How Automated Flow Cytometry Solutions Can Save Lives and Laboratory Resources
The arrival of new flow cytometry platforms finally puts powerful automation within reach for many labs.
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For the past 65 years, more than 1.5 million people with blood cancers such as leukemia and lymphoma have been saved by transplants of hematopoietic stem cells (HSCs) from bone marrow and peripheral blood cells.1 While the cancer treatment landscape has been transformed over these decades, HSC transplants continue to form a major component of therapy. The success of HSC transplants, due to better immunosuppressant medications and improvements in transplant engraftment methods, has raised 5-year survival rates of many blood cancers from 40% to 60%, and expanded the use of HSC transplants from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) to chronic lymphocytic leukemia and other bone marrow disorders.2
The success of an HSC transplant relies on the accurate diagnosis of disease, as well as ensuring that the appropriate amounts and types of HSCs have been harvested. If stem cell numbers are estimated incorrectly, the donor may need additional treatments to mobilize peripheral blood stem cells, or the patient needs to undergo an additional transplantation procedure.
This is why flow cytometry laboratories follow guidelines from the International Society of Hematotherapy and Graft Engineering (ISHAGE) and other region-specific agencies. Many laboratories rely on in vitro diagnostic kits and software packages to perform their cell enumerations, running their flow cytometry assays and instruments manually. This lengthy process requires highly trained technicians to prepare, acquire and analyze cells and data. Errors can occur at each step, as can contamination.
Until recently, the standardization and automation of these processes have seemed out of reach for even the most advanced labs. But the arrival of new flow cytometry platforms finally puts powerful automation within reach for many labs, allowing operators to enumerate stem cells more quickly and accurately for transplant. It’s an advance that saves money – and lives.
Next to the first bone marrow transplant itself, which was performed between identical twins in 1957,3 one of the most important advancements in HSC transplants has been the discovery of CD34 in the mid-1980s. A phosphorylated glycoprotein found on the surface of HSCs and progenitor cells, CD34 is key in helping these cells attach to the extracellular matrix in bone marrow or directly to cells of the hematopoietic niche in the bone marrow.4,5 The first assay to quantify CD34+ cells was developed in 1989, using fluorescently tagged anti-CD34 antibodies to count HSCs using flow cytometry.6
Subsequent experiments showed that the ideal HSC transfusion amount of CD34+ cells is around 5×106 cells per kilogram, depending on the transplantation scheme used. Too few cells, and the transplant won’t engraft; too many cells can extend hospital stays and create other harmful side effects.7 It's why ISHAGE developed a set of guidelines in 1996 to create an assay that was quick, reliable, highly sensitive, reproducible and accurate.8 Over the years, additional parameters were added to the test, all of which have been combined into commercial single-platform flow cytometry assay kits that can be purchased from Beckman Coulter Life Sciences and other companies.
Mistakes are costly
CD34 enumeration is just one part of the applications for flow cytometry where samples are particularly precious. Research aside, a recent report indicates up to 70% of hospitalization and discharge decisions are tied to laboratory results, putting increased pressure on accuracy in the general usage of flow cytometry.9 Perhaps even more alarming, that same report indicates an overall 6.3% error rate in the samples that were processed.
One laboratory we worked with shared the number of reagent tests ordered from us versus the number of tests reported out (all tests performed; not limited to CD34 enumeration). Here, we’ve seen a difference of up to 20%. This doesn’t necessarily mean that 20% of samples were lost, but probably that additional reagents were required to compensate for pipetting errors. If that laboratory processed 500 samples, that could mean as many as 100 samples would have to be re-examined – or worse, thrown out – a huge expense and setback for the research at hand.
No one wants to return to a patient and request another sample draw, even if it’s “only” peripheral blood. I learned quickly that no matter the patient, it is not a small ask. When I was part of the team developing a solution to diagnose HIV-AIDS patients in Africa, I heartbreakingly noticed that some people would not return to be re-tested because they could not afford the $3 bus fare.
The request is even more taxing when the sample isn’t blood-based. Bone marrow samples are incredibly difficult on the patient: having the needle pierce through the hip, collecting up to 200 ml of fluid for processing.10 The recovery time can take several weeks – just imagine having to go back to them one week later for another collection because of an error.
Evolving patient scenarios also can make re-testing prohibitive, regardless of whether it involves CD34 enumeration or other scenarios.
“Samples are very precious,” James Hutchinson of the Department of Surgery, University Hospital Regensburg in Germany explained. “Usually, we have the opportunity to sample a patient once at any given time point. For instance, in our immunotherapy study we sample the patient immediately before they have been given drugs. At that point when the drugs are given to the patient, you potentially change the outcome of your measurements, and so there's no opportunity to go back and resample the patient in that case.”
How automation saves lives
Back to CD34+ enumeration: for many patients, an HSC transplant is the last treatment option, making accurate diagnostic information key. Since the success of a stem cell transplant often depends on the number of viable CD34+ cells infused, laboratories need to analyze these cells quickly and accurately. Automated, load and go flow cytometry solutions can reduce error and increase throughput, all of which help to save lives.
For time-critical tests like CD34+ enumeration, those systems with complete automation can reduce hands-on time required by technicians by up to 95%. They can also reduce error-prone steps by 87.5% and shorten turnaround time.
All told, this solution can dramatically free up laboratory staff and resources, reduce manual strain, maintain precious sample integrity, and decrease the risk of errors and contamination... all while speeding up the overall patient journey and getting them back on track toward a regular life – the ultimate goal of why so many of us chose this rewarding career.
1. Niederwieser D, Baldomero H, Bazuaye N, et al. One and a half million hematopoietic stem cell transplants: continuous and differential improvement in worldwide access with the use of non-identical family donors. Haematologica. 2022;107(5):1045–1053. doi: 10.3324/haematol.2021.279189
2. Kanate AS, Majhail NS, Savani BN, et al. Indications for hematopoietic cell transplantation and immune effector cell therapy: guidelines from the American Society for Transplantation and Cellular Therapy. Biol Blood Marrow Transplant. 2020;26(7):1247–1256. doi: 10.1016/j.bbmt.2020.03.002.
3. Thomas ED, Lochte HL Jr, Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med. 1957;257(11):491–496. doi: 10.1056/NEJM195709122571102
4. Civin CI, Strauss LC, Brovall C, et al. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol. 1984;133(1):157–165. PMID: 6586833
5. Tindle RW. BI-3C5 (CD34) defines multipotential and lineage restricted haemopoietic progenitor cells and their leukemic counterparts. In: McMichael A (ed). Leucocyte typing III: white cell differentiation antigens, p654. Oxford University Press 1987. ISBN 978-0192615527.
6. Siena S, Bregni M, Brando B, et al. Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor. Blood. 1989;74(6):1905–1914. PMID: 2478216
7. Scheid C, Draube A, Reiser M, et al. Using at least 5×106/kg CD34+ cells for autologous stem cell transplantation significantly reduces febrile complications and use of antibiotics after transplantation. Bone Marrow Transplant. 1999;23(11):1177–1181. doi: 10.1038/sj.bmt.1701748
8. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother. 1996;5(3):213-226. doi: 10.1089/scd.1.1996.5.213
9. Abdollahi A, Saffar H, Saffar H. Types and frequency of errors during different phases of testing at a clinical medical laboratory of a teaching hospital in Tehran, Iran. N Am J Med Sci. 2014;6(5):224-228. doi: 10.4103/1947-2714.132941
10. Chahla J, Mannava S, Cinque ME, Geeslin AG, Codina D, LaPrade RF. Bone marrow aspirate concentrate harvesting and processing technique. Arthrosc Tech. 2017;6(2):e441-e445. Published 2017 Apr 10. doi: 10.1016/j.eats.2016.10.024
About the author:
Dr. Andreas Boehmler is part of the flow cytometry leadership team at Beckman Coulter Life Sciences. He has held positions of increasing responsibility in the field of flow cytometry for nearly two decades. He earned his PhD in Cell Biology and Immunology from the University of Tübingen.