TY - JOUR
T1 - Alkaline hemolysis fragility is dependent on cell shape
T2 - Results from a morphology tracker
AU - Ionescu-Zanetti, Cristian
AU - Wang, Lee Ping
AU - Di Carlo, Dino
AU - Hung, Paul
AU - Di Blas, Andrea
AU - Hughey, Richard
AU - Lee, Luke P.
PY - 2005/6
Y1 - 2005/6
N2 - Background: The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration-driven hemolysis or for the relation between cell shape and fragility. Methods: Hydroxide, a porating agent, was generated in a microfluidic enclosure containing RBCs in suspension. Automatic cell recognition, tracking, and morphometric measurements were done by using a custom image analysis program. Cell area and circular shape factor (CSF) were measured over time for individual cells. Implementations were developed in MATLAB and on Kestrel, a parallel computer that affords higher speed that approaches real-time processing. Results: The average CSF went through a first period of fast increase, corresponding to the conversion of discocytes to spherocytes under internal osmotic pressure, followed by another period of slow increase until the fast lysis event. For individual cells, the initial CSF was shown to be inversely correlated to cell lifetime (linear regression factor R = 0.44), with discocytes surviving longer than spherocytes. The inflated cell surface area to volume ratio was also inversely correlated to lifetime (R = 0.43) but not correlated to the CSF. Lifetime correlated best to the ratio of cell inflation volume (Vfinal - Vinitial) to surface area (R = 0.65). Conclusions: RBCs inflate at a rate proportional to their surface area, in agreement with a constant flux model, and lyse after attaining a spherical morphology. Spherical RBCs display increased alkaline hemolysis fragility (shorter lifetimes), providing an explanation for the increased osmotic fragility of RBCs from patients who have spherocytosis.
AB - Background: The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration-driven hemolysis or for the relation between cell shape and fragility. Methods: Hydroxide, a porating agent, was generated in a microfluidic enclosure containing RBCs in suspension. Automatic cell recognition, tracking, and morphometric measurements were done by using a custom image analysis program. Cell area and circular shape factor (CSF) were measured over time for individual cells. Implementations were developed in MATLAB and on Kestrel, a parallel computer that affords higher speed that approaches real-time processing. Results: The average CSF went through a first period of fast increase, corresponding to the conversion of discocytes to spherocytes under internal osmotic pressure, followed by another period of slow increase until the fast lysis event. For individual cells, the initial CSF was shown to be inversely correlated to cell lifetime (linear regression factor R = 0.44), with discocytes surviving longer than spherocytes. The inflated cell surface area to volume ratio was also inversely correlated to lifetime (R = 0.43) but not correlated to the CSF. Lifetime correlated best to the ratio of cell inflation volume (Vfinal - Vinitial) to surface area (R = 0.65). Conclusions: RBCs inflate at a rate proportional to their surface area, in agreement with a constant flux model, and lyse after attaining a spherical morphology. Spherical RBCs display increased alkaline hemolysis fragility (shorter lifetimes), providing an explanation for the increased osmotic fragility of RBCs from patients who have spherocytosis.
KW - Alkaline lysis
KW - Cell tracking
KW - Colloid-osmotic lysis
KW - Erythrocyte
KW - Morphometric analysis
KW - Parallel computer acceleration
KW - Red blood cell
KW - Spherocytosis
UR - http://www.scopus.com/inward/record.url?scp=19444362234&partnerID=8YFLogxK
U2 - 10.1002/cyto.a.20135
DO - 10.1002/cyto.a.20135
M3 - Article
C2 - 15849725
AN - SCOPUS:19444362234
SN - 1552-4922
VL - 65
SP - 116
EP - 123
JO - Cytometry Part A
JF - Cytometry Part A
IS - 2
ER -