Evaluation of a tryptase depletion index for better pathologic identification of mast cell activation syndrome.

BACKGROUND: Laboratory evidence supporting diagnosis of the prevalent condition of mast cell activation syndrome (MCAS) currently includes elevated levels in blood or urine of mediators relatively specific to mast cells (MCs) and/or increased numbers of MCs in luminal gastrointestinal (GI) tract tissues. However, identification of elevated mediators is technically challenging and expensive, and controversy persists regarding the normal ranges of numbers/counts of MCs in various GI tract segments, let alone challenges in determining how many of the visualized MCs are activated. To aid diagnosis of MCAS, we developed a potential new approach for the pathologist to identify the extent of GI tract MC activation easily and inexpensively. PARTICIPANTS AND METHODS: Visualization of MCs in gastrointestinal biopsies from 251 patients vs. 95 controls using antibodies against CD117 and tryptase; MC counting per mm2; calculation of the difference between the CD117-positive MCs (identifying all MCs) vs. tryptase-positive MCs (identifying non-activated tryptase-containing MCs), which we define as the tryptase depletion index (TDI). RESULTS: Mean total MC counts did not differ significantly between patients and controls, but mean TDIs differed significantly. Non-overlapping confidence intervals at the 99.9% level identified cut-offs of TDIs between patients vs. controls of 26, 45 and 32 MCs/mm2 in gastric antrum, duodenum, and colon, respectively. CONCLUSIONS: The TDI may discriminate between MCAS patients vs. controls. If this preliminary work can be independently confirmed, the TDI may become a useful additional minor diagnostic criterion for MCAS.

Effects of Primary Mast Cell Disease on Hemostasis and Erythropoiesis.

Mast cell disease is an epigenetically and genetically determined disease entity with very diverse clinical manifestations in potentially every system and tissue due to inap pro priate release of variable subsets of mast cell mediators together with accumulation of either morphologically normal or altered mast cells. Easy bruising, excessive bleeding, and aberrancies of erythropoiesis can frequently be observed in patients with mast cell disease. A thorough history, including a family history, will guide the appropriate work-up, and laboratory evaluations may provide clues to diagnosis. In recent years, our understanding of the involvement of coagulation and anticoagulant pathways, the fibrinolytic system, and erythropoiesis in the pathophysiology of mast cell disease has increased considerably. This review summarizes current knowledge of the impact of the disturbed hemostatic and erythropoietic balance in patients with mast cell disease and describes options of treatment.

Small intestinal dysmotility in cirrhotic patients: correlation with severity of liver disease and cirrhosis-associated complications.

INTRODUCTION: Altered small intestinal motility has been observed in various manometry studies in patients with cirrhosis. Since small bowel manometry is available only in a few centers, interpretation of dysmotility in cirrhosis is controversial. PATIENTS AND METHODS: In this study, both fasting and postprandial manometric tracings of 24-hour antroduodenojejunal manometries were analyzed using both visual analysis and computer-aided analysis. RESULTS: In 34 patients (83 %), the mean migrating motor complex (MMC) cycle length was different compared with healthy controls. Phase II was prolonged in 27 patients (66 %), while phase I showed a reduced duration in 23 (56 %) and in phase III in 13 individuals (32 %). We also observed special motor patterns, e. g., migrating clustered contractions (MCCs) or retrograde clustered contractions (RCCs), which were present during fasting (69 %) and postprandial (92 %) motility, while none of the healthy controls showed any special motor patterns. Special motor patterns showed a significant correlation with the severity of cirrhosis (Child-Score; p > 0.05) and the existence of ascites (p < 0.05). DISCUSSION: This study in a large cohort of patients with cirrhosis by using 24-hour, solid state portable manometry showed in most individuals disturbances of cyclic fasting motility. Special motor patterns like RCCs during fasting and postprandial motility could be observed exclusively in the cirrhosis group, showing a significant correlation with severity of cirrhosis and the occurence of associated complications.

Laboratory Limitations of Excluding Hereditary Protein C Deficiency by Chromogenic Assay: Discrepancies of Phenotype and Genotype.

Protein C (PC) deficiency is associated with an increased risk for venous thromboembolism (VTE). In daily practice, exclusion of a hereditary PC deficiency is often based on a single determination of PC activity, by either clotting time-based or mostly chromogenic assay. However, diagnosis of hereditary PC deficiency is challenging due to several laboratory and clinical limitations. We compared the potential of PC activity values measured by either chromogenic or clotting time-based assay to predict a variation in the PROC gene. One hundred one (35%) of 287 patients carried variations within the PROC gene, including 2 previously not published variations. In 20 (20%) patients with identified variation, PC activity, determined by chromogenic assay, was within the reference range. For prediction of an underlying genetic defect determined by chromogenic and clotting time-based assay, sensitivity was 80% versus 99%, specificity 75% versus 18%, positive predictive value 64% versus 39%, and negative predictive value (NPV) 88% versus 97%. The lower NPV of chromogenic versus clotting time-based PC assay can be mainly explained by the presence of PC deficiency type IIb. Following our proposed diagnostic algorithm, additional measurement of PC activity by clotting time-based assay in case of a positive VTE history improves detection of this subtype of PC deficiency. Considering potential therapeutic consequences for primary and especially for secondary VTE prophylaxis, genetic analysis is required not only for confirmation but also for clarification of PC deficiency.

Evaluation of a semi-automated von Willebrand factor multimer assay, the Hydragel 5 von Willebrand multimer, by two European Centers.

BACKGROUND: The phenotypic diagnosis of von Willebrand disease (VWD) is a multistep process with classification dependent on the quantification of von Willebrand factor (VWF) multimeric structure. VWF multimer analysis is a technically challenging, lengthy and non-standardised assay, usually performed in specialist laboratories. Recently, a new semi-automated multimer assay, the Hydragel 5 von Willebrand multimers (H5VWM) has become available. OBJECTIVES: This study, performed in two European centres, compared existing in-house multimer assays to the H5VWM in individuals with and without VWD. RESULTS: Overall agreement of 91.1% was observed in 74 individuals with normal VWF levels, 57 patients grouped as type 1 VWD, 33 type 2A, 16 type 2B, 28 type 2M, 11 type 2N. Patients tested following Desmopressin or VWF concentrate, with thrombotic thrombocytopenic purpura and acquired von Willebrand syndrome were also evaluated. Many of the discrepancies between methods were in patients with genetic mutations linked to more than one type of VWD including p.R1374C/H and p.R1315C. Quantifiable multimer results were available within one working day. Densitometry improved the interpretation of the multimers with slight structural variations that were not apparent by visual inspection of the in-house method. CONCLUSIONS: 5VWM was a rapid, sensitive, standardised assay which used existing technology and could be included as an initial screen of VWF multimers in a VWD diagnostic algorithm in conjunction with traditional multimer analysis.

Monitoring of argatroban and lepirudin anticoagulation in critically ill patients by conventional laboratory parameters and rotational thromboelastometry - a prospectively controlled randomized double-blind clinical trial.

BACKGROUND: Argatroban or lepirudin anticoagulation therapy in patients with heparin induced thrombocytopenia (HIT) or HIT suspect is typically monitored using the activated partial thromboplastin time (aPTT). Although aPTT correlates well with plasma levels of argatroban and lepirudin in healthy volunteers, it might not be the method of choice in critically ill patients. However, in-vivo data is lacking for this patient population. Therefore, we studied in vivo whether ROTEM or global clotting times would provide an alternative for monitoring the anticoagulant intensity effects in critically ill patients. METHODS: This study was part of the double-blind randomized trial "Argatroban versus Lepirudin in critically ill patients (ALicia)", which compared critically ill patients treated with argatroban or lepirudin. Following institutional review board approval and written informed consent, for this sub-study blood of 35 critically ill patients was analysed. Before as well as 12, 24, 48 and 72 h after initiation of argatroban or lepirudin infusion, blood was analysed for aPTT, aPTT ratios, thrombin time (TT), INTEM CT,INTEM CT ratios, EXTEM CT, EXTEM CT ratios and maximum clot firmness (MCF) and correlated with the corresponding plasma concentrations of the direct thrombin inhibitor. RESULTS: To reach a target aPTT of 1.5 to 2 times baseline, median [IQR] plasma concentrations of 0.35 [0.01-1.2] µg/ml argatroban and 0.17 [0.1-0.32] µg/ml lepirudin were required. For both drugs, there was no significant correlation between aPTT and aPTT ratios and plasma concentrations. INTEM CT, INTEM CT ratios, EXTEM CT, EXTEM CT ratios, TT and TT ratios correlated significantly with plasma concentrations of both drugs. Additionally, agreement between argatroban plasma levels and EXTEM CT and EXTEM CT ratios were superior to agreement between argatroban plasma levels and aPTT in the Bland Altman analysis. MCF remained unchanged during therapy with both drugs. CONCLUSION: In critically ill patients, TT and ROTEM parameters may provide better correlation to argatroban and lepirudin plasma concentrations than aPTT. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00798525 , registered on 25 Nov 2008.

Monitoring of Argatroban and Lepirudin: What is the Input of Laboratory Values in "Real Life"?

Monitoring of direct thrombin inhibitors (DTIs) in patients with heparin-induced thrombocytopenia (HIT) is primarily performed using the activated partial thromboplastin time (aPTT). This assay is poorly standardized, reagent dependent, and not DTI specific. We compared aPTT, thrombin time (TT), and prothrombin time (PT) to drug levels obtained by the ecarin chromogenic assay (ECA). We analyzed 495 samples of patients with confirmed or suspected HIT on treatment with either argatroban (n = 37) or lepirudin (n = 80). Mean DTI levels ± standard deviation (SD) were 0.41 ± 0.36 µg/mL for argatroban and 0.20 ± 0.21 µg/mL for lepirudin. Results of aPTT were highly variable: 67 ± 22 seconds for argatroban and 55 ± 20 seconds for lepirudin. Significant correlations ( P < .01) were found between ECA-based DTI level and TT (argatroban, r = .820 and lepirudin, r = .830), PT (argatroban, r = -.544), and aPTT (lepirudin, r = .572). However, there was no correlation of aPTT with argatroban or PT with lepirudin concentration. Multiple regression analyses revealed that the TT predicted 54% of argatroban and 42% of lepirudin levels, but no significant impact was seen for PT or aPTT. The aPTT-guided monitoring of DTI therapy leads to a high percentage of patients with inaccurate plasma levels, hence resulting to either undertreatment or overtreatment. Knowledge of baseline values prior to DTI therapy and inclusion of clinical settings are essential for dosing DTIs when using aPTT. However, due to several limitations of aPTT, monitoring according to exact plasma concentrations as obtained by specific tests such as ECA may be more appropriate.

Lower prevalence of vitamin D insufficiency in German patients with systemic mast cell activation syndrome compared with the general population.

Introduction The common mastocytosis variant systemic mast cell activation syndrome (MCAS) may underlie at least a subset of patients with irritable bowel syndrome (IBS). A critical role of vitamin D (VD) in the stabilization of mast cells (MCs) with deficiency of VD resulting in MC activation has been demonstrated. If so, supplementation of VD would be a potential therapeutic approach in the treatment of those IBS patients. Methods We investigated in the present study for the first time systematically whether the VD level in 100 MCAS patients differed from that in the German general population (Ggp) and made a first attempt to elucidate potential reasons for possible differences by simultaneously determining the blood levels of heparin and cholesterol. Results In contrast to the Ggp, the VD level was detected in a sufficient range (> 30 ng/mL) in 53 % of the MCAS patients (Ggp 8 %), and only 34 % had values in the range of deficiency (< 20 ng/mL; GgP 75 %). There was no correlation between VD blood level and heparin and cholesterol blood levels. Conclusions The demonstration that in the majority of MCAS patients the VD level is not in a deficient range argues against an essential contribution of VD deficiency to the high prevalence of MCAS in Germany. Our findings do not exclude the possibility of smaller effects of VD level on MC activation in vivo. However, if such effects are present, the effect sizes seem to be too small to become identifiable in the multifactorial process of disease development.

High on-treatment platelet reactivity in transcatheter aortic valve implantation patients.

Dual antiplatelet therapy (DAPT) is recommended early after transcatheter aortic valve implantation (TAVI) procedure at the moment despite the lack of evidence. Two small randomized trials failed to demonstrate DAPT to be superior to aspirin alone in TAVI patients. However, it is known that there are substantial response variabilities to antiplatelet medication. We aimed to investigate high on-treatment platelet reactivity (HTPR), low on-treatment platelet reactivity (LTPR) to clopidogrel as well as HTPR to aspirin in patients undergoing TAVI procedure. We analyzed data of 140 TAVI patients in a real world observational study. Platelet function assays (clopidogrel-vasodilator-stimulated protein phosphorylation assay; aspirin-light-transmission aggregometry) have been performed during hospital course. Clinical complications were investigated during 30 days follow-up and defined using the valve academic research consortium standardized criteria. HTPR to clopidogrel occurred in 87 (62%) patients and LTPR in 9 (6.4%) patients. Aspirin antiplatelet effects were insufficient in 25 (18%) patients. Clinical complications were observed in 35 (25%) patients. Ischemic events occurred in 6 (4%), bleeding complications in 28 (20%) patients. There were no differences regarding the incidence of HTPR/LTPR in patients with overall complications, ischemic events or bleeding events. HTPR to clopidogrel is very frequent in TAVI patients. However bleeding complications are frequent and ischemic events are rare. Therefore, future clinical trials investigating the optimal antithrombotic regiment in TAVI patients should consider this high incidence of HTPR to clopidogrel and monitor clopidogrel antiplatelet effects carefully.

Multicentric validation of a rapid assay for heparin-induced thrombocytopenia with different specimen types.

The diagnosis of heparin-induced thrombocytopenia type 2 (HIT) requires a combination of clinical and laboratory data. Rapid exclusion of HIT is supported by sensitive immunoassays. Citrated plasma or whole blood are more often available in the laboratory, and may thus, have practical advantages as compared to serum. In this study, various specimens from patients with suspected HIT were compared using a rapid lateral flow immunoassay (LFI-HIT). Serum, citrated whole blood and plasma were obtained from patients in whom HIT was suspected (n = 211) and were tested with LFI-HIT and several other assays. The comparison of plasma vs. serum in 210 samples showed agreement of 93.6% and between whole blood and serum in 183 samples was 96.7%. All serum-positive cases were correctly detected in plasma. Comparison of samples, which were positive in one of the immunoassays with the functional test heparin-induced platelet aggregation assay revealed a negative predictive value of 98.4% for serum, 97.8% for plasma and 96.2% for citrated whole blood. Performance in whole blood was slightly inferior. These data demonstrate that plasma and serum can be used in LFI-HIT with equivalent performance and that negative test results in LFI-HIT exclude HIT with high probability. The use of whole blood is not recommended by the authors.

Methotrexate concentrations in cerebrospinal fluid and serum, and the risk of central nervous system relapse in children with acute lymphoblastic leukaemia.

The aim of the study was to characterize the relationship between the pharmacokinetics of methotrexate in serum and concentrations in the cerebrospinal fluid, and to analyse the association to risk of a central nervous system relapse in children with acute lymphoblastic leukaemia. In this retrospective study, 353 children with acute lymphoblastic leukaemia treated with high-dose methotrexate according to the Nordic Society of Pediatric Haematology and Oncology-92 acute lymphoblastic leukaemia protocol were studied. Data from 18 patients with a subsequent central nervous system relapse and 335 event-free patients were available. In 34 patients the methotrexate concentrations were monitored repeatedly during each 24-h methotrexate intravenous infusion and a cerebrospinal fluid sample was taken at the end of the infusion. Using statistics separating interindividual and intraindividual variability, methotrexate concentration in cerebrospinal fluid was found to be significantly dependent upon both serum concentrations at the end of infusion and the area under the concentration curve in serum (P<0.0017 and <0.002, respectively). The logistic regression analysis revealed that high patient median serum methotrexate concentrations at the end of infusion were significantly associated with decreased risk for a central nervous system relapse in the standard risk group (P=0.02) and the number of courses with a calculated cerebrospinal fluid concentration of more than 1 mumol/l (P=0.048) with a decreased risk of a central nervous system relapse in the combined (standard risk and intermediate) risk group. In conclusion, methotrexate concentrations in cerebrospinal fluid are dependent on methotrexate concentrations in serum and serum area under the concentration curve. Multivariate analysis indicates that an increased exposure to methotrexate is related to decreased risk for central nervous system relapse.