Immunomagnetic Bead 및 Avidin-Biotin Column을 이용한 CD34+ 조혈간세포 분리

Abstract

Background: CD34 cell surface antigen(MY10, HPCA-1) is expressed selectively onthe hematopoietic stem cells and CD34+ cells are supposed to have the characteristics of clonogenic hematopoietic progeniton. CD34+ cells would be an ideal population for clinical transplantation as well as for basic research on stem cells and offer an advantage for current clinical gene transfer methodology.

Methods: CD34+ cells were separated from the bone marrows of 9 normal persons and 13 patients with acute myelogenous leukemia on diagnosis by immunomagnetic bead method and avidinbiotin immunoadsorption column method and efficacy of the two separation methods were assesed.

Results: 1) CD34+ cells were separated from bone marrows of 5 normal persons and 10 patients with acute myelogenous leukemia on diagnosis by immunomagnetic bead and after separation, CD34+ cell purity of AML marrow had a higher tendency compared with that of normal marrow, as 82.9土6.3%, 73.4±12.4%, respectively, and enrichment of CD34+ cell of normal marrow, which had lower preseparation purity, was 41.9±12.5X, significantly higher than AML marrow\'s 10.3±7.5X(P<0.05) and yields of normal and AML marrow were comparable, as 59.1±11.8 and 41.2±9.3%, respectively.

2) Total 15 sample\'s purity, enrichment and yield of CD34+ cells after separation by immunomagnetic bead were 79.8±5.7%, 20.0±7.4X and 47.2 ±7.4%, respectively.

3) CD34+ cells were separated from bone marrows of 4 normal persons and 3 patients with acute myelogenous leukemia on diagnosis by avidin-biotin column and after separation, CD34+ cell purity of AML marrow had a higher tendency compared with that of normal marrow, as 89.3±3.9%, 55.6±10.2%, respectively, and enrichment of CD34+ cell of normal marrow, which had lower preseparation purity, was 64.2±28.9X, significantly higher than AML marrow\'s 3.0±1.4X, and yields of normal and AML marrow were comparable, as 19.6±8.8 and 10.7±4.0%, respectively.

4) Total 7 sample\'s purity, enrichment and yield of CD34+ cells after separation by avidin-biotin column were 70.3±8.9%, 38.0±19.8X and 15.8±5.3%, respectively.

5) In normal marrow, CD34+ cell purity of immunomagnetic bead had a higher tendency compared with that of avidin-biotin column, as 73.4±12.4% and 55.6±10.2% respectively, but yield of immunomagnetic bead was significantly higher than that of avidin-biotin column(47.2±7.4% and 17.8±5.8, respectively)(P=0.05).

6) In AML marrow, purity and enrichment of CD34+ cell were comparable between immunomagnetic bead and avidin-biotin column, but yield of immunomagnetic bead had a higher tendency compared with that of avidin-biotin column(41.2±9.3% and 10.7±4.0, respectively).

7) Overall purity and enrichment of CD34+ cell were comparable between immunomagnetic bead and avidin-biotin column, but yield of immunomagnetic bead was significantly higher than that of avidin-biotin column(47.2±7.4% and 15.8±5.3, respectively)(P=0.02).

Conclusion: immunomagnetic bead method was effective CD34+ cell separation method with high CD34+ cell purity and less target cell loss. Further evaluation for clinical application of CD34+ cell separation may be warranted.