An interesting question from Stephanie Morgan, MT (ASCP) Chief Technologist at the Children’s Cancer Clinic, University of Mississippi Medical Center, Jackson, Mississippi. Her question was originally sent to Bernadette (Bunny) Rodak at Indiana University Medical Center. Bunny forwarded it to me to post here.
Bunny, I have been asked to aggregate a two-year old with possible leukocyte adhesion deficiency III (LAD-III) to identify the defect in her platelets. It appears that LAD-III patients present with severe bleeding similar to Glanzmann thrombasthenia. I can run a low-dose RIPA on her if she starts looking like a von Willebrand disease type 2B. I will also do a PFA-100, fibrinogen, thrombin time, flow for neutrophil markers CD11a, CD11b and CD18, platelet markers P-selectin (CD62) and glycoprotein IIb (CD41). I could also do a clot retraction and possibly EMs as well. If I use thrombin as an agonist, what concentration and supplier should I use? Can you think of anything else?
Stephanie, thank you for your question, which drove me to the books. The best LAD-III description I found is in Pasvolsky R, Feigelson SW, Kilic SS, et al. A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets, J Exp Med 2007;204: 1571-82. LAD-III is a severe defect inbeta-integrin activation that prevents leukocytes from adhering to endothelial cells. LAD-III also reduces platelet aggregation because the fibrinogen receptor glycoprotein IIb/IIIa is affected, and the few patients who have been described have severe Glanzmann-like mucocutaneous bleeding. The integrin name for GP IIb/IIIa is alphaIIb betaIII, and beta activation is necessary for the receptor to adhere to fibrinogen, the final step in platelet activation prior to the start of aggregation. Beta activation requires activation of membrane-related G-protein-coupled receptors, which respond to agonists.
I suggest you use your regular panel of agonists at their standard concentrations: ADP, collagen and arachidonic acid, all of which you would expect to generate no aggregation. You may also choose to aggregate with 1 unit/mL thrombin, although thrombin receptor activating peptide (TRAP) activates G-protein dependent pathways same as thrombin, and avoids the problem of thrombin-generated clotting. If you have a lumi-aggregometer it would be interesting to learn if the patient’s platelets secrete. My guess is the deficiency prevents aggregation but not secretion.
I don’t know of any value in using agonists at lower than standard concentrations. If the regular concentrations returned normal aggregation and low concentrations did not, you really couldn’t use the results to differentiate LAD-III from normal platelets.
According to the Pasvolsky article, ristocetin aggregation is normal in LAD-III. This makes sense as ristocetin relies on the von Willebrand factor receptor GP Ib/V/IX and not on platelet activation. The low-dose RIPA detects a gain-of-function defect in the von Willebrand factor molecule so it may not provide any additional information in your patient’s case.
Flow cytometry for CD62 and CD41 would give interesting results, especially CD41. You may also want to test for CD61, which measures the beta molecule directly.
Stephanie and Bunny, I hope this has been helpful. I’d love to hear from some platelet experts with experience in LAD-III symptoms and labs. Please post your information here in the comments section. Geo.