Platelets are non-nucleated cellular fragments that circulate in the peripheral blood. They derive from megakaryocytes and are critical for maintaining hemostasis. Alterations in platelet function are associated with a range of clinical conditions including Bernard–Soulier syndrome (BSS), Glanzmann’s thrombasthenia, and storage pool disease . Monitoring platelet number and function in blood via flow cytometry can provide useful diagnostic and prognostic information . Thrombocytopenia, a condition defined by significant loss of platelets in circulating blood, can lead to excessive bleeding. Conversely, conditions characterized by excessive platelet activation can lead to thrombosis and associated cardiovascular complications including myocardial infarction and stroke . In addition to their well-characterized role in hemostasis, platelets also function in regulating vascular endothelial permeability and leukocyte extravasation through the release of cytokines, growth factors, and proteases from storage granules . A growing body of evidence suggests that platelets can directly modulate signaling to innate immune cells and mediate recognition of pathogens through their surface expression of toll-like receptor 4 (TLR4) . Basic, translational, and clinical researchers are now studying the role of platelets beyond hemostasis and thrombosis, such as in areas of inflammation, immune response, wound healing, angiogenesis, metastasis, antibacterial activity, megakaryopoiesis, and platelet production, and in therapies, such as stem cell replacement and platelet transfusion .
Resting platelets, at 2–3 µm in diameter, are the smallest cellular component of peripheral blood. They have a concentration range of 1–3 x 108 platelets/mL in peripheral blood. Upon activation, platelets undergo rapid changes in cell surface receptor expression that lead to altered adhesive properties and changes in morphology that promote the formation of a platelet plug at the site of vascular disruption . These properties can make the interrogation of platelets by flow cytometry challenging, especially in the context of light scatter detection. One of the most widely used markers to identify and study platelets via flow cytometry is CD41 (glycoprotein IIb), a member of the integrin family of receptors that mediate cell-matrix interactions .
Platelets are typically studied in platelet-rich plasma (PRP) where they are the dominant population, and can be identified by light scattering properties alone. However, the procedure to make PRP requires considerable sample manipulation that may affect platelet health and function, a significant drawback of this method. Other common methods for the study of platelets use techniques such as selective lysis to remove red blood cells (RBCs) from the sample. With lysis of RBCs by ammonium chloride or a similar osmotic treatment, discrimination of platelets by light scatter alone is confounded by the cellular debris from RBCs that overlaps with and obscures the platelet population. The treatment to remove the RBCs from the sample can also have a detrimental effect on platelet health and function. Detection of platelets in intact whole blood is desirable because minimal sample manipulation helps keep platelets in a healthy, inactivated state. However, resolving the platelet population poses a difficult challenge since RBCs outnumber platelets by more than an order of magnitude, and the broad scatter distribution produced by the nonspherical shape of RBCs can overlap the platelets.
This application note features two different methods for the detection of platelets in human blood using the Invitrogen Attune NxT Flow Cytometer. The first method (Method 1) employs the use of CD41 immunophenotyping in lysed whole blood. This method uses both a forward scatter (FSC) threshold and a fluorescence threshold in the detection of platelets. The second method (Method 2) presents a new technique with minimal sample processing for the detection of platelets in unprocessed whole blood by utilizing a dual-laser light scatter approach to distinguish platelets from RBCs and leukocytes. A dual FSC and side scatter (SSC) threshold, and a fluorescence threshold using phycoerythrin (PE) anti-CD41 antibody are both used for the detection of platelets. Using the latter method also presents the opportunity to identify the platelet population using the dual-laser light scatter technique in whole blood without using a marker such as CD41. The default threshold channel for most commercial flow cytometers, including the Attune NxT Flow Cytometer, is FSC. The common practice of using FSC is largely a consequence of the utility of this channel in excluding small particles, including platelets and debris from white blood cell analysis. This feature is particularly useful in lysed blood where small particles from the RBC lysis greatly outnumber the cells to be analyzed, even after washing.
When using scatter thresholds for platelets or other small particles, it is often preferable to use SSC or a Boolean combination of SSC and FSC to differentiate the platelets from debris and instrument noise. In either case, care must be taken not to exclude cellular events from analysis. For small particles, FSC is a noisier parameter than SSC owing to high background autofluorescence, and a greater contribution from small particles in sheath fluid to the signal. With small scatter signals, this noise may be sufficient to make threshold setting with FSC alone more difficult.