Surface Adhesion and Growth of Microaggregates under Flow Condition (II): Clinical Findings in Patients with Thrombosis and Thrombocytopenia.

BACKGROUND. Ideally, thrombophilic states should be evaluated under in vitro conditions as close as possible to in vivo. We have been evaluating cell adhesion to surface and subsequent growth of microaggregates employing a new instrument intended to approach that ideal. We utilized the Diamed Impact-R, in which whole blood is added to a cone-and-plate analyzer (CPA) equipped with an image analyzer, and the resulting microaggregates adhering to the plastic plate after shearing are measured by three parameters: number of object adhering, average size of aggregate, and percent surface coverage (SC) by aggregates. (A companion abstract reports in vitro findings indetifying variables affecting results.)

METHODS: Blood was drawn in citrate Vacutainers and 130 uL was applied to the instrument ≤3hr after drawing. The CPA was operated at shear rate 1800 sec⁻¹ with 2min run time. Subjects were also assayed for cell-derived microparticles (MP) from leukocytes (LMP, CD45+), platelets (PMP, CD41+), red cells (RMP, glycophorin+) and endothelium (EMP, CD62E+ or CD31+/CD41−) by flow cytometry. MP which bound FITC-annexin V (AnV) were also measured. We investigated normal healthy controls and 2 groups of patients. Subjects consisted of

1. n=33 normal controls (NC);

2. n=38 with recent thrombosis (TBS) but stable for at least 4 wk, of various subgroups (DVT, APS, TIA, MI);

3. n=35 patients with thrombocytopenia (TP, mostly ITP).

RESULTS:

1. In NC, the means ±SD for SC, size, and number of aggregates were 10.3±2.5%, 41.4±14.1 um², and 1540±381, respectively.

2. In patients with TBS, SC and size were significantly higher (SC: 13.0±3.1%, p=0.0002; size: 54.5±22.9 um², p=0.006) compared to NC. Interestingly, however, there was no difference in number of aggregates between these groups.

3. In patients with TP, SC and number were significantly lower (SC, 6.3±4.0%, p=0.0001; number, 793±417, p=0.00001) than NC, but there was no difference in size of aggregates.

4. When TBS vs. TP groups were compared, all 3 parameters were higher in TBS (SC, p=0.000001; size, p=0.02; number, p=0.000001).

To gauge if the method might be useful clinically, we determined how many patients departed from the NC mean by ≥2SD: best was parameter SC, for which 11/38 (29%) TBS were >2 SD above the mean. On the other hand, 18/35 (51%) TP were >2SD below the mean. We attribute this moderate degree of sensitivity to the large SD (high variability) of the NC group. Lastly, we found that AnV+ MP correlated strongly with parameter SC (R=0.65, p<0.0001), as did EMP_CD31+ (R=0.60, p<0.001). The other MP measured did not correlate convincingly with SC, size or number.

CONCLUSIONS: Our studies show that in thrombotic patients, parameters SC and size of aggregates but not number of objects were significantly elevated compared to NC. The underlying mechanism of this effect is currently unknown. Our data suggest that cell-derived microparticles, specifically AnV+ MP or EMP, may be involved with increased surface coverage and aggregate size. On the other hand, in TP patients, we observed a lower surface coverage and number of objects. This appears to be due to lower platelet counts. These tests appear promising for evaluating thrombophilic or thrombocytopenic states. However it is required to reduce the high variability by evaluating variables such as shear rate, run time, surface coating, platelet and MP count.