Abstract
In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R=0.98, P≤0.001), negatively with fetal hemoglobin (HbF) levels (R=-0.558, P=0.027), negatively with pH (R=-0.688, P=0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R=-0.766, P=0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R=0.799, P=0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 598-603 |
| Number of pages | 6 |
| Journal | American Journal of Hematology |
| Volume | 89 |
| Issue number | 6 |
| DOIs | |
| State | Published - 2014 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Hematology
Cite this
Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease. / Van Beers, Eduard J.; Samsel, Leigh; Mendelsohn, Laurel; Saiyed, Rehan; Fertrin, Kleber Y.; Brantner, Christine A.; Daniels, Mathew P.; Nichols, James; McCoy, J. Philip; Kato, Gregory J.
In: American Journal of Hematology, Vol. 89, No. 6, 2014, p. 598-603.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease
AU - Van Beers, Eduard J.
AU - Samsel, Leigh
AU - Mendelsohn, Laurel
AU - Saiyed, Rehan
AU - Fertrin, Kleber Y.
AU - Brantner, Christine A.
AU - Daniels, Mathew P.
AU - Nichols, James
AU - McCoy, J. Philip
AU - Kato, Gregory J.
PY - 2014
Y1 - 2014
N2 - In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R=0.98, P≤0.001), negatively with fetal hemoglobin (HbF) levels (R=-0.558, P=0.027), negatively with pH (R=-0.688, P=0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R=-0.766, P=0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R=0.799, P=0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.
AB - In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R=0.98, P≤0.001), negatively with fetal hemoglobin (HbF) levels (R=-0.558, P=0.027), negatively with pH (R=-0.688, P=0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R=-0.766, P=0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R=0.799, P=0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.
UR - http://www.scopus.com/inward/record.url?scp=84900864121&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84900864121&partnerID=8YFLogxK
U2 - 10.1002/ajh.23699
DO - 10.1002/ajh.23699
M3 - Article
C2 - 24585634
AN - SCOPUS:84900864121
VL - 89
SP - 598
EP - 603
JO - American Journal of Hematology
JF - American Journal of Hematology
SN - 0361-8609
IS - 6
ER -