Page 1719 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 95 Practical Aspects of Hematologic Stem Cell Harvesting and Mobilization 1529

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at the time of chemotherapy mobilization were likely to mobilize populations of other blood cells, and can be identified using a
tumor cells into the blood. The most disturbing finding of this study variety of commercially available antibodies. If antibodies directly
is that the tumor cells were detected in the peripheral blood at the conjugated with dyes are used, the technique requires only about 1
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same time as CD34 cells. Similarly, Pecora and colleagues found hour of preparation time. Cell viability using propidium iodide or
a relationship between the ability to detect tumors in PBSC compo- 7-aminoactinomycin D exclusion can simultaneously be determined
nents and in bone marrow samples, but they also found a higher if the cells are analyzed while still fresh, or the cells can be fixed after
incidence of positive PBSC components for patients who required staining for analysis at a later date. Other antibodies can be added
greater numbers of apheresis procedures to achieve the target dose of for analysis of CD34 subsets if desired (and if the flow cytometer has
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CD34 cells. Investigators at The Johns Hopkins Oncology Center proper detectors to detect the different emission wavelengths of the
found no difference in the incidence of tumor contamination of fluorochromes used). A strong correlation exists between the numbers
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PBSC components between patients treated with chemotherapy and of CD34 cells and CFU-GM in the sample, but with ratios of about
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cytokines and those treated with cytokines alone. Ex vivo purging 5 : 1 to 20 : 1. Thus, CD34 analysis will provide data similar to that
of tumor cells from PBPC products has not been shown to reduce obtainable with cell cultures, except that the latter demonstrates the
the risk for relapse after autologous transplantation, either because functional viability of the progenitor cells. Mobilized PBPC products
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the techniques are not adequate in depleting minimal residual disease contain a heterogeneous mixture of cells including CD34 cells
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or because patients relapse primarily from endogenous disease surviv- belonging to different cell subsets. Subset analysis of CD34 cells will
ing the pretransplant conditioning regimen. Patients with marrow provide additional information regarding early and sustained engraft-
involvement may benefit from several cycles of debulking (in vivo ment after autologous PBPC transplantation but does not appear to
purging) chemotherapy before collection of HSCs, with the caveat be clinically useful for the patient who easily meets the PBSC collec-
that extensive chemotherapy will also decrease the subsequent yield tion goal.
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of PBSCs. Tumor cells in the HSC inoculum likely will not benefit The major difficulty with analysis of CD34 cells is the low fre-
the patient, but until further data demonstrating a deleterious effect quency of these cells. Clinical decisions to initiate apheresis are being
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on transplant outcomes are available, it is advisable that reports of made for CD34 cell levels as low as 10/µL. This may represent a cell
tumor contamination in the collections for individual patients be frequency that is 0.01% or less of the nucleated cells in the specimen.
interpreted with caution. This enumeration is possible because of multidimensional measure-
ments obtained by flow cytometry. Most cytometers can measure at
Microbial Contamination of Hematopoietic least five characteristics of each cell, including size, granularity, and
the presence of up to three different fluorochromes. Thus, the cells of
Stem Cell Components interest can be separated in five-dimensional space, achieving discrimi-
nation of cells as rare as 1 : 10,000. The difficulties arise from develop-
Bacterial culture is an essential quality-control component in HSC ing an adequate technique that makes optimal use of the cytometer
collection and transplantation used to identify errors and breakdowns to measure these rare cells. Sources of errors include (A) sampling of
in manufacturing technique. Skin flora are the bacteria usually iso- the HSC product, (B) cell counting, (C) cytometer calibration and
lated, and infusion of culture-positive HSC products is generally operation, (D) choice of antibody and fluorochrome, (E) lysis tech-
without clinical sequelae, although serious infections have occurred nique, and (F) gating strategy. Moreover, cytometry provides a propor-
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after infusion of HSC products contaminated during processing. tion of cells that must be multiplied by the cell count to obtain an
Culture-positive products need not be automatically destroyed. Any absolute number. The steps involved in preparing a specimen for
decision regarding the disposition of a culture-positive HSC product cytometry may alter the proportion of cells in the sample, and this
must be made by the patient’s transplant physician after considering error will be translated into an error in the absolute number. Clinically,
the type of contamination, the anticipated risks from use of the this imprecision may explain some of the range in engraftment kinetics
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component, and the ability to replace the culture-positive product(s) observed for patients receiving low doses of CD34 cells.
in a timely manner.
The incidence of culture-positive PBSC components is consider-
ably less than that for marrow. The actual incidence of contamination SUGGESTED READINGS
for all HSC products is likely several times higher than reported,
however, because most laboratories will culture only a very small Barker JN, Byam C, Scaradavou A: How I treat: the selection and acquisition
volume of product (≈1 mL). In a retrospective review of 2935 HSC of unrelated cord blood grafts. Blood 117(8):2332–2339, 2011.
products processed and transplanted at one center, positive microbial Bosi A, Bartolozzi B: Safety of bone marrow stem cell donation: a review.
products were reported for 1.3% of bone marrow products, 0.7% of Transplant Proc 42(6):2192–2194, 2010.
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PBSC products, and 2.0% of UCB products. Coagulase-negative Boulais PE, Frenette PS: Making sense of hematopoietic stem cell niches.
Staphylococcus and Bacillus species accounted for 23 of the 38 positive Blood 125(17):2621–2629, 2015.
cultures, but Escherichia coli, Klebsiella pneumonia, and Pseudomonas Broxmeyer HE: Chemokines in hematopoiesis. Curr Opin Hematol
cepacia were cultured from one product each. The recipient of the 15(1):49–58, 2008.
Pseudomonas-contaminated product subsequently died of complica- Buell JF, Beebe TM, Trofe J, et al: Donor transmitted malignancies. Ann
tions of Pseudomonas sepsis, but no adverse sequelae could be docu- Transplant 9(1):53–56, 2004.
mented for any of the other 34 recipients of culture-positive products. Confer DL, Shaw BE, Pamphilon DH: WMDA guidelines for subsequent
The highest rate of contamination occurred in the UCB products donations following initial BM or PBSCs. Bone Marrow Transplant
collected for related donor transplantation (5 out of 18, 27.8%), 46(11):1409–1412, 2011.
likely a reflection of the collection techniques in place at the time, Dettke M, Buchta C, Wiesinger H, et al: Anticoagulation in large-volume
and illustrating the need for strict quality control in the collection leukapheresis: comparison between citrate versus heparin-based antico-
and processing of HSC products. agulation on safety and CD34+ cell collection efficiency. Cytotherapy
14(3):350–358, 2012.
DiPersio JF, Micallef IN, Stiff PJ, et al: Phase III prospective randomized
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Quantitation of CD34 Cells double-blind placebo-controlled trial of plerixafor plus granulocyte
colony-stimulating factor compared with placebo plus granulocyte
Quantification of CD34 antigen-positive cells by flow cytometry has colony-stimulating factor for autologous stem-cell mobilization and
become the standard of care for management of the PBSC donor transplantation for patients with non-Hodgkin’s lymphoma. J Clin Oncol
because it provides a rapid and clinically relevant assessment of HSC 27(28):4767–4773, 2009.
content in the peripheral blood or in the PBSC product. This antigen Duong HK, Savani BN, Copelan E, et al: Peripheral blood progenitor
is found on HSCs (including a variety of subpopulations) and limited cell mobilization for autologous and allogeneic hematopoietic cell