Evaluation of Cell Types and Morphologies in Sickle Cell Disease with an Imaging Flow Cytometer

Sickle cell disease (SCD) is a hemoglobinopathy characterized by vaso-occlusive episodes and hemolysis. Hemoglobin S is prone to polymerize at low oxygen tension, causing the red cell to become sickle shaped, more rigid and sticky. Evaluation of blood cell morphology, counts and activation are important components of the patient evaluation. This is usually accomplished by evaluation of the blood film, performing a complete blood count (CBC), and with the use of flow cytometry. A typical blood film from an SCD patient shows anisocytosis, poikilocytosis, polychromasia, nucleated erythrocytes, sickled cells, and irregular contracted cells.

The methods of blood cell evaluation all have disadvantages. Preparation of the blood film is laborious and its evaluation is highly subjective and requires extensive experience.  Some CBC counters (e.g., Siemens - ADVIA 2120) are able to detect dense cells (increased hemoglobin content-high MCHC cells) by their volume and hemoglobin concentration after the red blood cells (RBC) are swelled to spheres with a hypotonic solution. Dense cells resist becoming spheres and are detected by their low volume and high hemoglobin concentration. However, the number of dense cells might be underestimated because reversibly sickled cells are capable of undergoing the sphering and will not be detected. In addition, the hypotonic solution can lyse the cells. Finally, RBC counters cannot detect cells on the basis of specific cell markers, which can be used to define cell types and cell morphology and activation status (platelets).

Conventional flow cytometry can detect cell markers, but yields little information on morphology and cannot detect dense cells.

Here, we used the ImagestreamX Flow Cytometer (Amnis) to analyze SCD blood. In addition to providing information available with conventional cytometers, this instrument provides an image of each cell analyzed, thus allowing for detailed morphological assessment of a large population of cells. We analyzed 5 patients. All were outpatients not suffering from acute complications. Blood was collected by venipuncture into citrate anticoagulant, stained with antibodies or other reagents, and then fixed in 4% paraformaldehyde. We evaluated the blood for cell numbers and morphology, reticulocytes, dense cells, platelet-monocyte aggregates, phosphatidylserine exposure, and platelet activation status. The blood from all of the SCD patients displayed characteristics not found in control blood. We could clearly distinguish RBC morphologies corresponding to sickle cells, dense cell and reticulocytes. Reticulocytes, identified by CD71 positivity, often displayed a “puckered” morphology, as previously seen in electron micrographs. We calculated the percentage of RBCs that were sickled based on the shape ratio of > 2 (length along the long axis/maximum thickness along the short axis). The sickle cell percentage was 1.4±0.5% (normal 0%) out of total RBC population. We also evaluated dense cell morphology after separating the cells on a percoll density gradient. The cells appeared flattened and “deflated”, clearly indicating their loss of intracellular fluid.  We also analyzed platelet activation status based on staining for P-selectin, the activated form of integrin a_IIbb₃ (PAC-1 antibody), and phosphatidylserine exposure. Platelets staining positively for these markers also demonstrated morphological evidence of activation: shape change and filopodia extension. Platelet-monocyte aggregates were higher in all of the patients than in controls (0.036% vs 0%) and were easily distinguished from coincident events by morphology. The number of platelets associated with individual monocytes varied from 1 to 3. Other heterotypic cell aggregates were rare.

In summary, imaging flow cytometry provides a powerful tool for the analysis of blood in SCD. The technique allows cell population analysis like conventional cytometry, while yielding detailed morphological information on many parameters of relevance in the disease. Further, the morphological assessment avoids many of the potential artifacts arising from blood film preparation and allows an unbiased assessment of the results.