Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban.

The effect of direct oral anticoagulants (DOACs) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa (FXa), is a well-known problem and can cause both false positive and negative results. Therefore, the correct interpretation of tests performed in patients receiving DOACs is necessary to avoid misclassification and subsequent clinical consequences. However, even with significant experience, there are situations where it is not possible to assess the influence of some methods. Particularly important is the situation in the diagnosis of lupus anticoagulants using the dilute Russell viper venom timetest, which is based on direct FXa activation. A very promising solution to this situation is offered by the DOAC laboratory balancing procedure DOAC-Stop. For evaluating the effectiveness of this procedure, 60 (20 apixaban, 20 dabigatran, and 20 rivaroxaban) patients treated with DOACs were enrolled. All patient samples were analyzed for the presence of individual DOAC types and subsequently subjected to the DOAC-Stop procedure.We evaluated its effectiveness by our own high-performance liquid chromatography-coupled tandem mass spectrometrymethod, which simultaneously sets all high-sensitivity DOACs. Unlike coagulation tests based on the determination of the residual effects of DOACs on target enzymes, which is complicated by extensive interindividual variation, this methodology is highly specific and sensitive.The DOAC-Stop procedure eliminated dabigatran from 99.5%, rivaroxaban from 97.9%, and apixaban from 97.1% of participants in our group. Residual amounts did not exceed 2.7 ng/mL for dabigatran, 10.9 ng/mL for rivaroxaban, or 13.03 ng/mL for apixaban, which are safe values that do not affect either screening or special coagulation tests.

Influence of fixation method and duration of archiving on immunohistochemical staining intensity in embryonic and fetal tissues.

INTRODUCTION: In this study we detail the effect of different fixation agents and the duration of storage has on the immunohistochemical staining positivity of samples of archival embryonic and fetal tissues. MATERIALS AND METHODS: The samples were stained by indirect two-step immunohistochemistry (IHC) method for Ki-67, cyclin A and ß-actin. RESULTS: Irrespective of the length of tissue archiving, tissue fixation with 10% neutral buffered formalin had better IHC intensity results in all cases when compared to methacarn-fixed tissues. In the case of ß-actin, this difference was statistically significant, while differences in Ki-67 and cyclin A were not. The second aspect studied was which effect tissue block archiving duration has on the IHC reactivity. We demonstrated a statistically significant decrease in IHC positivity for all studied antigens between the samples that were archived for 10-19 or 20-45 years, regardless the fixative solution. CONCLUSION: To the best of our knowledge, the influence that the duration of tissue block archiving has on IHC positivity in human embryo and fetal tissue material has not yet been studied. Although the causes of the IHC positivity decline in archived tissue blocks are not well understood, a possible decrease in IHC over time should be considered, particularly in retrospective studies.

Time-dependent expression pattern of cytochrome P450 epoxygenases and soluble epoxide hydrolase in normal human placenta.

CYP2C and CYP2 J enzymes, commonly named as cytochrome P450 (CYP) epoxygenases, convert arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs), biologically active eicosanoids with many functions in organism. EETs are rapidly hydrolysed to less active dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). We investigated spatio-temporal expression pattern of CYP2C8, CYP2C9, CYP2 J2 and sEH in normal human placenta by immunohistochemical method. In the villous trophoblast, CYP2C8 was the most abundant protein. Its expression is higher than the CYP2C9 and CYP2 J2 in the cytotrophoblast in the embryonic stage of development and remains higher in syncytiotrophoblast of term placenta. Unlike to CYP2C8, CYP2C9 and CYP2 J2 expression decrease in term placenta. sEH expression increases with gestation age and is strictly limited to cytotrophoblast in embryonic and foetal stages of the development. Moreover, CYP2C8 shows more intensive staining than the other protein monitored in Hofbauer cells in villous stroma. Specific information regarding the exact role of EETs and DHETs functions in a normal placenta is still unknown. Based on CYP epoxygenases and sEH localization and well known information about the functions of placental structures during development, we suggest that these enzymes could play different roles in various cell populations in the placenta. As the placenta is absolutely crucial for prenatal development, arachidonic acid is essential part of human nutrient and CYP epoxygenases expression can be affected by xenobiotics, further investigation of the exact role of CYP epoxygenases, sEH, and their metabolites in normal pregnancy and under pathological conditions is needed.

Parkinsonian syndrome complicating systemic lupus erythematosus.

Two girls with florid extrapyramidal parkinsonism complicating systemic lupus erythematosus (SLE) are reported. One patient (15 years old) presented with extreme rigidity, irritability, and mutism initially diagnosed as acute psychosis. Examination revealed severe extrapyramidal akinetic mutism, along with marked restlessness. CT and MRI imaging of the brain were unremarkable. EEG revealed moderate generalized disturbance of background activity. 99mTc-HmPAO SPECT cerebral scanning detected decreased regional cerebral blood flow at the basal ganglia. Dopamine-agonist drugs led to complete recovery after 3 months, along with normalization of EEG and SPECT alterations. The second patient (16 years old) was assessed for progressive bradykinesia and apathy impeding her active daily activities, and she was suspected to have developed depression. Neurologic assessment revealed a parkinsonian syndrome that was less severe than that of the first patient. The EEG showed mild disturbance of background activity, and 99mTc-HmPAO SPECT demonstrated impaired regional cerebral blood flow over the basal ganglia. A parkinsonian extrapyramidal syndrome complicating SLE should therefore be taken into account in any patient with SLE presenting with marked behavioral alterations, rigidity, or akinetic mutism.

[PCNA (proliferating cell nuclear antigen) in the tissues and organs of the human fetus. II]. / PCNA (proliferating cell nuclear antigen) in den Geweben und Organen der menschlichen Keimlinge. II.

The expression of PCNA was proved in some organs of human fetuses aged from 10 to 25 weeks of the intrauterine development. The distinct expression of PCNA was demonstrated in the nuclei of the myoblasts of the ventricles and atria of the fetal heart. The high expression of PCNA was found in the nuclei of the epithelial cells of the fetal bronchial tree, surrounding mesenchyme and in the nuclei of the endothelial cells of the primitive vessels. The highest expression of PCNA was demonstrated in the nuclei of the cells of the "neogene zone" of the primitive kidney. The distinct expression of PCNA exhibited the nuclei of the cells of the surface zone of the primitive adrenal cortex and the cells of the primitive islets of the adrenal medulla. The expression of PCNA was demonstrated in the primitive hepatocytes, endothelial cells of the liver sinusoids and epithelial cells of the bile ducts and in the epithelial cells of the intestinal anlage.

[Histochemical differentiation of lymphatic organs during intrauterine development]. / Histochemische Differenzierung der lymphatischen Organe während der intrauterinen Entwicklung.

The activities of some enzymes (phosphatases, esterases peptidases, and dehydrogenases) were studied. The activities of the enzymes in the youngest embryos (4-8 weeks) were relatively low (ALP-activity on membranes of the primitive capillary endothelium, ACP and UE activities in the cytoplasm of the epithelial reticulum cells). The DPP IV activity was observed on membranes of the epithelial reticulum cells. The activities of GPDH and SDH were relatively strong in the epithelial cells of the primitive cytoreticulum. In the fetal period, the activities of the studied enzymes are gradually increasing. The immunohistochemical findings demonstrate that 95% of the lymphocytes in the thymus anlage (in the fetal period) are T-lymphocytes. The enzyme activities in other lymphatic organs (spleen, lymph nodes) were significantly lower.

[Expression of BCL-2 protein in tissues and organs of the human embryo]. / Expression des BCL-2 proteins in den geweben und organen menschlicher keimlinge.

The bcl-2 gene product is a 24-kD protein localized in the nuclear envelope, endoplasmic reticulum, and mitochondrial membranes. Protein BCL-2 prolongs survival cells by blocking programmed cell death--apoptosis. The role of this protein in the regulation of mammalian embryo development is also suggested. BCL-2 is widely expressed early in mouse fetal development in tissue derived from all three germ layers and this expression becomes restricted with maturation. The expression of BCL-2 was studied in 20 human embryos from the 4 to 12 weeks gestation. Immunoperoxidase staining was performed using the mouse primary monoclonal antibody clone 124 (Dako, Danmark). We found the expression of BCL-2 in many organs of gastrointestinal tract, in mesenchymal cells surrounding primitive bronchial epithelium, in the cells of the metanephronic blasteme and ureteric buds. Our results indicate that bcl-2 gene could play an important role in the human embryonal tissue development.

Proliferating cell nuclear antigen in tissues and organs of human embryos.

The expression of proliferating cell nuclear antigen (PCNA) was demonstrated in some organs in the course of the early intrauterine development. The high expression of PCNA was found in the primitive embryonic myocardium, lungs, primitive gut, liver, pancreas, in the area of "neogene zone" of metanephros (primitive kidney), in the narrow surface zone of the primitive adrenal cortex and in the surface coeloma epithelium, in medullary strips and in the mesenchyme surrounding gonads.

Hepatic, cutaneous and hematologic manifestations in juvenile chronic arthritis.

Two patients suffering from systemic onset juvenile chronic arthritis (JCA) with a polyarticular course developed an acute illness shortly after commencing a new second line drug (gold and penicillamine). The clinical picture consisted of fever, rash, lymphadenopathy and hepatomegaly with elevated liver enzymes and hematological changes. The possible relationship of the disease to second line drugs is discussed.

Inhibition of metalloendopeptidases by 2-mercaptoacetyl-dipeptides.

A series of 2-mercaptoacetyl-dipeptides, a potential group of metalloendopeptidase inhibitors, has been synthesized by coupling the N-hydroxysuccinimide ester of S-acetyl-2-mercaptoacetic acid with hydrophobic dipeptide methyl ester hydrochlorides, followed by hydrolysis with NaOH in aqueous methanol and acidification with HCl. Thus, the 2-mercaptoacetyl derivatives of L-phenylalanyl-L-leucine, L-leucyl-L-phenylalanine and L-leucyl-D-phenylalanine were prepared. The first two compounds inhibit effectively thermolysin from Bacillus thermoproteolyticus and a metalloendopeptidase isolated from Streptomyces griseus, with Ki values in the micromolar range or below. The third compound inhibits the two enzymes only poorly, showing the stereospecificity of the inhibition process. These inhibitors should provide a useful tool for the study of bacterial and mammalian metalloendopeptidases (or dipeptidyl carboxypeptidases) and for the assessment of their physiological role.

Successful treatment of neonatal Flavobacterium meningosepticum infection.

An 8-day-old, 2.48-kg, 35-week gestation infant developed neonatal sepsis and meningitis due to Flavobacterium meningosepticum serotype F. Treatment with a new antibiotic, azlocillin, in combination with chloramphenicol, led to complete recovery.

Prediction of respiratory distress syndrome by the fetal lung maturity analyzer microviscosimeter on newborn gastric aspirate.

To predict the development of respiratory distress syndrome (RDS), the authors determined the fluorescent polarization values on gastric aspirates obtained from 67 premature infants within 30 minutes of birth. In 29 cases these results were also compared with the fluorescent polarization values measured on the corresponding amniotic fluid samples. Measurements for microviscosity were made by fetal lung maturity analyzer. Among 15 of 67 premature infants who developed RDS, the fluorescent value measured on gastric aspirates in all 15 infants was greater than 0.320. The fluorescent polarization values were less than 0.320 in all 52 infants in whom RDS did not develop, a predictability of 100%. Direct comparison found fluorescent polarization values measured on gastric aspirates to be somewhat lower than the corresponding amniotic fluid fluorescent polarization values. The results indicate that gastric aspirate obtained within 30 minutes of birth contains swallowed amniotic fluid. In cases where amniotic fluid samples were not available for surfactant evaluation prenatally, the determination of fluorescent polarization values on the newborn's gastric aspirate may accurately predict the development of RDS. The use of the fetal lung maturity analyzer microviscosimeter provides a simple, reliable, and rapid (45 minutes) method for assessment of surfactant in premature infants.