Angel Hernandez

Assessment of platelet activation in several different anticoagulants by the Advia 120 Hematology System, fluorescence flow cytometry, and electron microscopy

In vivo platelet activation results are often confounded by activation induced in vitro during the preparative procedures. We measured ex vivo (basal) and in vitro (thrombin-induced) platelet activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and Citrate Theophylline Dipyridamole Adenosine (CTAD) whole blood specimens. Determinations were made by measurements of platelet density (mean platelet component: MPC concentration) on the Advia 120 Hematology System. The MPC has been previously shown to correlate with a fluorescence flow cytometric method, also determined in this study, using the surface expression of CD62P. Moreover, platelet shape and structure changes in EDTA and CTAD anticoagulated whole blood specimens were characterized by transmission electron microscopy (TEM). Observations made using the Advia 120 Hematology System platelet density parameter, MPC, in the absence of thrombin were 25.7 +/- 0.9 g/dl, 27.9 +/- 0.9 g/dl and 24.8 +/- 1.2 g/dl in sodium citrate, EDTA and CTAD whole blood specimens, respectively. Addition of thrombin induced a significant change in platelet MPC for sodium citrate (21.9 +/- 1.9 g/dl; p<0.0001) and EDTA (23.2 +/- 0.9 g/dl; p<0.0001) whole blood specimens. In contrast, thrombin had no effect on MPC measured in whole blood taken into CTAD tubes. In vitro fluorescence flow cytometric platelet activation experiments measuring the percentage of platelets expressing anti-CD62P showed increase in sodium citrate specimens from 9.2 +/- 7.0 to 55.5 +/- 23.1 % (p<0.0001) and in EDTA specimens from 1.9 +/- 1.7 to 64.6 +/- 12.4 % (p<0.0001) after addition of thrombin. However, in blood taken into CTAD tubes, there was no significant change. Studies on platelets isolated from whole blood in CTAD showed activation by thrombin indicating that platelets in CTAD, while protected in its presence remained functional upon its removal. When observed by TEM over time, platelets in EDTA appear more activated and contain fewer granules than platelets in CTAD. We conclude that CTAD demonstrates in vitro platelet activation inhibition and may be useful in stabilizing ex vivo platelet activation. The novel platelet activation parameter, MPC, measured by an automated routine hematology system, using customized proprietary software, may be used in conjunction with CTAD, a stabilizing anticoagulant, to measure the ex vivo platelet activation state in whole blood specimens. TEM studies verify shape modifications and simultaneous retention of intracellular granules at early post-venipuncture time periods in CTAD specimens.

FECHTNER SYNDROME VARIANT: A NEW FAMILY WITH MILD ALPORT'S MANIFESTATIONS

Kocca et ai (1993) have recently reported a variant of the Fechtner syndrome with a reduced expression of Alport’s manifestations and have suggested the existence of mild E’echtner variants which might mimic the Sebastian syndrome. We present another family which could also correspond to a mild Fechtner variant. The patients were a 70-year-old woman and her two daughters (44 and 3 3 years old) with macrothrombocytopenia and inclusion bodies in the granulocytes. It was not possible to study the parents and sisters of the mother, or her son who died at birth. The two children of the elder daughter and the son of the younger one did not have thrombocytopenia. All three patients had mild haemorrhagic diathesis and their bleeding times were only prolonged in the daughters (12 and 1 7 min). Moreover, the mother had suffered, since her youth, from a progressive bilateral neurosensorial deafness affecting the entire frequency range, which was more severe for high frequencies as described in the Alport syndrome (Shuknecht, 1974). Microscopic platelet counts were about 70-100 x 10y/l in the three cases and the blood films revealed the presence of giant elements which sometimes were larger than erythrocytes, especially in the younger daughter. There were greyish-blue Fechtner-like cytoplasmic inclusions in about 3 5-70% of the granulocytes. Platelet membrane glycoproteins were normal and the aggregation curves were comparable to those of control platelets diluted to the same final platelet count. Bone marrow megakaryocytes were in normal numbers and showed relative increases in elements with low ploidy or naked nuclei. Some megakaryocytes showed abnor-

New types of granulocyte inclusions in hereditary macrothrombocytopenias

ment d’Hematologia, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; Núria Pujol-Moix, Departament de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain; Mercè Durfort, Departament de Biologia Cellular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain. Correspondence to: Dr. Núria Pujol-Moix, Hospital de la Santa Creu i Sant Pau, Departament d’Hematologia, Sant Antoni M. Claret 167, 08025-Barcelona, Spain. Tel.: +34-93-2919246; Fax: +34-93-2919192. E-mail: npujol@hsp.santpau.e s Letter to the Editor