Cell analysis system based on immunomagnetic cell selection and alignment followed by immunofluorescent analysis using compact disk technologies.
BACKGROUND
Although the flow cytometer has become the standard in cell analysis, it has limitations. Recently, we introduced a new cell analysis method based on immunomagnetic selection and aligning of cells. No flow system is needed and cell analysis can be performed in whole blood.


METHODS
Whole blood is incubated with fluorescent labels and immunomagnetic nanoparticles. The blood is injected into a capillary that is in a strong magnetic field. The immunomagnetic-labeled cells move upward and align themselves along ferromagnetic lines present on the upper surface of the capillary. An optical focus and tracking system analogous to that used in a conventional compact disk player focuses a 635-nm laser-diode on the magnetically aligned cells. The emitted fluorescence signals are projected on two photomultipliers. Allophycocyanin (APC)-labeled CD4 (CD4-APC) and Cyanin5.5 (Cy5.5)-labeled CD8 (CD8-Cy5.5) antibodies and Oxazine750, all red excited, are used as fluorescent labels.


RESULTS
A differential white blood cell count performed in whole blood is obtained using the CD4-APC in combination with Oxazine750. The results are compared with the Technicon-H1 hematology analyzer. Correlation coefficients of 0.91 for neutrophilic granulocytes, 0.93 for lymphocytes, 0.93 for monocytes, and 0.96 for eosinophilic granulocytes were obtained. Immunofluorescence is demonstrated using CD4-APC and CD8-Cy5.5. The absolute counts obtained for CD4+ and CD8+ are compared with the Coulter Epics XL flow cytometer. Correlation coefficients of, respectively, 0.91 and 0.94 were obtained.


CONCLUSIONS
We conclude that our system is as capable as a standard flow cytometer or hematology analyzer for a reliable routine white blood cell analysis, including immunophenotyping, and can be used as an easy-to-handle disposable white blood cell test.
Tibbe
A G
AG
Twente University, Enschede, The Netherlands. A.G.J.Tibbe@tn.utwente.nl
de Grooth
B G
BG
Greve
J
J
Liberti
P A
PA
Dolan
G J
GJ
Terstappen
L W
LW
D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D016250:Compact Disks; D005434:Flow Cytometry; D005453:Fluorescence; D005455:Fluorescent Antibody Technique / Q000295:instrumentation* / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods*; D016130:Immunophenotyping / Q000295:instrumentation / Q000379:methods; D007834:Lasers; D007958:Leukocyte Count / Q000295:instrumentation / Q000379:methods*; D007962:Leukocytes / Q000166:cytology; D010798:Phycocyanin / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D015704:CD4 Antigens; D016827:CD8 Antigens; D005456:Fluorescent Dyes; C002497:allophycocyanin; D010798:Phycocyanin