Stem cells have the capability to proliferate and differentiate into various cells of the body. Few stem cell sources have been approved for transplantation, among them are the hematopoietic progenitor cells which are progenitors of the myeloid and erythroid lineage in the hematopoietic system, that continually provides mature blood cells during the lifespan of the individual. These well-characterized stem cells are clinically relevant in the treatment of diseases such as breast cancer, leukemias, and congenital immunodeficiencies. Peripheral blood stem transplantation is a standard procedure after its first successful transplantation more than 35 years ago. The minimum intended dose of stem cells given to the patient is
Peripheral blood-derived stem cells (PBSCs) have been used in bone marrow transplantation ever since its first report was published in the late ‘70s [
Hematopoietic stem cells (HSCs) belong to a group of multipotent precursors that have a self-renewal capacity and the ability to generate different cell types that comprise of the blood-forming system [
PBSC transplantation (PBSCT) has become increasingly common with PBSCs largely replacing bone marrow (BM) as the preferred stem cell source due largely to quicker engraftment kinetics and ease of collection. In the peripheral blood, stem cells are found in limited numbers (less than 0.1% of all nucleated cells). Stem cell progenitor cells circulate in the periphery, as this ensures an even distribution of hematopoiesis within the bone marrow.
PBSCs consist of a subpopulation of hematopoietic progenitor cells (CD34+), which is morphologically difficult to identify. These cells can be distinguished by their immunophenotypic patterns as CD34+/CD38−. They do not express a full complement of either myeloid or lymphoid lineage-specific markers (Lin−) but do express the Thy-1 differentiation antigen. The CD34+/CD38−/Lin−/Thy-1+ cells are responsible for initiating long-term culture initiating colony (LTC-IC) assays [
There are many methods for stem cell quantification after collection but the most common method used today is the flow cytometric evaluation of CD34+ cell numbers. Enumeration of CD34+/CD38−, CD34+/CD33−, and CD34+/Thy-1+ cell subsets has proven to be a useful technique in the estimation of stem cell numbers [
Hematopoietic stem cells have an inherent property to constantly leave the bone marrow and penetrate tissues thereafter returning to the BM or peripheral niches via the blood or lymphatic system [
Levels of pluripotent hematopoietic stem cells rise up to 50-fold in the recovery phase after myelosuppressive chemotherapy and can be collected for autologous transplantation. In order to achieve circulating levels high enough to ensure a harvest capable of reconstituting a mature hematopoietic system after allogeneic donation, healthy donors must be “primed” with hematopoietic growth factors, using either rHuG-CSF or rHuGM-CSF. G-CSF is thought to stimulate HSC mobilization by decreasing SDF-1
The successful transplantation of hematopoietic stem/progenitor cells (HSPCs) is based on their ability to home to the BM niche and on their engraftment capacity. Interactions between HSPCs and their niches are altered during mobilization and must be reestablished during BM homing and repopulation. The homing of HSPCs to BM is a rapid process that takes place during the hours after transplantation and is an essential and necessary requirement for repopulation and engraftment [
Transplantation of hematopoietic progenitor cells (HPC) has become a widely accepted therapeutic option, particularly for patients with chemotherapy-sensitive hematological malignancies. Transplantation of HPC offers several advantages compared to bone marrow. Collection of HPC can be performed without general anesthesia, engraftment is faster, and supportive care and costs are reduced. HPC are harvested by leukapheresis after mobilization with chemotherapy and/or G-CSF. The use of mobilized peripheral blood is now the method of choice in autologous transplantation for various reasons, including an elevated production of immature cells, and, in comparison to the utilization of BM, the shorter time period required for a satisfactory repopulation, the more rapid engraftment, fewer technical difficulties, lower risk, and considerably less pain [
Although BM and peripheral blood are both still considered a source of stem/progenitor cells for this purpose [
A decisive factor for patients being transplanted in an autologous setting is the dose of transplanted HPC usually determined by measurement of CD34+ cells. Some data suggests that transplantation with less than 2 million of CD34+ cells/kg body weight (bw) is associated with a prolonged hematologic engraftment and worse outcome, whereas a dose of more than 5 million CD34+ cells/kg bw was of benefit [
Number of publications state that the minimum level of CD34+ enumerated from the peripheral blood is around 14 cells/
The study was approved by the local ethics committee for research. This study was part of the data of hematopoietic stem cell transplantation (HST) done at our institution. Transplantation involving peripheral blood stem cells is carried out on patients as part of the bone marrow transplantation procedure. Identity of patients is kept confidential and every sample in the laboratory is denoted by the laboratory identification number (Lab id). The data is taken from 20 subjects of different kg body weights to establish a uniform collection strategy.
The minimum sample that is accepted is 100 mL. A cut off CD34+ level of 14 cells/
We use cytokines such as filgrastim (glycosylated granulocyte colony-stimulating factor [G-CSF] which is a common mobilizing agent for haematopoiesis. This cytokine initiates the cascading sequence to produce committed mature blood cell components. Stem cell harvesting begins with enticing the CD34+ rich cell population out of the bone marrow niche environment as done earlier [
Success of a stem cell transplant depends on the mobilization of blood-forming CD34+ cells from the bone marrow. This level has to be analyzed so as to have an optimal level of CD34+ cells collected. We use an optimal value of 14 cells/
After the apheresis procedure is completed in the hospital, the stem cell unit is brought to the laboratory for plasma reduction as part of the plasma reduction strategy. The concentration of the isolated CD34+ cells is calculated, thereby calculating the CD34+ cells isolated per kilogram body weight of the patient.
The postprocessing analysis is done as a quality control analysis of the isolated stem cells. 1 mL of the postprocessing nucleated cell rich plasma is taken for the QC analysis. 500 The formula is as follows: CD34+ cells present/ Total CD34+ cells to be Transplanted/kg body weight:
where TDF is total dilution factor = dilution factor of the leukocyte for hematological evaluation × difference in the dilution factor between hematological and flow cytometry evaluation.
A comparative analysis is carried out on three factors, namely, the number of CD34+ cells enumerated in the peripheral blood, the total CD34+ cells isolated per kg body weight of the patient, and the body weight of the patient (Table
Comparison study of the enumeration of stem cells isolated from peripheral blood from the donor, the body weight of the patient, and the total CD34+ cells enumerated.
Serial number | Peripheral blood CD34+ cells/ |
Total CD34+ cells isolated | Body weight of the patient | CD34+ cells isolated/kg body weight |
---|---|---|---|---|
1 | 22.5 | 86.6 × 106 | 76 kgs | 1.1 × 106 |
2 | 74.97 | 358.1 × 106 | 76 kgs | 4.7 × 106 |
3 | 10.96 | 115.58 × 106 | 87 kgs | 1.3 × 106 |
4 | 8.4 | 24.74 × 106 | 87 kgs | 0.3 × 106 |
5 | 15.2 | 14.84 × 106 | 87 kgs | 0.2 × 106 |
6 | 25 | 83.28 × 106 | 31.2 kgs | 2.7 × 106 |
7 | 30 | 157.56 × 106 | 31.2 kgs | 5.1 × 106 |
8 | 86 | 532.54 × 106 | 21 kgs | 11.5 × 106 |
9 | 55 | 288.43 × 106 | 90 kgs | 3.2 × 106 |
10 | 15 | 38.12 × 106 | 12.3 kgs | 3.1 × 106 |
11 | 679 | 1961.56 × 106 | 60 kgs | 32.7 × 106 |
12 | 413 | 1321.89 × 106 | 40.1 kgs | 33 × 106 |
13 | 15 | 93.34 × 106 | 63 kgs | 1.5 × 106 |
14 | 15 | 136.91 × 106 | 63 kgs | 2.2 × 106 |
15 | 20 | 161.96 × 106 | 63 kgs | 2.6 × 106 |
16 | 153 | 909.4 × 106 | 85 kgs | 10.7 × 106 |
17 | 74 | 225.62 × 106 | 49 kgs | 4.6 × 106 |
18 | 18 | 87.28 × 106 | 73.5 kgs | 1.2 × 106 |
19 | 13 | 83.41 × 106 | 74 kgs | 1.1 × 106 |
20 | 12 | 80.36 × 106 | 74 kgs | 1.1 × 106 |
Graphical representation of the peripheral blood CD34+ level isolated from peripheral blood versus the final CD34+ level enumerated after processing.
We next plotted a graph that correlated the CD34+ level of the peripheral blood isolated per
Graphical representation of the peripheral blood CD34+ level isolated from peripheral blood versus the CD34+ level enumerated/kg body weight.
Successful bone marrow transplantation using hematopoietic stem/progenitor cells (HSPCs) is based on homing of HSPCs to BM and is a rapid process that takes place after transplantation. This process is called as engraftment which is the basis in haematopoiesis [
The aim of this study is to establish a suitable factor that can be multiplied by so as to get the desired CD34+ cells enumerated in the peripheral blood thus ensuring more than
We next plotted a graph of peripheral blood CD34+ cells enumerated from the peripheral blood versus CD34+ cells isolated per kg body weight (Figure
Graphical representation of the total CD34+ cells isolated after each collection procedure versus the body weight of the patient in kgs. A mean point is plotted from patients of different kg body weights but gives almost the same number of CD34+ progenitor cells enumerated per kg body weight.
We thus have come up with a factor of 0.8 CD34+ cells/kg of the body weight that has to be considered before collection. The factor of 0.8 can be a suitable conversion factor to enumerate the minimum CD34+ cells required prior to transplantation. Thus, a 20 kg individual will need a baseline value of
This paper assumes significant importance as, at the moment, the only accepted methodology in transplantation is minimal manipulation as per the standards. Expansion of HSCs does not come under the purview of an unmanipulated cell so we are forced to increase the efficiency of transplantation by limiting our strategy to an increased cell dosage as observed in the enumeration of the peripheral blood stem cells. We next plan to expand our study to include all the patients that are registered for stem cell transplantation.
Most of the stem cell transplants that have been carried out in our hospital are autologous cases. The patients are administered with premedication drugs such as ondansetron, dexamethasone, and lorazepam before the administration of cyclophosphamide (2000 mg/m2) followed by G-CSF (10 mcg/kg/day) until the day of apheresis.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors acknowledge the effort of staff members Tareeq Anwar Mohammed, Romelyn Castronueva, and Myra Gamboa from the Stem Cell section who were instrumental in conducting this project.