Flow cytometric analyses of adhesion marker–positive stress reticulocytes and double adhesion marker–positive erythrocytes from a representative patient with SCD.

For analysis of PS⁺ stress reticulocytes, dot plots of anti-CD71–PE fluorescence and annexin V–FITC fluorescence from an RBC preparation stained in the presence of EDTA and calcium are presented in panels A and B. PS⁻ (Q1) and PS⁺ (Q2) dot plot regions were set using erythrocytes stained in the presence of EDTA (A). The events in quadrants Q1 and Q2 (B) represent PS⁻ and PS⁺ stress reticulocytes, whereas Q3 and Q4 represent PS⁺ and PS⁻ nonreticulocytes. The following analyses were made: (1) Percent PS⁺ stress reticulocytes in the total stress reticulocyte fraction = (Q2 × 100)/(Q1 + Q2); (2) Percent PS⁺ stress reticulocytes in the total PS⁺ RBC fraction = (Q2 × 100)/(Q2 + Q3); (3) Percent PS⁺ stress reticulocytes in the total RBC fraction = (Q2 × 100)/(Q1 + Q2 +Q3 +Q4). Analyses of CD36⁺ stress reticulocytes (C,D) were performed in a manner similar to that described above for PS relationships. CD36⁻ (Q1) and CD36⁺ (Q2) dot plot regions were set using erythrocytes stained with anti-CD71–PE and FITC-labeled negative isotype (C). For analysis of erythrocytes positive for both PS and CD36, dot plots of mouse anti-CD36–pure plus goat anti–mouse IgG–TC fluorescence and annexin V–FITC fluorescence from an RBC preparation stained in the presence of EDTA and calcium are presented in panels E and F. The quadrants in panel F represent PS⁻ and CD36⁺ RBCs (Q1), erythrocytes positive for both PS and CD36 (Q2), CD36⁻ and PS⁺ RBCs (Q3), and erythrocytes negative for both PS and CD36 (Q4). Data were analyzed in a manner similar to that presented above for PS⁺ stress erythrocytes.