[Growth hormone of dried blood spot for the diagnosis of growth hormone deficiency]. / Hormona de crecimiento en sangre de papel filtro para el diagnóstico de deficiencia de hormona de crecimiento.

INTRODUCTION: The diagnosis of growth hormone deficiency (GHD) is difficult to determine, and could be associated with severe complications, especially in the neonatal period. The stimulation test of growth hormone (GH) secretion is considered the gold standard for diagnosis, but it has methodological complications and is associated with adverse effects. Neonates present physiological increased secretion of GH, representing a diagnostic window. OBJECTIVE: To evaluate if the dried blood spot on filter paper obtained in the neonatal period, as part of a neonatal screening for con genital hypothyroidism and phenylketonuria, allows differentiating patients with GHD from those who do not have it. PATIENTS AND METHOD: Study of cases and controls by measuring the GH concen tration in dried blood spot on filter paper obtained in the neonatal period, comparing controls with GHD with cases with discarded deficiency. The sample was extracted from the filter paper, obtaining two 0.125 inch discs per each patient from the center of the blood spot on the paper, for a highly sen sitive ELISA assay for human GH based on the use of polyclonal antibodies against 22 kDa recom binant human GH. RESULTS: Seven cases of GHD and ten controls were obtained. The median GH concentration of the dried blood spot in the cases is 2.0 ng/ml (Interquartile range 3.6 ng/ml) and 2.05 ng/ml (Interquartile range 2.0 ng/ml) in the controls, Mann-Whitney U test 30.5 (p = 0.68). The two cases with multiple pituitary-hormone deficiency (MPHD) present concentrations lower than 1 ng/ml. CONCLUSION: The dried blood spot sample did not differentiate GHD patients from control cases, although MPHD cases present much lower concentrations compared to isolated growth hor mone deficiency (IGHD).

Inherited disorders of platelet function and challenges to diagnosis of mucocutaneous bleeding.

SUMMARY: Platelets play a pivotal role in the arrest of bleeding at sites of vascular injury. Following endothelial damage, they respond rapidly by adhesion to subendothelial matrix proteins resulting in platelet activation, spreading, aggregation, secretion and recruitment of additional platelets to form the primary haemostatic plug. This mass provides a surface for thrombin generation and fibrin mesh formation that stabilizes the clot. Careful study of patients with inherited platelet disorders and, subsequently, of informative animal models, has identified structural platelet abnormalities that have enhanced our understanding of platelet function. The investigations of rare, but severe, inherited platelet disorders have led us to the discovery of causative molecular defects. One of the most informative is the rare autosomal recessive disorder Glanzmann thrombasthenia, caused by defect or deficiency in the platelet integrin alphaIIbbeta3, resulting in absent platelet aggregation and a significant clinical bleeding diathesis. Our new challenge is to understand the mechanisms underlying more common, but less well-defined, mucocutaneous bleeding (MCB) disorders. Present diagnostic testing for platelet function disorders and von Willebrand's Disease often fails to identify the cause of bleeding in individuals with inherited MCB.

Diagnostic challenges of inherited mild bleeding disorders: a bait for poorly explored clinical and basic research.

The best-known inherited mild bleeding disorders (MBDs), i.e. type 1 von Willebrand disease (VWD), platelet function disorders (PFDs), and mild to moderate clotting factor deficiencies, are characterized clinically by mucocutaneous bleeding, and, although they are highly prevalent, still pose difficult diagnostic problems. These include establishing the pathological nature of bleeding, and the uncertainties surrounding the clinical relevance of laboratory results. Furthermore, the high frequency of bleeding symptoms in the normal population and the subjective appraisal of symptoms by patients or parents makes elucidating the pathological nature of bleeding difficult. Standardized bleeding assessment tools and semiquantitative bleeding scores (BSs) help to discriminate normal from abnormal bleeding. However, as most MBDs have similar bleeding patterns, for example, bleeding sites, frequency, and severity, BSs are of little help for diagnosing specific diseases. Global tests of primary hemostasis (bleeding time; PFA-100/200) lack sensitivity and, like BSs, are not disease-specific. Problems with the diagnosis of type 1 VWD and PFD include assay standardization, uncertain definition of von Willebrand factor cut-off levels, and the lack of universal diagnostic criteria for PFD. Regarding clotting factor deficiencies, the bleeding thresholds of some coagulation factors, such as factor VII and FXI, are highly variable, and may lead to misinterpretation of the clinical relevance of mild to moderate deficiencies. Remarkably, a large proportion of MBDs remain undiagnosed even after comprehensive and repeated laboratory testing. These are tentatively considered to represent bleeding of undefined cause, with clinical features indistinguishable from those of classical MBD; the pathogenesis of this is probably multifactorial, and unveiling these mechanisms should constitute a fertile source of translational research.

Osler-Weber-Rendu Syndrome in Relation to Dermatology. / Síndrome de Osler-Weber-Rendu y su relación con la dermatología.

Osler-Weber-Rendu syndrome, also known as hereditary hemorrhagic telangiectasia, is a rare autosomal dominant disorder with an estimated worldwide prevalence of 1 case per 10,000 population. Its clinical manifestations are the result of arteriovenous malformations characterized by telangiectases that can affect the skin, mucous membranes, and solid organs and cause life-threatening conditions, such as liver disease, systemic emboli, and heart failure. Timely diagnosis is thus essential in order to prevent disease-related complications and offer genetic counseling to families. We review the clinical features of Osler-Weber-Rendu syndrome with a focus on mucocutaneous manifestations and their treatment.

Template bleeding time and PFA-100 have low sensitivity to screen patients with hereditary mucocutaneous hemorrhages: comparative study in 148 patients.

OBJECTIVES AND PATIENTS: We compared the template bleeding time (BT) and closure time (CT) in the PFA-100 as screening tests in 148 consecutive patients with unequivocal mucocutaneous bleeding and positive family history. EXCLUSION CRITERIA: drug intake, concomitant diseases including minor infections, low platelet count, diseases of secondary hemostasis. RESULTS: Type 1 von Willebrand disease (VWD-1) was diagnosed in 26 patients, primary platelet secretion defect (PSD) in 33, VWD-1 + PSD in nine, whereas 80 patients did not comply with the criteria for known hemostatic disorders (UD, unknown diagnosis). BT and CT were prolonged in 35.8% and 29.7% of all the patients, respectively (P = 0.23). Sensitivity increased to 48% if an abnormality of BT and/or CT was considered. Same comparisons for BT and CT in each diagnostic category were, respectively: 42 vs. 61.5% in VWD-1 (P = 0.18), 42 vs. 24% in platelet secretion defects (P = 0.11), 67 vs. 89% in VWD-1 + PSD (P = 0.50), and 27.5 vs. 15% in UD (P = 0.06). CONCLUSION: Both tests were relatively insensitive and not significantly different in detecting incoming patients with mucocutaneous hemorrhages. In patients with VWD-1, the PFA-100 performed slightly better, whereas the opposite occurred in those patients with platelet secretion defects. In the UD group, both tests lost sensitivity, but the BT detected 1.8 times more patients than the PFA-100. Given the large proportion of undiagnosed bleeders and the overall low sensitivity of these tests, clinical decisions still rely on the medical history and etiological diagnosis of the bleeding disorder.

Measurement of pleural fluid cholesterol and lactate dehydrogenase. A simple and accurate set of indicators for separating exudates from transudates.

OBJECTIVES: To evaluate the usefulness of diverse combinations of pleural cholesterol concentration, pleural or serum protein, and lactate dehydrogenase (LDH) levels for the differentiation of pleural exudates and transudates. DESIGN: Prospective laboratory study of pleural effusions. SETTING: Medical school hospital. PATIENTS: One hundred eighty consecutive internal medicine ward patients in whom the etiologic diagnosis of their pleural effusion was confirmed. MEASUREMENTS: Cholesterol concentration in pleural fluid and protein and LDH both in pleural fluid and blood serum. RESULTS: According to their etiology, 49 (27.2%) of the effusions were transudates and 131 (72.7%) were exudates. Using a cutoff point of 45 mg for pleural cholesterol and values for protein and LDH of Light et al, the best diagnostic power corresponded to the combination of pleural cholesterol and LDH: cholesterol level over 45 mg/dL and/or LDH over 200 IU/L identified exudates with a sensitivity of 99% and a specificity of 98%. All the other combinations showed inferior values and the criteria of Light et al reached 98 and 82%, respectively. CONCLUSIONS: The measurement of pleural cholesterol and LDH permits the separation of pleural exudates from transudates with an accuracy similar to the original report of Light et al, with the advantage of requiring only two laboratory determinations and no simultaneous blood sample.

Diagnosis of typhoid fever by two serologic methods. Enzyme-linked immunosorbent assay of antilipopolysaccharide of Salmonella typhi antibodies and Widal test.

Serum samples from 85 patients with proven typhoid fever, 11 patients with p-typhoidal fever, 101 patients with febrile non-typhoidal, and 130 healthy subjects were tested for immunoglobulin G (IgG), IgA, and IgM antilipopolysaccharide (LPS) of Salmonella typhi antibodies by enzyme-linked immunosorbent assay (ELISA) and Widal test. The levels of all three classes of immunoglobulin anti-LPS of S. typhi were higher in typhoid patients than in healthy or febrile nontyphoidal groups; we selected various combinations between the three classes of immunoglobulin to obtain the best combination of sensitivity and specificity. The sum of the absorbance values obtained from the ELISA assay for IgG+IgA+IgM (sigma lgs) was the best choice for diagnostic utility for typhoid fever. We selected a positive test at a decision level of sigma lgs > or = 1.2 with a sensitivity of 94% and a specificity of 92% with a frequency of false negative of 5.9%. The frequency of false positives for healthy controls was 7.7% and, for the febrile nontyphoidal group, it was 7.9%. We also compared receiver (or relative) operating characteristic (ROC) curves for the diagnostic usefulness of the ELISA with that of the Widal test, whose merits and limitations, especially in endemic regions, are discussed. The ELISA assay was much more sensitive and specific than any combination of the Widal test, and hence it could be a useful tool for the serologic diagnosis of typhoidal fever with a single blood sample.

Quantitative impact of using different criteria for the laboratory diagnosis of type 1 von Willebrand disease.

INTRODUCTION: Only ± 50% of patients with type 1 von Willebrand disease (VWD) have recognized molecular defects and diagnosis still rests on demonstrating low plasma von Willebrand factor (VWF) protein/function. However, no generalized consensus exists regarding the type and number of VWF variables that should be considered for diagnosis. AIM: To compare the quantitative impact of four different criteria to diagnose type 1 VWD. METHODS: We tested four laboratory criteria on 4298 laboratory studies during a 5-year period. The first was the National Heart, Lung, and Blood Institute recommendation, which diagnoses type 1 VWD with plasma VWF antigen (VWF:Ag) and VWF ristocetin cofactor (VWF:RCo) < 30 IU dL(-1) and possible VWD/'low VWF' with values between 30 and 50 IU dL(-1) . Second, diagnosis was established when two of three variables, VWF:Ag, VWF:RCo, VWF collagen binding assay (VWF:CB), were ≤ 2.5th percentile. Diagnostic criterion for possible VWD/'low VWF' using percentiles was also described. The third criterion (European Group on von Willebrand Disease, EUVWD), uses a plasma level of VWF:RCo (or VWF:CB) ≤ 40 IU dL(-1) for diagnosis. Finally, the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCBVWD) diagnoses VWD if VWF:Ag or VWF:RCo are ≤ 40 IU dL(-1) . RESULTS: The three assays had high correlation and excellent agreement at levels < 120 IU dL(-1) . The National Heart, Lung, and Blood Institute recommendation was followed to diagnose 122 (2.8%) patients with type 1 VWD and 704 (16.4%) with possible VWD/'low VWF.' Using percentiles, the diagnosis of type 1 VWD increased to 280 (6.5%) patients; 169 (3.9%) patients had possible VWD and 180 (4.2%) patients had 'low VWF.' Diagnoses using EUVWD and ZPMCBVWD criteria increased to 339 (7.9%) and 357 (8.3%) patients, respectively. DISCUSSION: Identical data, analyzed using different criteria, led to almost three-fold difference (2.8-8.3%) in diagnostic rate. This increase is mostly explained by increasing the cut-off values of VWF measurements from < 30 to ≈ 40 IU dL(-1) . Further refinement of the laboratory diagnosis of type 1 VWD is a priority.

Hormona de crecimiento en sangre de papel filtro para el diagnóstico de deficiencia de hormona de crecimiento / Growth hormone of dried blood spot for the diagnosis of growth hormone deficiency

INTRODUCCIÓN: El diagnóstico de deficiencia de hormona de crecimiento (DHC) es difícil de establecer, y se puede asociar a serias complicaciones, especialmente en el período neonatal. La prueba de estímulo de secreción de hormona de crecimiento (HC) se considera de elección para el diagnóstico, pero presenta complicaciones metodológicas y se asocia a efectos adversos. Los neonatos presentan aumento de la secreción de HC de forma fisiológica, siendo una ventana diagnóstica. OBJETIVO: Evaluar si la muestra de sangre en papel filtro tomada en el período neonatal, en contexto del tamizaje neonatal de hipotiroidismo congénito y fenilcetonuria, permite diferenciar pacientes con DHC, de los que no la presentan. PACIENTES Y MÉTODO: Estudio de casos y controles mediante determinación de concentración de HC en sangre de papel filtro extraída en período neonatal, comparando controles con DHC con casos con deficiencia descartada. Se realizó extracción de la muestra del papel filtro, obteniendo dos discos de 0,125 pulgada por cada uno de los pacientes desde el centro de la mancha de sangre del papel, para un ELISA de HC humana altamente sensible basado en el uso de anticuerpos policlonales dirigidos contra la HC humana recombinante de 22kDa de peso molecular. RESULTADOS: Se obtuvo un total de 7 casos de DHC y 10 controles. La mediana de concentración de HC de papel filtro en los casos es 2,0 ng/ml (Rango intercuartil 3,6 ng/ml) y controles 2,05 ng/mL (RIC 2,0 ng/ml), U de Mann-Withney 30,5 (p = 0,68). Los dos casos con deficiencia de hormonas hipofisarias múltiples (DHHM) presentan concentraciones menores a 1 ng/ml. CONCLUSIÓN: La muestra de papel filtro no permitió diferenciar a los pacientes con DHC de los casos controles, aunque los casos con DHHM presentaron concentraciones mucho menores, en comparación a la deficiencia de hormona de crecimiento aislada (DHCA).

INTRODUCTION: The diagnosis of growth hormone deficiency (GHD) is difficult to determine, and could be associated with severe complications, especially in the neonatal period. The stimulation test of growth hormone (GH) secretion is considered the gold standard for diagnosis, but it has methodological complications and is associated with adverse effects. Neonates present physiological increased secretion of GH, representing a diagnostic window. OBJECTIVE: To evaluate if the dried blood spot on filter paper obtained in the neonatal period, as part of a neonatal screening for con genital hypothyroidism and phenylketonuria, allows differentiating patients with GHD from those who do not have it. PATIENTS AND METHOD: Study of cases and controls by measuring the GH concen tration in dried blood spot on filter paper obtained in the neonatal period, comparing controls with GHD with cases with discarded deficiency. The sample was extracted from the filter paper, obtaining two 0.125 inch discs per each patient from the center of the blood spot on the paper, for a highly sen sitive ELISA assay for human GH based on the use of polyclonal antibodies against 22 kDa recom binant human GH. RESULTS: Seven cases of GHD and ten controls were obtained. The median GH concentration of the dried blood spot in the cases is 2.0 ng/ml (Interquartile range 3.6 ng/ml) and 2.05 ng/ml (Interquartile range 2.0 ng/ml) in the controls, Mann-Whitney U test 30.5 (p = 0.68). The two cases with multiple pituitary-hormone deficiency (MPHD) present concentrations lower than 1 ng/ml. CONCLUSION: The dried blood spot sample did not differentiate GHD patients from control cases, although MPHD cases present much lower concentrations compared to isolated growth hor mone deficiency (IGHD).

Reproducibility and performance of one or two samples of salivary cortisol in the diagnosis of Cushing's syndrome using an automated immunoassay system.

The purpose of this article is to evaluate the variability and reproducibility of late night salivary cortisol (LNSC) using electrochemiluminescence immunoassay (ECLIA) and compare the accuracy of one or two samples in diagnosis of Cushing's syndrome (CS). We prospectively included 64 healthy volunteers (HV), 35 patients with clinically suspected CS (S), and 26 patients with confirmed CS. Nine patients in the CS group had 24-h urinary free cortisol (UFC) less than two times the upper limit of normal (mild CS). UFC and two consecutive LNSC (LNSC1, LNSC2) were collected at home. All patients in the S group had normal UFC and low-dose dexamethasone suppression test. No differences were found between the HV and S groups in UFC, LNSC1, and LNSC2. Intra-individual variability between the two samples of LNSC was 22% in HV (1.6-91%), 32% in the S group (1.6-144%), and 51% (1.6-156%) in the CS group. Variability was higher in CS patients than those in the HV (P < 0.001) and S groups (P = 0.05). The AUC of LNSC1 was 0.945 (IC 95% 0.880-1.004); when considering the highest LNSC, the AUC was 0.980 (IC 95% 0.954-1.007) (P < 0.01). We found 23% of discordant LNSC in the S group and 11% in the CS group. Three patients with CS had only one elevated LNSC, all of them with mild CS. Our results suggest that LNSC is variable, and reproducibility is affected in both CS and S patients. We found significant improvements in the diagnostic accuracy of the LNSC measurement by obtaining two samples.

Diseño y simulación de un filtro de células de tipo linfocitos T en un sistema de microfluidos / Design and Simulation of a T-Type Lymphocyte Cells Filter on a Microfluidic System / Desenho e simulação de um filtro de células de tipo linfócitos T em um sistema de microfluidos

Este trabajo consistió en diseñar y validar, mediante simulación experimental por computadora, un sistema de filtraje de linfocitos T en un sistema basado en microfluidos para detección del virus del VIH. Materiales y métodos: se utilizó la herramienta de simulación AutoDesk® Inventor, con la cual se realizó el diseño del sistema de microfluídica. El sistema de filtraje se probó haciendo una simulación por computadora en la herramienta de simulación AutoDesk® Simulation cfd (computational fluid dynamics software) en la cual diferentes partículas con varios diámetros (5 µm, 10 µm, 15 µm) fluían por el sistema a probar. Resultados y conclusiones: los resultados demostraron que el sistema de filtraje permitió el paso de las partículas esperadas, sin embargo, se observó que permitió el paso de partículas más grandes que las deseadas, por lo cual hay que seguir trabajando en el perfeccionamiento del sistema. La eficiencia del sistema de filtraje fue de un 33,33 %.

This work consisted in designing and validating, by experimental computational simulation, a T-Lymphocites filtering system based on microfluidics for hiv virus detection. Material and methods: It was used AutoDesk® Inventor simulation tool was used with which the microfluidic system design was performed. The filter system was tested by a computer simulation in the AutoDesk® Simulation cfd (computational fluid dynamics software, simulation tool in which different particles with different diameters (5 µm, 10 µm, 15 µm) flow through the system to test. Results and conclusions: Results showed that this system allowed to pass the expected particles, however, it also was observed that it allows bigger particles than desired, for this reason it is necessary to keep on working on system perfectioning. Filtering system efficiency was of a 33.33 %.

Este trabalho consistiu em desenhar e validar, mediante simulação experimental por computador, um sistema de filtragem de linfócitos T em um sistema baseado em microfluidos para detecção do vírus do VIH. Materiais e metodos: utilizou-se a ferramenta de simulação AutoDesk® Inventor, com a qual se realizou o desenho do sistema de microfluídica. O sistema de filtragem provou-se fazendo uma simulação por computador na ferramenta de simulação AutoDesk® Simulation CFD (computational fluid dynamics software) na qual diferentes partículas com vários diâmetros (5 µm, 10 µm, 15 µm) fluíam pelo sistema a provar. Resultados e conclusaos: Os resultados demonstraram que o sistema de filtragem permitiu a passagem das partículas esperadas, no entanto, se observou que permitiu a passagem de partículas mais grandes que as desejadas, pelo qual deve-se seguir trabalhando no aperfeiçoamento do sistema. A eficiência do sistema de filtragem foi de 33,33 %.

[HLA A, B, C and DR antigens in a urban population from Santiago of Chile]. / Antígenos HLA-A, B, C y DR en una población urbana de Santiago de Chile.

HLA antigens vary in different ethnical groups and in Chile there are no reports on the frequency of these antigens in a normal representative population. The few existing studies are of indigenous populations and control groups, without including HLA-DR antigens. Therefore, the aim of this study was to study the frequency of HLA A, B and C antigens in 349 individuals and HLA-DR in 257, using the microlymphocytotoxicity method, and compared the results with those on normal caucasian populations (Europe and USA). Significant differences were found for 7 antigens of group A, 10 of group B, 4 of group C and 6 of group DR. The observed difference allow us to conclude that the population from Santiago has a distinct HLA antigen distribution. This fact must be bore in mind future studies in genetics, paternity or autoimmune diseases.

[von Willebrand's disease]. / Enfermedad de von Willebrand.

von Willebrand's disease is the most frequent congenital disorder of primary hemostasis; it is transmitted in an autosomal manner and according to type there is an alteration in structure, function and/or synthesis or release of von Willebrand's factor. Patients present with muco-cutaneous hemorrhages of varying severity. Significant advances in the understanding of the molecular defect and laboratory diagnosis of the different subtypes of the disease have been made in the last years, but we still face the emergence of new subtypes and the difficulties posed by the genetic, clinical and laboratory variability of the disorder.

[Diagnosis of von Willebrand's disease subtypes by the analysis of multimeric composition of plasma von Willebrand factor]. / Diagnóstico de los sub-tipos de enfermedad de von Willebrand mediante análisis de la composición multimérica del factor von Willebrand plasmático.

The accurate diagnosis of the type of von Willebrand's disease (vWD) is important for a better understanding of its pathogenesis and for adequate treatment decision. We assessed the usefulness of the analysis of the multimeric composition of von Willebrand factor (vWF) in the diagnosis of vWD variants. vWF is purified by gel filtration and polyclonal, heterologous antibodies are raised in rabbits, which are immunopurified and radioiodinated. These are used to reveal, by autoradiography, the bands of vWF of various molecular weights separated by electrophoresis in agarose gels. This procedure reveals that 74 (80%) out of 92 patients with vWD suffer the classic type (I) of the disease, 6 (6.5%) type as IIA, 6 (6.5%) as IIB and 6 (6.5%) as III. The test is necessary for the correct classification of the patients, but is complex, laborious and expensive; it should be performed in patients with high probability of vWD or after the generic diagnosis of vWD has been made.