[Perforation of the acalculous gallbladder in a renal transplant recipient with CMV infection]. / Perforacion de vesicula biliar en el se-no de una infeccion por citomegalovirus en trasplante renal.

We report the case of a renal transplant recipient who developed acute acalculous cholecyscitis resulting in gallbladder perforation. At admission CMV antigenemia was negative. Emergency laparatomy was performed and showed the gallbladder to be infarted with a perforation. The abdominal cavity contained two litres of sterile bilious fluid. The pathological report showed frequent endothelial cells contained intranuclear and intracitoplasmatic inclusion (fig. 1). Treatment with Ganciclovir iv was started after diagnosis, but a computerized tomography scan demonstrated severe acute pancreatitis (grade E. Baltazar). The patient developed multiorgan failure and died on 19th day after surgery. Necropsy showed cytomegalic inclusions in pancreas (fig. 2), gastrointerstinal tract, lung and graft. A necroticing pneumonia with Mycotic spores and hiphae was seen. Aspergillus was also observed in myocardium (fig. 3).

Perforación de vesícula biliar en el seno de una infección por citomegalovirus en trasplante renal / Perforation of the acalculous gallbladder in a renal transplant recipient with CMV infection

Se describe el caso de un receptor de trasplante renal con serología CMV positiva que desarrolló una infección por CMV que provocó una colecistitis alitiásica, con posterior perforación de la vesícula biliar. En el momento de su ingreso la antigenemia CMV era negativa, efectuándose el diagnóstico de infección por CMV al evidenciar numerosas inclusiones citomegálicas en las células endoteliales de la pieza de colecistectomía. Se inició tratamiento con Ganciclovir iv en el momento del diagnóstico, que fue ineficaz ya que posteriormente el paciente presentó una pancreatitis aguda necrotizante (grado E de Baltazar), también por la acción del CMV y posteriormente una sobreinfección por aspergillus falleciendo el enfermo en el seno de un fracaso multiórganico. En los estudios histológicos efectuados se objetiva la presencia de inclusiones citomegálicas en vesícula biliar y páncreas entre otros órganos, así como aspergilosis invasiva en pulmón y miocardio

We report the case of a renal transplant recipient who developed acute acalculous cholecyscitis resulting in gallbladder perforation. At admission CMV antigenemia was negative. Emergency laparatomy was performed and showed the gallbladder to be infarted with a perforation. The abdominal cavity contained two litres of sterile bilious fluid. The pathological report showed frequent endothelial cells contained intranuclear and intracitoplasmatic inclusion (fig. 1). Treatment with Ganciclovir iv was started after diagnosis, but a computerized tomography scan demonstrated severe acute pancreatitis (grade E. Baltazar). The patient developed multiorgan failure and died on 19th day after surgery. Necropsy showed cytomegalic inclusions in pancreas (fig. 2), gastrointerstinal tract, lung and graft. A necroticing pneumonia with Mycotic spores and hiphae was seen. Aspergillus was also observed in myocardium (fig. 3)

Seroprevalence of varicella among children and adolescents in Valencia, Spain. Reliability of the parent's reported history and the medical file for identification of potential candidates for vaccination.

This study assessed the seroprevalence of varicella antibodies in children and adolescents in Spain and evaluated the reliability of two methods for detecting susceptible individuals: (1) parental-reported history of varicella and (2) medically-documented histories maintained by the pediatrician. A total of 186 children (6 to 15 years of age) were recruited in 13 pediatric offices of Valencia, Spain. A brief case report form was completed including previous history of varicella referred by the parents, and a 5 mL blood sample was obtained. The pediatrician medical file was reviewed for antecedent of varicella. The overall prevalence of varicella antibodies was 84% and 88% in the 6-9 years and 10-15 years age brackets, respectively. The predictive value of a negative history of varicella disease was 48% by parental recall (52% "false negative"), and only 26% by medical record (74% "false negative"). However, the positive predictive value of a positive parental reported history or a positive medically-documented history was 95%. The most effective strategy for varicella vaccination of older children and adolescents in Spain will be to immunize those individuals with a lack of positive (unknown or negative) history of disease.

Molecular genetic analysis of HLA-DR and -DQ alleles in Spanish patients with melanoma.

Controversial data have been reported about HLA alleles and susceptibility to melanoma. The relationship between distribution of HLA alleles in patients with melanoma and susceptibility to tumour was analysed, to study the possible correlation between HLA class II DQA1, DQB1 and DRBI genes and melanoma in a Spanish population. Genomic DNA from 82 patients with melanoma and 367 random healthy donors, from the same geographic area, were typed by PCR-SSP (sequence specific primers). The patients were also divided into different groups according to the age and presence of cancer relatives, and compared with the controls. None of these HLA class II alleles showed significant positive or negative associations with either the overall population of patients with melanoma or the considered subgroups. Moreover, values for relative risk of DQB1*0301, DQB1*0302, DQB1*0303, DQB*05, DQA1*0401, DQA1*0101/0104 and DRB*08, which have been reported to be increased or decreased in patients with melanoma, were very low and of no statistical significance. Our results indicate that HLA class II alleles may not contribute to a strong susceptibility to melanoma in the Spanish population, although further studies on larger series are needed to corroborate this. Key words:

Rapid quantitative detection of BCR-ABL transcripts in chronic myeloid leukemia patients by real-time reverse transcriptase polymerase-chain reaction using fluorescently labeled probes.

BACKGROUND AND OBJECTIVES: The limited value of qualitative reverse transcription polymerase chain-reaction (RT-PCR) for monitoring chronic myeloid leukemia (CML) patients has prompted the development of quantitative assays. We have developed a quantitative real-time PCR (QC-PCR) method in the LightCycler, based on the use of fluorescently labeled probes (HybProbes), to estimate BCR-ABL fusion gene transcripts in samples from CML patients. DESIGN AND METHODS: Fifty-two samples (45 peripheral blood, five bone marrow, and two apheresis product samples) from nine patients with CML were analyzed. Seven patients were studied at diagnosis and during follow-up after hematopoietic stem cell transplantation (HSCT), whereas two were evaluated only after HSCT. The PCR reaction was carried out in capillary tubes in a final volume of 10 microL, using 2 microL cDNA, the Mensik et al. primers, and two HybProbes. The results for BCR-ABL were normalized with reference to ABL. The PCR program is completed in only 45 min. RESULTS: The sensitivity attained allowed the detection of rearrangements at dilutions of between 5-10(-4) and 10(-5) K562 cDNA. The within-assay coefficient of variation was 11% for BCR-ABL, and 9% for ABL. A greater than 2 log reduction in the BCR-ABL/ABL ratio was evident shortly after transplantation in all allografted patients. INTERPRETATION AND CONCLUSIONS: We may conclude that the TaqMan probe technology can be easily adapted to HybProbes with equivalent results. Besides, the results of BCR-ABL quantification in the follow-up of patients clearly confirm that real-time PCR with HybProbes is a reliable and sensitive method for monitoring minimal residual leukemia after HSCT in CML patients.

Pharmacokinetic-Pharmacodynamic Modelling of the antipyretic effect of two oral formulations of ibuprofen.

OBJECTIVE: To analyse the population pharmacokinetic-pharmacodynamic relationships of racemic ibuprofen administered in suspension or as effervescent granules with the aim of exploring the effect of formulation on the relevant pharmacodynamic parameters. DESIGN: The pharmacokinetic model was developed from a randomised, cross-over bioequivalence study of the 2 formulations in healthy adults. The pharmacodynamic model was developed from a randomised, multicentre, single dose efficacy and safety study of the 2 formulations in febrile children. PATIENTS AND PARTICIPANTS: Pharmacokinetics were studied in 18 healthy volunteers aged 18 to 45 years, and pharmacodynamics were studied in 103 febrile children aged between 4 and 16 years with bodyweight 225kg. METHODS: The pharmacokinetic study consisted of two 1-day study occasions, each separated by a 1-week washout period. On each occasion ibuprofen 400mg was administered orally as suspension or granules. The time course of the antipyretic effect was evaluated in febrile children receiving a single oral dose of 7 mg/kg in suspension or 200 or 400mg as effervescent granules. During the pharmacodynamic analysis, the predicted typical pharmacokinetic profile (based on the pharmacokinetic model previously developed) was used. RESULTS: The disposition of ibuprofen was described by a 2-compartment model. No statistical differences (p > 0.05) were found between the 2 formulations in the distribution and elimination parameters. Absorption of ibuprofen from suspension was adequately described by a first-order process; however, a model with 2 parallel first-order input sites was used for the drug given as effervescent granules, leading to time to reach maximum drug concentration (tmax) values of 0.9 and 1.9 hours for suspension and granules, respectively. The time course of the antipyretic effect was best described using an indirect response model. The estimates (with percentage coefficients of variation in parentheses) of Emax (maximum inhibition of the zero-order synthesis rate of the factor causing fever), EC50 (plasma concentration eliciting half of Emax), n (slope parameter) and k(out) (first order rate constant of degradation) were 0.055 (10), 6.16 (14) mg/L, 2.71 (18) and 1.17 (23) h(-1), respectively, where To is the estimate of the basal temperature, 38.8 (1) degrees C. No significant (p > 0.05) covariate effects (including pharmaceutical formulation) were detected in any of the pharmacodynamic parameters. CONCLUSIONS: Because of the indirect nature of the effect exerted by ibuprofen, the implications of differences found in the plasma drug concentration profiles between suspension and effervescent granules are less apparent in the therapeutic response.

Quality analysis of blood components obtained by automated buffy-coat layer removal with a top & bottom system (Optipress (R)II).

BACKGROUND AND OBJECTIVE: There are Council of Europe recommendations for the quality of blood components. We analyzed the quality of blood components processed by a top & bottom system (Optipress((R)) II), the routine method used in our blood bank, to test whether the components reached the recommended quality. DESIGN AND METHODS: Blood was collected in triple CPD-SAGM bags (Optipac((R)) Baxter). Whole blood (WB) was centrifuged at 4,158 g for 14 min before separation by an automated top & bottom system (Optipress((R) )II). Platelet concentrate (PC) was prepared by pooling four isogroup buffy-coat (BC) units before low-speed centrifugation, and transferring the supernatant (4 BC-PC) to a 5-day storage bag (PL732, Baxter). An alternative approach involved PC preparation from a single BC unit by adding approximately 70 mL of plasma before centrifugation, followed by transfer of the platelet concentrate (1BC-PC) to a 300 mL Teruflex((R)) transfer bag. Both 4 BC-PC and 1 BC-PC were stored in a flat agitator at 22 degrees C for up to 5 days after collection. Cell counts were determined, along with hemoglobin and hematocrit in a Sysmex K-800 cell counter. The pH was determined on day 5 at 22 degrees C. Weights were measured and volumes were calculated based on specific gravity. Statistical analyses were carried out using the Kolmogorov-Smirnov test as a normality distribution test, the t-test for parametric values and Wilcoxon's test as a non-parametric test. Statistical significance between samples was considered to have been reached when p<0.05. RESULTS: The best parameters for configuring the system were: strength 25; BC volume 33-55; level of BC 5.5. Red blood cell (n = 1,434) volume was 279+/-20 mL, with 54.92+/-7.16 g of hemoglobin. More than 96% of units had fewer than 1.2x10(9) white blood cells. Fresh plasma volume (n = 803) averaged 279+/-19 mL, with a white blood cell contamination of fewer than 0.1x10(9)/L in all samples examined (n = 23). Platelet recovery in BC was 92+/-9% of platelets present in WB; the percentage of removed leukocytes was 74+/-10%, and between 13 and 15% of RBCs were lost in the BC (95% confidence interval). The BC volume (n = 1,037) fitted the target volume of 60 mL, except for some devices, when Optipress II((R)) lost the configuration for this parameter. Of 4 BC-PCs 80.3% yielded more than 0.6x10(11) platelets per unit, whereas this criterion was only met by 59.7% of 1 BC-PCs, and a greater proportion of 1 BC-PCs (58.8%) showed pH values within the range of 6.5-7.4 after 5 days of storage in comparison with 4 BC-PCs (44.25%). INTERPRETATION AND CONCLUSIONS: Optipress II((R)) provides standardized, leukocyte-poor blood components. Council of Europe requirements were met in a large percentage of red-cell concentrates, with less than 92 and 74% of the original platelets and leukocytes, respectively, and a small hemoglobin loss per unit. The system gave an optimal yield in terms of plasma volume. The top & bottom technique allowed us to reduce the number of blood units per platelet concentrate from 6 to 4 units, with similar platelet yields compared with traditional procedures. Nevertheless, the storage conditions must be improved to satisfy all Council of Europe requirements for platelet concentrates.

Analysis of the CDKN2A and CDK4 genes and HLA-DR and HLA-DQ alleles in two Spanish familial melanoma kindreds.

Some confusion exists in the literature about which criteria should be used to define familial melanoma. This could explain the different reported frequencies of mutations in predisposing genes, mostly CDKN2A, in these patients. This study evaluated the human leucocyte antigen (HLA) class II genotype and the presence of mutations in CDKN2A and CDK4 genes in 2 families with very different clinical features. The family with a germinal mutation in exon 2 of CDKN2A (Gly101Try) presented the following clinical features: 3 first-degree affected members, 1 of whom had 2 melanomas, and all the melanomas appearing before 35 years of age. In contrast, the second family did not present any mutation in the studied genes and included 2 first-degree affected members diagnosed at over 45 years of age. Neither family showed an association with HLA genotype. Other genes are also involved in familial melanoma but, when the CDKN2A gene is affected, some clinical features seem to be uniform.

A new method for phenotyping red blood cells using microplates.

BACKGROUND AND OBJECTIVES: The supply of phenotyped red blood cells (RBC) for patients with several RBC antibodies presents a difficult task to hospital blood banks and regional blood centers. The aim of this study was to establish a low-cost typing system to allow extensive phenotyping of regular blood donors for clinically significant RBC antigens. MATERIALS AND METHODS: We developed a new buffer that greatly intensifies the antigen-antibody reaction and thus reduces the quantity of serum needed for phenotyping. The procedure was carried out on microplates. RESULTS: A total of 20,435 regular blood donors have been typed to date. For 752 units required for transfusion, 3,584 phenotyping tests were performed, validating the results by tube or gel typing methods; agreement was achieved in all cases. CONCLUSION: This technique seems adequate for phenotyping a large number of RBC units at very low cost, thus facilitating the availability of phenotyped blood.

[Analysis of the quality of hemoderivatives obtained using a buffy-coat extraction system with a top-and-bottom technique (Optipress II)]. / Análisis de calidad de hemoderivados obtenidos mediante un sistema de extracción de la capa leucoplaquetar con tecnología Top & Bottom (Optipress II).

PURPOSE: The aim of the present study is to know the results of the quality analysis of blood components processed with a Top & Bottom system (Optipress II) as a routine method in our blood bank, and compare it with the CE recommendations for quality of blood components. MATERIAL AND METHODS: Blood was collected in triple CPD-SAGM bags (Optipac, Baxter) and whole blood (WB) were centrifuged at 4,158 g, 14 min. Blood separation was performed by an automated Top & Bottom system (Optipress II), in which parameters were individually configured in preliminary trials. The buffy-coat (BC) layer was maintained within the configured levels during the separation process and remained into the original bag, whereas red cells (RBC) were collected into the bottom satellite bag (with 100 mL of SAGM) and fresh plasma (FP) was sent to the top satellite bag. Platelet concentrate (PC) was prepared by two different ways: 4 isogroup buffy-coats units were pooled by means of a sterile connector device (TSCD-201, Terumo) before a low centrifugation (1,040 g, 9 min) and the supernatant (4BC-PC) was transferred into a PL732 bag (Fenwal, Baxter); the other PC was prepared from one unit of BC by additioning approximately 70 mL of FP before centrifugation (321 g, 6 min) and following transference of the platelet concentrate (1BC-CP) into a 300 mL (Teruflex, Terumo) transfer bag. Both, 4BC-PC and 1BC-PC, were stored in a flat agitator at 22 degrees C to up five days after collection. We determined cell counts, haemoglobin, and hematocrit in a Sysmex K-800 cell counter in WB and blood components. Nageotte chamber was used when low white blood cells (WBC) counts were obtained. We also determined pH values on day five at 22 degrees C in a Crison 2000. Weights were measured and volumes were calculated using specificity gravity. Statistical analysis were carried out by Kolmogorov-Smirnov test as a normality distribution test, t-test for parametrical values and Wilcoxon-test as a no parametrical test (p < 0.05 was considered as Wilcoxon a significant value between different samples). RESULTS: The best parameters to configure the system were: strength: 25; BC volume: 33-35; level of BC: 5.5. RBCs (n: 1434) volume was 279 +/- 20 mL with 54.92 +/- 7.16 g of haemoglobin. More than 96% units had less than 1.2 x 10(9) WBC. FP volume (n: 803) averaged 279 +/- 19 mL with a WBC contamination less than 0.1 x 10(9)/L in all examined samples (n: 23). Platelet recovery in BC 92 +/- 9 percent of platelets present in WB, the percentage of removed leukocytes was 74 +/- 10 and between 13 and 15% of RBCs were lost in the BC (CI 95%). The BC volume (n: 1037) fitted the target volume of 60 mL (59-61 mL, CI 95%) except in some devices, where Optipress II lost the configuration for this parameter. 4BC-CPs (n: 325) showed a platelet yield per unit greater than 1BC-CPs (226). In addition, 80.3% of 4BC-CPs yielded more than 0.6 x 10(11) platelets per unit, whereas this criteria was only met in 59.7% of 1BC-CPs (p < 0.001). The ratio volume oper 10(9) platelets in 1 BC-CPs was significantly higher (1.57 mL) than 4BC-CPs (1.31 mL), and a greater level of 1BC-CPs (58.8%) showed pH values within 6.5-7.4 after 5 days of storage in comparison with 4BC-CPs (44.25%) (p < 0.001). CONCLUSIONS: Optipress II provides standardized and poor leukocytes blood components. CE requirements were met in a great percentage of red-cell concentrates with less than 92 and 74 percent of original platelets and leukocytes, respectively and a low loss of haemoglobin per unit. Plasma volume obtained with this system represents an optimal yield. Top and Bottom technique allowed us to reduce the number of blood units per platelet concentrate, from six to four units with similar platelet yield compared to traditional procedures. Nevertheless, we must improve the storage conditions, in orter to satisfy all the CE requirements for platelet concentrates.

A monolayer coagglutination microplate technique for typing red blood cells.

BACKGROUND AND OBJECTIVES: Serologic agglutination tests have the disadvantage of lack of an objective endpoint that can be easily read and quantitated. We have developed a new method for ABO and Rh typing based on producing a red blood cell (RBC) monolayer on microplates and the mixed hemagglutination reaction. MATERIALS AND METHODS: We have employed a new red cell fixation buffer to prepare the RBC monolayer. As the technique for grouping, we used a mixed agglutination reaction between the RBC monolayer and the RBCs to be typed. RESULTS: Compared with an automated hemagglutination system in 34,519 samples, the method is shown to be more sensitive, free of false positive reactions, and cost-effective. CONCLUSIONS: The microplate coagglutination method is accurate and lower in cost than conventional methods.

A new microplate red blood cell monolayer technique for screening and identifying red blood cell antibodies.

A new method has been developed to immobilize red blood cells in wells of microplates using a cell fixation buffer. This method has been employed for detecting and identifying red blood cell antibodies with greater sensitivity than haemagglutination antiglobulin tests, without loss of specificity. This method decreases the amount of test erythrocytes and anti-human globulin reagents employed per test, consequently lowering the cost.

Onychopathy in a patient with fetal hydantoin syndrome.

Hydantoin is an anticonvulsant drug with several side effects. A teratogenic potential has been suggested. The fetal hydantoin syndrome is an entity that consists of a broad range of morphologic and developmental disorders in children born of epileptic mothers exposed to hydantoin during pregnancy. We treated a girl in whom onychopathy was a monosymptomatic or mild form of this syndrome.

Modifications in cortical ependyma of the lizard, Podarcis hispanica, during postnatal development.

The ependymal surface, cell number, cell size and cell density were measured in adult and neonatal exemplars of the lizard, Podarcis hispanica. The ependymal monostratified surface increases with age, while that of sulcal areas decreases and the ependymocyte number remains constant. The mitotic activity found in the neonatal sulcal areas could explain the postnatal increase of neuronal number described for the cerebral cortex of the studied species.