Obtención de plasma rico en plaquetas (PRP) en el Laboratorio de Terapia Celular para uso como herramienta terapéutica en medicina regenerativa / Obtaining platelet rich plasma (PRP) in the Cell Therapy Laboratory for use as a therapeutic tool in regenerative medicine

Platelet rich plasma (PRP) is used to speed up tissue repair. Despite its widespread use, the therapeutic application of PRP generates controversies in clinical results due to the variability in methods of obtaining the different preparations and differences between the components of different types of PRP, so it's recommended to mention the type of platelet preparation used. In this article, we describe technical and biologics characteristics of our platelet product, and we compare them to different commercial preparations described in order to validate their clinical use. Our results determine that the preparation can be considered a platelet rich plasma with biological activity in vivo and in vitro, which supports its use as a valid therapeutic tool, alternative to products currently available in Regenerative Medicine. (AU)

Estandarización de la técnica Real Time PCR para la detección molecular de la mutación V617F en el gen de JAK-2 en neoplasias mieloproliferativas crónicas / Standardization of the Real Time PCR technique for the molecular detection of the V617F mutation in the JAK-2 gene in chronic myeloproliferative neoplasms

V617F mutation in exon 14 of Janus Kinase 2 gene (jak-2) is used as a molecular marker for the diagnosis of Philadelphia negative myeloproliferative neoplasms (Phi-) such as Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (MFP). To detect this mutation, we used conventional polymerase chain reaction technique (PCR), a simple and inexpensive technique, however, has some drawbacks that current technology allows to solve. During the last years, more sensitive molecular techniques have been incorporated in clinical practice to support the diagnosis, prognosis and follow-up of hematological patients. For its implementation in the clinical routine should be considered technical and economic aspects, so in this work, we evaluate the Real Time PCR technique as a diagnostic method for the detection of the Jak-2-V617F mutation, using in house primers design. Our result show that the technique implemented has a concordance index of 0.87 with the conventional PCR used in the molecular diagnosis of myeloproliferative neoplasms. In addition, it has the same specificity, greater sensitivity and, shorter execution time in relation to conventional PCR. The implementation of this diagnostic method in our Hospital is technically possible and commercially convenient. (AU)

Frecuencia génica de antígenos menores de histocompatibilidad en la población chilena y estimación de sus efectos inmunológicos en el trasplante alogénico de progenitores hematopoyéticos / Frequency of minor histocompatibility antigens among Chilean blood donors

Background: Minor histocompatibility antigens (mHAgs) play a critical role in the immune responses associated with allogeneic stem cell transplantation, such as graft versus host disease (GVHD) and graft-versus-tumor (GVT). Aim: To determine the gene frequencies of the mHAgs HA-1, HA-2 and HA-8 in Chilean Blood Bank donors. Material and Methods: Blood from 192 blood donors was analyzed. The presence of haplotype HLA-A*02 was determined by flow cytometry. The frequency of mHAgs was determined by allele specific polymerase chain reaction in genomic DNA. Results: Sixty one participants were carriers of the haplotype HLA-A*02. The relative allele frequency HA-1H was 45%, HA-Ir 55%, HA-2V 80.6%, HA-2M 19.4%, HA-8R 49.8% and HA-8P was 50.2%. Based on mHAgs disparity between HA-1, HA-2 or HA-8, the probability to generate a GVT response in HLA-A*02 individuals was 40%. Conclusions: The mHAgs frequency in Chilean population is under Hardy-Weinberg equilibrium and they are similar to those of other ethnic populations in the world.

Accidentes de riesgo biológico entre estudiantes de carreras de la salud: Cinco años de experiencia / Biological risk accidents among undergraduate healthcare students: Five years experience

Undergraduate healthcare students are exposed to bloodborne pathogens, and data from developing countries is scarce. We report the experience of a comprehensive program dedicated to the management of this risk. The program includes financial coverage, a 24-hour attention system, HIV, HBV, HCV testing, and free provisión of post-exposure antiretroviral drugs. During 2003-2007, incidence rates of these exposures reached 0.9 per 100 student-years. Events were only observed among medicine, nursing, and midwifery students, with rates highest among nursing students (RR 3.5 IC95 1.93 - 6.51). Cuts andneedle stick injuries predominated (74.7 percent of accidents). Three students were exposed to HIV patients (1.9 percent), all of them received prophylactic drugs, infection was discarded after follow up, and also discarded after exposures to HBV or HCV (0.6 percent of all accidents). Cost per 1000 student-year was less than 2000 USD. Healthcare students are exposed to biological risks during their studies and a comprehensive program is feasible in a developing country.

Los estudiantes de pregrado de las carreras de la salud están expuestos a riesgos biológicos con agentes de transmisión sanguínea. En este trabajo se reporta la experiencia acumulada con un programa integral para este tipo de accidentes y que incluye atención gratuita las 24 horas, estudio serológico de la fuente para VIH, VHC y VHB, y entrega de anti-retrovirales post-exposición a pacientes infectados por VIH. Desde el año 2003 al 2007 la tasa de incidencia alcanzó una cifra de 0,9 eventos por 100 estudiantes-año. Las exposiciones de riesgo fueron observadas sólo entre estudiantes de medicina, enfermería y obstetricia, siendo la mayor tasa en alumnos de enfermería (RR 3,5 IC95 1,93 a 6,51). Tres alumnos estuvieron expuestos a pacientes con infección por VIH (l,9 por cientoo de todos los accidentes), todos ellos recibieron profilaxis, descartándose seroconversión en el seguimiento, al igual que en casos con exposición ante VHB y VHC (0,6 por cientoo del total de accidentes). El costo del programa fue menor a US$ 2000 por 1.000 estudiantes-año. Los estudiantes de las carreras de la salud están expuestos a riesgos biológicos durante sus estudios y requieren de un programa de manejo, el que es posible de lograr en un país en desarrollo.

Respuesta inmune contra antígenos menores de histocompatibilidad en el trasplante alogénico de progenitores hematopoyéticos / Immunologycal response under antigens hematopoietic stem cell transplantation

Patients who receive allo hematopoietic stem cell transplantation (SCT) could develop graft versus host disease and/or graft versus tumour effect. These immunological responses can happen even with perfect fully HLA matched haematopoietic stem cells. Moreover, the engraftment of the donor’s cells depends on the immunological conditions of both donor and recipient. The development of alloreactivity occurs in the context of the polymorphisms of the human genome, these genomic differences results in proteins with antigenic properties which trigger immune responses. Considering this, the SCT is a powerful tool to heal the patient disease, because all of them become chimeras. In other words, into individuals with two different genomic sets, which will develop a strong immunological response that cannot exist in natural conditions.

Polimorfismo del gen de la tiopurina S-metiltransferasa en donantes de sangre de un hospital universitario / Thiopurine S-methyltransferase gene polymorphism in Chilean blood donors

Background: Thiopurine S- methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of 6-mercaptopurine and azathioprine. Lowactivity phenotypes are correlated with polymorphism in the TPMT gene. Patients with low or undetectable TMPT activity could develop severe myelosuppression when they are treated with standard doses of thiopurine drugs. Since ethnic differences in the TPMT gen polymorphism have been demonstrated worldwide, its assessment in the Chilean population is worthwhile. Aim: To investigate the TMPT gene polymorphism in a Chilean blood donor individuals. Subjects and Methods: The frequency of four allelic variants of the TPMT gene, *2 (G238C), *3A (G460A and A719G), *3B (G460A) and *3C (A719G) were analyzed in 210 Chilean blood donors, using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and allele-specific PCR-based assays. Results: TPMT variants associated to low enzymatic activity, were detected in 16 subjects (8%), who had a heterozygous genotype (*3A in 12; *3C in three and *2 in one subject). No TPMT*3B allelic variant was found. The normal allele (wild-type) was found in 92% of studied individuals. Conclusions: The allele TPMT*3A, is the most prevalent in this group of Chilean blood donors, as in Caucasian populations.

Terapia transfusional: criterios de indicaciones de componentes sanguíneos / Transfusion therapy: criteria for indications of blood components

The new development in transfusion medicine has allowed that blood component would be available safely for patients. However, like other medical therapies, we should establish clearly the indications based on the best benefit/risk relationship. In the following paragraphs the indications for red cells, platelets, fresh frozen plasma and cryoprecipitate will be shown. Finally, the risks and adverse events to blood transfusion will be reviewed.

Experiencia del Hospital Clínico de la Universidad de Chile en el uso de tecnicas de laboratorio en el lugar de atención del paciente (point of care) / Experience of the Hospital Clínico de la Universidad de Chile in the use of techniques of laboratory in the place of attention of the patient

La tecnología de Point of Care o Near-patient testing, permite acercar el laboratorio a las áreas asistenciales críticas, las cuales requieren minimizar al máximo el tiempo de respuesta en la obtención del resultado para la toma de decisiones. En este trabajo se presenta la correlación entre los equipos de Point of Care (i-STAT y HemoCue), respecto de las técnicas habituales utilizadas en el Laboratorio Central del Hospital Clínico de la Universidad de Chile, para los parámetros pH, PO2, PCO2, sodio (Na+), potasio (K+), hematocrito y hemoglobina. Los resultados obtenidos en los equipos Point of Care evaluados muestran una adecuada confiabilidad respecto de los métodos tradicionales utilizados en el Hospital. La determinación de gases sanguíneos, evidenció una muy buena correlación entre los equipos i-STAT y AVL, como para la determinación de hemoglobina en equipo HemoCue y Celldyn 3700. Los resultados obtenidos de electrolitos en el equipo i-STAT e Hitachi 912 igualmente se relacionan bien, a pesar de que las mediciones de sodio y potasio en i-stat fueron realizadas en sangre completa y la técnica tradicional, Hitachi 912, utiliza suero. Las correlaciones en las determinaciones de hematocrito y hemoglobina al utilizar el equipo i-STAT, no fueron tan óptimas, debido probablemente a diferencias en las metódicas de detección. Los resultados obtenidos nos hace concluir que las técnicas de Point of Care constituyen herramientas útiles para los médicos de las unidades críticas, ya que entregan resultados de laboratorio confiables y oportunos.

Point of Care or Near-patient testing diagnostic techniques allow to approach the laboratory to critical care unit, with the goal of reducing the total response time of the analytical procedure. In this paper, we compare the results obtained in the Point of care equipment (i-STAT and Hemocue) with the common techniques used in the Central Laboratory of the University of Chile Hospital, for the determination of pH, PO2, PCO2, sodium, potassium, hematocrit and hemoglobin. In general, the results obtained in the point of care equipment showed a good correlation respect to the traditional techniques. The measurement of arterial blood gases showed a good correlation between i-STAT and AVL equipment and also for measurement of hemoglobin with Hemocue. The electrolyte analysis by i-STAT equipment also exhibited a good correlation with the traditional method, despite of the measurement of sodium and potassium were performed with whole blood instead of serum like the traditional method. The correlation in the hematocrit/hemoglobin levels by i-STAT were not that good, due probably to difference in the method of detection. The results obtained allow us to conclude that the Point of care techniques constitutes useful tools for physicians in critical care units since they give reliable and rapid results.

Estudio de quimerismo: aplicación en pacientes con trasplante de progenitores hematopoyéticos / Chimerism study: application in patients with allogeneic progenitor cell transplantation

El trasplante alogénico de progenitores hematopoyéticos (TAPH) es una técnica que ha cambiado el pronóstico de muchas enfermedades hematológicas malignas y no malignas. En determinados tiempos post trasplante coexisten células hematológicas de receptor y dador, por lo cual el individuo posee dos sistemas hematopoyéticos. El término Quimera se utiliza para indicar el origen dual de las células hematológicas. Este análisis es indispensable para saber si existe prendimiento o rechazo del trasplante. La determinación de quimerismo se realiza mediante la amplificación por PCR de secuencias cortas repetidas tandem (STRs del ingles short tandem repeat sequence). Este examen es fácil de realizar, reproducible, altamente sensible y específico. El resultado del quimerismo es de vital importancia a la hora de tomar decisiones clínicas, sobre todo si se utilizan técnicas no mieloablativas de acondicionamiento, infusión de linfocitos del donante, o modificaciones en los protocolos de inmunosupresión, donde existe una lata variabilidad en el prendimiento del injerto y el desarrollo de enfermedad de injerto contra huésped (EICH) e injerto contra tumor (ICT).

Allogeneic progenitor cell transplantation (ALoPCT) is a procedure that has changed the prognosis of many malignant and non-malignant hematologic diseases. For a period of time post-trasplant, cellular subtypes from the donor and the host coexist, giving the patient two hematopoietic systems. The term Chimera is used to indicate the dual origin of blood cells. This analysis is important to determine whether engrafment or rejection has occurred. The determination of chimerism is based on PCR pf Short Tandem Repeat sequences (STRs). The PCR technique is easy to perform, reproducible, highly sensitive and specific. Chimerism determination helps to improve the clinical approach to the patient, specifically when non-mieloablative conditioning, lymphocyte donor infusion or modification of the immunosuppressive protocols have employed. All of these manipulations produce a high variability in engraftment, development of graft versus host disease (GVHD) and the likelihood to eliminate tumor cells through graft versus tumor (GVT) effects.

Autotrasplante (AT) de progenitores hematopoyéticos en mieloma múltiple: Experiencia clínica / Autologous transplant (AT) with peripheral-blood stem-cell rescue for multiple myeloma: A clinical experience

Background:Multiple myeloma is rarely curable. Advances in high dose chemotherapy and stem cell transplantation have improved overall survival and event-free disease periods, but relapses are inevitable. Aim: To report our experience with AT in multiple myeloma, between 1994 and 2003. Material and Methods: Retrospective analysis of 20 patients (12 women), with a mean age of 51.1 years. VAD (vincristine, doxorubicin and dexamethasone) was used as initial therapy in 19 patients. High dose cyclophosphamide (11 patients) and variations of VAD regimen (7) associated with granulocyte colony stimulating factor were used for peripheral-blood stem cell harvest. The conditioning regimen consisted of melphalan 200 mg/m2 followed by the reinfusion of peripheral-blood stem cells 24 hours later. The median number of CD34 cells infused was 3,3x106/kg. Three patients were subjected to a second auto graft and one to a non-myeloablative transplant. Mean follow up was 35.5 months. Results: Mucositis and febrile neutropenia were common complications. The median number of days for neutrophyl engraftment was 9 (range 8-11) and for platelets, 10 (range 7-13). No patient died. Complete remission was obtained in 60% (12/20), progession-free survival was 30 months and overall median survival, 47 months. Conclusions: The AT with high-dose melphalan is a safe procedure in our hospital, without mortality and engraftment in all the patients. Complete remission and progression free survival were similar to those reported abroad but the overall median survival was lower.

Linfoma intravascular tratado con anticuerpos monoclonales anti CD20: descripción de un caso clínico / Intravascular lymphoma treated with anti CD20 monoclonal antibodies: report of one case

We report a 78 year old male with prostatism, that was subjected to a prostate biopsy. The pathological study showed a microvascular lymphocytic infiltration. Four months later, the patients presentd with reduced alertness, cough, dyspnea, fever and elevation of lactic dehydrogenase and erythrocyte sedimentation rate. Chest and abdominal CAT scans, bone marrow aspirate, protein electrophoresis and prostate specific antigen were normal. A re-evaluation of prostate biopsy showed an intravascular lymphoid infiltration, positive for CD45 and CD20, compatible with the diagnosis of intravascular lymphoma. Chemotherapy was started, but it was not tolerated by the patient and the response was partial. Therefore, treatment with monoclonal antibodies anti CD20 (Rituximab) was started. The tumor had a complete and prolonged (24 months) remission after the treatment.