Figure 4
Flow cytometric and serologic testing of the 2 Apae individuals. The y-axis displays the number of cells on a linear scale whereas the x-axis shows the fluorescence intensity on a logarithmic scale. Flow cytometry line color codes: Sheep RBCs (green), dog RBCs (purple), Apae #1 (red), Apae #2 (blue) group A1 (light blue), and group O RBCs (black). (A) MAb anti-Fs with goat anti-rat IgM labeled with phycoerythrin as secondary antibody. Sheep RBCs were used as a strong positive control and human RBCs of various ABO groups as negative controls (only A1 and O are shown for clarity). (B) Reactivity of plasma antibodies from a group B donor against Apae RBCs and visualized with rabbit anti-human IgM labeled with FITC as secondary antibody. Group O RBCs were used as a negative control. Similar reactions could be obtained with O plasma (not shown). Because of the expected, strong agglutination of group A RBCs, that is the positive control, it was not possible to run the A RBCs on the flow cytometer hence they are not included in the histogram. (C) Reactivity of an eluate prepared from group B plasma antibodies adsorbed to and eluted from Apae#2 RBCs and visualized with rabbit anti-human IgM FITC secondary antibody. Group A1 and O RBCs were included as negative controls. (D) Gel column hemagglutination test between the eluate [prepared from Apae#2 cells, also used in (C)] against RBCs of various phenotypes. Negative reactions were observed with the common human blood groups (A1, A2 and O), whereas positive reactions were seen with RBCs from the Apae#1 individual and both dog and sheep RBCs known to express Fs antigen. (E-F) The hemolysin test was performed with 2 strongly Apae-reactive sera. Lysis patterns for group O and B serum are displayed in panels E and F, respectively. Results for RBCs of Apae and 3 common ABO phenotypes (A1, B, and O) are shown. ABO-compatible and ABO-incompatible combinations serve as negative and positive controls, respectively. Incubations were performed with both native (gray) and papain-treated (black) RBCs. Diagrams show hemoglobin levels (g/L) in all the tubes tested, whereas the photos show the results with papainised cells.

Flow cytometric and serologic testing of the 2 Apae individuals. The y-axis displays the number of cells on a linear scale whereas the x-axis shows the fluorescence intensity on a logarithmic scale. Flow cytometry line color codes: Sheep RBCs (green), dog RBCs (purple), Apae #1 (red), Apae #2 (blue) group A1 (light blue), and group O RBCs (black). (A) MAb anti-Fs with goat anti-rat IgM labeled with phycoerythrin as secondary antibody. Sheep RBCs were used as a strong positive control and human RBCs of various ABO groups as negative controls (only A1 and O are shown for clarity). (B) Reactivity of plasma antibodies from a group B donor against Apae RBCs and visualized with rabbit anti-human IgM labeled with FITC as secondary antibody. Group O RBCs were used as a negative control. Similar reactions could be obtained with O plasma (not shown). Because of the expected, strong agglutination of group A RBCs, that is the positive control, it was not possible to run the A RBCs on the flow cytometer hence they are not included in the histogram. (C) Reactivity of an eluate prepared from group B plasma antibodies adsorbed to and eluted from Apae#2 RBCs and visualized with rabbit anti-human IgM FITC secondary antibody. Group A1 and O RBCs were included as negative controls. (D) Gel column hemagglutination test between the eluate [prepared from Apae#2 cells, also used in (C)] against RBCs of various phenotypes. Negative reactions were observed with the common human blood groups (A1, A2 and O), whereas positive reactions were seen with RBCs from the Apae#1 individual and both dog and sheep RBCs known to express Fs antigen. (E-F) The hemolysin test was performed with 2 strongly Apae-reactive sera. Lysis patterns for group O and B serum are displayed in panels E and F, respectively. Results for RBCs of Apae and 3 common ABO phenotypes (A1, B, and O) are shown. ABO-compatible and ABO-incompatible combinations serve as negative and positive controls, respectively. Incubations were performed with both native (gray) and papain-treated (black) RBCs. Diagrams show hemoglobin levels (g/L) in all the tubes tested, whereas the photos show the results with papainised cells.

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