Pseudothrombocytopenia: an immunologic study on platelet antibodies dependent on ethylene diamine tetra-acetate

Abstract

Antibodies specifically reacting with platelets only in the presence of EDTA, by the platelet immunofluorescence test, were found in the serum of 20 patients with pseudothrombocytopenia due to in vitro EDTA- dependent platelet agglutination. These antibodies reacted optimally at 0–4 degree C. In 19 patients, IgG antibodies were detected; in 8 patients, IgM or IgA antibodies were also found. In one patient, only IgM antibodies were found. In 14 patients, the IgG antibodies were IgG1, but IgG2, IgG3, and IgG4 antibodies were also seen in 7 patients. The reaction of platelets with the antibodies was detectable in the presence of Na2EDTA, the K, Ca, and Mg salts of EDTA, and K2EGTA. F(ab')2 or F(ab') fragments of the IgG antibodies reached as strongly as the intact antibodies, indicating that the reaction is dependent on the antibody-combining site. The EDTA-dependent antibodies did not show platelet-group specificity. However, platelets from patients with Glanzmann disease did not react with the antibodies.

Spurious counts and spurious results on haematology analysers: a review. Part I: platelets

“…Abnormal PLT from myeloproliferative diseases have been shown to be much more sensitive than normal ones to clumping in presence of EDTA (Norberg & Nilsson, 1987). Agglutinins were shown to be IgG, IgM, or IgA in 33-50%, 10-63%, and 4-40% of cases, respectively (Watkins & Shulman, 1970;Shreiner & Bell, 1973;Onder, Weisntein & Hoyer, 1980;Pegels et al, 1982;Imai et al, 1983;van Vliet, Kappers-Klunne & Abels, 1986;Casonato et al, 1994;Bizzaro & Brandalise, 1995;van der Meer et al, 2002). Agglutinins react more strongly at room temperature or below (« cold » agglutinins), but some are temperature independent or react even better at 37°C.…”

“…HA do not enumerate PLT from the large clumps and the number printed corresponds to that from a mixture of small clumps and unaggregated PLT, leading to PLT counts as low as 20 · 10 9 /l, whereas the accurate number is within normal ranges (Cohen et al, 2000). Anticoagulants other than EDTA (citrate, oxalate and heparin) are also concerned in several reports (Watkins & Shulman, 1970;Shreiner & Bell, 1973;Onder, Weinstein & Hoyer, 1980;Pegels et al, 1982;Savage, 1984;Payne, 1985;Lombarts & de Kieviet, 1988;Cunningham & Brandt, 1992;Bizzaro, 1995). As thrombocytopenia discovered in a patient may induce several procedures including unnecessary bone marrow aspiration or/and PLT transfusion, recognition of this phenomenon is important (Payne & Pierre, 1984;Vicari, Banfi & Bonini, 1988;Foresti et al, 1990;Berkman et al, 1991;Bizzaro & Brandalise, 1995;Cohen et al, 2000).…”

“…But, in the latter instance, PLT aggregates may be generated in vivo and enumerated on HA if the relevant blood samples are anticoagulated with sodium citrate (Shimizu et al, 2003). The first important observation concerning the mechanism of aggregation in EDP was that serum or EDTA-plasma not only induced agglutination of PLT from the patient but also induced agglutination of EDTA-PLT from nearly all normal individuals (Watkins & Shulman, 1970;Shreiner & Bell, 1973;Veenhoven et al, 1979;Onder, Weinstein & Hoyer, 1980;van der Lelie, van der Plas-Van Dalen & von dem Borne, 1984), with the exception of PLT from patients with Glanzmann's disease, suggesting that the fibrinogen receptor, Glycoprotein (GP) alpha IIb beta IIIa (GPIIb/IIIa), was involved in EDP (Pegels et al, 1982;Casonato et al, 1994;Ryo et al, 1994;Bizzaro, 1995;Schrezenmeier et al, 1995). Indirect evidence is shown with artifactual pseudothrombocytopenia that develops frequently in patients following exposure to PLT GPIIb/IIIa receptor antagonists (Christopoulos & Machin, 1994;Stiegler et al, 2000).…”

A comparison of platelet parameters in EDTA‐ and citrate‐anticoagulated blood in dogs

“…2,3 Similar estimates are not available for dogs. Another cause of platelet aggregation that has been reported in humans [4][5][6] and (rarely) in horses, 7,8 is an antibody-mediated EDTA-dependent pseudothrombocytopenia. EDTA is the preferred anticoagulant for hematology testing because of optimal preservation of cell morphology.…”

“…Collection of blood into citrate anticoagulant prevented platelet aggregation and provided accurate counts in these patients. [4][5][6][7][8] Although, to our knowledge, EDTA-dependent pseudothrombocytopenia has not been reported in dogs, it has become common practice in our hospital for clinicians to submit citrate-anticoagulated blood samples from canine patients with low platelet counts in EDTA samples, particularly if platelet clumps are observed in blood smears and reported with the hemogram results. However, in our experience, higher platelet counts are not obtained with the citrate-anticoagulated blood samples.…”