Stem Cells, Vol. 18, No. 2, 76-86,
March 2000
© 2000 AlphaMed Press
Technical Aspects and Clinical Impact of Hematopoietic Progenitor Subset Quantification
John Baecha,
Hans E. Johnsenb
a The Department of Clinical Immunology and Transfusion Medicine, Aalborg Hospital;
b The Department of Hematology, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark
Key Words. CD34 • Analysis • Flow cytometry • Differentiation antigens • Cytokine receptors • Adherence molecules • Hematopoietic stem cell transplantation • Prediction of engraftment
John Baech, M.D., The Department of Clinical Immunology and Transfusion Medicine, Aalborg Hospital, Postbox 561, DK-9100 Aalborg, Denmark. Telephone: 45-99-32-11-11; Fax: 45-99-32-11-39; e-mail: john{at}dadlnet.dk
 |
Abstract
|
|---|
As high-dose therapy for malignancies is now being applied to newly diagnosed patients as adjuvant therapy, it has become a requirement that quality and safety assessment of hematopoietic stem cell grafts be evidence-based. This process has developed a new institution in medicine, the stem cell laboratory. In most cases this speciality has evolved from or within hematological research laboratories. However, the increased routine technologies applied in quality evaluation, ex vivo manipulation and safety assessment in stem cell handling naturally places this activity in transfusion medicine.
Multiparametric flow cytometry can identify progenitor subsets in normal human bone marrow and peripheral blood, and such subset quantification has been used retrospectively to predict three-lineage engraftment following high-dose therapy for malignancies. Published single center data have suggested an impact on clinical outcome, and a standardized technique for subset enumeration needs to be established before prospective multicenter trials can be initiated to document the prognostic value of such quality assessment in autografting.
Based on experiences of CD34 enumeration, which we consider to be the first step in quality assessment of hematopoietic stem cell grafts, this review discusses flow cytometry subset identification by lineage-specific differentiation markers, stromal-dependent adherence molecules, and regulatory growth factor receptors from a technical point of view. The aim of this review is:
- To recommend a simple method based on the experiences of the Nordic workshop III on subset identification;
- To present new molecular genetic-based methods for future use in quality assessment; and
- To propose new endpoints necessary for validation of the likely clinical impact of subsets in prospective trials.
As sample differences between blood and marrow result in technical difficulties, this review only focuses on the methodology of identifying subsets in blood and leukapheresis products. Methods for subset analysis in diagnostic bone marrow samples will be covered in a forthcoming review.
|
Introduction
|
|---|
Background and Rationale
The CD34 antigen is stage-specific and identifies cells in the early stages of hemopoietic differentiation. This population, therefore, contains progenitors committed to the myeloid, erythroid, megakaryoid, and lymphoid lineages, as well as primitive progenitors and stem cells capable of long-term reconstitution [1-4]. Enumeration of CD34+ cells has been shown to be useful in the harvest procedure of stem cells from blood for transplantation, as it is important for the prediction of fast and delayed three-lineage engraftment following high-dose therapy [5-10]. However, it has been suggested that this prediction does not apply to autografts with CD34+ cell numbers between 2-10 million CD34+ cells/kg patient weight. Most transplanted patients receive such numbers, which has forced us to focus on other factors enabling us to predict the success of a three-lineage engraftment [8, 11].
A recent approach defining progenitor compartments within the CD34 population has been to use multicolor flow cytometry to identify antigens whose expression subdivides the CD34+ population [6, 12-17]. Such studies have identified committed or lineage-specific progenitor populations as well as uncommitted or multipotential progenitors and have been useful for defining normal hematopoietic differentiation patterns. Although the measurement of minor subsets of CD34 has been reported to have some utility in estimating stem cells in autografts, the value of determining CD34+ subsets for clinical use and the role of various CD34+ subsets in hematopoietic recovery are not well understood and have not been well documented.
The purpose of this review is to describe the experimental background for the identification and enumeration of early-stage stem cells as well as uncommitted progenitor cells and lineage-specific cells. We will discuss the technical problems related to flow cytometry-dependent enumeration and present the likely clinical impact known from retrospective analysis, and finally propose future endpoints for clinical trials on standardized flow cytometry techniques for quality assessment of autografts. The background for these goals is our experiences of CD34 enumeration, which we consider the first step in quality assessment of hematopoietic stem cell grafts.
The History of a New Discipline in Laboratory Medicine
Current indications for high-dose therapy as adjuvant are now being used on newly diagnosed patients. This development has required evidence-based quality and safety assessment of hematopoietic stem cell grafts and introduced a new institution in medicine, the stem cell laboratory. In most cases this speciality has evolved from or within hematological research laboratories. However, the increased routine technologies applied in quality evaluation, ex vivo manipulation, and safety assessment in stem cell handling naturally place this activity of progenitor cell processing in transfusion medicine.
More than a decade ago, the CD34 antigen was identified as a marker of hematopoietic stem and progenitor cells [18, 19]. A few years later, the clinical implication was documented by the Milan Group reporting that CD34+ cells circulate in blood during hematopoietic recovery or expansion following pancytopenia induced by chemotherapy and/or growth factor administration [5].
In those years the shift in clinical autografting from marrow to blood revealed an urgent need for one or more simple and reproducible assays documenting the engraftment potential by enumeration of hematopoietic peripheral blood stem and progenitor cells (PBSPC) [7, 8, 11-13, 20-25]. The major step forward in meeting the need for such quality assessment was the general acceptance of flow cytometry as the standard method for the identification and enumeration of the CD34+ progenitor compartment.
|
Technical Aspects of CD34 and Subset Analysis
|
|---|
Immunophenotyping of Uncommitted and Committed Progenitors
Uncommitted progenitors that can initiate long-term cultures, or can repopulate immunodeficient mice are CD34+ cells that do not express, or express very low levels of lineage-specific antigens including CD33, CD7, and CD10. They express either no or low levels of CD38, CD71, HLA-DR antigens, and the Thy1 antigen [26-33]. They are quiescent and express a high level of p-glycoprotein, which allows their identification and selection on the basis of the absence of staining with the mitochondrial dye, rhodamine [34-37]. It is thought that human hematopoietic stem cells are also present in this population.
In contrast, more mature progenitors express CD34 antigens coexpressed with the lineage-specific antigens CD38, CD71, HLA-DR, CD13, CD33, glycophorine A, CD61, and CD19 [2, 6, 12-17, 22, 38]. Accurate determination by flow cytometry of such small subpopulations of CD34+ cells is difficult and needs special strategies as discussed below.
CD34 Enumeration as the First Step in Quality Assessment
Culture assays of colony-forming cells were originally applied, but due to the inconvenience of this method several laboratories focused on flow cytometry analysis in quality assessment. The first steps were taken in 1991 by S. Serke of Berlin, S. Siena of Milan, and G. Fritsch of Vienna [5, 6, 39, 40]. This development was promoted by the farseeing Mulhouse Group when, in early 1992, they hosted the First European Workshop on stem cell determination and standardization. The resulting manual is still a valuable procedure reference in laboratory practice [41, 42].
The subsequent years have resulted in several publications about the standardization of the flow cytometry analysis [13, 25, 43-50] as well as reports on the clinical implication of the enumeration of CD34+ cells in blood and autografts on the timing of apheresis and time to engraftment [7-10, 50, 51].
We do not yet know the minimum safe number of CD34+ cells needed for clinical engraftment of all lineages, as this may vary depending on the CD34+ cell subset composition in a given patient or autograft [38, 52-54]. However, we know that a graft content of more than 2 ml per kg bodyweight is safe, resulting in fast three-lineage engraftment in >90% of patients and most important, involves a minor risk for engraftment failures [7, 8, 55].
Reference Protocols on CD34 Enumeration
Recently several reports presented new reference protocols on flow cytometry enumeration, and several companies announced standard kits for absolute enumeration of CD34+ cells. The most important change in relation to the Milan/Mulhouse protocol is the recommendation of a "lyse no wash" technique. Such a technique eliminates any potential cell loss caused by cell washing which has been shown to reduce the number of CD34+ events [56].
One very important step in such a strategy exemplified by the Nordic protocol (Table 1
) is to add the pan CD45 antibody in order to quantify leukocytes and discriminate these from erythroblasts and non-lysed erythrocytes in the live gate, i.e., in obtaining an accurate denominator for the CD34% calculation [12, 22, 43]. The number of CD34+ events is still calculated on the basis of a population of a minimum in the range of 50 to 100 events positively stained by a CD34 class III antibody and present in the low side scatter region [5, 6, 12, 23, 42].
The percentage of CD34+ cells can then be calculated by the number of CD34 events divided by the denominator (= CD45+ leukocytes). The total number of CD34+ cells per volume can subsequently be calculated by multiplication of the CD34% and the leukocyte count obtained from a hematology analyzer (the dual platform technique). However, a more accurate determination may require a volumetric measurement added into the analysis which can be obtained, e.g., by introducing a known number of reference particles per volume of sample—the so-called single platform technique [57]. This technique has the advantage of being independent of variation in leukocyte enumeration by the hematology analyzer, however, one has to be very precise in pipetting the sample volume to get a correct absolute count.
CD34 class III antibodies with sufficient activity, independent of flourochrome conjugation, can be used if they have been titrated appropriately before use. The isotype control should also be selected after sufficient testing. Class I and II antibodies are not recommended [2, 3, 41, 43].
The most important step, however, may be choosing the red blood cell lysing reagent as this is not an "innocent by-stander" in sample preparation. The different chemicals, particularly fixatives, may affect the cell and the staining intensity.
Introduction of reference protocols (Table 1
) will allow each stem cell laboratory to choose their own methodology, including commercial kits available on the market. The result may be that it will be impossible to diminish interlaboratory variation in future analyses. On the other hand, it may be that various methods used in different laboratories in accordance with a reference protocol will produce interlaboratory variations which are comparable by a strictly standardized method. Future workshops may give the answer to such questions.
However, it is an open question whether such work will be worth the effort as more important clinical questions have been forwarded to the stem cell laboratories regarding the prediction of "poor mobilizers" and the prediction of "insufficient autografts" with the risk of delayed platelet engraftment.
The Challenge of CD34+ Subset Analysis
Such predictive tests may very likely include subset analyses of uncommitted and lineage-specific progenitors before harvest and/or autografting [8, 11, 52-54]. In a recent multicenter study from the Nordic Stem Cell Laboratory Group (NSCL-G) of more than 700 transplantations, prolonged thrombocytopenia (i.e., <20,000 platelets/µl blood day +20) was recognized in less than 10% of patients, most of whom had lymphomas [8]. There may be several explanations for this phenomenon, including severe infections and regimen-related toxicity or, alternatively, an insufficient autograft due to impaired progenitor and stem cell quality.
In the attempt to identify the group of patients at risk for prolonged thrombocytopenia, several reports have studied a series of autografts with the aim of evaluating the effect of CD34+ lineage-specific subset numbers on time to platelet engraftment. The most significant correlation was recognized for the CD34/CD61+ megakaryocytic cell [11, 53, 58-62]. Furthermore, in analysis of uncommitted progenitors, we discovered an inverse correlation between the CD34/CD38– or CD34/CD33– subset and platelet engraftment, indicating that high numbers of CD34/CD38– cells, i.e., early uncommitted progenitors, may increase the risk of delayed engraftment [11, 52, 53, 58-69].
Generally, studies of CD34+ subsets have used two- or three-color flow cytometry to define subsets within the CD34+ population. Subset enumeration by double marking of CD34+ cells is a new challenge for the stem cell laboratory as current recommendations (Table 2) do not seem to work optimally in an interlaboratory setting [8]. Workshops are needed to focus on the two most important problems related to the definition of: A) the acquisition gate for sampling of the double-stained CD34+ cells and B) the negative population by the isotype control or other strategies, including blocking with excess unlabeled CD34 monoclonal antibody. Extremely careful analysis will be required in prospective multicenter studies to correlate well-defined minor subsets with clinical features such as prior chemotherapy and rapidity and durability of engraftment to confirm the present single-center studies.
View this table:
[in this window]
[in a new window]
|
Table 2. The revised Nordic Stem Cell Laboratory Group recommendation for CD34-subset enumeration in blood and leukapheresis products [8, 11]
|
|
Flow Cytometry-Dependent Enumeration of CD34+ Subsets
Quantification of the absolute number of circulating CD34+ subsets in whole blood, leukapheresis products, or stem cell grafts depends on the accurate determination of two values (Table 2). The first value is the absolute number of CD34+ cells determined by the currently used one- or two-platform laboratory protocols. The number is derived from the mean of two replicate CD34+ determinations, by CD45-flourescein isothiocyanate (FITC)/CD34-phycoerythrin (PE)-specific staining of more than 100 positive events from a lysed but not washed fresh sample. The negative control should only contain a minor number of events representing nonspecific staining, as determined by the CD45-FITC/isotype-PE control (Table 1). Second is the value of the relative number of the specific CD34 subset in question. Cells are incubated and double-stained with CD34 monoclonal antibodies plus one with specificity against one (CDX) of the subsets described in Table 3. The absolute number of lineage-specific progenitors is calculated by the formula: 
.
View this table:
[in this window]
[in a new window]
|
Table 3. Summary of published studies of subset enumeration and clinical outcome in high-dose therapy and autografting
|
|
Flow Cytometry Identification of CD34+ Subsets
The sample is incubated and double stained with anti-CD34-PE or -FITC-conjugated monoclonal antibodies plus one of the specific antibodies selected for CDX+ subset identification. The analytic strategy is given in Table 2 and illustrated in Figure 1. The first step is to set a CD34+ gate and subsequent acquisition of a minimum of 5,000-10,000 CD34+ double-stained cells. The second step is through the use of quadrant statistics or histogram analysis to identify the positive lineage-specific subset population by help of the test antibody in question and the relevant negative control antibody. The sensitivity of such a two-step analysis will be about 1% based on a minimum of 50-100 events positively stained by the anti-CDX antibody.
View larger version (135K):
[in this window]
[in a new window]
|
Figure 1. The two-step analytic strategy for subset enumeration in accordance with the Nordic Stem Cell Laboratory Group recommendation. SSC = side scatter.
|
|
Cytokine Receptors and Adherence Molecules—New Markers
Hematopoietic commitment is likely to be regulated by extrinsic molecules including matrix and cellular molecules as well as growth factors, indicating that CD34+ progenitors have adherence molecules and receptors. Recent flow cytometry studies have identified such progenitor subsets as described in Table 3 [11, 52, 53, 59, 60, 63, 65, 66]. These potential candidate molecules may be closely related to the putative complex mechanisms of progenitor cell mobilization and engraftment.
These complementary phenomena may reflect changes in the expression and function of cell adherence molecules of PBPC, either as a direct effect of the mobilizing agent through cytokine receptors or as an indirect response to secondary signals from stromal cells.
During physiological conditions, a differentiation-dependent pattern of adherence molecule expression seems to be essential for binding, proliferation, and differentiation of CD34+ cells in the marrow, and up- or downregulation may influence retention and release of stem cells. CD34+ blood circulating cells expressed ICAM-1, leukocyte function-associated antigen-3 (LFA-3), PECAM, very late activation antigen-4 (VLA-4), VLA-5, LFA-1, L-selectin, c-kit, Thy-1, and finally CD34 itself is suggested to act as an adhesion molecule. Obviously, quantitative analysis of such a subset should include quantitation of a membrane-binding site in an attempt to describe density changes during regulation as a useful marker [2, 16, 70-73].
Erythropoietin, G-CSF, and thrombopoietin (TPO), as well as the corresponding receptor-positive progenitors, are considered essential for development of the lineage-specific progenitors [36, 74-79]. Recently, TPO has been demonstrated to be of importance for early stem cells [80].
Table 3 is a summary of known studies of the influence and subset analysis on blood cell recovery following transplantation. The 15 studies contain data from up to 1,058 patients analyzed. These data strongly suggest that subset analysis is of major clinical impact. However, multicenter collaboration is necessary to establish recommendations for the flow cytometry method taking into account the multiparametric possibilities for three to four-color staining.
|
Quantitative Molecular Genetic Analysis
|
|---|
Molecular Genetic Analysis of Minor Subpopulations by Single-Cell Polymerase Chain Reaction (PCR) and Limiting Cell Dilution—A New Instrument in Specificity Evaluation
In studies of CD34+ cells, a single-cell reverse transcriptase (RT)-PCR for CD34-mRNA+ cells has been established, and frequency of CD34-mRNA+ cells has been estimated by single-cell flow cytometry sorting and limiting dilution [81].
The technology combining the RT-PCR and flow cytometry methods has been a breakthrough for identification, isolation, and functional analysis of minor subsets as described in recent reports on pathogenesis in multiple myeloma [82]. We strongly recommend that this technology be used for documentation of the specificity of minor CD34+ subsets identified by monoclonal antibodies as discussed below. Therefore, we describe this new instrument in detail.
First, the principle of the method was based on isolation and identification of specific CD34 transcripts in one single flow-sorted whole cell in one well. This was optimized in sorted cells from the CD34+ leukemic cell line KG-1. Second, the sorting step has to be optimal as does the placing of the numbers of cells from one to hundreds of thousands into one single well by using a fluorescence-activated cell sorter (Vantage sorter). According to the manufacturer's documentation, the automated cell deposition unit permitted single-cell sorting with an accuracy greater than 99% as recognized by the presence of a genomic CD34 PCR product serving as a positive control for the sorting procedure. Third, the general method to calculate the total number of initial targets present in a single sample using limiting dilution and an all-or-none PCR reaction against low numbers of DNA targets is based on the single-hit Poisson model. This model assumes that only one cell of one defined cell subset is necessary for the positive response, which should be the case for the present analysis of single sorted whole cells [49, 81, 82].
The progenitors and stem cells are contained in a compartment expressing the CD34 surface membrane protein, which is a complex stage-specific molecule with a density highest on early progenitors and stem cells. This density decreases progressively as cells mature [2]. The CD34bright population includes cells with the possibility of long-term hematopoiesis and the CD34dim population contains lineage-committed progenitors without the ability to provide long-term hematopoiesis [14, 38, 83, 84]. Recent data have indicated that CD34 plays a role in the maintenance of primitive cells by inhibiting differentiation since downregulation is necessary for end-stage differentiation. It is assumed that the downregulation is correlated to messenger RNA (mRNA) reduction following gene inactivation. The half-life of membrane molecules is much longer than for mRNA, predicting that only a subpopulation of CD34 antigen-positive cells could be expected positive for CD34 mRNA. Indeed, it was found that only 50% of CD34 antigen-positive cells had an actively transcribed CD34 gene [85]. Furthermore, in accordance with the model, CD34 mRNA transcripts were found in up to 100% of CD34+/CD38– putative uncommitted cells significantly reduced in CD34+/CD38+ lineage-specific progenitors. Such findings may in part explain the varying engraftment capability among autografts with an identical number of CD34 antigen-positive cells.
One more confusing factor is the evidence from mice, where some stem cells may also be present in the CD34 antigen-negative bone marrow compartment. Studies in humans have also shown that some cells present in the CD34– or CD34 very dim cell population can initiate hematopoiesis when transplanted into immunodeficient mice or preimmune sheep fetuses [86-90]. These CD34– cells cannot, however, generate mature progeny when cultured ex vivo. It is possible that this CD34 antigen-negative cell is a more primitive precursor of the CD34+ compartment, capable of engrafting in vivo but unable to generate progeny in culture. If such a cell has CD34 transcripts present, the frequency is below 1 in 1,000 quantified by the method described above [82].
Real-Time RT-PCR Assays May be the Assay for the Future
Real-time quantitative analysis of CD34 mRNA has been established, and future studies need to compare flow cytometry and real-time RT-PCR for CD34 subsets found to be of clinical importance [82]. Real-time quantitation refers to a kinetic quantitative PCR method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time. The amplification reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components are limited during the exponential phase, meaning that values are highly reproducible in reactions starting with identical numbers. This greatly improves the precision of cDNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in a much faster and higher sample throughput.
The real-time PCR method has been used for quantitation of full-length CD34 (CD34F) and truncated (CD34T) mRNA by primers and probes designed on the basis of the differential splicing pattern of CD34 mRNA [82].
The use of this new simple technique will make molecular analysis of minor subpopulations simpler and maybe more reliable, and should be studied to find the applications in clinical and research settings. The technique offers a unique opportunity to standardize assays and to develop rigorous standards and controls. It is our belief that real-time RT-PCR may become a routine and robust basis for clinical decision-making due to its sensitivity, which may allow us to quantitate the very minor subsets of immature and mature progenitors of importance for engraftment.
|
Clinical End Points and Impact on Hematopoietic Recovery
|
|---|
Phenotyping by In Vitro Assays Is a Surrogate Marker
The only conclusive method to assay stem and progenitor cells is to follow their ability to repopulate conditioned recipients, which makes it difficult to study such cells. Phenotypic characterization of primitive subpopulations of CD34+ cells, in vitro culture assays and transplantation of hematopoietic stem cells into immunodeficient mice or preimmune sheep, are different surrogate assays that attempt to determine the number of progenitors and stem cells present in a given human hematopoietic cell population. In this short review, we have discussed the advantages and disadvantages of using flow cytometry to determine the number of subsets in a reinfused graft following marrow-ablative therapy. It remains to be determined, however, if this technology is a useful adjunct in a well-defined clinical setting.
Engraftment and Hematopoietic Recovery as Clinical Endpoints?
Supportive reinfusion of hematopoietic stem and progenitor cells following marrow ablative therapy has the ultimate goal to reestablish hematopoiesis following an initial recovery of end-stage blood circulating cells to a level necessary to reduce the risk of side effects such as infections, bleeding, or anemia. This level has traditionally been >0.5 x 109 neutrophils/l, >20 x 109 platelets/l and >2 x 10–3 reticulocytes. So far no levels have been established for recovery of the immune system, i.e., B cells, T cells, natural killer cells, and monocytes.
Consequently, engraftment has most often been evaluated by the time of three-lineage recovery by calculating the time-dependent probability reaching the above-given levels following blood cell nadir after transplantation. By means of Kaplan-Meyer statistics, a simple description of the observed data can be analyzed by log-rank statistics to evaluate the prognostic value of different factors, e.g., CD34+ cell numbers, subset numbers, as well as other factors of proposed importance. However, this strategy for analysis was established in the early days of allogeneic and autologous transplantation and needs to be reevaluated in actual practice. The use of blood progenitor grafts and growth factor administration may have changed the initial correlation between blood cell recovery and the clinical events.
Future Clinical Endpoints
In the last decade, hundreds of reports have based their conclusions on quality assessment of hematopoietic grafts on surrogate markers, and it seems to be time for a move towards evaluating present practice on the basis of clinically relevant factors. Obviously such data have to be generated in a prospective manner.
It is worth mentioning that the introduction of peripheral blood autografting has not changed the risk of documented infections compared to the use of conventional bone marrow grafts, although a faster neutrophil recovery to 0.1, 0.5, and 1.0 x 109/l is substantially documented [89, 90]. Furthermore, the risk of thrombocytopenic bleedings has not been evaluated and formally documented as being reduced by an increase from below to above 10, 20, or 50 x 109 platelets/l.
In future clinical trials, the following factors (Table 4) need to be considered:
- The primary clinical endpoints should be events of clinical importance, i.e., the risk of severe infections and bleedings, including antibiotic administration and transfusion of platelets as well as erythrocytes.
- The secondary endpoints should be a toxicity evaluation in accordance with, for example, Common Toxicity Criteria (CTC), including grading of hematological toxicity.
- The tertiary endpoints should be the risk of regimen-related death within the first 90 days following graft reinfusion.
|
Future Directions: Time and Technology are Ready For the Next Step
|
|---|
Quality Assessment of Hematopoietic Stem Cell Grafts
The success of the stem cell laboratories during the last decade is documented by the elimination of the risk of engraftment failure—not by the introduction of PBSPC grafting—but very likely due to the quality assessment procedures. This has been achieved by hard work on a very simple technology, allowing us to identify patients at risk of engraftment failure due to either lack of mobilization or insufficient harvest outcome [55].
|
Conclusion and Future Goals
|
|---|
This work has given us the experience needed to move on to the next step in quality assessment of autografts—subset enumeration by standardized flow cytometry techniques based on recommendations given in this review and clinically validated by carefully planned prospective trials on efficacy and toxicity, not only blood cell recovery. Finally, when such data are available, the international community can reach a consensus on guidelines and reference protocols for subset identification and enumeration in an attempt to make high-dose therapy a safe procedure [55].
|
Acknowledgments
|
|---|
We gratefully acknowledge the support from the Danish Cancer Society, the Nordic Cancer Union, the Meyer Foundation, the Ydes Foundation, and the PC Petersens Foundation.
|
References
|
|---|
-
Haas R, Witt B, Mohle R et al. Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support. Blood 1995;85:3754-3761.[Abstract/Free Full Text]
-
Steen R, Tjonnfjord GE, Gaudernack G et al. Differences in the distribution of CD34 epitopes on normal haemopoietic progenitor cells and leukaemic blast cells. Br J Haematol 1996;94:597-605.[CrossRef][Medline]
-
Sutherland DR, Keating A. The CD34 antigen: structure, biology, and potential clinical applications. J Hematother 1992;1:115-129.[Medline]
-
Civin CI, Almeida-Porada G, Lee MJ et al. Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo. Blood 1996;88:4102-4109.[Abstract/Free Full Text]
-
Siena S, Bregni M, Brando B et al. Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients. Blood 1991;77:400-409.[Abstract/Free Full Text]
-
Serke S, Sauberlich S, Huhn D. Multiparameter flow-cytometrical quantitation of circulating CD34(+)-cells: correlation to the quantitation of circulating haemopoietic progenitor cells by in vitro colony-assay. Br J Haematol 1991;77:453-459.[Medline]
-
Weaver CH, Hazelton B, Birch R et al. An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 1995;86:3961-3969.[Abstract/Free Full Text]
-
Johnsen HE, Baech J, Nicolaisen K, for the Nordic Stem Cell Laboratory Group. Validation of the Nordic flow cytometry standard for CD34+ cell enumeration in blood and autografts: report from the third workshop. J Hematother 1999;8:15-28.[CrossRef][Medline]
-
Knudsen LM, Gaarsdal E, Jensen L et al. Improved priming for mobilization of and optimal timing for harvest of peripheral blood stem cells. J Hematother 1996;5:399-406.[Medline]
-
Knudsen LM, Gaarsdal E, Jensen L et al. Evaluation of mobilized CD34+ cell counts to guide timing and yield of large-scale collection by leukapheresis. J Hematother 1998;7:45-52.[Medline]
-
Knudsen LM, Jensen L, Jarlbaek L et al. Subsets of CD34+ hematopoietic progenitors and platelet recovery after high dose chemotherapy and peripheral blood stem cell transplantation. Haematologica 1999;84:517-524.[Abstract/Free Full Text]
-
Bender JG, Unverzagt KL, Walker DE et al. Characterization of CD34+ cells mobilized to the peripheral blood during the recovery from cyclophosphamide chemotherapy. Prog Clin Biol Res 1992;377:575-582.[Medline]
-
Brecher ME, Sims L, Schmitz J et al. North American Multicenter Study on flow cytometric enumeration of CD34+ hematopoietic stem cells (see comments). J Hematother 1996;5:227-236.[Medline]
-
Tjonnfjord GE, Steen R, Veiby OP et al. Lineage commitment of CD34+ human hematopoietic progenitor cells. Exp Hematol 1996;24:875-882.[Medline]
-
Sutherland DR, Marsh JC, Davidson J et al. Differential sensitivity of CD34 epitopes to cleavage by Pasteurella haemolytica glycoprotease: implications for purification of CD34-positive progenitor cells. Exp Hematol 1992;20:590-599.[Medline]
-
Kawashima I, Zanjani ED, Almaida-Porada G et al. CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells. Blood 1996;87:4136-4142.[Abstract/Free Full Text]
-
Voura EB, Billia F, Iscove NN et al. Expression mapping of adhesion receptor genes during differentiation of individual hematopoietic precursors. Exp Hematol 1997;25:1172-1179.[Medline]
-
Krause DS, Fackler MJ, Civin CI et al. CD34: structure, biology, and clinical utility. Blood 1996;87:1-13.[Free Full Text]
-
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;133:157-165.[Abstract]
-
Inaba T, Shimazaki C, Ashihara E et al. Two-color flow cytometric analysis of CD34-positive peripheral blood stem cells mobilized by recombinant human granulocyte-colony stimulating factor for autotransplantation. Prog Clin Biol Res 1992;377:561-567.[Medline]
-
Read EJ, O'Shaughnessy JA, Yu MY et al. Flow cytometric quantitation of circulating hematopoietic progenitor cells in breast cancer patients on chemotherapy. Prog Clin Biol Res 1992;377:523-530.[Medline]
-
Bender JG, Williams SF, Myers S et al. Characterization of chemotherapy mobilized peripheral blood progenitor cells for use in autologous stem cell transplantation. Bone Marrow Transplant 1992;10:281-285.[Medline]
-
Sovalat H, Wunder E, Tienhaara A et al. Commentary: prospects for standardization of stem cell determination within Europe. J Hematother 1993;2:293-296.[Medline]
-
Di Nicola M, Siena S, Bregni M et al. Quantization of CD34+ peripheral blood hematopoietic progenitors for autografting in cancer patients. Int J Artif Organs 1993;16(suppl 5):80-82.
-
Johnsen HE, Knudsen LM. Nordic flow cytometry standards for CD34+ cell enumeration in blood and leukapheresis products: report from the second Nordic Workshop. Nordic Stem Cell Laboratory Group (NSCL-G). J Hematother 1996;5:237-245.[Medline]
-
Andrews RG, Singer JW, Bernstein ID. Human hematopoietic precursors in long-term culture: single CD34+ cells that lack detectable T cell, B cell, and myeloid cell antigens produce multiple colony-forming cells when cultured with marrow stromal cells. J Exp Med 1990;172:355-358.[Abstract/Free Full Text]
-
Galy A, Travis M, Cen D et al. Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity 1995;3:459-473.[CrossRef][Medline]
-
Terstappen LW, Huang S, Safford M et al. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38– progenitor cells. Blood 1991;77:1218-1227.[Abstract/Free Full Text]
-
Issaad C, Croisille L, Katz A et al. A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38– progenitor cells in long-term cultures and semisolid assays. Blood 1993;81:2916-2924.[Abstract/Free Full Text]
-
Lansdorp PM, Dragowska W. Long-term erythropoiesis from constant numbers of CD34+ cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow. J Exp Med 1992;175:1501-1509.[Abstract/Free Full Text]
-
Brandt J, Baird N, Lu L et al. Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro. J Clin Invest 1988;82:1017-1027.
-
Sutherland HJ, Eaves CJ, Eaves AC et al. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 1989;74:1563-1570.[Abstract/Free Full Text]
-
Verfaillie C, Blakolmer K, McGlave P. Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J Exp Med 1990;172:509-512.[Abstract/Free Full Text]
-
Baum CM, Weissman IL, Tsukamoto AS et al. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA 1992;89:2804-2808.[Abstract/Free Full Text]
-
Shibagaki T, Inoue T, Kubota N et al. Fraction of pluripotent hemopoietic stem cells in DNA synthesis varies with generation age. Exp Hematol 1986;14:794-797.[Medline]
-
Chaudhary PM, Roninson IB. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 1991;66:85-94.[CrossRef][Medline]
-
Zijlmans JM, Visser JW, Kleiverda K et al. Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability. Proc Natl Acad Sci USA 1995;92:8901-8905.[Abstract/Free Full Text]
-
Tjonnfjord GE, Steen R, Evensen SA et al. Characterization of CD34+ peripheral blood cells from healthy adults mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1994;84:2795-2801.[Abstract/Free Full Text]
-
Fritsch G, Emminger W, Buchinger P et al. CD34 analysis in peripheral blood correlates with colony forming capacity—an update. Prog Clin Biol Res 1992;377:531-536.[Medline]
-
Ravagnani F, Siena S, Bregni M et al. Methodologies to estimate circulating hematopoietic progenitors for autologous transplantation in cancer patients. Haematologica 1991;76(suppl 1):46-49.
-
Wunder E, Sovalat H, Henon P et al., eds. Hematopoietic Stem Cells. The Mulhouse Manual. Dayton, OH: AlphaMed Press, 1994:1-317.
-
Wunder E, Sovalat H, Fritsch G et al. Report on the European workshop on peripheral blood stem cell determination and standardization—Mulhouse, France, February 6-8 and 14-15, 1992. J Hematother 1992;1:131-142.[Medline]
-
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;5:213-226.[Medline]
-
Serke S, Arseniev L, Watts M et al. Imprecision of counting CFU-GM colonies and CD34-expressing cells. Bone Marrow Transplant 1997;20:57-61.[CrossRef][Medline]
-
Macey MG, McCarthy DA, van Agthoven A et al. How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures. J Immunol Methods 1997;204:175-188.[CrossRef][Medline]
-
Lowdell MW, Bainbridge DR. External quality assurance for CD34 cell enumeration—results of a preliminary national trial. Royal Microscopical Society Clinical Flow Cytometry Group QA Schemes. Bone Marrow Transplant 1996;17:849-853.[Medline]
-
Urashima M, Ohkawara J, Hoshi Y et al. Peripheral blood progenitor cell transplantation estimated by three-colour (CD34, HLA-DR, CD33) flow cytometry. Acta Haematol 1994;92:23-28.[Medline]
-
Knape CC. Standardization of absolute CD34 cell enumeration (Letter; comment). J Hematother 1996;5:211-212.[Medline]
-
Lumley MA, McDonald DF, Czarnecka HM et al. Quality assurance of CD34+ cell estimation in leucapheresis products. Bone Marrow Transplant 1996;18:791-796.[Medline]
-
Remacha AF, Martino R, Sureda A et al. Changes in reticulocyte fractions during peripheral stem cell harvesting: role in monitoring stem cell collection. Bone Marrow Transplant 1996;17:163-168.[Medline]
-
Zimmerman TM, Lee WJ, Bender JG et al. Quantitative CD34 analysis may be used to guide peripheral blood stem cell harvests. Bone Marrow Transplant 1995;15:439-444.[Medline]
-
Dercksen MW, Weimar IS, Richel DJ et al. The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P-selectin-mediated binding of platelets. Blood 1995;86:3771-3782.[Abstract/Free Full Text]
-
Dercksen MW, Rodenhuis S, Dirkson MK et al. Subsets of CD34+ cells and rapid hematopoietic recovery after peripheral-blood stem-cell transplantation. J Clin Oncol 1995;13:1922-1932.[Abstract/Free Full Text]
-
Bender JG, Unverzagt K, Walker DE et al. Phenotypic analysis and characterization of CD34+ cells from normal human bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell transplantation. Clin Immunol Immunopathol 1994;70:10-18.[CrossRef][Medline]
-
To LB, Haylock DN, Simmons PJ et al. The biology and clinical uses of blood stem cells. Blood 1997;89:2233-2258.[Free Full Text]
-
Menendez P, Redondo O, Rodriguez A et al. Comparison between a lyse-and-then-wash method and a lyse-non-wash technique for the enumeration of CD34+ hematopoietic progenitor cells. Cytometry 1998;34:264-271.[CrossRef][Medline]
-
Gratama JW, Orfao A, Barnett D et al. Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. European Working Group on Clinical Cell Analysis. Cytometry 1998;34:128-142.[CrossRef][Medline]
-
Dercksen MW, Gerritsen WR, Rodenhuis S et al. Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation. Blood 1995;85:3313-3319.[Abstract/Free Full Text]
-
Feng R, Shimazaki C, Inaba T et al. CD34+/CD41a+ cells best predict platelet recovery after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 1998;21:1217-1222.[CrossRef][Medline]
-
Maharaj D, Steinberg JP, Gouvea JV et al. Changes in endogenous TPO levels during mobilization chemotherapy are predictive of CD34+ megakaryocyte progenitor yield and identify patients at risk of delayed platelet engraftment post-PBPC transplant. Bone Marrow Transplant 1999;23:539-548.[CrossRef][Medline]
-
Pecora AL, Preti RA, Gleim GW et al. CD34+CD33– cells influence days to engraftment and transfusion requirements in autologous blood stem-cell recipients. J Clin Oncol 1998;16:2093-2104.[Abstract]
-
Watanabe T, Dave B, Heimann DG et al. Cell adhesion molecule expression on CD34+ cells in grafts and time to myeloid and platelet recovery after autologous stem cell transplantation. Exp Hematol 1998;26:10-18.[Medline]
-
Gonzalez-Requejo A, Madero L, Diaz MA et al. Progenitor cell subsets and engraftment kinetics in children undergoing autologous peripheral blood stem cell transplantation. Br J Haematol 1998;101:104-110.[CrossRef][Medline]
-
Sampol MA, Besalduch VJ, Galmes LA et al. CD34+ cell dose and CD33– subsets: collection and engraftment kinetics in autologous peripheral blood stem cells transplantation. Haematologica 1998;83:489-495.[Abstract/Free Full Text]
-
Stewart DA, Guo D, Luider J et al. Factors predicting engraftment of autologous blood stem cells: CD34+ subsets inferior to the total CD34+ cell dose. Bone Marrow Transplant 1999;23:1237-1243.[CrossRef][Medline]
-
Henon P, Sovalat H, Becker M et al. Primordial role of CD34+38– cells in early and late trilineage haemopoietic engraftment after autologous blood cell transplantation (see comments). Br J Haematol 1998;103:568-581.[CrossRef][Medline]
-
Millar BC, Millar JL, Shepherd V et al. The importance of CD34+/CD33– cells in platelet engraftment after intensive therapy for cancer patients given peripheral blood stem cell rescue. Bone Marrow Transplant 1998;22:469-475.[CrossRef][Medline]
-
Teofili L, Iovino MS, Sica S et al. Characterization of peripheral blood CD34+ progenitor cells mobilized with chemotherapy and granulocyte colony-stimulating factor. Exp Hematol 1994;22:990-995.[Medline]
-
Papayannopoulou T, Craddock C. Homing and trafficking of hemopoietic progenitor cells. Acta Haematol 1997;97:97-104.[Medline]
-
Papayannopoulou T, Priestley GV, Nakamoto B. Anti-VLA4/VCAM-1-induced mobilization requires cooperative signaling through the kit/mkit ligand pathway. Blood 1998;91:2231-2239.[Abstract/Free Full Text]
-
Zeller W, Kroger N, Berger J et al. Expression of the adhesion molecules CD49d and CD49e on G-CSF-mobilized CD34+ cells of patients with solid tumors or non-Hodgkins and Hodgkins lymphoma and of healthy donors is inversely correlated with the amount of mobilized CD34+ cells. J Hematother 1999;8:539-546.
-
Olweus J, Terstappen LW, Thompson PA et al. Expression and function of receptors for stem cell factor and erythropoietin during lineage commitment of human hematopoietic progenitor cells. Blood 1996;88:1594-1607.[Abstract/Free Full Text]
-
Zeigler FC, Bennett BD, Jordan CT et al. Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells. Blood 1994;84:2422-2430.[Abstract/Free Full Text]
-
Moore KA, Pytowski B, Witte L et al. Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs. Proc Natl Acad Sci USA 1997;94:4011-4016.[Abstract/Free Full Text]
-
McKinstry WJ, Li CL, Rasko JE et al. Cytokine receptor expression on hematopoietic stem and progenitor cells. Blood 1997;89:65-71.[Abstract/Free Full Text]
-
Shimoda K, Okamura S, Harada N et al. High-frequency granuloid colony-forming ability of G-CSF receptor possessing CD34 antigen positive human umbilical cord blood hematopoietic progenitors. Exp Hematol 1995;23:226-228.[Medline]
-
Nichol JL, Hokom MM, Hornkohl A et al. Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia. J Clin Invest 1995;95:2973-2978.
-
Solar GP, Kerr WG, Zeigler FC et al. Role of c-mpl in early hematopoiesis. Blood 1998;92:4-10.[Abstract/Free Full Text]
-
Rasmussen T, Honoré L, Johnsen HE. Identification and characterisation of malignant cells using RT-PCR on single flow-sorted cells. Med Oncol 1998;15:96-102.[Medline]
-
Rasmussen T, Jensen L, Honoré L et al. Circulating clonal cells in multiple myeloma do not express CD34 mRNA, as measured by single-cell and real-time RT-PCR assays. Br J Haematol 1999;107:818-824.[CrossRef][Medline]
-
Sutherland DR, Yeo EL, Stewart AK et al. Identification of CD34+ subsets after glycoprotease selection: engraftment of CD34+Thy-1+Lin– stem cells in fetal sheep. Exp Hematol 1996;24:795-806.[Medline]
-
DiGiusto D, Chen S, Combs J et al. Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34. Blood 1994;84:421-432.[Abstract/Free Full Text]
-
Knudsen LM, Rasmussen T, Jensen L et al. Reduced bone marrow stem cell pool and progenitor mobilization in multiple myeloma after melphalan treatment. Med Oncol 1999;16:245-254.[Medline]
-
Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242-245.[Abstract]
-
Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997;3:1337-1345.[CrossRef][Medline]
-
Morel F, Galy A, Chen B et al. Equal distribution of competitive long-term repopulating stem cells in the CD34+ and CD34– fractions of Thy-1lowLin–/lowSca-1+ bone marrow cells. Exp Hematol 1998;26:440-448.[Medline]
-
Zanjani ED, Almeida-Porada G, Livingston AG et al. Human bone marrow CD34– cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells [see comments]. Exp Hematol 1998;26:353-360.[Medline]
-
Bhatia M, Bonnet D, Dick JE. Identification of a novel CD34 negative population of primitive human hematopoietic cells capable of repopulating NOD/SCID mice. Blood 1997;90:1134a.
-
Larsson K, Bjorkstrand B, Ljungman P. Faster engraftment but no reduction in infectious complications after peripheral blood stem cell transplantation compared to autologous bone marrow transplantation. Support Care Cancer 1998;6:378-383.[CrossRef][Medline]
-
Schiødt I, Bergman OJ, Johnsen HE et al. Early infections after autologous transplantation for haematological malignancies. Med Oncol 1998;15:103-108.[Medline]
accepted for publication December 22, 1999.
Top
Abstract
Introduction
