La Ricerca in clinica e in laboratorio最新文献

Von Willebrand factor (vWf) is a multimeric and multivalent adhesive protein which is essential for platelet adhesion to subendothelium and for stabilization of factor VIII procoagulant activity in circulation. The quantitative measurement of vWf involves essentially two different approaches. The first is based on the interaction between vWf and Gp Ib of the platelet membrane in presence of ristocetin (ristocetin cofactor activity, RiCof) and depends not only on the amount of the factor but also on its ability to bring about this interaction, large multimers being more active. The second approach involves the immunological quantitation of vWf (vWf:Ag) by its interaction with specific polyclonal or monoclonal antibodies as measured by several methods, i.e., electroimmunoassay, immunoradiometric assay and immunoenzymatic assay. Although in the majority of type II von Willebrand disease (vWd) with dysfunctional vWf there is a discrepancy between RiCof and vWf:Ag, it should be emphasized that RiCof activity does not entirely reflect the 'true' activity of vWf since it does not explore all the functions of this factor; furthermore, the relationship between degree of multimerization and RiCof level is not always tenable, as for example in vWd 'Vicenza'. For the diagnosis of congenital and acquired vWd RiCof assay together with family investigation is the eligible test, with an estimated ability to detect at least 50% of the carriers of the abnormal gene, including mildly affected patients; vWf:Ag appears less sensitive and, on the basis of studies carried out in our laboratory, a relative sensitivity of 64% is proposed. Both assays require the definition of separate normal ranges for children and adults and for 0 and non-0 blood group subjects; a nonparametric approach in a large sample of normal subjects is advisable. With RiCof assay performed by an aggregometric method using formalin-fixed platelets an interassay variability of 6% and 8.5% respectively for high- and low-control plasma was found in our laboratory. With vWf:Ag assayed by an ELISA method a variability of 7% for low- and 6% for high-control plasma was found. Thus, both methods appear sufficiently precise for clinical use. The use of an internal pool calibrated against an international standard allows to perform comparable interlaboratory measurements. To further improve standardization of these assays, collaborative studies seem urgently required.

The development of international standards over the last 15-20 years has led to improved interlaboratory agreement on assays of factor VIII and factor IX. In the most recent international collaborative study, the coefficient of variation for one-stage assays (26 laboratories) was 5.6%. However, in quality assurance surveys, carried out in the UK and USA, agreement between laboratories is much less good, with coefficients of variation ranging from 30% to over 50%. Improvements in agreement between clinical laboratories could be obtained by increasing the amount of testing on each sample, especially the number of dilutions, and reducing the number of reagent systems used. A large number of laboratories now use immunodepleted plasmas instead of congenitally deficient plasmas as substrates for one-stage assays. These plasmas may give satisfactory assays, but many of them have not been thoroughly evaluated in comparison with congenitally deficient plasma. In assessment of potency of very high purity (VHP) factor VIII concentrates, some immunodepleted plasmas were found to give lower potencies than hemophilic plasma. This is partly due to the fact that VHP concentrates contain little or no von Willebrand factor (vWF), and most immunodepleted plasmas are also deficient in vWF. In recent collaborative studies, assays of VHP factor VIII concentrate were much more variable, both within and between laboratories, than assays of intermediate purity concentrates. Standardization of these new products will require careful attention to methodological detail.

A decreased expression of major histocompatibility complex (MHC) class I antigens is a common feature of many experimental and human tumors and can often be correlated with malignancy grade. In fact, reduction of class I antigens is associated in most tumors with an enhanced ability to elude immune surveillance. Loss of HLA-A,B,C antigens ranges from a decrease in the percentage of A,B,C-positive cells to selective loss of particular antigens and total loss of class I molecule expression. In man, this has been documented in melanomas, carcinomas, lymphomas, neuroblastoma and acute leukemias. The reduction in membrane antigens is generally associated with a parallel fall in immunoprecipitable intracellular proteins and the corresponding mRNAs in the absence of structural changes in the coding genes. The literature concerning the above mentioned topics is reviewed and discussed.

An important plasminogen activator-inhibitor (PAI-1) is present in plasma and concentrated in alpha-granules of platelets. PAI-1 is released during platelet stimulation in vitro. It is presently unknown to what extent the treatment with acetylsalicylic acid (ASA) inhibits platelet PAI-1 release and if release inhibition has an effect on plasma PAI-1 levels. We therefore investigated by a double-blind placebo-controlled crossover study the effects of oral ASA (500 mg/day for 3 days) on platelet PAI-1 release and on plasma PAI-1 levels of healthy male volunteers. The PAI-1 release from platelets stimulated by arachidonic acid and collagen was significantly reduced by ASA: from average values of 88 and 80% to 14 and 17%, respectively (p less than 0.01). However, plasma PAI-1 levels were not modified by this treatment. We can conclude that platelet PAI-1 release does not play a role in modulating the plasma PAI-1 levels in healthy volunteers.

Specific tests for the measurement of protein C antigen and activity and protein S antigen are used in the clinical laboratory for the routine diagnosis of hereditary protein C and protein S deficiency. The performance of these tests is reviewed and discussed. Special attention is paid to the application of these tests for the analysis of patients on oral anticoagulant therapy.

The serum levels of beta 2-microglobulin (beta 2-m) were determined in 80 intravenous drug addicts (IVDA) with acquired immunodeficiency syndrome (AIDS), in 128 HIV-positive IVDA with persistent generalized lymphadenopathy (PGL) and in 44 HIV-seronegative IVDA. Seventy-two out of 80 (90%) AIDS patients had elevated serum beta 2-m levels and high levels of beta 2-m were also found in 105 of 128 (82%) HIV-infected subjects without AIDS. The mean beta 2-m level was significantly higher in HIV-infected patients with PGL than in HIV-negative IVDA. Nine out of 64 (14%) PGL patients developed AIDS in a period of 24-54 months. In these patients the mean beta 2-m level (5.16 +/- 2.37 mg/l), obtained from sera stored at the first observation, was significantly higher than in the other PGL patients (3.40 +/- 1.03 mg/l); in particular, 5 out of 7 PGL patients with beta 2-m levels greater than 5.0 mg/l showed an advanced disease.

Fibrinogen levels are considered a useful indicator in several pathological conditions and recent epidemiological studies have indicated a relationship between fibrinogen levels and increased risk of cardiovascular disease. An accurate measurement of this protein is therefore recommended and the Italian Committee for Standardization of Methods in Hematology and Laboratory has carried out a collaborative study to determine accuracy, precision and comparability of results obtained by six different methods, i.e., 1. Blombäck and Blombäck method, 2. clotting assay according to von Clauss, 3. radial immunodiffusion according to Mancini et al., 4. total amount of clottable fibrinogen by means of turbidimetric assay according to Ellis and Stransky, and 5. with ChromotimeSystem, 6. prothrombin time (PT)-derived fibrinogen assay on ACL coagulometer. The most accurate resulted the von Clauss method, but only if calibrated with an internal standard; in fact, when the manufacturer's tables are used, the method proved to be highly inaccurate. The best precision, both intra- and between-laboratory, was obtained by the PT-derived test on ACL. On the basis of this still incomplete evaluation of the CISMEL study data, we can conclude that: i. some methods used in clinical laboratories give accurate results only after adequate calibration; ii. a reference standard pool may be a valid tool for calibration and for a better between-laboratory comparability; iii. a predilution of the samples with high fibrinogen levels seems indicated; iv. automation markedly increases the precision of methods.

The results of a cooperative study on the quality of treatment of patients with prosthetic heart valves (PHV) on long-term oral anticoagulants (OA) are reported. The study was carried out on 695 patients bearing PHV (with an overall 8,340 checks carried out in 1988) through a computer-assisted system for monitoring the laboratory and treatment follow-up. The specifications of the software are described in detail in the text. The mean INR slightly differed in the two participating centers (Rome and Parma) (3.25 vs 3.19) as did the weekly mean dosage (18.2 vs 20.7 mg) of oral anticoagulant, which was always Acenocoumarol. More than 70% of the checks were within the range in both centers as evaluated by a check randomly chosen once a month for each single patient. Considering the whole bunch of data, about 80% of the patients had greater than 50% of their checks within the therapeutic range and more than 30% had greater than 75% of the checks within the range. This study demonstrates that the availability of a computerized program for monitoring oral anticoagulant therapy allows to perform multicenter studies and to obtain data which can improve the quality of treatment of patients on OA.

The INR/ISI system has been validated in multicenter trials. The system provides a reliable scale for the intensity of oral anticoagulation. It has been recognized that the ISI is slightly dependent on the instrument used for prothrombin time determination. External quality assessment (EQA) of the INR in national programs should be stimulated. EQA results suggest that the precision of the INR can be improved. Many physicians prescribing coumarin congeners are not familiar with the INR system. Improvement of the situation can only be achieved by continuous education.

Growing interest is observed in chromogenic substrate assays, because of their better precision, performance and possibilities for automation. Applied to a Cobas Bio centrifugal analyzer, we compared Nycotest-Chrom (N-test) and Thromboquant-PT (Tbq) with Thrombotest (TT). Precision tests were performed with samples from 4 plasma pools: normal (45 sec), low (100 sec), middle (150 sec) and high (200 sec) segments of the therapeutic range of TT. N-test had the best precision profile in both intraassay and interassay determinations compared with Tbq. Both chromogenic substrate assays were better than TT in this respect. By orthogonal regression a provisional therapeutic range was calculated from 312 determinations and later adjusted in a confirmation experiment with natural logarithm regression in 946 determinations. Compared with a TT range of 105-180 sec, the therapeutic ranges were 71-120 sec for N-test and 63-103 sec for Tbq. In a clinical therapeutic control phase, 110 patients were randomized in equal proportions to two groups A and B. Every 2 weeks for a period of 16 weeks, blood samples were tested for N-test, Tbq and TT. In group A, dose adjustment was based on N-test, and in group B on Tbq. The monitoring physician was blinded for Tbq and TT in group A and for N-test and TT in group B. No differences were found between the groups for mean TT, N-test, Tbq or mean dosage, nor differences were found in complication rate. A definite therapeutic range was complied using sensitivity/specificity curves together with their 95% confidence limits. Given TT 105-180 sec, the therapeutic range of N-test is from 80 (95% confidence limits: 77-82) to 110 (95% confidence limits: 108-115) sec, and of Tbq from 68 (95% confidence limits: 66-71) to 95 (95% confidence limits: 91-98) sec. It was concluded that the two chromogenic substrate assays performed equally safe in the monitoring of patients on oral anticoagulant therapy, despite differences in diagnostic correspondence with the reference TT.