New method for cord blood processing and CD34+ cell separation

by Alexey Bersenev on February 26, 2013 · 3 comments

in cell separation, clinical lab, cord blood

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Hematologist Jay Mehrishi from U of Cambridge published a very interesting review, where he criticizes the current methods of cord blood processing and CD34+ cell separation methodology. This is very interesting and unique review and absolutely worth a look!

He discusses the use of different reagents and temperature during typical cord blood processing and highlights the flaws. His review focuses on such methodological issues as use of appropriate temperature, HESPAN (red blood cell sedimentation agent), DNase and other enzymes. He went through possible issues with magnetic positive selection of CD34+ cells by commercially available Miltenyi Biotech reagents and CliniMACS procedure.

Miltenyi particles internalized by cells could release iron that accumulating in liver or spleen would then risk toxicity.
… Omitting the positive selection with antibody-linked Miltenyi particles obviates the use of harsh reagents to release the cells. Internalized Miltenyi particles are a toxicity hazard that needs investigations.

Mehrishi think that the current methodologies, widely used for cord blood processing, lead to great loss of CD34+ cells, damage cells and could compromise their function. He noted, that most of current methods allow to enrich CD34+ cells only as much as 0.13 – 0.3% of total mononuclear cells.

In the following original study, Mehrishi with a co-author propose a novel methodology for cord blood processing and CD34+ cell enrichment. They used so-called physicochemical charge-based methods, including (1) nylon wool column (NWC), (2) direct rosetting with sheep red blood cells (SRBCs) and (3) avoiding incubation in the cold. Importantly, new methodology allows to avoid magnetic (Miltenyi) particles and HESPAN.

The authors were able to get much better recovery of CD34+ cells than by current (positive selection) methods:

CD34+ cell yields (approx. 5.12%) were 39 times greater than 0.13% (Korean study, 11,098 UCB units) and 10 to 20 times greater than 0.25% to 0.3% harvested by anti-CD34 Miltenyi particles.

Interestingly, SRBCs associated with significant reduction of T-cells:

The physicochemical approach, specifically the direct SRBC rosetting that ensures retaining immature preprogenitors, reduced the number of mature CD3+ T cells below 10% (Table 2, bottom row, Experiments 2, 3, and 5, Columns 5, 6, and 7). This is important because one of the gravest hazards and risks in transplantation arises from mature T cells responsible for GVHD.

In the discussion, the authors also noted:

The procedure described herein is manageable in a closed-bag system under GMP conditions, such as already developed.

In conclusion:

Achieving approximately 5% yields of CD34+ cells (153 × 105/110 mL cord-placenta blood) is a major advance holding great promise, for the first time increasing the prospect of stem cell therapy of 70-kg adults, using a single UCB donation (with dose of 1.5 × 105 cells/kg) and considerably cheaper cultured red blood cells manufacture (multiple packs/2 × 1012).

I think, these data are impressive and very interesting. I wonder if rosetting with sheep red blood cells was ever cleared as clinically grade (for human use)? What do you think folks? I’d value your opinions!

{ 3 comments… read them below or add one }

Dobrila Veljkovic March 18, 2013 at 6:38 am

Would you be so kind to tel me about cell count in leukapheresis product before alfa , beta T cells and B cells immunomagnetric removal using Mylteni CliniMACS for haploidentical PBSC transplantation. Is it possible to harvest product with 60x10e9 WBC, 0.6x10e9 CD34+ cells in volume of 200mL. Please give me references about harvesting. I have experience in harvesting PBSC in children and adults but not in healthy donors mobilized with G-CSF. Thanks in advance.


Alexey Bersenev March 22, 2013 at 1:42 pm

Since I don’t work with with the same products as you, I don’t know the answer. CliniMACS have different programs and different tubing sets, which you can customize for your needs. 60 billions of WBC is not a problem for device, but I don’t think you will end up with volume < 200 ml. Again, depends on kit, program and populations that you're depleting or enriching, fractions that you're collecting (positive of negative).


Jeanne Adiwinata Pawitan May 18, 2014 at 8:50 am

I wonder, whether they can 100% eliminate the sheep red blood cells that are used for rosetting. If elimination is incomplete, there will be xeno-material in the product, which may be harmful.


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