefore, EGFR-directed radioimmunotherapy using (90)Y-Y-CHX-A″-DTPA-cetuximab appears to be a powerful tool that can be used to inactivate tumors with strong EGFR overexpression, which are often characterized by a pronounced radioresistance.

Dual targeting of EGFR and HER3 with MEHD7945A overcomes acquired resistance to EGFR inhibitors and radiation.

EGF receptor (EGFR) inhibition is efficacious in cancer therapy, but initially sensitive tumors often develop resistance. In this study, we investigated the potential to overcome acquired resistance to EGFR inhibitors with MEHD7945A, a monoclonal antibody that dually targets EGFR and HER3 (ErbB3). In cancer cells resistant to cetuximab and erlotinib, we found that MEHD7945A, but not single target EGFR inhibitors, could inhibit tumor growth and cell-cycle progression in parallel with EGFR/HER3 signaling pathway modulation. MEHD7945A was more effective than a combination of cetuximab and anti-HER3 antibody at inhibiting both EGFR/HER3 signaling and tumor growth. In human tumor xenograft models, we confirmed the greater antitumor potency of MEHD7945A than cetuximab or erlotinib. MEHD7945A retained potent activity in tumors refractory to EGFR inhibitor alone. Furthermore, MEHD7945A also limited cross-resistance to radiation in EGFR inhibitor-resistant cells by modulating cell-cycle progression and repair processes that control apoptotic cell death. Taken together, our findings confirm an important role of compensatory HER3 signaling in the development of acquired resistance to EGFR inhibitors and offer preclinical proof-of-concept that MEHD7945A can effectively overcome EGFR inhibitor resistance.

Cellular and tumor radiosensitivity is correlated to epidermal growth factor receptor protein expression level in tumors without EGFR amplification.

PURPOSE: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. METHODS AND MATERIALS: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. RESULTS: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. CONCLUSIONS: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

The epidermal growth factor receptor modulates DNA double-strand break repair by regulating non-homologous end-joining.

In mammalian cells repair of radiation-induced DNA damage appears to be also controlled by the epidermal growth factor receptor (EGFR) with a special impact on DNA double-strand break (DSB) repair. Aim of this study was to demonstrate this interaction between EGFR signalling and DNA DSB repair and to identify the underlying downstream pathways. We especially wanted to know in how far non-homologous end-joining (NHEJ) as the most important DSB repair pathway is involved in this interaction. Overall DSB repair was determined by counting gammaH2AX foci remaining 24 after irradiation, while NHEJ activity was monitored by using a specially designed repair construct stably integrated into the genome. The overall DSB repair capacity was clearly enhanced when EGFR was activated by its natural ligand EGF and, vice versa, was reduced when EGFR was blocked either by the specific antibody Cetuximab or the tyrosine kinase inhibitor erlotinib, whereby reduction was clearly stronger for erlotinib. There was also a difference in the pathways affected. While erlotinib lead to a block of both, MAPK as well as AKT signalling, Cetuximab only affected MAPK. As demonstrated by specific inhibitors (PD98059, AKTIII) EGFR interacts with DSB repair mostly via MAPK pathway. Also for NHEJ activity, there was a substantial increase, when EGFR was activated by EGF as determined for two different reporter cell lines (A549.EJ and H1299.EJ) and, vice versa, a reduction was seen when EGFR signalling was blocked by Cetuximab or erlotinib. There was, however, no difference for the two inhibitors used. This regulation of NHEJ by EGFR was only blocked when ERK was affected by siRNA but not when AKT was knocked down. These data indicate that EGFR modulates DSB repair by regulating NHEJ via MAPK signalling.