Does increased platelet aggregation have a prognostic value in the deterioration of background diabetic retinopathy? The Damad Study Group.

Many case-control studies have suggested that increased platelet aggregation (PA) could be involved in the pathogenesis of diabetic microangiopathy. However, longitudinal data are needed to support this hypothesis. We consider here such an approach in the placebo group (93 diabetic patients) of a controlled clinical trial on the effect of PA inhibitors in the treatment of diabetic retinopathy (Damad program). We have measured spontaneous PA and PA induced by ADP, collagen and arachidonic acid before treatment and yearly during a 3-year period. The assessment of retinopathy was based on the changes in the number of microaneurysms present in the macular field as seen on fluorescein angiograms during follow-up. PA was estimated by maximal decrease in optical density. The lowest ADP concentration still able to induce irreversible aggregation was also determined. No significant correlations between any baseline PA measurements and end point criterion were found (all correlation coefficients lower than 0.20). No significant changes in mean PA were observed during follow-up. Within-subject variation of PA was markedly large accounting for 61% to 98% of the total variance of various measurements. Allowances for the main characteristics of diabetes made no substantial difference to the results. These negative findings can be partly attributed to the lack of reliability of PA tests. In our study, we conclude that PA tests are not useful measures for the prediction of evolution of background diabetic retinopathy.

A fatal low-molecular-weight heparin-associated thrombocytopenia after hip surgery: possible usefulness of PF4-heparin ELISA test.

In 37 patients undergoing total hip replacement, a prophylactic treatment by a low-molecular-weight heparin (LMWH) was conducted for 2 weeks. They belonged to a group of 499 patients included in a multicenter clinically controlled trial comparing two LMWHs. Blood was collected 1 day before surgery (D-1) and at D+1 or D+2 and D+5 or D+6 as well as D+10 through D+14 after surgery for determinations of platelets counts and anti-Xa. Bilateral venography was performed between D+10 and D+14. A fatal heparin-associated-thrombocytopenia (HAT) occurred on D+9 in one patient and was associated with a positive platelet aggregation test. This finding was confirmed with a recent ELISA test which evidenced a high concentration of PF4-heparin dependent antibodies 72 h before the detection of thrombocytopenia. This led us to study retrospectively PF4-heparin ELISA results by testing the plasma samples of 36 other surgical patients treated under the same conditions and during the same period (four measurements per patient). Among these patients, seven had a venous thrombotic event as a treatment failure. Although some authors claimed that some post-operative thromboses may be facilitated by the presence of heparin-dependent antibodies associated with or without thrombocytopenia, no thrombocytopenia and no positive PF4-heparin ELISA test was observed in this group. Out of the 144 tests performed in these 36 patients for the detection of PF4-heparin complexes dependent antibodies, 15 results were borderline in ten patients and three results in two patients were positive. No relation was evidenced between a positive ELISA test and the occurrence of venous thrombosis. This study points out the possible usefulness of the PF4-heparin ELISA test for HAT-antibodies detection. A daily platelet count in a postoperative patient under heparin therapy, showing thrombocytopenia associated with the detection of heparin-dependent antibodies could allow an earlier and more reliable diagnosis of HAT.

The role of platelet factor 4 in platelet aggregation induced by the antibodies implicated in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment. Recent studies using immunological methods demonstrated that antibodies contained in plasma, or in purified total immunoglobulin (Ig)G from patients suffering HIT, recognize as target antigen the complex heparin/platelet factor (PF4). In the present study, the role of PF4 in in-vitro platelet aggregation induced by purified total IgG or platelet-poor plasma from patients suffering HIT was investigated. In order to demonstrate the functional role of PF4, an anti-PF4 antibody that specifically blocked PF4 was used. In an experimental system composed of washed platelet suspension, incubation of F(ab')2 fragments (0.125 microg/ml) of the polyclonal anti-PF4 antibody resulted in complete inhibition of platelet aggregation triggered by purified total IgG from patients suffering HIT and heparin. In platelet-rich plasma, a significantly higher concentration (4.25 microg/ml) of the anti-PF4 F(ab')2 was required to inhibit platelet aggregation induced by HIT-PPP and heparin. Intermediate concentrations of the anti-PF4 antibody partially inhibited platelet aggregation. In plasma milieu, the concentration of PF4 was about five-fold higher in comparison with that measured in the purified system. The intensity of platelet aggregation depended on the concentration of HIT-IgG. Platelet aggregation was abolished in the presence of high concentrations of heparin (superior or equal to 10 IU/ml). The present study shows that PF4 is essential for platelet aggregation triggered by the antibodies related to HIT in the presence of heparin. The concentration of PF4 that is available to bind with heparin or with the HIT-related antibodies is critical for platelet aggregation induced by HIT antibodies.

[Platelet collection by apheresis with reduced use of citrate: quantitative and qualitative evaluation of three protocols]. / Prélèvement de plaquettes par aphérèse avec réduction de l'utilisation de citrate: évaluation quantitative et qualitative de trois protocoles.

Three protocols of platelet collection by apheresis on Cobe Spectra with different citrate concentrations were compared. First we determined until what level we could diminish the ratio anticoagulant/whole blood flow to enhance the efficiency without clotting in the cell separator by platelet aggregates. We found that protocol 2 had a better platelet collection efficiency than the standard protocol 1: respectively 59% versus 52%. Protocol 3 had even a better efficiency but, due to the risk of clotting (5% of the procedures) we rejected it for routine use. We compared in vitro platelet functions of protocols 1 and 2 by aggregometry in vitro. The results with the different aggregating agents indicated that the less citrated environment kept the platelets more functional in vitro. A disadvantage of protocol 2 compared to the standard protocol is the more important number of platelet concentrates that contain more than 10(6) leucocytes.

[Platelet aggregation tests in 26 cases of heparin-induced thrombopenia. Methodological, diagnostic problems and therapeutic aspects]. / Tests d'agrégation plaquettaire dans 26 cas de thrombopénies induites par l'héparine (TIH). Problèmes méthodologiques, diagnostiques et aspects thérapeutiques.

A diagnosis of heparin induced thrombocytopenia (HIT) in the 26 patients was based on: 1. normal platelet count prior to heparin administration; 2. its fall to less than 100 Giga/l (m = 46 +/- 23) at time of first sample collection for test to detect a platelet aggregation factor (PAF); 3. restoration of normal values after discontinuation of heparin treatment during which the thrombocytopenia had appeared. The result of the first PAF test in these 26 patients was positive in 22 cases, negative in 2; twice the plasma provoked platelet control aggregation without in vitro addition of heparin. The origin, dose and mode of administration of the heparin did not appear determinant in the production of the thrombocytopenia: 16 of the patients were later treated with a low molecular weight heparin (LMWH). An in vitro "compatibility" test was able to be performed 8 times previously and was negative in 7 cases in the 16 patients. Samples were collected during LMWH treatment and were negative in 13 cases, and this in agreement with the increased platelet count after 7 +/- 3 days and the clinical improvement. In 3 patients the test was positive: in one case the count did not return to normal, in the second case this did occur, but slowly (21 days); the PAF test in the last patient was positive prior to LMWH treatment and remained so while the count became normal. Concordance exists therefore between negativity of the test practiced with LMWH and the increase in count when this heparin is administered; inversely, the positivity of the test does not exclude normalization of the platelet count.

[Demonstration of spontaneous platelet aggregation and research on circulating platelet aggregates: methodology, results and significance]. / Mise en évidence de l'agrégation plaquettaire spontanée et recherche des agrégats plaquettaires circulants: méthodologie, résultats et signification.

136 control subjects and 131 patients, consisting of 23 diabetics with severe retinopathy, 43 cases of valvular disease with or without a prosthesis and 65 patients who had a cerebral vascular accident, were systematically investigated for the presence of spontaneous platelet aggregation 31 controls and 108 patients were examined for reversible circulating platelet aggregates using the technique of Wu and Hoak. Frank spontaneous aggregation was observed in 6 of the 136 control subjects, 6 of the 21 patients without a valvular prosthesis and 2 of the 22 patients with such a valvular prosthesis and 2 of the 22 patients with such a prosthesis, only 1 of the 23 diabetics and 5 of the 65 patients with old or recent cerebral vascular accidents. The incidence of spontaneous aggregation seems to be directly related to certain operative conditions: the type of machine used, the number of platelets, and to the treatment administered, the Wu and Hoak test. No statistically significant correlation was demonstrated between spontaneous aggregation and the Wu and Hoak test. The exact clinical significance of the presence of spontaneous aggregation is still disputed. However, the examination for this abnormality should be routine as its presence can alter the interpretation of the results of aggregation induced by various aggregating agents.

[Heparin-induced thrombopenia: significance and difficulties of precise identification of the immunologic mechanism]. / Thrombopénies induites par l'héparine: intérêt et difficultés de l'identification précise d'un mécanisme immunologique.

Heparin-induced thrombocytopenia (HIT) is a drug induced immunohematologic adverse reaction which is a rare but potentially very severe accident. Its diagnosis is important for epidemiologic and drug surveillance studies and in order to decide the most appropriate treatment. Its importance is enhanced since there is no gold standard diagnostic criteria. In clinical practice the diagnosis is based on a group of criteria related to clinical events and laboratory tests. We have established a score based on anamnestic criteria which allowed us to evaluate and compare two different laboratory tests: a platelet aggregation test (PAT) and a test for the detection of heparin dependent antibodies (Heparin Platelet Induced Antibodies or HPIA). The functional test PAT which is commonly used in expert laboratories detects antibodies inducing platelet aggregation in the presence of heparin. The HPIA test more recently developed is an ELISA test which detects antibodies directed at heparin-platelet factor 4 complexes. The relative value of theses two methods for the diagnosis of HIT is not well documented. We have analysed the results of these two tests in 273 consecutive patients with a suspicion of HIT. The results were concordant in 70% of patients. In selecting the patients with the lowest and the highest probability of HIT according to the score, PAT was found a more sensitive and HPIA a more specific test than the other. At low probability PAT is more often positive than HPIA 18% and 9% respectively. No test is 100% reliable, the specificity being limited for both tests since in about 20% of cases one or both tests are negative contrasting with a highly probable HIT. In this last group of patients, PAT was more frequently positive (86%) than HPIA (72%). Both tests are negative in 6% of patients suggesting the existence of presently unknown antigenic targets. Considering a group of 19 patients with a high probability of HIT, we have found antibodies against IL-8 or NAP-2 in only 7 patients. The discrepancy between a HPIA positive and a PAT negative encountered in 8% of patients may be explained by the existence of IgA or IgM immunoglobulins since in contrast to IgG they are unable to promote platelet aggregation via the CD32 platelet membrane receptor. This work suggests than neither test is 100% reliable and that they play a complementary role in the diagnosis of HIT. The potential advantage of using both tests should be confirmed in complementary studies

[Quantitative determination of Willebrand factor activity in Willebrand's disease]. / Kolichestveno opredeliane aktivnostta na faktora na Vilebrand pri bolestta na Vilebrand

Certain new data about the nature of Willebrand factors are discussed, its activity being considered as part of the activity of the antihemolytic factor VIII. An one-stage method for its determination is described based on the absence of thrombocytic aggregation in the presence of ristocetin in the patients with Willebrand disease as well as the illustration of one case.