65 Years of stem cell transplantation: insights into improvements and current challenges

/ in E-News

By Dr Andreas Boehmler

Accounting for nearly 10% of all cancer diagnoses, blood cancers remain a threat to people of all ages. Since the first successful transplant in 1956, stem cell transplantation has become prevalent in leukemia treatment. Today, quantitative measures of stem cell transplants, such as enumeration of viable CD34-positive hematopoietic stem cells, inform the success rate of engraftment. However, preventing variability and lab errors prove challenging. Standardization of CD34+ enumeration can help improve transplant outcomes.

Shifting trends in stem cell transplantation since the 1980s

Blood cancers such as leukemia and lymphoma can affect the bone marrow. Transplanting hematopoietic stem cells (HSCs) from healthy bone marrow or peripheral blood can help patients regenerate functional blood cells. The regenerative capability of bone marrow cells was unveiled in the 1950s by Thomas et al. [1]. The first breakthrough in this technique took place in New York, USA, in 1956, involving a bone marrow transplantation from one twin to another. Between 1957 and 2019, 1.5 million HSC transplant (HSCT) procedures were carried out [2]. Not only HSC research but also cancer healthcare have evolved in many aspects throughout the past 65 years.

One of the shifts can be observed in the source of stem cells. HSCTs can come from the patients themselves (autologous HSCT) or another donor (allogeneic HSCT). Although the number of both types of transplants increased between 1988 and 2018, autologous HSCT surpassed allogeneic, nearly doubling in numbers [3]. This could be attributed to the advantages of autologous over allogeneic transplants, the obvious reason being that the likelihood of donor cell rejection decreases. Furthermore, there are fewer chances of infection than if the cells came from another person. That said, allogeneic stem cells boast unique benefits, such as the graft versus cancer effect, where the donor immune cells might prevail over the endogenous immune system in killing cancer cells. Although the best transplant option varies across subtypes and patients, both HSCT types steadily increased over the last 30 years.

Another change concerns the location of cell harvesting. Until the 1990s, bone marrow was the primary source of HSCs. In 1993, an acute lymphocytic leukemia patient was the first to receive an allogeneic transplant from peripheral blood stem cells (PBSCs) with successful engraftment [4]. Owing to their advantages over bone marrow HSCs, such as faster engraftment, ease of harvesting, and faster recovery, PBSCs replaced bone marrow HSCs as the primary stem cell resource. Whereas only 30% of allogeneic transplants were PBSC-based in 1996, the percentage increased to 71% in 2009 [5]. A recent statistical analysis revealed that peripheral blood became the main graft source in 2016, accounting for 99.7% of autologous and 72.8% of allogeneic HSCT [2]. Nevertheless, there is still controversy around the disadvantages of peripheral blood-derived HSCs, such as a higher risk of graft-vs-host disease. Further research is needed to shed light on the conditions in which bone marrow is more suitable than PBSC for the patient.

The applications of HSCT have continued to diverge with the advancements in transplantation. Over the last 15 years, there have been dramatic increases (over 100%) in the use of HSCTs, mainly in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), as well as non-malignant blood disorders, such as bone marrow deficiencies [2]. Besides the disease types, the age groups needing and receiving HSCTs shifted as well. For example, the upper age limit of allogeneic HSCT for AML increased from 45 to 75 over the last 30 years [6]. At the same time, we have seen a steady increase in the number of Baby Boomers needing HSCT owing to myeloid leukemia and chronic lymphocytic leukemia [7]. Fortunately, multiple studies deemed HSCT suitable for people over 70, as the result of less invasive engraftment techniques, the development of more tolerable immunosuppressive drugs, and improvements in post-transplant patient care. These advancements also led to increases in five-year survival rates from 40% to 60% over the last 30 years, and the non-relapse mortality (NRM) risk has been halved over the same period [8].

Overall, HSCT research and blood cancer patient care have progressed dramatically, with several accomplishments achieved over the last four decades. That said, more work is needed to improve the outcomes even further.

CD34 enumeration: a new era in stem cell transplantation

The 1980s was a turning point in HSC research. Studies by Civin et al. and Tindle et al. were the first to describe CD34, a cell surface glycoprotein that mediated the attachment of HSCs to the extracellular matrix [9,10]. They employed monoclonal antibodies to detect CD34 as a cell surface antigen on HSCs and progenitor cells. This discovery was a milestone in documenting the enrichment of HSCs in bone marrow transplants.

Today, CD34+ cell enumeration is a crucial checkpoint in determining the reconstitutive capacity of the transplant. The optimum CD34+ count is 5×10^6 cells. Numbers below this optimum cause delayed or incomplete engraftment, and other studies associate excessive CD34+ count with adverse effects and prolonged hospital stays [11].

Flow cytometry has been the benchmark for CD34+ enumeration since the development of the first assay in 1989 [12]. However, these assays did not provide accurate measurements because of the rare occurrence of CD34+ in peripheral blood and interference from other lymphocytes and monocytes.

In the 90s, significant progress was made to establish a more accurate and robust transplant setting. The first improvement was the employment of another transmembrane glycoprotein called CD45 as a counterstain in flow cytometry. Since HSCs expressed lower levels of CD45, differential CD45 expression helped isolate HSCs further [13]. Furthermore, distinct light scattering properties of CD34+ have been unveiled. This gave rise to a multiparameter flow methodology, laying the foundations of a standardized CD34+ enumeration.

In light of these findings, the International Society of Hematotherapy and Graft Engineering (ISHAGE) launched a guideline for CD34+ cell quantitation in PBSC products [14]. The protocol emphasized four parameters: CD34 staining, CD45 staining, and forward and side light scatter. It also delineated an algorithm for clustering the CD34+ cell populations of interest based on these parameters – sequential Boolean gating, where cells were separated into subpopulations based on parameter-associated logic gates. Additional parameters were incorporated into the protocol in subsequent years, including absolute cell count (number of fluorescent counting beads = CD34+ cell count) and 7-amino-actinomycin D (7-AAD) to isolate only the viable cells. The combination of these parameters and gating protocols forms the widely-accepted single-platform flow cytometry assays [15].

Today, many flow cytometry laboratories perform CD34+ enumeration using commercial single-platform flow cytometry assay kits from companies including Beckman Coulter Life Sciences.

Automation and standardization to reduce lab errors in CD34+ enumeration

CD34+ stem cell enumeration is a critical checkpoint for harvesting PBSCs at the optimum time. Incorrect estimation of absolute CD34+ count can give rise to a need for repeated mobilization therapy or a need for re-engraftment due to insufficient cell numbers. Both scenarios create an extra financial burden on healthcare providers while prolonging the hospital stay and causing discomfort for the patient. That’s why flow cytometry laboratories are under immense pressure to follow the ISHAGE guidelines correctly while adhering to their region-specific mandates.

A 2011 survey conducted by UK NEQAS revealed the incorrect implementation of the ISHAGE gating strategy had twice the risk of failing an external quality assessment (EQA), while 43% of the participants had an incorrect set-up for the gating strategy [16]. The various sources of error included the omission of lymphocyte gating regions that helped identify platelet aggregates and dead cells, incorporation of additional antibodies, and parameter alteration. Each error created discrepant CD34+ enumeration, mainly overestimating the numbers. It was also observed that rectifying these errors improved the laboratories’ EQA performance. This clearly shows that tailoring a well-established protocol to specific research needs can obstruct CD34+ enumeration workflows, making it challenging to deduce the number of viable stem cells.

Laboratories currently rely on existing in vitro diagnostic (IVD) solutions for enumeration; however, most of these kits and software packages are too rigid to adapt to different purposes and regulations. First, they don’t take into account some of the regional mandates. For example, European Pharmacopoeia indicates the compulsory use of a negative control, despite ISHAGE making it optional [17]. Unfortunately, current IVD-based software solutions do not possess the flexibility to select/deselect the negative control option while remaining IVD compliant. Furthermore, these packages are not suitable for adapting the panel to switch from duplicate to single test mode, an essential quality control step for highly regulated laboratories. Without extensive quality control, data traceability of specimens and reagents becomes cumbersome, hindering the documentation for instrument malfunction, expired reagents and precision reports.

To add to these issues, the lack of automation capability is a serious nuisance. Many laboratories still perform the critical flow cytometry steps manually, which requires significant training and continuous monitoring of the process. Manual washing, labelling, pipetting, acquisition and analysis make CD34+ enumeration error-prone. In addition, laboratories use red blood cell lysing agents that require thorough sample preparation, making automation challenging to implement.

Despite the gold standard status, flow cytometry workflows for CD34+ enumeration present several challenges. These can be mitigated by automating the entire workflow while introducing new means of flexibility. A huge responsibility falls on the shoulders of flow cytometer manufacturers. New generation flow cytometers must feature a load-and-go system to automate sample handling and minimize contamination risk. Sample preparation, testing and analysis must take place in a single platform to reduce the requirement for trained operators to drive automation flow cytometry. To accompany upgrades to flow cytometers and assay kits, software solutions need to introduce more flexibility, such as adaptable acquisition panels for different reagents and regulations, while ensuring IVD compliance at all times. Improvements in flow cytometry software must also focus on thorough process control, reagent tracking and parameter verification so that QC failures can be detected immediately.

Finally, there is also a critical need for full-scale assay kits containing reagents for the monoclonal antibody, absolute count, negative control, lysing and cell viability. It is important to note the emergence of lysing reagents that can be stored at room temperature, exempt from the need for daily dilution from stock solutions, and non-destructive towards cell viability. Incorporating these reagents will help laboratories unlock full automation while adhering to ISHAGE Guidelines.

References

1. 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.
2. 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.
3. D’Souza A, Fretham C, Lee SJ et al. Current use of and trends in hematopoietic cell transplantation in the United States. Biol Blood Marrow Transplant 2020;26(8):e177–e182. doi: 10.1016/j.bbmt.2020.04.013.
4. Russell NH, Hunter A, Rogers S et al. Peripheral blood stem cells as an alternative to marrow for allogeneic transplantation. Lancet 1993;341(8858):1482. doi: 10.1016/0140-6736(93)90929-b.
5. Baldomero H, Gratwohl M, Gratwohl A et al. The EBMT activity survey 2009: trends over the past 5 years. Bone Marrow Transplant 2011;46(4):485–501. doi: 10.1038/bmt.2011.11.
6. Zuckerman T. Allogeneic transplant: does age still matter? Blood 2017;130(9):1079–1080. doi: 10.1182/blood-2017-07-795948.
7. Hao T, Li-Talley M, Buck A, Chen W. An emerging trend of rapid increase of leukemia but not all cancers in the aging population in the United States. Sci Rep 2019;9(1):12070. doi: 10.1038/s41598-019-48445-1.
8. 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.
9. 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.
10. 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.
11. Scheid C, Draube A, Reiser M et al. Using at least 5×10(6)/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.
12. 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.
13. Sutherland DR, Keating A, Nayar R et al. Sensitive detection and enumeration of CD34+ cells in peripheral and cord blood by flow cytometry. Exp Hematol 1994;22(10):1003–1010.
14. Sutherland DR, Anderson L, Keeney M et al. 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.
15. Keeney M, Chin-Yee I, Weir K et al. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. Cytometry 1998;34(2):61–70.
16. Whitby A, Whitby L, Fletcher M et al. ISHAGE protocol: are we doing it correctly? Cytometry B Clin Cytom 2012;82(1):9–17. doi: 10.1002/cyto.b.20612.
17. European Pharmacopoeia, 10th edn 2019–2022. EDQM Council of Europe (https://pheur.edqm.eu/home).

Further resources

1. Anasetti C, Logan BR, Lee SJ et al. Peripheral-blood stem cells versus bone marrow from unrelated donors. N Engl J Med 2012;367(16):1487–1496. doi: 10.1056/NEJMoa1203517.
2. Flowers ME, Parker PM, Johnston LJ et al. Comparison of chronic graft-versus-host disease after transplantation of peripheral blood stem cells versus bone marrow in allogeneic recipients: long-term follow-up of a randomized trial. Blood 2002;100(2):415–419. doi: 10.1182/blood-2002-01-0011.
3. Mehta RS, Saliba RM, Alsfeld LC et al. Bone marrow versus peripheral blood grafts for haploidentical hematopoietic cell transplantation with post-transplantation cyclophosphamide. Transplant Cell Ther 2021;27(12):1003.e1-1003.e13. doi: 10.1016/j.jtct.2021.09.003.

The author

Andreas Boehmler PhD
Beckman Coulter Life Sciences, Krefeld, Germany
e-mail aboehmler