Pilot study on the use of basophil activation tests and skin tests for the prevention of allergic transfusion reactions.

Background and objectives: Management of severe allergic transfusion reactions (ATR) is challenging. In this study, we investigate the usefulness of skin tests and basophil activation tests (BAT) in chronically transfused patients for the prevention of future ATR. Materials and methods: BAT and skin tests were carried with the supernatant of red blood cell (RBC) units for a sickle-cell disease patient under chronic exchange transfusion who has presented a severe ATR, in order to prevent potential future ATR. If the results for both BAT and skin tests were negative, the RBC units could be transfused to the patient. If either one of the results was positive, the tested RBC unit was discarded for the patient. Results: 192 RBC units were tested with both tests. The level of results concordance between the two tests was 95%. Out of the 169 negative units with both tests, 118 units were transfused to the patient for which he presented no ATR. Conclusion: In our study, combining both BAT and skin tests was associated with a good negative predictive value since we were able to safely transfuse our patient. Further studies are still necessary to confirm this result but this pilot study indicates that skin tests and BAT might help prevent ATR. When BAT is not available, skin tests may also be useful in preventing ATR.

Tocilizumab-Induced Unexpected Increase of Several Inflammatory Cytokines in Critically Ill COVID-19 Patients: The Anti-Inflammatory Side of IL-6.

Early evidence during the coronavirus disease 2019 (COVID-19) pandemic indicated high levels of interleukin (IL)-6 in patients with severe COVID-19. This led to the off-label use of tocilizumab (TCZ) during the first wave of the pandemic. While the monoclonal antibody blocks IL-6 pathway, its effect on other inflammatory cytokines remains poorly described. To better understand the effect of TCZ on the biological inflammatory profile, we monitored a large panel of inflammatory cytokines in critically ill COVID-19 patients receiving off-label TCZ. Twenty-three patients with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were included in the study, among which 15 patients received TCZ and 8 patients did not. Serum samples were collected for 8 days, before and following TCZ administration or hospital admission for the control group. Serum profile of 12 cytokines (IL-1ß, -2, -4, -6, -8, -10, -12, -13, -17, -18, tumor necrosis factor α (TNF-α), interferon-gamma (IFN-γ), and sIL-6R were assessed in these two groups. Although the increased IL-6 concentrations after TCZ infusion were expected, we observed an unexpected increase in IL-1ß, -2, -4, -10, -12p70, -18, and sIL-6R levels in the treated patients with maximal values reaching 2 to 4 days after TCZ. In contrast, no change in cytokine levels was observed in the control group. Our results suggested that some inflammatory pathways escape IL-6R blockade and even appeared amplified. This finding highlights an old observation of the anti-inflammatory effects of IL-6 as already suggested over 20 years ago. Clinical Trial Registration number: NCT04346017.

Belgian rare diseases plan in clinical pathology: identification of key biochemical diagnostic tests and establishment of reference laboratories and financing conditions.

BACKGROUND: One objective of the Belgian Rare Diseases plan is to improve patients' management using phenotypic tests and, more specifically, the access to those tests by identifying the biochemical analyses used for rare diseases, developing new financing conditions and establishing reference laboratories. METHODS: A feasibility study was performed from May 2015 until August 2016 in order to select the financeable biochemical analyses, and, among them, those that should be performed by reference laboratories. This selection was based on an inventory of analyses used for rare diseases and a survey addressed to the Belgian laboratories of clinical pathology (investigating the annual analytical costs, volumes, turnaround times and the tests unavailable in Belgium and outsourced abroad). A proposal of financeable analyses, financing modalities, reference laboratories' scope and budget estimation was developed and submitted to the Belgian healthcare authorities. After its approval in December 2016, the implementation phase took place from January 2017 until December 2019. RESULTS: In 2019, new reimbursement conditions have been published for 46 analyses and eighteen reference laboratories have been recognized. Collaborations have also been developed with 5 foreign laboratories in order to organize the outsourcing and financing of 9 analyses unavailable in Belgium. CONCLUSIONS: In the context of clinical pathology and rare diseases, this initiative enabled to identify unreimbursed analyses and to meet the most crucial financial needs. It also contributed to improve patients' management by establishing Belgian reference laboratories and foreign referral laboratories for highly-specific analyses and a permanent surveillance, quality and financing framework for those tests.

A score combining early detection of cytokines accurately predicts COVID-19 severity and intensive care unit transfer.

OBJECTIVES: We aimed to explore cytokine profile in patients as it relates to Coronavirus Disease 2019 (COVID-19) severity, and to establish a predictive cytokine score to discriminate severe from non-severe cases and provide a prognosis parameter for patients that will require intensive care unit (ICU) transfer. METHODS: Serum samples of 63 patients diagnosed with SARS-CoV-2 infection were collected early after hospital admission (day 0-3). Patients were categorized in five groups based on the clinical presentation, the PaO2/FiO2 ratio and the requirement of mechanical ventilation. RESULTS: Three cytokines, IL-6, IL-8 and IL-10, were markedly higher in severe forms (n = 44) than in non-severe forms (n = 19) (p < 0.005). A score combining levels of these three cytokines (IL-6*IL-8*IL-10) had the highest performance to predict severity: sensitivity of 86.4% (95% CI, 72.4-94.8) and specificity of 94.7% (95% CI, 74.0-99.9) for a cutoff value of 2068 pg/mL. Elevated levels of IL-6, IL-8 and IL-10 were also found in critically ill patients. The combination of IL-6*IL-10 serum levels allowed the highest predictability for ICU transfer: AUC of 0.898 (p < 0.0001). CONCLUSION: The combinatorial IL-6*IL-8*IL-10 score at presentation was highly predictive of the progression to a severe form of the disease, and could contribute to improve patient triage and to adapt therapeutic strategy within clinical trials more accurately and efficiently.

Variability of Faecal Calprotectin in Inflammatory Bowel Disease Patients: An Observational Case-control Study.

BACKGROUND AND AIMS: Several factors have been reported to affect faecal calprotectin [FC] values, and significant variation in FC concentrations has been observed in inflammatory bowel disease [IBD] patients. We aimed to evaluate FC variability in IBD patients, and to assess the robustness of a single stool punch. METHODS: This is a single-centre observational case-control study. Disease activity was assessed using endoscopic and clinical activity scores, as well as C-reactive protein levels. Stool samples were collected twice within a 1 to 6 days interval, and FC was measured on punches and homogenates by fluorometric enzyme immunocapture assay. RESULTS: In all, 260 stool samples were collected from 120 patients. Intrastool variability was low, with an intraclass correlation coefficient for single measures between three punches from a single stool sample of 0.91, and median coefficient of variation [CV] of 17%. CV of two stool samples a few days apart [intra-individual variability] were significantly higher [p <0.01] with median CV of 36%. FC standard deviations correlated with mean FC levels either for intrastool or for intra-individual variability, with a Spearman's coefficient of rank correlation of 0.85 and 0.78, respectively [p <0.01]. Disease type, location, activity, and FC levels did not influence variability. CONCLUSIONS: A single stool punch is reliable for FC measurement, considering that intrastool variability is low. Intra-individual variability a few days apart is significantly higher. Therefore, decision-making strategies based on single measurements should consider this variability, to determine the minimum optimal variation to be achieved, rather than a cut-off, especially in high FC levels.

Disulfiram inhibition of cyanide formation after acetonitrile poisoning.

CONTEXT: Cyanide poisoning may be caused by acetonitrile, a common industrial organic solvent and laboratory agent. OBJECTIVE: To describe the potential use of disulfiram in treating acetonitrile poisoning in a human clinical case and to further study its effect in human liver microsomes in vitro. CASE DETAILS: A 30-year-old man initially presented with a cholinergic toxic syndrome following ingestion of aldicarb. Toxicological analysis revealed coingestion of ethanol. He subsequently developed severe metabolic acidosis caused by the cyanogenic compound acetonitrile which was erroneously interpreted as acetone in the chromatogram. After three treatments with hydroxocobalamin (5 g i.v.) and sodium thiosulfate (12.5 g i.v.) on days 2, 3, and 5, he had transient improvement but recurrent lactic acidosis. Treatment with disulfiram was associated on day 7 with resolution of metabolic acidosis and slowing of the decrease in acetonitrile concentration. He recovered from acetonitrile toxicity completely. The time course of acetonitrile, thiocyanate, and cyanide concentrations suggested that disulfiram inhibited cyanide formation. RESULTS: In vitro experiments with human liver microsomes showed the cyanide concentration was significantly lower after incubation with acetonitrile and disulfiram than acetonitrile alone (a mean 60% reduction in cyanide level). DISCUSSION: Although disulfiram was given late in the course of the poisoning it is possible that it contributed to the recovery.

Pneumococcal aetiology and serotype distribution in paediatric community-acquired pneumonia.

Community-acquired pneumonia (CAP) is a major cause of morbidity in children. This study estimated the proportion of children with pneumococcal CAP among children hospitalised with CAP in Belgium and describes the causative serotype distribution after implementation of the 7-valent pneumococcal conjugate vaccine. Children 0-14 years hospitalised with X-ray-confirmed CAP were prospectively enrolled in a multicentre observational study. Acute and convalescent blood samples were collected. Pneumococcal aetiology was assessed by conventional methods (blood or pleural fluid cultures with Quellung reaction capsular typing or polymerase chain reaction [PCR] in pleural fluid), and recently developed methods (real-time PCR in blood and World Health Organization-validated serotype-specific serology). A total of 561 children were enrolled. Pneumococcal aetiology was assessed by conventional methods in 539, serology in 171, and real-time PCR in blood in 154. Pneumococcal aetiology was identified in 12.2% (66/539) of the children by conventional methods alone but in 73.9% by the combination of conventional and recently developed methods. The pneumococcal detection rate adjusted for the whole study population was 61.7%. Serotypes 1 (42.3%), 5 (16.0%), and 7F(7A) (12.8%) were predominant. In conclusion, Streptococcus pneumoniae remains the predominant bacteria in children hospitalised for CAP in Belgium after implementation of 7-valent pneumococcal conjugate vaccine, with non-vaccine-serotypes accounting for the majority of cases. The use of recently developed methods improves diagnosis of pneumococcal aetiology.

Different T cell memory in preadolescents after whole-cell or acellular pertussis vaccination.

To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines.

Evaluation of a WHO-validated serotype-specific serological assay for the diagnosis of pneumococcal etiology in children with community-acquired pneumonia.

BACKGROUND: The etiologic diagnosis of community-acquired pneumonia (CAP) remains challenging in children because blood cultures have low sensitivity. Novel approaches are needed to confirm the role of Streptococcus pneumoniae. METHODS: In this study, pneumococcal etiology was determined by serology using a subset of blood samples collected during a prospective multicentre observational study of children <15 years of age hospitalized in Belgium with radiogram-confirmed CAP. Blood samples were collected at admission and 3-4 weeks later. Pneumococcal (P)-CAP was defined in the presence of a positive blood or pleural fluid culture. Serotyping of S. pneumoniae isolates was done with the Quellung reaction. Serological diagnosis was assessed for 9 serotypes using World Health Organization-validated IgG and IgA serotype-specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: Paired admission/convalescent sera from 163 children were evaluated by ELISA (35 with proven P-CAP and 128 with nonproven P-CAP). ELISA detected pneumococci in 82.8% of patients with proven P-CAP. The serotypes identified were the same as with the Quellung reaction in 82% and 59% of cases by IgG ELISA and IgA ELISA, respectively. Overall, ELISA identified a pneumococcal etiology in 55% of patients with nonproven P-CAP. Serotypes 1 (51.6%), 7F (19%) and 5 (15.7%) were the most frequent according to IgG ELISA. CONCLUSIONS: In conclusion, the serological assay allows recognition of pneumococcal origin in 55% of CAP patients with negative culture. This assay should improve the diagnosis of P-CAP in children and could be a useful tool for future epidemiological studies on childhood CAP etiology.

The influence of bypass procedures and other anatomical changes in the gastrointestinal tract on the oral bioavailability of drugs.

The gastrointestinal (GI) tract plays an important role in the absorption of orally administered drugs. However, in some cases the anatomy of the GI tract is changed due to GI surgery, which has the potential of influencing drug bioavailability. In this review, we aim to compile, review, and comment the existing but sometimes fragmented scientific data regarding the impact of GI surgery on the oral bioavailability of drugs. Relevant reports were gathered through the PubMed database from database inception through January 2012. Drugs for which at least one trial or case report suggested a change in oral bioavailability or absorption caused by GI surgery are discussed in detail. Major methodological differences, such as study design, number of subjects and choice of reference group, were observed in the reported studies. Predicting the impact of GI surgery on the oral bioavailability was therefore difficult to perform, even the most sophisticated classification systems could not be used for predicting purposes.

Switch from intravenous to enteral moxifloxacin in critically ill patients: a pilot study.

Critically ill patients generally receive moxifloxacin intravenously to achieve rapid bacterial killing. An early switch from intravenous to enteral moxifloxacin may be considered because of its good oral bioavailability in healthy volunteers. Since bioavailability may be altered in critically ill patients due to pathophysiological changes, this study aimed to investigate whether enteral moxifloxacin is bioequivalent to intravenous moxifloxacin in such patients. Blood samples were obtained from 4 critically ill patients before and at serial time-points after intravenous and enteral administration. In all patients, lower maximum plasma concentration (C(max)) and area under the plasma concentration-time curve during the 24-h observation period (AUC(24h)) values were observed after enteral administration compared to those after intravenous administration. This resulted in lower C(max)/minimum inhibitory concentration (MIC) and AUC(24h)/MIC values, which are 2 indices predicting the antibacterial efficacy of moxifloxacin. Despite the limited number of subjects, we conclude that a switch from intravenous to enteral moxifloxacin is not recommended in these patients, because the 2 administration forms are not bioequivalent.

Impact of corneal cross-linking on drug penetration in an ex vivo porcine eye model.

PURPOSE: To analyze the influence of corneal cross-linking (CXL) using ultraviolet-A and riboflavin on corneal drug penetration of topically applied drugs. METHODS: In an ex vivo porcine eye model, eyes were randomly assigned to CXL or control treatment. Central corneal thickness and anterior chamber depth were measured with a Pentacam device. In the CXL group, eyes were treated with CXL using ultraviolet-A (370 nm) and riboflavin, whereas in the control group only riboflavin was applied without irradiation. Subsequently, 0.3% ofloxacin (n = 40 eyes) or 1% voriconazole (n = 40 eyes) eye drops were applied to the cornea every 5 minutes for 30 minutes. Aqueous humour samples were obtained performing an anterior chamber tap. The concentrations of ofloxacin and voriconazole were determined with high-pressure liquid chromatography. Groups were compared performing a Mann-Whitney test. RESULTS: In the CXL group, the mean concentration of ofloxacin (13.33 ± 4.67 µg/mL) and voriconazole (52.70 ± 8.76 µg/mL) was significantly lower than in the untreated control group (ofloxacin: 18.51 ± 6.08 µg/mL, P = 0.005; voriconazole: 62.43 ± 13.5 µg/mL, P = 0.01). This corresponds to a reduction in permeability of 27.98% for ofloxacin and 15.59% for voriconazole. Central corneal thickness and anterior chamber depth were comparable in the CXL and control groups (P > 0.05, each). CONCLUSIONS: CXL reduces the corneal permeability of ofloxacin and voriconazole. This may be of clinical significance, for example, in keratitis treatment.

Development and validation of a fast and sensitive UPLC-MS/MS method for the quantification of six probe metabolites for the in vitro determination of cytochrome P450 activity.

A fast and sensitive UPLC-MS/MS method was developed and validated for the simultaneous quantification of six probe metabolites for the in vitro cytochrome P450 activity determination in hepatic microsomes from patients with hepatic impairment. The metabolites acetaminophen (CYP1A2), 4'-hydroxy-mephenytoin (CYP2C19), 4-hydroxy-tolbutamide (CYP2C9), dextrorphan (CYP2D6), 6-hydroxy-chlorzoxazone (CYP2E1) and 1-hydroxy-midazolam (CYP3A4), together with the internal standard chlorpropamide, were separated on a Waters Acquity UPLC BEH C18 column (50 mm × 2.1mm, 1.7 µm particle size) with VanGuard pre-column (5 mm × 2.1mm, 1.7 µm particle size). A short gradient elution (total run time of 5.25 min), using water with 0.1% formic acid (eluent A) and acetonitrile with 0.1% formic acid (eluent B) at a flow rate of 400 µl/min, was used. The metabolites were detected with a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Two runs, one in the positive ionization mode and one in the negative mode, were necessary for the detection of all metabolites. The method was selective and showed good accuracy (84.59-109.83%) and between-day (RSD%<5.13%) and within-day (RSD%<9.60%) precision. The LOQ was in full accordance with the intended application, and no relative matrix effects were observed. Also, the sample incubation extracts were stable after three freeze-thaw cycles. The usability of the method was demonstrated by the incubation of pediatric microsomes with subsequent quantification of the formed metabolites and CYP activity calculation.

Oral bioavailability of moxifloxacin after Roux-en-Y gastric bypass surgery.

OBJECTIVES: Roux-en-Y gastric bypass surgery is the most commonly performed procedure for the treatment of morbid obesity. This anatomical alteration may affect the absorption and consequently the bioavailability of oral drugs. This study aims to investigate the oral bioavailability of moxifloxacin in 12 healthy volunteers who underwent gastric bypass surgery. PATIENTS AND METHODS: In this randomized crossover study, each subject received two single standard doses of 400 mg of moxifloxacin orally or intravenously administered on two occasions separated by a washout period of 1 week. Serial venous blood samples were drawn up to 72 h after dosing and moxifloxacin plasma levels were measured by a validated HPLC method with fluorescence detection. [clinicaltrials.gov database (identifier: NCT01130922).] RESULTS: After oral dosing, moxifloxacin plasma concentrations reached a maximum (C(max)) of 3.38 ± 1.41 mg/L after 1.75 h (0.75-4.00). After intravenous dosing, C(max) and T(max) were 4.53 ± 1.43 mg/L and 1.03 h (0.75-2.50), respectively. The mean areas under the plasma concentration time curve extrapolated to infinity (AUC(∞)) were 46.2 ± 1.4 mg ·â€Šh/L after oral dosing and 52.3 ± 1.3 mg ·â€Šh/L after intravenous dosing, resulting in a mean oral bioavailability of 88.32% [90% confidence interval (CI) 85.64%-91.08%]. CONCLUSIONS: This study confirms that exposure to moxifloxacin is equivalent for oral and intravenous administration of 400 mg dosages in healthy volunteers who underwent gastric bypass surgery. But these exposures were more than 50% higher than those described for subjects without gastric bypass. This may suggest a higher enterohepatic recirculation of moxifloxacin after gastric bypass.

Pharmacokinetics of two formulations of omeprazole administered through a gastrostomy tube in patients with severe neurodevelopmental problems.

AIMS: Omeprazole is often administered through a gastrostomy tube as either (i) a Multiple Unit Pellet System (MUPS®) tablet disintegrated in water (MUPS® formulation), or (ii) a suspension in 8.4% sodium bicarbonate (suspension formulation). This bioavailability study evaluates this practice in tube-fed patients with severe neurodevelopmental problems. METHODS: Nonblinded, two-phase cross-over trial. RESULTS: In seven of 10 patients, bioavailability was higher for the suspension formulation than for the MUPS® formulation. Median (90% confidence interval) area under the plasma concentration-time curve ratio (MUPS® over suspension) was 0.5 (0.06-2.37). CONCLUSIONS: In this population, omeprazole MUPS® formulation has no apparent advantage over the more easily administered suspension formulation.

Antibody deficiency due to a missense mutation in CD19 demonstrates the importance of the conserved tryptophan 41 in immunoglobulin superfamily domain formation.

Immunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca(2+) influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.

CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency.

Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.

A bio-analytical hydrophilic interaction LC-MS/MS method for the simultaneous quantification of omeprazole and lansoprazole in human plasma in support of a pharmacokinetic omeprazole study in children.

A hydrophilic interaction LC method with MS/MS was developed and validated for the simultaneous quantification of omeprazole and lansoprazole in human plasma. Chromatographic separation was achieved on a Betasil silica column using a high organic mobile phase (eluent A: ACN/formic acid 997.5:2.5 v/v; eluent B: water/formic acid 997.5:2.5 v/v) and gradient elution. The mass spectrometer was operated in the Multiple Reaction Monitoring mode. Prior to chromatography, liquid-liquid extraction with ethyl acetate was used and the organic layer was diluted with ACN, allowing direct injection on column. The method showed acceptable linearity, high precision (RSD%<10.5%), accuracy (88.9-109.3%) and selectivity in the two concentration ranges studied: 1.5-100 and 5-2000 ng/mL. The LOQ was established at 1.5 and 5 ng/mL for the two concentration ranges. Lack of variability in matrix effects was demonstrated and mean extraction recovery for omeprazole and lansoprazole was determined in the low (56.3-67.7%) and high (45.3-44.3%) concentration range, respectively. Additionally, plasma samples were found to be stable after three freeze-thaw cycles and for at least 15 h after extraction. This assay was successfully applied to a pharmacokinetic omeprazole study in children with cerebral palsy and mental retardation.