Serial measurement of circulating calprotectin as a prognostic biomarker in COVID-19 patients in intensive care setting.

OBJECTIVES: Circulating calprotectin (cCLP) has been shown to be a promising prognostic marker for COVID-19 severity. We aimed to investigate the prognostic value of serial measurements of cCLP in COVID-19 patients admitted to an intensive care unit (ICU). METHODS: From November 2020 to May 2021, patients with COVID-19, admitted at the ICU of the OLV Hospital, Aalst, Belgium, were prospectively included. For sixty-six (66) patients, blood samples were collected at admission and subsequently every 48 h during ICU stay. On every sample (total n=301), a cCLP (EliA™ Calprotectin 2, Phadia 200, Thermo Fisher Scientific; serum/plasma protocol (for Research Use Only, -RUO-) and C-reactive protein (CRP; cobas c501/c503, Roche Diagnostics) analysis were performed. Linear mixed models were used to associate biomarkers levels with mortality, need for mechanical ventilation, length of stay at ICU (LOS-ICU) and medication use (antibiotics, corticosteroids, antiviral and immune suppressant/modulatory drugs). RESULTS: Longitudinally higher levels of all biomarkers were associated with LOS-ICU and with the need for mechanical ventilation. Medication use and LOS-ICU were not associated with variations in cCLP and CRP levels. cCLP levels increased significantly during ICU hospitalization in the deceased group (n=21/66) but decreased in the non-deceased group (n=45/66). In contrast, CRP levels decreased non-significantly in both patient groups, although significantly longitudinally higher CRP levels were obtained in the deceased subgroup. CONCLUSIONS: Serial measurements of cCLP provides prognostic information which can be useful to guide clinical management of COVID-19 patients in ICU setting.

Resolving DOAC interference on antithrombin activity testing on a FXa based method by the use of activated carbon.

BACKGROUND: Direct oral anticoagulants (DOACs) may cause falsely increased levels of antithrombin (AT) activity depending on the AT activity method and the specific target of the DOAC. Activated carbon (AC) has proven to remove DOAC interference on PT, aPTT and LA assays. We evaluate whether AC could be useful to resolve DOAC interference on AT assays. METHODS: Normal pooled plasma (NPP) was diluted to obtain AT activity of 25 %, 50 % and 75 % respectively. The diluted NPPs were spiked with DOACs (apixaban, edoxaban, dabigatran and rivaroxaban) in concentrations of respectively 100, 250 and 500 ng/ml. DOAC concentrations and AT activity were tested at baseline and after treatment with 20 mg/ml AC. AT activity was measured with a FXa-based method (HemosIL Liquid Antithrombin®, Werfen). RESULTS: All DOAC concentrations were below the limit of quantification (LoQ) after addition of AC. DOAC interference on AT activity testing was removed by adding AC, resulting in correctly diagnosing low levels of AT for all dilutions. The influence of DOACs on AT activity was directly correlated to the concentration of the DOAC. As expected, only the anti-FXa DOACs influenced the used assay. CONCLUSIONS: AC effectively removes anti-FXa DOAC interference on FXa-based AT assays.

Saline-induced coronary hyperemia with continuous intracoronary thermodilution is mediated by intravascular hemolysis.

BACKGROUND AND AIMS: Absolute coronary flow can be measured by intracoronary continuous thermodilution of saline through a dedicated infusion catheter (RayFlow®). A saline infusion rate at 15-20 mL/min induces an immediate, steady-state, maximal microvascular vasodilation. The mechanism of this hyperemic response remains unclear. We aimed to test whether local hemolysis is a potential mechanism of saline-induced coronary hyperemia. METHODS: Twelve patients undergoing left and right catheterization were included. The left coronary artery and the coronary sinus were selectively cannulated. Absolute resting and hyperemic coronary flow were measured by continuous intracoronary thermodilution. Arterial and venous samples were collected from the coronary artery and the coronary sinus in five phases: baseline (BL); resting flow measurement (Rest, saline infusion at 10 mL/min); hyperemia (Hyperemia, saline infusion at 20 mL/min); post-hyperemia (Post-Hyperemia, 2 min after the cessation of saline infusion); and control phase (Control, during infusion of saline through the guide catheter at 30 mL/min). RESULTS: Hemolysis was visually detected only in the centrifugated venous blood samples collected during the Hyperemia phase. As compared to Rest, during Hyperemia both LDH (131.50 ± 21.89 U/dL [Rest] and 258.33 ± 57.40 U/dL [Hyperemia], p < 0.001) and plasma free hemoglobin (PFHb, 4.92 ± 3.82 mg/dL [Rest] and 108.42 ± 46.58 mg/dL [Hyperemia], p < 0.001) significantly increased in the coronary sinus. The percentage of hemolysis was significantly higher during the Hyperemia phase (0.04 ± 0.02% [Rest] vs 0.89 ± 0.34% [Hyperemia], p < 0.001). CONCLUSIONS: Saline-induced hyperemia through a dedicated intracoronary infusion catheter is associated with hemolysis. Vasodilatory compounds released locally, like ATP, are likely ultimately responsible for localized microvascular vasodilation.

Prognostic value of circulating calprotectin levels on the clinical course of COVID-19 differs between serum, heparin, EDTA and citrate sample types.

INTRODUCTION: During the recent SARS-CoV-2 pandemic, circulating calprotectin (cCLP) gained interest as biomarker to predict the severity of COVID-19. We aimed to investigate the prognostic value of cCLP measured in serum, heparin, EDTA and citrate plasma. MATERIALS AND METHODS: COVID-19 patients were prospectively included, in parallel with two SARS-CoV-2 negative control populations. The prognostic value of cCLP was compared with IL-6, CRP, LDH, procalcitonin, and the 4C-mortality score by AUROC analysis. RESULTS: For the 136 COVID-19 patients, cCLP levels were higher compared to the respective control populations, with significantly higher cCLP levels in serum and heparin than in EDTA or citrate. Higher cCLP levels were obtained for COVID-19 patients with i) severe/critical illness (n = 70), ii) ICU admission (n = 66) and iii) need for mechanical ventilation/ECMO (n = 25), but iv) not in patients who deceased within 30 days (n = 41). The highest discriminatory power (AUC [95% CI]) for each defined outcome was i) CRP (0.835 [0.755-0.914]); ii) EDTA cCLP (0.780 [0.688-0.873]); iii) EDTA cCLP (0.842 [0.758-0.925]) and iv) the 4C-mortality score (0.713 [0.608-0.818]). CONCLUSION: Measuring cCLP in COVID-19 patients helps the clinician to predict the clinical course of COVID-19. The discriminatory power of EDTA and citrate plasma cCLP levels often outperforms heparin plasma cCLP levels.

Resolving DOAC interference on aPTT, PT, and lupus anticoagulant testing by the use of activated carbon.

BACKGROUND: Direct oral anticoagulants (DOACs) affect laboratory coagulations tests. Activated carbon (AC) can be used for adsorption of DOACs during acute human intoxications. OBJECTIVES: This study evaluates whether AC can also be used to resolve DOAC interference on in vitro clotting tests (prothrombin time [PT], activated partial thromboplastin time [aPTT], and lupus anticoagulant [LA] assays). PATIENTS/METHODS: Interference on PT, aPTT, Liquid anti-FXa, DTI, and LA screening/confirmation (SCT and dRVVT) was determined by spiking citrated plasma from 5 adult controls with 0, 20, 40, 80, 120, or 160 mg/mL AC. DOAC concentrations, PT, and aPTT were compared before and after AC addition to citrated plasma from patients receiving DOACs (n = 29), low molecular weight heparin (n = 10), and coumarin (n = 10) therapy. Samples from 69 LA screened patients were compared before and after AC addition. RESULTS: A concentration of 20 mg/mL AC had the lowest interference and was selected for further experiments. After AC addition, all DOAC concentrations were below the limit of quantification in the 29 treated patients, except for 2 apixaban samples. AC removed DOAC interference on PT and aPTT but had no impact on results obtained during coumarin or low molecular weight heparin therapy. Of 15 LA samples with interference resulting from DOAC therapy, 14 samples became negative and 1 positive after AC addition. Interference from coumarin therapy was not resolved. All 19 LA negative samples remained negative. AC treatment of the negative pooled plasma was required to avoid false-negative LA results in 21 known LA-positive samples. CONCLUSIONS: AC selectively removes DOAC interference on PT, aPTT, and LA assays.

Analytical performance evaluation of four cartridge-type blood gas analyzers.

BACKGROUND: The immediate impact of blood gas test results on patient care favors the use of blood gas analyzers as point-of-care-testing (POCT) devices. We performed an analytical performance evaluation of four cartridge-type blood gas analyzers for the determination of pH, partial carbon dioxide pressure (pCO2), partial oxygen pressure (pO2), ionized calcium (iCa2+), potassium (K+), glucose, lactate and total hemoglobin (tHb), in comparison with a traditional blood gas analyzer. METHODS: The analyzers included in the study are: RP405, GEM Premier 4000, ABL90 FLEX and Cobas b 123. The ABL700 served as comparator. For each instrument the imprecision was estimated according to the CLSI EP5-A2. Based on the CLSI EP9-A2 evaluation protocol, a method comparison was performed using patient samples. Obtained data were compared against preset quality specifications, based on ABL700 performance and biological variation. RESULTS: The precision of the RP405 and ABL90 FLEX was remarkably better than the preset criteria based on ABL700 performance. The GEM appears to have the worst precision. The RP405 yielded the best overall performance, with exception for tHb and iCa2+. Noteworthy is the very good performance of the glucose determination on RP405. The ABL90 FLEX showed the best performance for pH, K+ and iCa2+ measurements. For tHb determination the Cobas b123 revealed the best results. Regarding practicability, all instruments were found to be user friendly. CONCLUSIONS: Globally, all four cartridge-type blood gas analyzers produced clinically acceptable results. The analytical performance, together with the ease of use and low maintenance time of the instruments, demonstrate that these analyzers are perfectly suitable for both POCT and laboratory use.

Chronic lymphocytic leukemia and prolymphocytic leukemia with MYC translocations: a subgroup with an aggressive disease course.

Translocations involving MYC are rare in chronic lymphocytic leukemia (CLL), and up to now, their prognostic significance remains unclear. We report the characteristics of 21 patients with CLL and nine patients with prolymphocytic leukemia (PLL), diagnosed in multiple centers (n = 13), which showed an MYC translocation demonstrated by fluorescence in situ hybridization. The prevalence was estimated to be <1%. Advanced age and male predominance were observed. Morphological analysis frequently revealed the presence of prolymphocytes. A typical "CLL-immunophenotype" was found in four of nine cases with PLL. Moreover, CD5 and CD23 were frequently expressed in PLL. The latter findings are atypical for PLL and may suggest transformation or progression of an underlying CLL. MYC translocations were frequently observed with concomitant adverse cytogenetic markers, such as del(11q) (n = 8/30) and/or del(17p)/monosomy 17 (n = 7/30). In addition, the presence of unbalanced translocations (n = 24 in 13/30 cases) and complex karyotype (n = 16/30) were frequent in cases with MYC translocations. Altogether, del(17p)/monosomy 17, del(11q), and/or complex karyotype were observed in 22 of 30 patients. Survival outcome was poor: the median time to treatment was only 5 months, and overall survival (OS) from clinical diagnosis and from genetic detection was 71 and 19 months, respectively. In conclusion, CLL/PLL with MYC translocations is a rare entity, which seems to be associated with adverse prognostic features and unfavorable outcome.

Relationship between peripheral arterial reactive hyperemia and residual platelet reactivity after 600 mg clopidogrel.

Clopidogrel reduces long-term ischemic events in patients with acute coronary syndrome or stable angina (SA) undergoing percutaneous coronary intervention (PCI). Endothelial function improvement has been proposed, among other factors, for this beneficial effect of clopidogrel, but whether this might be associated to its anti-platelet action remains unclear. We tested the hypothesis that clopidogrel improvement of peripheral vascular endothelial function might be associated with inhibition of platelet aggregation. Endothelial function was evaluated before and at least 12 h after 600 mg clopidogrel in 43 SA pts undergoing elective PCI by: (a) reactive hyperemia peripheral arterial tonometry (measuring the Endoscore); (b) circulating endothelial microparticles (EMPs). Response to clopidogrel was measured with point-of-care VerifyNow P2Y12 assay and expressed as platelet reaction unit (PRU) and percent platelet inhibition (%PI). High platelet reactivity after clopidogrel was defined as PRU ≥ 240. Endothelial function improved after clopidogrel in 20 pts. Changes in Endoscore (Δ Endoscore) were significantly correlated with both PRU (r = -0.61, P < 0.001) and %PI (r = 0.57, P < 0.001). Endoscore significantly increased after clopidogrel in pts with PRU < 240 (0.38 ± 0.26 to 0.57 ± 0.33, P < 0.001), but did not in pts with PRU ≥ 240 (0.53 ± 0.31 to 0.40 ± 0.37, P = 0.12). EMPs were also significantly reduced in pts with PRU < 240 (222 [140-593] to 142 [83-371]/µl, P = 0.001), while no changes were observed in pts with PRU ≥ 240 (256 [178-531] to 388 [238-499]/µl, P = 0.55). In patients with stable coronary artery disease, a single 600 mg clopidogrel loading dose improves vascular endothelial function. This improvement is associated with optimal platelet inhibition and it is not observed in patients with post-clopidogrel high platelet reactivity.

The kinase inhibitor TKI258 is active against the novel CUX1-FGFR1 fusion detected in a patient with T-lymphoblastic leukemia/lymphoma and t(7;8)(q22;p11).

We report a patient with T-lymphoblastic leukemia/lymphoma and a t(7;8)(q22;p11). CUX1 was identified as the fusion partner of FGFR1 by fluorescence in situ hybridization and 5' RACE-PCR. We further investigated this novel FGFR1 fusion using the interleukin-3 (IL-3) dependent Ba/F3 cell line and demonstrated IL-3 independent cell growth of CUX1-FGFR1 expressing cells. TKI258 and PKC412 potently inhibited proliferation of CUX1-FGFR1 transformed Ba/F3 cells. This growth inhibition was shown to be mediated by inhibition of CUX1-FGFR1 kinase activity for TKI258 but not PKC412. In summary, we identified a novel CUX1-FGFR1 fusion oncogene in a patient with the 8p11 myeloproliferative syndrome and demonstrated its transforming potential in the Ba/F3 cell line. Our in vitro data support the further investigation of TKI258 for the treatment of constitutively active FGFR1 fusion proteins.