"Zeus" a new oral anticoagulant therapy dosing algorithm: a cohort study.

The demand for oral anticoagulant therapy (OAT) has constantly increased during the last ten years with an extended use of computer assistance. Many mathematical algorithms have been projected to suggest doses and time to next visit for patients on OAT. We designed a new algorithm: "Zeus". A "before-after" study was planned to compare the efficacy and safety of this algorithm dosing OAT with manual dosage decided by the same expert physicians according to the target of International Normalized Ratio (INR). The study analysed data of 1876 patients managed with each of the two modalities for eight months, with an interval of two years between them. The aim was to verify the increased quality of therapy by time spent in INR target and efficiency and safety of Zeus algorithm. Time in therapeutic range (TTR) was significantly (p < 0.0001) higher during the algorithm dosing period in comparison with the TTR during manual management period (62.3% vs 50.3%). The number of PT/INR tests above 5 was significantly (p < 0.001) reduced by algorithm suggested prescriptions in comparison with manual those (254 vs 537 times). The anticoagulant drug amount prescribed according to the algorithm suggestions was significantly (p < 0.0001) lower than that of the manual method. The number of clinical events observed in patients during the algorithm management time was significantly (p < 0.05) lower than that in those managed with the manual dosage. This study confirms the clinical utility of the computer-assisted OAT and shows the efficacy and safety of the Zeus algorithm.

Performance of recalibrated ReFacto laboratory standard in the measurement of FVIII plasma concentration via the chromogenic and one-stage assays after infusion of recalibrated ReFacto (B-domain deleted recombinant factor VIII).

The use of ReFacto Laboratory Standard (RLS) in the one-stage clotting assay was proposed to reduce the underestimation of factor VIII (FVIII) plasma concentration after the infusion of 'ReFacto' (B-domain deleted recombinant FVIII) in haemophilia A patients. Both ReFacto and RLS were recently recalibrated, with the resulting materials containing approximately 20% more protein than the previous products. The aim of this study was to evaluate the performance of recalibrated RLS in the measurement of FVIII plasma concentration after the infusion of recalibrated ReFacto. In 13 severe haemophilia A patients, 25 IU kg(-1) of ReFacto were injected intravenously. Venous blood samples were collected at 0.25, 0.5, 1, 3, 6, 9, 24, 28 and 32 h after the end of the infusion. Pharmacokinetic parameters were measured for the chromogenic and one-stage assays using International Plasma Standard (IPS) and RLS for both assays and assuming a non-compartmental drug disposition. Comparisons among assays and standards were performed using anova. Pharmacokinetic estimates obtained with the chromogenic method were in agreement with those published in the literature. The one-stage method was confirmed to be more sensitive to lower plasma concentrations of FVIII. The measured maximum plasma concentration (C(max)) was slightly higher than theoretical values and independent of the assay used. C(max), area under the curve (AUC) and volume of distribution at steady state (V(ss)) presented non-significant differences among the methods and standards used. The clinical utility of RLS in the evaluation of FVIII concentration after the infusion of ReFacto seems to be reduced since recalibration of the product.

Immune tolerance induction with a high purity von Willebrand factor/VIII complex concentrate in haemophilia A patients with inhibitors at high risk of a poor response.

Immune tolerance induction (ITI) is effective in approximately 70% of haemophilia patients with inhibitors. Poor prognostic factors are age >6 years, ITI started >1 year from inhibitor development, inhibitor peaks >200 BU, inhibitor titre >10 BU when ITI is started and previously failed ITI. The objective of this study was to identify the effectiveness in ITI of a high purity von Willebrand factor/factor VIII (VWF/FVIII) complex concentrate in inhibitor patients at high risk of failure. Patients with severe or moderate haemophilia A and high responding inhibitors who had at least one poor prognostic factor for ITI failure were prospectively followed-up. Success was defined by undetectable inhibitor, recovery and half life >66% of expected values. ITI dose regimens were chosen by each haemophilia centre. Seventeen haemophiliacs (16 severe, one moderate), aged 4-54 years (median 23) were followed-up for 6-71 months. Poor prognostic factors were delayed-onset ITI (n = 16), age >6 years (n = 16), previously failed ITI (n = 4), inhibitor peak >200 BU (n = 2) and inhibitor >10 BU when ITI was started (n = 4). Complete success was obtained in nine patients (53%) after 4-30 months of treatment (median 24), including two of four patients who had previously failed ITI. Seven patients achieved a partial success, with sustained low inhibitor titres (median 1.5 BU, range 1.1-2.8) but abnormal recovery and/or half-life, while the remaining patient withdrew ITI after 12 months when the inhibitor titer was still 70 BU. These findings suggest that high purity VWF/FVIII complex concentrates are effective in ITI, even in patients at high risk of failure.

Detection of TT virus in lymph node biopsies of B-cell lymphoma and Hodgkin's disease, and its association with EBV infection.

Human TT virus (TTV) recently isolated from the serum of a patient with post-transfusion hepatitis does seem to have only hepatopathic effect. The virus can also infect the serum, peripheral blood mononuclear cells (PBMC) and bone marrow cells (BMC ). Additional evidence has indicated that TTV is also present in the serum of people with hematopoietic malignancies. A significant increase in the incidence of lymphoma has recently been observed worldwide. We have investigated the presence of TTV DNA in lymph node biopsies of Italian patients affected with the most common lymphoma types in Western Countries: follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and nodular sclerosis Hodgkin's disease (NS-HD). The possible role of a co-infection with Epstein-Barr virus (EBV) has also been investigated. DNA was extracted from 73 paraffin-embedded and 38 snap-frozen tissue specimens. From these, only 67 samples (29 paraffin-embedded and 38 snap-frozen tissues) from a total of 56 patients, were suitable for PCR analysis. TTV and EBV were detected by PCR using primers from two different conserved region in TTV and EBV genomes respectively. TTV DNA was detected in 30.0-50.0% of FL, 30.8% of DLBCL and 30.0-50.0% of NS-HD cases, depending on the primers used. All cases of non-specific reactive lymphoid hyperplasia (RLH), used as a putative control, were negative. The two major TTV genotypes circulating in Italy (G1 and G2) were detected in the analysed lymphoid neoplasms. EBV DNA was detected in 40.0% of FL, in 72.7%of DLBCL, in 80.0% of SN-HD and in 40.0% of RLH cases. EBV co-infection was found in 90% of TTV positive cases. The in situ hybridization assay was performed in TTV positive frozen samples. The significant prevalence of TTV DNA in lymphocytes circulating in the lymph nodes of both B-cell lymphomas and HD reported herewith suggests an implication of TTV infection in the development of these lymphoproliferative disorders.

p53 over-expression identifies a subset of nodal peripheral T-cell lymphomas with a distinctive biological profile and poor clinical outcome.

Peripheral T-cell lymphomas (PTCL) are usually characterized by aggressive clinical behaviour and poor clinical outcome, but their biological background has not been extensively investigated to date, due to their low incidence, about 10% of all non-Hodgkin's lymphoma cases in Western countries, and also to the paucity of specific molecular-genetic abnormalities. Neverthless, there is increasing biological and clinical evidence that primary nodal PTCL should be considered separately from extra-nodal cases, but little is known about biological factors of possible clinical and prognostic impact. This immunohistochemical study has analysed the expression of p53, Mdm2, p21(WAF1), BCL-2 and p-glycoprotein (MDR-1 gene product) in a series of 45 cases of nodal peripheral T-cell lymphomas (PTCL) with 'high-grade' histology. The immunohistochemical findings were then correlated with proliferative activity and clinical outcome. p53 was over-expressed in 13 cases (28.9%). p53 positive cases showed significantly higher proliferative activity (p<0.01), more frequent expression of Bcl-2 (p<0.01) and less frequent expression of p21(WAF1) than p53 negative cases. Mdm2 and p-glycoprotein were expressed in 4/13 (30.8%) and 8/13 (61.5%) p53 positive cases respectively, and in none (0%) of the p53 negative cases (p<0.01). Analysis of the survival curves showed that p53 positive cases were associated with a significantly poorer clinical outcome than p53 negative cases, in terms of both overall survival (p=0.0032) and event-free survival (p=0.0004). Furthermore, multivariate analysis showed that p53 expression was the most important independent prognostic variable. These findings indicate that p53 over-expression identifies a subset of nodal PTCL cases with a distinctive biological profile (higher proliferative activity, less frequent expression of p21(WAF1) and more frequent expression of Bcl-2, Mdm2 and p-glycoprotein than p53 negative cases) and poor clinical outcome. The immunohistochemical analysis of p53 expression is a simple, rapid and low-cost method which may provide information of potential clinical and prognostic value in nodal peripheral T-cell lymphomas.

Hodgkin/Reed-Sternberg cells and Hodgkin's disease in patients with B-cell chronic lymphocytic leukaemia: an immunohistological, molecular and clinical study of four cases suggesting a heterogeneous pathogenetic background.

We report the immunohistological, molecular and clinical findings in four patients affected by B-cell chronic lymphocytic leukaemia (CLL) who developed "Richter's syndrome with Hodgkin's disease (HD) features" or "CLL with Hodgkin's transformation", all characterised by the presence of typical Hodgkin/Reed-Sternberg (H/RS) cells in lymph node biopsies. In three cases the nodal involvement by CLL was demonstrated both by the presence of a predominant background of CD5/CD19/CD23+ small lymphocytes and an IgH monoclonal rearrangement revealed by PCR analysis. Conversely, in the remaining case there was neither immunohistological nor molecular evidence of lymph node involvement by CLL. In all four cases H/RS cells were Epstein-Barr virus (EBV) latent membrane protein (LMP-1) positive. These findings suggest that the presence of H/RS cells in the first three patients, who had CLL/HD nodal involvement, might be related to transformation or clonal evolution of CLL cells in H/RS cells, which is in keeping with use of the term "CLL with Hodgkin's transformation". In the fourth case a de novo HD may be postulated, representing a second malignancy presumably not clonally related to CLL. In all cases a key pathogenetic role of EBV is suggested by the expression of LMP-1 in H/RS cells. Our findings indicate that the presence of typical H/RS cells in lymph node biopsies in CLL patients may reflect a heterogeneous pathogenetic background. The different clinico-pathologic settings should be taken into consideration because of their possible implications for patients' treatment and prognosis.

"Composite" lymphoma, lymphoplasmacytoid and diffuse large B-cell lymphoma of the spleen: molecular-genetic evidence of a common clonal origin.

We describe here the first well-characterized case of "composite" lymphoma of the spleen in which the two components were a low-grade and a high-grade B-cell non-Hodgkin's lymphomas. The patient was an elderly man with prominent splenomegaly and multiple hypoechogenic lesions of the spleen. A splenectomy was performed, and the macroscopic and histological findings showed the simultaneous presence of a "low-grade" B-cell lymphoma, lymphoplasmacytoid (immunocytoma) and a "high-grade" B-cell lymphoma (immunoblastic), which were spatially separated. The two lesions expressed the same immunoglobulin light chain (lambda), but the Southern blot analysis showed different patterns of immunoglobulin heavy chain (IgH) clonal rearrangement. PCR analysis followed by direct sequencing of the IgH-amplified rearrangement products provided molecular-genetic evidence that the two components of the composite lymphoma had the same clonal origin. Since both EBV LMP-1 and p53 were negative by immunohistochemistry, it is unlikely that EBV and p53 were involved in the neoplastic progression in this case. PCR analysis and direct sequencing of IgH-amplified rearrangement products are useful tools to investigate clonality in cases in which Southern blot analysis cannot be performed or does not provide conclusive findings.

Expression of p53 and retinoblastoma gene in high-grade nodal peripheral T-cell lymphomas: immunohistochemical and molecular findings suggesting different pathogenetic pathways and possible clinical implications.

The expression of p53 and the retinoblastoma gene has been investigated by immunohistochemical and molecular analysis in 45 cases of nodal peripheral T-cell lymphoma with high-grade histology. Most cases (73.3 per cent) were primary nodal lymphomas without any extra-nodal site involvement. Most of them (75.6 per cent) were histologically classified as pleomorphic, small, medium, and large cell type. Immunohistochemistry detected p53 in nine cases (20 per cent). In each of these cases, the polymerase chain reaction (PCR)/heteroduplex analysis did not show the presence of mutations, this finding being consistent with an alteration of the p53 functional pathway, in the presence of a wild-type protein. The retinoblastoma gene product was detected by immunohistochemistry in 35 cases (77.8 per cent) and not detected in ten cases (22.2 per cent). In the latter cases, the reverse transcription (RT)-PCR analysis showed the presence of a specific retinoblastoma gene transcript in six cases and was negative in the remaining four cases. The immunohistochemical and molecular findings seem to be consistent with abnormalities of retinoblastoma gene expression at either the transcriptional or the post-transcriptional level. Since all nine p53-positive cases by immunohistochemical analysis were also retinoblastoma gene product-positive, and all ten retinoblastoma gene product-negative cases were also p53-negative, two different and mutually exclusive pathways of possible pathogenetic significance may be suggested, the former involving abnormalities of the functional pathway of p53 in the absence of mutations and the latter abnormalities of retinoblastoma gene expression at the transcriptional and/or post-transcriptional level. Finally, the clinico-pathological correlations showed that p53 immunohistochemical expression is significantly associated with a poorer response to intensive chemotherapy.

The role of high resolution pulsed and color Doppler ultrasound in the differential diagnosis of benign and malignant lymphadenopathy: results of multivariate analysis.

BACKGROUND: The role of high resolution pulsed and color Doppler ultrasound in the differential diagnosis of benign and malignant lymphadenopathy is still unclear. METHODS: High resolution pulsed and color Doppler ultrasound was used prospectively to investigate superficial lymph node enlargement in 71 patients undergoing surgical biopsy at the onset of lymphadenopathy. The aim of this study was to define, in multivariate analysis, the ultrasonographic parameters useful in the differential diagnosis of benign and malignant lymphadenopathy. RESULTS: Volume, vascularization score, pulsatility index, and resistive index were significantly higher in the 53 malignant lymph nodes studied than in the 18 benign lymph nodes studied. The long-to-short axis ratio was significantly lower in neoplastic lymph nodes than in reactive lymph nodes. Stepwise logistic regression selected only the long-to-short axis ratio and the vascularization score as parameters that independently and significantly contributed to the differentiation of neoplastic from reactive lymph nodes. The diagnostic efficiency of the combined criteria evaluated by the area under the receiver operating characteristic curve was 0.8339. CONCLUSIONS: High resolution pulsed and color Doppler ultrasound may provide information that is useful in making correct differential diagnoses of malignant or benign lymphadenopathy.

Diagnostic relevance of peripheral blood immunocytochemistry in hairy cell leukaemia.

AIMS--(1) To assess the diagnostic relevance of peripheral blood immunocytochemistry in hairy cell leukaemia (HCL); (2) to compare the immunostaining of bone marrow biopsy specimens with bone marrow and peripheral blood cytospins; (3) to evaluate the sensitivity of the different markers used; (4) to identify the ultrastructural localisation of DBA.44 in HCL variant. METHODS--Immunoenzymatic staining procedures, immunoperoxidase and immunoalkaline phosphatase, were used with a panel of monoclonal antibodies directed to HCL associated antigens. Ultrastructural immunostaining was performed using colloidal gold conjugated antibodies. RESULTS--HCL showed strong cytoplasmic reactivity for CD22, CD25, CD103, DBA.44, kappa, or lambda light chains. Peripheral blood diagnostic hairy cells were found in all the cases with absolute counts ranging from 0.11 x 10(9)/l up to 6.4 x 10(9)/l and values increasing with the size of the spleen. A median of 36.5% of leukaemic cells was found in bone marrow aspirates and 70% in bone marrow trephine specimens. The monoclonal antibodies CD22 and DBA.44 showed the highest and the lowest percentage of positive hairy cells, respectively; this difference was statistically significant (p = 0.0025). Ultrastructural immunolabelling with DBA.44 showed a cytoplasmic membrane localisation of the antigen in one case of HCL variant. CONCLUSIONS--(1) Immunocytochemistry is a useful technique which enhances the accuracy of diagnosis in HCL; (2) peripheral blood immunocytochemistry is recommended because it highlights hairy cells in all cases; (3) CD22 appears to be the most sensitive of the markers tested; (4) ultrastructural analysis is a useful tool in selected cases of HCL variant.

Immunohistology of bone marrow: a modified method of glycol-methacrylate embedding.

We describe here a modified glycol-methacrylate embedding technique for bone marrow biopsies which allows high-quality visualization of morphological details and optimal antigen preservation. By the use of this technique it is possible to recognize haemopoietic and stromal cells which are known to play a key role in the regulation of haemopoiesis.