Multiplex fluorescent analysis of four short tandem repeats for rapid haemophilia A molecular diagnosis.

Indirect molecular diagnosis of hemophiliaA (HA) is carried out by analyzing intragenic polymorphic markers described along the coagulation factorVIII (FVIII) gene. Several studies have demonstrated that the two commonly used intronic short tandem repeats (STR13 and STR22) located in the FVIII gene are highly informative for this task. Two extragenic markers closely linked to FVIII (DXS1073 and DXS1108) have also been described as valuable tools for gene tracking.

[Oral iron and folic acid supplements in a preoperative autologous blood collection program: a randomized study].

Background And Objective: We evaluated the capacity of oral iron with or without oral folic acid administration to improve the accomplishment of our scheduled preoperative autologous blood collection program in patients with baseline hemoglobin > 115 g/l.

Patients And Method: Patients were enrolled in a randomized trial. The control group received no vitamin supplements.

A case of non-lethal pulmonary air embolism after leukapheresis catheter removal.

Mononuclear cell leukapheresis requires good-quality venous access. Catheter placement and removal of the catheter may be associated with life-threatening local or systemic complications. Thus, prompt recognition of these complications and appropriate therapy can be life-saving.

Von Willebrand gene tracking by single-tube automated fluorescent analysis of four short tandem repeat polymorphisms.

Molecular diagnosis of von Willebrand disease (VWD) has been hampered by the large size and complex genomic characteristics of the gene involved. For this reason, indirect methods using intragenic polymorphic markers described along the von Willebrand factor (VWF) gene are valuable tools for gene monitoring and linkage analysis. Several studies have demonstrated the four commonly utilized short tandem repeats (STRs), three located in intron 40 and one in the promoter region of the VWF gene, to be highly informative for this task.

[Therapeutic applications of stem-cells].

In the last years stem cells (SC) have generated huge expectations and have become a new hope for the development of novel cell therapies in the context of regenerative medicine. So far, the hypothetic therapeutic effects of SC, both of embryonic and adult origin, have been demonstrated only in a very few cases. Embryonic SC are pluripotential and have, in theory, more plasticity to differentiate into a wide range of cell or tissue types.

Development of a new HLA-DRB real-time PCR typing method.

Polymerase chain reaction (PCR)-based human leukocyte antigen (HLA) typing methods currently used in most histocompatibility laboratories, such as PCR-sequence-specific primers (PCR-SSP) and PCR-sequence-specific oligonucleotide probes (PCR-SSO), are time-consuming and are at risk of contamination during the post-PCR process. The aim of this study was to develop a real-time PCR (rtPCR)-based HLA-DRB1 and -DRB3/4/5 low-medium resolution typing method to avoid these problems. This new method combined the use of specific primers and probes for HLA-DRB alleles.

Thrombotic thrombocytopenic purpura and pregnancy: a review of ten cases.

Background And Objectives: A series of women with pregnancy-associated thrombotic thrombocytopenic purpura, is presented. This study will focus on the relationship between thrombotic thrombocytopenic purpura and pregnancy and on maternal and neonatal outcomes.

Materials And Methods: Among forty-six consecutive patients with thrombotic thrombocytopenic purpura, nine pregnant patients were identified.

Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs.

Background: To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively.

Methods: ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates, and a series of potential inhibitors of the transporter.

Results: ABCG2 expression was detected in all cell lines.

Retroviral vectors: new applications for an old tool.

Retroviral vectors (RVs) have been used for stable gene transfer into mammalian cells for more than 20 years. The most popular RVs are those derived from the Moloney murine leukaemia virus (MoMLV). One of their main limitations is their inability to transduce noncycling cells.

One thousand seventy antibodies detected only by a 2-stage papain test: wanted and unwanted positive reactions.

Despite the wide use of the antibody detection test for unexpected antibodies, controversy still remains regarding the use of enzyme-treated red blood cells. Over a 6-year period, 72,573 samples from 49,863 patients submitted for pretransfusion compatibility testing were examined for unexpected antibodies. The antibody detection tests included a low-ionic-strength solution (LISS) indirect antiglobulin test and a two-stage papain (2SP) test.

Autoanti-D in a patient after cladribine treatment for lymphoplasmocytic lymphoma.

We report the case of a 62-year-old woman who developed an autoanti-D after cladribine treatment. In May 2000, the patient underwent splenectomy for a stage IV-B lymphoplasmocytic lymphoma. She was transfused with ABO- and Rh(D)-matched blood.

The direct antiglobulin test in a hospital setting.

To evaluate the current use of the DAT in our hospital,we reviewed the charts of all patients who had a DAT performed in our laboratory. The collected data included DAT results and a previously completed laboratory evaluation of suspected hemolytic anemia. Four hundred sixty-three DATs were performed in our laboratory from April 1999 to October 2001.

HLA-B27 genotyping by fluorescent resonance emission transfer (FRET) probes in real-time PCR.

Several polymerase chain reaction (PCR)-based human leukocyte antigen (HLA) genotyping methods are in use, but none is fully satisfactory. The introduction of real-time PCR (rt-PCR) with fluorescence resonance energy transfer (FRET) probes provides a powerful tool to overcome the drawbacks of current methods such as the long processing time and the requirement for post-PCR manual procedures. Here we present evidence that the FRET-fluorotyping principle may resolve HLA-B27 variants, providing a higher resolution in less time than the techniques currently in use.

First molecular characterization of an unequal homologous alu-mediated recombination event responsible for hemophilia.

The large number of Alu repeats in the human genome provides abundant opportunities for unequal homologous recombination events that are responsible of several human diseases. We here describe a novel large FVIII gene deletion from a severe hemophilia A patient in which Alu-repetitive elements are directly involved in the origin of the mutation. Using a long-fragment PCR method, a approximately 23 kb deletion was delimited between introns 24 and 25.

FK506 in the maturation of dendritic cells.

Background And Objectives: FK506 (tacrolimus) is a potent immunosuppressive agent that inhibits interleukin-2 (IL-2) and interferon-g production by CD4+ cells. The effect of this agent on dendritic cells (DCs), the highly professional antigen-presenting cells for T-cells, has not been completely defined. We investigated the effect of FK506 on DC differentiation from monocytes, and on the shift from immature to mature immunophenotypes.

Absence of anti-D alloimmunization in hematologic patients after D-incompatible platelet transfusions.

Background: Rh antigens are not present on the platelet surface. However, platelet concentrates may contain enough RBCs to elicit an anti-D response. Thus, D status must be considered in platelet transfusion.

[Promotion of the blood donation in a hospital: comparison of two approaches].

Background: Our purpose was to evaluate the results of the promotion of blood donation in patients undergoing stem cell transplantation (SCT) and to compare it with this promotion in remaining in-patients.

Patients And Methods: The hematologist and the blood bank staff informed the patient undergoing SCT about the need for transfusions. Donors were selected for blood or platelet donation.

Identification of a novel HLA-DQB1*06 allele, DQB1*0618.

We report here the identification of a novel DQB1*06 allele, DQB1*0618, found in a bone marrow donor. The new allele was detected during routine DNA-based HLA typing by an ambiguous pattern of probe hybridization, obtained by polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO). Molecular cloning and sequencing confirmed that the new allele is identical to DQB1*0609 at exon 2 except for 3 nucleotide substitutions at positions 353, 356 and 367, also found in other alleles.

Identification of a new HLA-DRB1*04 allele, DRB1*0437.

In this report we describe the identification of a novel DRB1*04 allele, DRB1*0437, found in a Spanish individual. The routine HLA typing, in the context of bone marrow transplantation, by polymerase chain reaction-sequence-based typing (PCR-SBT) made possible the identification of this new allele. This allele is identical to DRB1*0402 except for a single nucleotide substitution at position 286 (A-->C), changing the encoded Isoleucine to a Leucine.

Interferon and ribavirin combination therapy for chronic hepatitis C in human immunodeficiency virus-infected patients with congenital coagulation disorders.

We have conducted an open, prospective trial to assess the safety and efficacy of interferon alfa-2b and ribavirin in combination for the treatment of chronic hepatitis C in human immunodeficiency virus (HIV)-infected hemophiliacs. Twenty hemophiliacs coinfected with HIV and hepatitis C virus (HCV), 18 of them under highly active antiretroviral therapy (HAART), with a mean CD4(+) cell count of 490 +/- 176 cells/mm(3) and undetectable (n = 9) or low-level HIV RNA (<10,000 copies/mL; n = 11), were treated with interferon-alfa2b (3 MU thrice weekly) and ribavirin (800 mg/d) for 6 or 12 months according to virologic response. Patients were monitored for tolerance and response at 4, 8, 12, 24, 36, and 48 weeks during treatment and every other month thereafter.

Identification and sequence characterization of a novel HLA-A11 serological variant (HLA-A*1108).

In this report we describe the identification of a novel HLA-A*11 allele, HLA-A*1108, found in two individuals of a Spanish family. This new allele was detected during routine HLA typing by an atypical serological reactivity pattern and by inconclusive patterns obtained in DNA-based typing methods. The nucleotide sequence of exons 2 and 3 of HLA-A*1108 was identical to HLA-A*11011 except for two nucleotide substitutions at codons 152 (GCG-->GAG) and 156 (CAG-->CGG).

Factor IX gene sequencing by a simple and sensitive 15-hour procedure for haemophilia B diagnosis: identification of two novel mutations.

We have developed a simple, sensitive and cost-effective direct DNA sequencing procedure for the molecular diagnosis of haemophilia B. All factor IX gene essential regions were amplified under identical thermocycling parameters allowing mutation identification in less than 15 h from blood sample collection. Identical results in terms of accuracy and speed were obtained when using a single hair as the source of DNA.

[Immunohematologic study of ABO hemolytic disease].

Aim: ABO-hemolytic disease occurs when ABO-incompatibility is present between the mother and the fetus. The objective of the present study was to analyze ABO-hemolytic disease in our center, to compare two serologic tests to diagnose it and to evaluate hematocrit values in cord blood samples.

Methods: ABO and Rh(D) group was found in 881 blood samples.