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Abstract Background Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy (TMA) requiring urgent treatment. Standardization of its diagnosis and optimal management is challenging. This study aimed to evaluate the role of centralized, rapid testing of ADAMTS13 in patients experiencing acute TMAs requiring plasma-exchange (PEX) and to estimate the incidence of TTP in a large Italian Region. Methods We perfomed a cohort study in the frame of the project “Set-up of a Lombardy network for the study and treatment of patients undergoing apheresis”, including 11 transfusion centers in the Region. Consecutive patients referred from 2014 to 2016 with acute TMAs requiring PEX were enrolled. Centralized ADAMTS13 activity testing was performed at the Milan Hemophilia and Thrombosis Center within 24 h. Results Forty-three TMA patients (44 events) were enrolled, of whom 35 (81%) had severe ADAMTS13 deficiency. Patients with severe ADAMTS13 deficiency were younger, mainly women, with a higher prevalence of autoimmune disorders and a lower prevalence of cancer. Clinical and laboratory characteristics of patients with and without severe ADAMTS13 deficiency largely overlapped, with a lower platelet count being the only baseline marker that significantly differed between the two patient groups (ADAMTS13 activity

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy (TMA) requiring urgent treatment. Standardization of its diagnosis and optimal management is challenging. This study aimed to evaluate the role of centralized, rapid testing of ADAMTS13 in patients experiencing acute TMAs requiring plasma-exchange (PEX) and to estimate the incidence of TTP in a large Italian Region., Methods: We perfomed a cohort study in the frame of the project "Set-up of a Lombardy network for the study and treatment of patients undergoing apheresis", including 11 transfusion centers in the Region. Consecutive patients referred from 2014 to 2016 with acute TMAs requiring PEX were enrolled. Centralized ADAMTS13 activity testing was performed at the Milan Hemophilia and Thrombosis Center within 24 h., Results: Forty-three TMA patients (44 events) were enrolled, of whom 35 (81%) had severe ADAMTS13 deficiency. Patients with severe ADAMTS13 deficiency were younger, mainly women, with a higher prevalence of autoimmune disorders and a lower prevalence of cancer. Clinical and laboratory characteristics of patients with and without severe ADAMTS13 deficiency largely overlapped, with a lower platelet count being the only baseline marker that significantly differed between the two patient groups (ADAMTS13 activity < 10% vs ≥ 10%: median difference of -27 × 10 9 /l, 95% CI - 37 to - 3). PEX treatment was initiated in all patients, but soon discontinued in cases without severe ADAMTS13 deficiency. In this group, the mortality rate was higher and no episode exacerbations or relapses within 6 months occured. The estimated average annual incidence of acute acquired TTP events was 1.17 [0.78-1.55] per million people., Conclusions: Severe ADAMTS13 deficiency distinguished two groups of patients with largely overlapping clinical features but different treatment and disease course. This study provides a feasible model implemented in a large Italian region for the practical clinical approach to TMAs and underlines the importance of urgent ADAMTS13 activity testing for an accurate differential diagnosis and therapeutic approach., (© 2022. The Author(s).)

Unusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entire ABO coding sequence, splice junctions, proximal promoter, and intron 1 enhancer. Amplification products were sequenced by next-generation or Sanger dideoxy methods, either directly or after cloning into a bacterial plasmid vector. Eight unreported alleles were found in the 15 cases analyzed. Alleles ABO*A(28+1C) and ABO*A(29-5G) harbor variants that alter the consensus sequence at the intron 1 donor and acceptor splice sites, respectively. The other alleles harbor variants that alter the consensus sequence at transcription factor-binding sites in the intron 1 enhancer: specifically, ABO*A(28+5792T), ABO*A(28+5859A) , and ABO*A(28+5860G) at GATA-1 sites; ABO*B(28+5877T) and ABO*B(28+5878G) at a RUNX1 site; and ABO*A(28+5843A) at or near a C/EBP site. Molecular and serologic characterization of ABO alleles can help in their future identification and in the resolution of discrepancies., Unusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entire ABO coding sequence, splice junctions, proximal promoter, and intron 1 enhancer. Amplification products were sequenced by next-generation or Sanger dideoxy methods, either directly or after cloning into a bacterial plasmid vector. Eight unreported alleles were found in the 15 cases analyzed. Alleles ABO*A(28+1C) and ABO*A(29–5G) harbor variants that alter the consensus sequence at the intron 1 donor and acceptor splice sites, respectively. The other alleles harbor variants that alter the consensus sequence at transcription factor–binding sites in the intron 1 enhancer: specifically, ABO*A(28+5792T), ABO*A(28+5859A) , and ABO*A(28+5860G) at GATA-1 sites; ABO*B(28+5877T) and ABO*B(28+5878G) at a RUNX1 site; and ABO*A(28+5843A) at or near a C/EBP site. Molecular and serologic characterization of ABO alleles can help in their future identification and in the resolution of discrepancies.

Aggressive non-Hodgkin lymphoma is associated with poor long-term survival after relapse or resistance to chemotherapy. We report a case of aggressive non-Hodgkin lymphoma refractory to first-line R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) and second-line R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin) chemotherapy treatments. The patient achieved remission with single-agent pixantrone, and received a consolidation with high-dose BEAM (BCNU, etoposide, cytarabine, and melphalan) chemotherapy and autologous stem cell transplantation. He received consolidation radiotherapy on the site of bulky disease. At 20 months from transplant, the disease is in continuous complete remission. The successful use of pixantrone as a bridge to transplant is highlighted, together with the absence of serious side effects., (© 2017 S. Karger AG, Basel.)

Background: Recommendations on eligibility criteria for donation of haematopoietic stem cells, management of collection of the cells and follow-up mainly concern unrelated donors. The aim of this study was to analyse the screening of related donors and collection practices at different Italian apheresis centres., Materials and Methods: A questionnaire regarding eligibility criteria for related haematopoietic stem cell donors, their peripheral blood collections and early follow-up was sent to several apheresis units. Data from the full charts of 500 candidates, screened between May 2005 and December 2009, were retrospectively evaluated., Results: The donors' records, eligibility criteria, collections and follow-up are managed differently in each centre. Of the 500 evaluable candidates (51.2% male, 49.8% female; median age 47 years, range 13-77), 26.4% underwent thorough screening according to Italian Bone Marrow Donor Registry standards, while local protocols were applied to 73.6%; 91 candidates (18.2%) proved ineligible for donation. In the end, 352 donors (53.4% male, 46.6% female; median age 45 years, range 16-76) underwent 508 leukaphereses. Central venous catheters were used in 8.0% of donors, mainly in one centre. Unsuitable pre-apheresis peripheral blood parameters were reported in 38.7% of the aphereses. Leukapheresis-related adverse events were recorded in 23.0% of the procedures, with a drop-out rate of 0.2% for severe events. No donation-related fatalities occurred. The CD34+ cell yield was <2×10(6)/kg of recipient's body weight from 1.1% of donors ≥70 years old., Discussion: More uniformity in donor screening procedures, management of peripheral blood collection and follow-up should be planned at a national level to maximise the safety of related donors.

Reticulated platelets are the youngest marker of megakaryopoiesis. We monitored 13 haematological patients undergoing autologous peripheral haematopoietic progenitor cell transplantation. We used a flow cytometric method based on thiazole orange platelet staining. Patients showed a fall in reticulated platelet percentage at a mean of 5 days before a nadir of the platelet count. In all cases, platelet recovery is preceded by a rise of reticulated platelets; in 10 cases patients received platelet prophylactic transfusions after RP rise. The rise in reticulated platelets is an early sign of engraftment and may represent an easy parameter for transfusion management of transplanted patients.

Wound healing is a specific host immune response for restoration of tissue integrity. Experimental studies demonstrated an alteration of growth factors activity due to their reduced synthesis, increased degradation and inactivation. In wound healing platelets play an essential role since they are rich of alpha-granules growth factors (platelet derived growth factor--PDGF; transforming growth factor-beta--TGF-beta; vascular endothelial growth factor--VEGF). Topical use of platelet gel (PG), hemocomponent obtained from mix of activated platelets and cryoprecipitate, gives the exogenous and in situ adding of growth factors (GF). The hemocomponents are of autologous or homologous origin. We performed a technique based on: multicomponent apheretic procedure to obtain plasma rich platelet and cryoprecipitate; manual processing in an open system, in sterile environment, for gel activation. Every step of the gel synthesis was checked by a quality control programme. The therapeutic protocol consists of the once-weekly application of PG. Progressive reduction of the wound size, granulation tissue forming, wound bed detersion, regression and absence of infective processes were considered for evaluating clinical response to hemotherapy. 24 patients were enrolled. They had single or multiple cutaneous ulcers with different ethiopathogenesis. Only 3 patients could perform autologous withdrawal; in the others homologous hemocomponent were used, always considering suitability and traceability criteria for transfusional use of blood. Complete response was observed in 9 patients, 2 were subjected to cutaneous graft, 4 stopped treatment, 9 had partial response and are still receiving the treatment. In each case granulation tissue forming increased following to the first PG applications, while complete re-epithelization was obtained later. Pain was reduced in every treated patient. Topical haemotherapy with PG may be considered as an adjuvant treatment of a multidisciplinary process, useful to enhance therapy of cutaneous ulcers.

The need for standardization criteria and result reproducibility in immunophenotyping hematological diseases has increased along with their clinical importance. Our group "Policentric Study Group on Immunological Markers", is composed of 40 laboratories. Its aim, over recent years, has been to find a standardized way of immunophenotypic analysis applicable to various hematological diseases. The objective of this study is to contribute to the debate concerning standardization of monoclonal antibody panels and immunophenotypic analysis procedures in acute leukemia (AL) and myelodysplastic syndrome (MDS), with the following targets: to improve interlaboratory reproducibility of the immunophenotyping data, and interpretative results; to study, with improved feasibility, correlation between immunophenotype and clinical or biological findings on a large number of AL and MDS cases; to verify the utility of the proposed monoclonal antibody panels for proper AL and MDS classification, and to detect minimal residual disease. In the field of AL and MDS our experience is based on about 1800 and 700 cases respectively analyzed over the last five years. Starting from these experiences and data of the literature we have elaborated the proposed panels of monoclonal antibodies and the methods of analysis. We have suggested a standardized immunophenotypic approach to study AL and MDS. In particular our work has focused on the gating strategy. This aims at drawing a gate of analysis having high purity and recovery, and on the choice of monoclonal antibody combinations for multiparametric analysis, particularly the normal antigen expression on each step of lineage differentiation or their clinically relevant aberrant expressions. A standardized criteria has become a necessary starting point in any kind of analytical process. In the field of acute leukemias and myelodysplastic syndromes the work of this polycentric group has focused on the pre-analytical and analytical steps to be taken in cytometric evaluation of hematological malignancies. The results obtained may contribute to reaching intra and inter-laboratory reproducibility.

Background: Cutaneous T-cell lymphoma (CTCL) includes several lymphoproliferative disorders involving mature T-lymphocyte proliferation initially confined to the cutis. These affections, after variable periods, may progress to the blood, limph nodes and visceral organs. Mycosis fungoides (MF) is the most frequent form of CTCL and has an indolent clinical course. The therapy of CTCL depends on the stage of the disease and the patient's general conditions. For advanced cases it includes chemotherapy, retinoids, and interferon-alpha. Since 1987 extracorporeal photochemotherapy (ECP), a novel immunomodulatory approach based on apheresis and photoirradiation of leukocytes, has been successfully introduced for the treatment of advanced CTCL. It can prolong survival of patients with erythrodermic CTCL without significant side effects., Objective: To review our five-year experience with ECP in CTCL., Methods: Since June 1994, 33 CTCL patients have been recruited for ECP, using two different regimens: two procedures on two consecutive days at four-week intervals for six months, or at two-week intervals for three months with progressive tapering in the second three-month period for the more severe forms. Six patients received ECP with IFN-alpha. ECP was done using the photopheresis UVAR system and UVAR XTS (Therakos, West Chester, Pa) and always with 8-MOP liquid formulation injected directly into the buffy coat bag. Lymphocytes in peripheral blood were immunophenotypically characterized for each patient and every ECP session., Results: All patients tolerated ECP well, without significant side effects. Thirty patients are clinically evaluable (at least three ECP cycles). A favourable clinical response was obtained in 80.9% (16/21) of MF patients (complete response 33%, partial response 47.6%) and in 66% (6/9) of patients in the Sézary's syndrome phase (complete response 33.3%, partial response 33.3%). Five of the six patients given IFN-alpha as adjunctive therapy had a PR and one a CR. Four patients are in CR without therapy at follow-ups of 46, 20, 10 and 8 months. There have been no changes in the peripheral lymphocyte immunophenotype during the follow-up. In 19/30 patients the CD95 antigen, correlated with cellular apoptosis, was expressed and was frequently associated with a good clinical response., Conclusions: In our experience ECP achieved favourable clinical responses in 73% of patients, in monotherapy or in combination with IFN-alpha, without significant side effects.

Photopheresis is an extracorporeal photochemotherapy (ECP) used for the treatment of oncological and autoimmune diseases. Lymphocytes are drawn from the patients by leukapheresis, treated with 8-methoxypsoralen (8-MOP) and ultraviolet light A (UVA) in an extracorporeal system; then, reinfused to the host. Because skin exposure to 8-MOP and UVA (PUVA) has been shown to improve cutaneous GVHD, we evaluated in a pilot study, if ECP might be beneficial for patients with GVHD unresponsive to conventional protocols. In this study, we enrolled 9 children or young adults, with acute (no = 1) or chronic extensive GVHD (no. = 8). A significant improvement was observed in three of the 5 patients with scleroderma-like lesions and in one patient with severe liver involvement. Karnofsky performance score improved from 30-50% to 90% in the 4 responders. The better control of GVHD in these patients allowed a reduction of the immunosuppressive therapy that was, finally, discontinued in two. No significant side effects were observed during ECP. Our results suggest that ECP is a nonaggressive treatment that may benefit patients with c-GVHD unresponsive to standard immunosuppressive therapies.

We report a case of biclonal component in lymphoplasmacytic/lymphoplasmacytoid non-Hodgkin's lymphoma, in which two Ig with different light chains were found, because not many cases have been reported. In our case in conjunction with the presence of an IgM-K protein, which was in accordance to both cytomorphologic aspects similar to WM and monoclonal population (K), another protein that showed different light chain was expressed (IgG-lambda). Therefore it is possible that in a neoplastic clone a subsequent neoplastic change could verify, not closely related to the first, and subsequently the presence of a subclone with the possibility to rearrange for a new protein with light chain different from the first protein. Demonstration of the isotypic difference in case of lymphoid malignancies is of critical importance in developing therapeutic protocols involving use of anti-idiotype antibodies.

Anti-HCV was tested in 77 uremic patients, 48 on hemodialysis (HD), 29 on CAPD, by immunoenzymatic 1st and 2nd generation assays (ELISA I, II) and 4-antigen (4-RIBA) immublotting. The investigation was extended to the staff (n = 29) and to HCV-positive patients' families (n = 30). The prevalence using 2nd generation tests was double (21%) that in 1st generation tests (11%). A greater incidence in the HD than in the CAPD group (23 vs. 17%) and a highly significative correlation to dialytic age were observed. No one among the sanitary personnel and only 2 family members were found HCV positive, suggesting a low infectivity via the parenteral inevident route. Extracorporeal circulation and particularly the exposure time to the treatment seem to be the main risk factors.

The beta 2-Microglobulin is a polypeptide present on the surface membrane of both B and T cells and is integrated into the structure of HLA antigenes. The beta 2-Microglobulin concentration have been used as a reliable indicator of glomerular and tubular function of the kidney. Increased serum concentration of beta 2-Microglobulin are observed also in lymphoproliferative disorders with high cell proliferation rates. More recently, increased concentration of beta 2-Microglobulin was shown in patients with anti-HIV antibodies with or without symptomatic AIDS. We have determined beta 2-Microglobulin in 61 subjects: 40 between the ages of 25 and 35 and seemingly healthy, 21 patients between the ages of 22 and 32 and intravenous drug abuser with anti-HIV antibodies and at high-risk for AIDS. In all subjects we have tested: BUN, creatinine, beta 2-Microglobulin and T4/T8 ratio. In 40 subjects as normal controls, beta 2-Microglobulin average was means = 1.07 mg/L (SD = 0.39), T4/T8 ratio average: means = 1.06 (SD = 0.119). In 21 patients drug abuser with anti-HIV antibodies, the beta 2-Microglobulin average was cleanly increased: means = 4.72 mg/L (SD = 2.23), the T4/T8 ratio average cleanly decreased: means = 0.54 (SD = 0.21). We believe the beta 2-Microglobulin quantitation, even if not specific for patient with symptomatic AIDS, used in conjunction with other laboratory tests, principally T4/T8 ratio, will be a useful marker for recognizing persons with possible asymptomatic AIDS who are members of populations known to be at high-risk for AIDS.

The appearance of hypercalcemic syndrome during the course of lymphoma is not only an unusual event, but it puts a very difficult diagnostic and therapeutic question too. There are three aetiologic moments: the diffuse osseus metastases, the paraneoplastic syndrome and concomitant primitive hyperparathyroidism. The pathogenesis of these questions has lately stimulated a new kind of research, even if there are still a lot of unknown points. The Authors suggest, as their own contribution, the revision of the literature on this subject and they also suggest an acute hypercalcemic syndrome case brought about a primitive hyperparathyroidism in a patient suffering from Hodgkin's lymphoma.