High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. Cells derived from DC patients present short telomeres at early ages, as a result of mutations in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). However, mutations have been identified only in around 50% of the cases, indicating that other genes could be involved in the development of this disease. Indeed, mutations in TCBA1 or chromosome segment C16orf57 have been described recently. We have used HRM technology to perform genetic analysis in the above mentioned genes, in Spanish patients showing both, some clinical features of DC and short telomeres. The mutations have been identified by PCR amplification of DC genes followed by high resolution melting (HRM) and direct DNA sequencing analysis. We have identified seven new families with DC, three with X-linked DC and four with autosomal dominant DC, in which we have found two novel mutations in DKC1 (p.His68Arg and p.Lys390del) and four novel mutations in TERT gene (p.Pro530Leu, p.Arg698Trp, p.Arg971His and p.Arg698Gln). The results show that the use of HRM analysis enables a rapid and inexpensive identification of mutations in dyskeratosis congenita associated genes.

Usefulness of severity scales for cardiogenic shock in-hospital mortality. Proposal for a new prognostic model.

Cardiogenic shock (CS) is a condition comprising multiple etiologies, which associates high mortality rates. Some scoring systems have been shown to be good predictors of hospital mortality in patients admitted to Critical Care Units (CCU). The main objective of this study is to analyze their usefulness and validity in a cohort of CS patients. METHODS: Observational unicentric study of a cohort of CS patients. SOFA, SAPS II and APACHE II scores were calculated in the first 24 h of CCU admission. RESULTS: 130 patients with CS were included. SOFA, SAPS II and APACHE II scores revealed good discrimination for hospital mortality: (AUC) ROC values (AUC: 0.711, 0.752 and 0.742 respectively; P = .6). Calibration, estimated by the Hosmer-Lemeshow test, was adequate in all cases. Acute coronary syndrome, lactate serum values, SAPS II score and vasoactive inotropic score (VIS) were found to be independent predictors for mortality, upon ICU admission. With these variables, a specific prognostic indicator was developed (SAPS-2-LIVE), which improved predictive capability for mortality in our series (AUC) ROC, 0.825 (95% CI 0.752-0.89). CONCLUSION: In this contemporary CS cohort, the aforementioned scores have been shown to have good predictive ability for hospital mortality. These findings could contribute to a more accurate risk stratification in CS.

Late Onset Graft Plasmacytoma-Like PTLD Presenting as Acute Hyperglycemia in a Kidney-Pancreas Transplant Recipient.

Allograft infiltration has been described in up to 20% of all patients with posttransplant lymphoproliferative disorder (PTLD), most representing EBV-positive B-cell lymphomas. Plasma cells are often observed in humoral rejection biopsies, but graft infiltration by plasmacytoma-like PTLD is rare. We report the case of a 54-year-old simultaneous pancreas-kidney transplant recipient (immunosuppression: OKT3, methylprednisolone, cyclosporine, and azathioprine), diagnosed with an IgG-kappa monoclonal gammopathy of undetermined significance eighteen years after transplant. Nine months later, pancreas allograft biopsy performed due to new-onset hyperglycemia (HgA1C 8.6%, C-peptide 6.15ng/mL and anti-GAD 0.9UI/mL) revealed a monotypic plasma cell infiltrate, CD19, CD79a, CD138 positive, with IgG-kappa light chain restriction, and EBV negative. PET-scan FDG uptake was limited to pancreas allograft. Tumor origin could not be established (using DNA microsatellite analysis). Despite treatment with bortezomib and dexamethasone, patient eventually died one month later. This is the first report of a late onset extramedullary plasmacytoma involving a pancreas allograft.

Sustained release of bioactive glycosylated glial cell-line derived neurotrophic factor from biodegradable polymeric microspheres.

Glial cell-line derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinson's disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30 microm, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6% to 100% depending on the microsphere composition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly(vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive.

Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Purification of bioactive glycosylated recombinant glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) neuroprotective effect on dopaminergic neurons has been described in vitro and in vivo, turning up as a promising drug for the treatment of Parkinson's disease. Unglycosylated bacteria-obtained GDNF has already been successfully delivered for a long period of time through an infusion pump directly to the putamen of Parkinsonian patients. Nevertheless, improved distribution and safety issues need to be solved and alternative strategies to long-term delivery seem necessary. The use of glycosylated GDNF could eliminate some safety concerns regarding the presence of antibodies against exogenous unglycosylated GDNF used for the treatment. Therefore, we have chosen a mammalian expression system as a source of glycosylated GDNF. In the present work, we describe the purification of recombinant rat GDNF from the culture media of baby hamster kidney (BHK) cells through several purification steps. Highly pure N-glycosylated recombinant GDNF has been obtained similar to the endogenous protein. Furthermore, the purified protein is biologically active when tested its ability to induce PC12 neurite outgrowth.

Laser technique for the fabrication of blood vessels-like models for preclinical studies of pathologies under flow conditions.

In this work a method for fabricating functionalized preclinical devices is presented. The manufacturing process combines a laser indirect writing technique to fabricate a soda-lime glass master and soft-lithography methods to obtain the final structure in polydimethylsiloxane (PDMS). The roughness of the device is modified in a controlled manner by applying a post-thermal treatment to the master, and thus devices with different roughness values are created. The PDMS devices are fully covered with human umbilical vein cells in a two-step process. In order to determine the most suitable device to perform bioassays, the cell attachment to the channel is evaluated with regards to the walls roughness when flow experiments are carried out.

G-CSF increases the number of peripheral blood dendritic cells CD16+ and modifies the expression of the costimulatory molecule CD86+.

Dendritic cells (DC) play a key role in initiating immune reactions after allogeneic stem cell transplantation. The two main peripheral blood DC populations are myeloid (DC1) and lymphoplasmacytoid (DC2). A new subset of myeloid DC, expressing CD16, has been identified. We analyzed the number and CD86 expression of DC subsets in peripheral blood of 18 healthy donors, before and after granulocyte colony-stimulating factor (G-CSF) and in the inoculum of allogeneic peripheral blood transplants (allo-PBT; n=100) and allogeneic bone marrow transplants (allo-BMT; n=22). Granulocyte colony-stimulating factor administration increased the median number of DC1 (P=0.0007), of DC2 (P<0.0001) and of DC CD16+ (P=0.0001). Granulocyte colony-stimulating factor administration was also associated with a significant decrease of CD86 expression on DC1 (P=0.0003) and with a trend for an increase on DC CD16+ (P=0.07). Recipients of allo-PBT received similar quantities of DC1 and higher doses of DC2 and DC CD16+ than recipients of allo-BMT (P=0.5; P=0.0001; P<0.0001, respectively). Granulocyte colony-stimulating factor modifies the number of DC in peripheral blood and the expression of the costimulatory molecule CD86. This resulted in a different composition of DC2 and especially of DC CD16+ in the harvests, which might explain some of the differences observed in allogeneic reactions after allo-PBT with respect to allo-BMT.

[Neuroprotective and neurorestorative therapies for the treatment of Parkinson's disease]. / Terapias neuroprotectoras y neurorestauradoras en el tratamiento de la enfermedad de Parkinson.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Current therapies are symptomatic and, although these therapies are efficacious during the early stages of the disease, they present important side effects when they are used for a long time. The ideal therapy would be the one that would slow down or stop the progression of the disease. This can be achieved, for instance, with neuroprotective and neurorestorative therapies. Among them, cell therapy and therapy with trophic factors such as glial cell line derived neurotrophic factor (GDNF) are the most challenging and promising ones for the scientific community. Although the use of GDNF as a treatment for Parkinson s disease was proposed several years ago, it is necessary to develop alternative strategies to deliver GDNF appropriately to concrete areas of the brain. Here, the use of microspheres as the most suitable tool for the administration of this neurotrophic factor is discussed.

Serial quantification of lymphoid and myeloid mixed chimerism using multiplex PCR amplification of short tandem repeat-markers predicts graft rejection and relapse, respectively, after allogeneic transplantation of CD34+ selected cells from peripheral blood.

We used multiplex amplification of nine microsatellite sequences (PCR-STR) to analyse chimerism in pure populations of T cells and neutrophils from peripheral blood from 40 patients submitted to an allogeneic transplant, 22 having received a T-cell depleted (TCD) peripheral blood graft by means of CD34(+) selection (allo-PBT/CD34(+)), and 18, an unmodified graft (allo-SCT; 13 allogeneic bone marrow transplants and five allo-PBT). T-cell mixed chimerism (TcMC) was observed in 16 of the 22 (72.3%) patients receiving an allo-PBT/CD34(+), but in only one of the 18 (5.5%) patients receiving an allo-SCT (P=0.0001). TcMC was transient (n=6), stable (n=7), and associated with poor haematopoietic engraftment (n=4). All patients with TcMC who developed graft failure had more than 30% of host T cells. Myeloid MC (MyMC) was observed in four (19%) allo-PBT/CD34(+) patients and in three (17%) allo-SCT patients (P=NS). Five out of seven (71%) patients with MyMC relapsed, all of them diagnosed with myeloid malignancies, as compared with two of the 20 (10%) patients with complete donor chimerism (P&<0.0001). In conclusion, TcMC appears in a significant number of allo-PBT/CD34(+) patients and may be associated with poor engraftment when the percentage of host T cells is >30%; likewise, MyMC appears in a small percentage of recipients of both allo-PBT/CD34(+) and allo-SCT patients, and is associated with leukaemia relapse in myeloid malignancies.

The γ-secretase inhibitor PF-03084014 combined with fludarabine antagonizes migration, invasion and angiogenesis in NOTCH1-mutated CLL cells.

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), especially for the poor prognostic subgroup of NOTCH1-mutated patients. Here, we report that the γ-secretase inhibitor PF-03084014 inhibits the constitutive Notch activation and induces selective apoptosis in CLL cells carrying NOTCH1 mutations. Combination of PF-03084014 with fludarabine has a synergistic antileukemic effect in primary NOTCH1-mutated CLL cells, even in the presence of the protective stroma. At transcriptional level, PF-03084014 plus fludarabine treatment induces the upregulation of the proapoptotic gene HRK and the downmodulation of MMP9, IL32 and RAC2 genes that are related to invasion and chemotaxis. PF-03084014 also overcomes fludarabine-mediated activation of nuclear factor-κB signaling. Moreover, this combination impairs angiogenesis and CXCL12-induced responses in NOTCH1-mutated CLL cells, in particular those related to tumoral migration and invasion. Importantly, all these collaborative effects are specific for NOTCH1 mutation and do not occur in unmutated cases. In conclusion, we provide evidence that Notch is a therapeutic target in CLL cases with NOTCH1-activating mutations, supporting the use of Notch pathway inhibitors in combination with chemotherapy as a promising approach for the treatment of these high-risk CLL patients.

A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.

Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinicobiologic CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to classify CLL patients accurately into the three subgroups, namely naive B-cell-like, intermediate and memory B-cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV (immunoglobulin heavy-chain locus) mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time to first treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinicobiologic features and outcome.

Malignant transformation in IgM monoclonal gammopathy of undetermined significance.

Monoclonal gammopathy of undetermined significance (MGUS) is a frequent disorder characterized by the presence of a small serum M-protein in individuals with no evidence of multiple myeloma (MM), Waldenstrom's macroglobulinemia (WM), or primary amyloidosis (AL). Although one fourth of these individuals will develop a malignant disease, there are no well-established predictors of outcome, particularly in the IgM type MGUS. Among 434 patients diagnosed with MGUS from 1970 to 2001 with a minimum follow-up of 1 year, 52 (27 men and 25 women; median age, 67 years) of IgM type were identified. After a median follow-up of 5 years, five patients (9.6%) have developed WM. The risk of transformation was 13.3% (95% confidence interval [CI], 0 to 27) and 27.7% (95% CI, 0.3 to 55.1) at 10 and 20 years, respectively. The variables significantly associated with transformation were the proportion of bone marrow plasma cells (BMPC) and the percentage of bone marrow lymphocytes (BML). No significant differences in the risk of transformation were found between IgM MGUS and the remaining MGUS types. Thus, in IgM MGUS the rate of transformation was similar to the risk observed in other MGUS types, the percentage of BMPC and BML being the features significantly associated with evolution into WM.

Evidence for pigment epithelium-derived factor receptors in the neural retina.

PURPOSE: The neurotrophic activity of pigment epithelium-derived factor (PEDF), an extracellular factor present in the retina, is mediated by binding to cell-surface receptors in responsive cell cultures. In the present study, the expression of PEDF receptors in native neural retinas from adult steers was examined. METHODS: Binding reactions were performed with (125)I-PEDF and fluoresceinated PEDF using plasma membranes, detergent-soluble membrane proteins, or cryosections of retina from adult bovine eyes. Radioligand-binding and competition analyses were performed with a computer-assisted program. Ligand blot analysis of detergent-soluble membrane proteins was performed with (125)I-PEDF followed by autoradiography. Ligand-affinity column chromatography of detergent-soluble membrane proteins was performed with PEDF-coupled resin followed by SDS-PAGE. Binding of fluoresceinated PEDF to retina cryosections was detected by confocal microscopy. RESULTS: Radioligand-binding assays showed that (125)I-PEDF bound in a specific and saturable fashion to one class of sites on retina membranes (K(d) = 2.5-6.5 nM; maximum binding [B(max)] = 1-48 x 10(10) sites/retina). A peptide of 44 amino acids (44-mer), identified as the receptor-binding region of PEDF, competed efficiently for (125)I-PEDF binding to retina membranes with kinetics similar to the full-length PEDF. Ligand blot analysis and ligand-affinity chromatography revealed a specific and high-affinity PEDF-binding protein of approximately 85 kDa in retina plasma membranes. Confocal microscopy showed that fluorescein-conjugated PEDF stained exclusively the inner segments of photoreceptors and cells of the ganglion cell layer in retinal cryosections. CONCLUSIONS: Altogether, these data conclusively demonstrate the existence of PEDF receptors discretely distributed on the surface of cells from the adult neural retina of bovine eyes. Furthermore, they provide evidence for the direct action of PEDF on photoreceptor and ganglion cell neurons and an anatomic basis for studies to assess PEDF neurotrophic effects on the adult retina.

Management of Helicobacter pylori in duodenal ulcer: a cost-effectiveness analysis.

BACKGROUND: Empirical eradication therapy of H. pylori has been proposed as a therapeutic alternative for duodenal ulcer. AIM: To identify the cost-effectiveness of empirical eradication therapy vs. test-and-treatment for the management of patients already diagnosed with a duodenal ulcer. METHODS: A decision analysis was performed to compare the cost-effectiveness of empirical eradication therapy of H. pylori diagnosed duodenal ulcer vs. eradication therapy after confirmatory diagnosis of Helicobacter pylori infection by means of several diagnostic tests. RESULTS: The empirical eradication therapy of duodenal ulcer was found to be the most effective and cost-effective strategy of all the alternatives. Amongst the alternatives, which included the previous performance of confirmatory diagnostic tests, the best cost-effectiveness ratio used a serology test. The model was robust in the face of changes in the values of therapeutic effectiveness, sensitivity and specificity of the diagnostic tests, prevalence of H. pylori infection in duodenal ulcer, duration of the antisecretory therapy, and number of medical visits. CONCLUSIONS: Based on our cost-effectiveness analysis, a treat approach is more effective and cost-effective than a test-and-treat approach in the clinical management of already diagnosed duodenal ulcer.

Rapid diagnosis of acute promyelocytic leukemia by analyzing the immunocytochemical pattern of the PML protein with the monoclonal antibody PG-M3.

The fusion protein, promyelocytic leukemia-retinoic acid receptor (PML-RAR)alpha, generated by the t(15;17) translocation has an abnormal cellular distribution with colocalization of RARalpha and PML proteins. We analyzed the immunostaining pattern of PML protein using the PG-M3 monoclonal antibody directed against the amino terminal portion of PML (retained in wild-type PML and PML-RARalpha fusion protein) in the diagnosis of acute promyelocytic leukemia (APL). In addition, we compared this test with other methods for detecting the PML-RARalpha fusion gene. A normal immunostaining pattern was observed in nonmyeloid disorders and in 78 of 111 acute myeloid leukemias (AMLs). A microgranular pattern was observed in 25 AMLs, all corresponding to APL. These results were concordant with the reverse transcriptase-polymerase chain reaction results for PML-RARalpha fusion gene. Only 1 case positive for the PML-RARalpha transcript showed a normal protein pattern by immunocytochemistry. PML immunostaining was helpful to rapidly differentiate 7 cases with borderline characteristics and to obtain the diagnosis in 2 cases with scarce material. The effectiveness and low cost of this technique support its routine use as a first-line procedure in the differential diagnosis of AML.

Functionality of enterococci in meat products.

The presence of enterococci in meat fermentation is a constant as reported in the literature. Despite the concern about pathogenicity of enterococci, recent studies point out that food and meat enterococci, especially Enterococcus faecium have a much lower pathogenicity potential than clinical strains. Enterococci possess a competitive advantage over other microbiota in meat fermentations, and many enterococci isolated from sausages have the ability to produce enterocins harbouring antimicrobial activity against pathogens and spoilage microorganisms of meat concern. The application of enterocins producing enterococci or their purified metabolites, as extra hurdles for preservation in sausage fermentation and in sliced-vacuum packed cooked meat products can be beneficial, preventing the outgrowth of Listeria monocytogenes and slime-producing lactic acid bacteria. Enterocins and bacteriocinogenic enterococci hold considerable promise as alternatives to traditional chemical preservatives and they could be exploited for the control of emergent pathogens in meat products. Their inhibitory effect can be increased when used in conjunction with particular physical and chemical processes, but current regulation is hampering the application of purified bacteriocins.

Technological and sensorial evaluation of Lactobacillus strains as starter cultures in fermented sausages.

The performance of several lactobacilli strains isolated from naturally fermented sausages as starter cultures was evaluated. Microbiological, physical and chemical changes, in addition to sensorial aspects were studied. From the 12 different strains tested, 10 were capable of leading the fermentation in every batch throughout the process. The monitoring of the inoculated strains was easily carried out by plasmid profiles and by checking the pH rate drop of the sausages, which was slower for the non-inoculated lots. Treatment using natural fermentation resulted in a product where hydrogen sulphide odours, which could be related to the higher content of Enterobacteriaceae throughout the ripening process, diminished its overall acceptability. The lots seeded with different L. sake strains were found to be low in acid in sensory evaluation, correlating with a low lactic acid content. In contrast, the L. plantarum lot gave rise to an overacidified product related to having the highest amount of lactic acid at the end of the process. As a general rule lactobacilli strains isolated from meat origins are good candidates as starter cultures in the manufacture of dry-fermented sausages and produce satisfactory products depending on the specific strains used more than on the species.

[Efficacy and safety of thrombolytic therapy in pulmonary embolism: meta-analysis of randomized controlled trials]. / Eficacia y seguridad del tratamiento trombolítico en la tromboembolia pulmonar: metaanálisis de ensayos controlados y aleatorizados.

BACKGROUND: The role of thrombolytic agents in the treatment of pulmonary thromboembolism (PTE) remains a controversial issue. The objective of this study is to assess the efficacy and safety of thrombolytic therapy in the treatment of PTE by means of a meta-analysis of randomized controlled trials (RCT). METHODS: A bibliographic search of the main biomedical bibliographic databases was carried out and eight randomized controlled trials that fulfilled the inclusion criteria were found. Two blinded and independent evaluators assessed the quality of RCT according to Jadad scale, and selected the necessary data to fulfill the objective of this study. RESULTS: The selected trials were heterogeneous regarding the type of thrombolytic agent, the administration schedule, and the efficacy measures used. The methodological quality was 2 points in the Jadad scale as an average. No statistically significant differences in mortality nor in risk of PTE relapse were found between the group of patients receiving thrombolytic agents and the group not receiving them. Significant differences were found, however, between these two groups as regards the risk of bleeding events (OR = 2.62; CI 95%: 1.56-4.38). CONCLUSION: The results of these meta-analyses do not suggest the use of thrombolytic therapy in PTE in everyday clinical practice since measurable risks overcome potential benefits.

[Abnormal changes in erythrocyte membrane proteins in hereditary spherocytosis and their relation to clinical and biological aspects of the disease]. / Alteraciones proteicas de la membrana eritrocitaria en la esferocitosis hereditaria y su relación con la expresividad clínica y biológica de la enfermedad.

BACKGROUND: In the present paper we report a study of 20 patients with hereditary spherocytosis (HS) performed with the aim of provide further information on the electrophoretic abnormalities of red blood cell (RBC) membrane proteins and their putative relationship with the clinical, biological and genetic aspects of the disease. METHODS: General hematological parameters, reticulocyte count, osmotic fragility test and erythrocyte morphology analysis, were performed by routine procedures. Membrane proteins of erythrocyte were analyzed by SDS-polyacrylamide gradient gel electrophoresis (SDS-PAGE) using the Laemmli and Fairbanks methods. RESULTS: In 8 out of 20 cases (40%) a defect of band 3, alone or associated with a slight deficiency of protein 4.2, was observed. In addition to the presence of spherocytes, in all these 8 patients, a peculiar morphologic RBC alteration called pincered RBCs was also observed. Moreover, 2 cases showed a deficiency of protein 4.2, 2 cases a deficiency of ankyrin and 2 cases a deficiency of spectrin. In 6 cases (30%) the electrophoretical pattern of the erythrocyte membrane proteins was normal. A significant (r = -0.6; p < 0.01) correlation between the protein 4.2 (pallidin) and the mean corpuscular haemoglobin concentration (MCHC) was found. Also, the multiple regression analysis showed a correlation (r2 = 0.6; p < 0.0001) between the amount of protein 2.1 (ankyrin) and two hematological parameters: the mean corpuscular volume (MCV) and the red cell distribution width (RDW). CONCLUSIONS: The defect of band 3 is the most frequent membrane protein abnormality associated with HS.