The Impact of Erythrocyte Lysing Procedures on the Recovery of Hematopoietic Pro - 《stemcells》干细胞学杂志干细胞之家



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作者：Burkhard Grevea, Constanze Bellerc, Uwe Cassensb, Walter Sibrowskib, Wolfgang Ghdea ' i4 \# }7 l; f4 c& Y# j
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      【摘要】+ H, X/ }  R5 q! X- F6 a% K$ b
   Since preanalytic lysing of erythrocytes remains critical in flow cytometry, we investigated the influence of four lysing procedures on the quantification of leukocyte and CD34  cells in hematopoietic cell transplants (HCTs). Samples were derived from stem cell¨Cenriched mobilized whole blood collected by apheresis (unselected) and immunologically purified stem cell products (selected) and were measured using the dual-platform (2-PF) method with two flow cytometric systems. Additionally, cells were measured by a volume-based technique (single platform ). Results were identical in the 2-PF mode (unselected HCTs, r = 0.998; selected HCTs, r = 0.999). In comparison with the 2-PF results, the single-platform (1-PF) measurements revealed a mean decrease of 59.5% for CD34  cells (50.8% for CD45  cells) in unselected HCTs and a mean decrease of 52% for CD34  cells (49.8% for CD45  cells) in selected HCTs. In order to check the accuracy of cell quantification using the 1-PF method, leukocyte reference values from hematology counter results were compared with flow cytometric (1-PF)¨Ccounted nucleated cells. That analysis revealed good congruency, with r = 0.998 for unselected HCTs and r = 0.999 for selected HCTs. In conclusion, all lysing procedures that we used induced substantial loss of leukocytes and CD34  cells. As demonstrated by the high accuracy of the 1-PF technique, all erythrocyte lysing procedures caused significant cell loss, which led to inconsistent counting of CD34  cells in nonvolumetric flow cytometric (2-PF) protocols.
      【关键词】 Hematopoietic cell transplants Leukapheresis Flow cytometry Erythrocyte lysing+ S& b( }+ X- x4 \
   INTRODUCTION& t$ q) o- i) F; K  c/ T

The reconstitution of hematopoiesis after autologous or allogeneic stem cell transplantation (SCT) is performed by progenitor cells (PCs) from bone marrow (BM), umbilical cord blood (UCB), or peripheral blood (PB). PB has virtually replaced BM as the primary source of PCs for autologous and allogeneic transplantation because of the better availability of peripheral blood progenitor cells (PBPCs) and faster hematopoietic reconstitution. However, independent of the source of collected cells, engraftment potential correlates with both the integrity and the number of hematopoietic progenitor cells (HPCs). It is well known that unmanipulated HPC transplants from all sources are highly contaminated with leukocytes, platelets and erythrocytes. These populations may interfere with the accurate determination of HPCs. Accordingly, there is a demand for high-quality standards in the quantification of HPCs, as demonstrated by numerous established protocols and guidelines for determination of CD34  cells ) were not the point of these studies.9 c% M) s) c; y
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From a technical view, two basic flow cytometric systems, known as dual-platform (2-PF) and single-platform (1-PF) techniques, are used for this purpose .# `) l' y! ~% ~4 S: S
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Preanalytic sample preparation errors can also influence results. Although most protocols for quantification of CD34  cells are standardized in terms of sample collection, storage, and staining procedures (including antibody clones, dyes, etc.), some other aspects remain critical , and selected (purified) PBPCs without contaminating white blood cells (WBCs), RBCs, and platelets, in order to simulate the influence of lysing procedures specifically on the determination of PBPCs.
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MATERIALS AND METHODS9 Q6 b( U6 {3 n; _  d( b

Collection of Stem Cells

The effect of erythrocyte-lysing procedures on the quantification of leukocytes and HPCs was investigated with two different flow cytometric techniques and systems. A total of 135 flow cytometric determinations of 10 unselected and five positively selected HPC products was performed. All samples were derived from representative aliquots of HPC products and were collected by apheresis (COBE-Spectra cell-separator; GAM-BRO-BCT, Martinsried, Germany, http://www.gambrobct.com) from patients undergoing autologous HPC transplantation or allogeneic transplantation from healthy volunteers. The positive selection of CD34  cells was performed using the large-scale device CliniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Positive selection was provided if required for clinical application. Characteristics of all autologous and allogeneic HPC donors are given in Table 1.

Table 1. Overview of patient data1 M$ C8 {- v% z& K! j( j

Flow Cytometry
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Immunological Staining. 2-PF measurements were performed using the FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) flow cytometer as the standard method (SM) and the particle-analyzing system (PAS) flow cytometer (Partec, M¨¹nster, Germany, http://www.partec.com) as the comparison method (CM) (30 determinations). The numbers of leukocytes (CD45  cells) in the HPC products were measured by flow cytometry (first platform), and the total leukocyte concentration was measured by the HC (second platform). The absolute number of leukocyte subsets was calculated according to a formula given in the next section for CD34  cells and described previously . The PAS device was also used for direct (absolute) volumetric determinations of CD45  and CD34  cell concentrations (1-PF, without beads) (60 determinations).
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Determination of CD34  and CD45  Cells. HPCs (CD34  cells) were quantified by both the 2-PF and 1-PF methods after labeling with a monoclonal antibody specific for HPCA-2 antigen (class III antibody, clone 8G12; Becton, Dickinson and Company) conjugated with phycoerythrin (PE). For the detection of CD45-expressing leukocytes, cells were labeled with CD45 monoclonal antibodies conjugated to fluorescein isothio-cyanate (FITC) (clone J33; Immunotech, Luminy, France, http://www.immunotech.com). Measurements were performed according to the German consensus protocol established by the German Society for Transfusion Medicine and Immunohematology and the German Society for Hematology and Oncology. This protocol for the quantification of CD34  cells is similar to the International Society for Hematotherapy and Graft Engineering protocol . The German consensus protocol was chosen as the reference method because it is the most common and accepted method for the determination of CD34  cells in Germany. The laboratory involved in this study uses this protocol and has passed all its internal and external proficiency tests. In detail, 100 µl of the unselected or selected product was incubated with a CD45-FITC¨C and a CD34-PE¨Cconjugated monoclonal antibody. After 15 minutes of incubation at room temperature in the dark, cells were fixed and lysed. In short, all CD45  cells were gated in a side-scatter (SSC)/CD45  plot. This gate was activated in a plot of SSC/CD34  cells, and CD34  cells were marked in a separate gate. Only events within both gates were considered as true CD34  cells. Both gates were taken for a backgating in a forward-scatter (FSC)/SSC and SSC/CD34  plot. In both cases, the CD34  cells should be visible as an endemic population (Fig. 1). In summary, 50,000 events (leukocytes) were collected as recommended by the 2-PF method. Using the volumetric 1-PF method, 200 µl sample volumes were analyzed, corresponding to more than 50,000 events. Whereas in the volumetric 1-PF method, the concentrations of progenitor cells and leukocytes were directly given without further calculations. The concentration of CD34  cells was calculated after the 2-PF measurement according to the following formula:
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Figure 1. Flow cytometric gating strategy for the determination of CD45  cells and CD34  cells in a representative blood sample. The example demonstrates the measurement with the particle-analyzing system (PAS) using lysis II, but analysis with the FACSCalibur system was performed in the same manner. Abbreviations: SSC, side scatter; FSC, forward scatter; FL-I, fluorescence I; FL-II, fluorescence II: Q1, quadrantgate 1; QA1, quadrantgate A1." M) c, K  k( t4 v( n; e4 @  r

The number of CD34  cells per µl (x) was calculated by taking the number of CD34  cells measured using the flow cytometer, multiplying this by the absolute number of leukocytes measured using the HC, and dividing by the number of CD45  cells measured using the flow cytometer (50,000  ¨C number of CD45  cells).

Nuclear Staining. Nuclear DNA was stained with a commercially available dye as a ready-to-use staining solution (CyStain UV; Partec). The buffer contains 4',6'-diamino-2-phenylindole (DAPI) and is able to fluorescently label nucleated cells in a one-step procedure. We added 0.9 ml of the staining solution to 0.1 ml of each sample to be analyzed. Samples were measured volumetrically with the PAS flow cytometer directly after the addition of the staining buffer (45 determinations). DAPI-containing nucleated cells were fluorescently excited by ultraviolet light derived from a mercury high-pressure lamp (Osram Sylvania, Munich, Germany, http://www.sylvania.com) .
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Determination of Leukocytes

The Sysmex K-1000 HC (Sysmex, Kobe, Japan, http://www.sysmex.co.jp) was used as a reference method (RM) for counting the leukocytes in unselected and selected HPC products and as the second platform method for the 2-PF evaluation with both flow cytometric devices. The manufacturer of the HC indicates a maximum deviation of 3% from the accurate value and precision with a maximum coefficient of variation of 3% for leukocytes in the range of 1.0¨C99.9 x 103 leukocytes/µl. The accuracy of the HC was regularly subjected to internal and external proficiency testing. In addition, a recent study proved a high correlation for counting results of leukocytes using this HC and the flow cytometer that we used ., i' {7 D% q) a) n& a' o! E# e. M
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Erythrocyte-Lysing Procedures; @- i$ \4 y% f1 S9 m8 X4 |

The influence of four different commercially available erythrocyte-lysing reagents on the recovery of HPCs and leukocytes was investigated in this study. We compared FACS-Lysing Solution (Becton, Dickinson and Company) (lysis I, a lysis agent based on ethylene glycol), Ortho-mune (Ortho¨CClinical Diagnostic Systems, Neckargm¨¹nd, Germany, http://www.orthoclinical.com) (lysis II, a lysis agent based on ammonium chloride), Uti Lyse (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) (lysis III, a lysis agent not further specified), and Cylyse (Partec) (lysis IV, a lysis agent based on a detergent). The erythrocyte-lysing reagents were applied to identical samples and were used according to the manufacturers¡¯ recommendations.# J3 u4 t# }" i" b

Statistical Analysis. v$ s4 K( V+ T4 x) f1 q( |" L

Identical samples treated with four different lysing reagents were measured. The results of two flow cytometry systems and the HC were compared. Pearson correlation coefficients and paired t-tests with a confidence level of 95% (p

RESULTS

The influence of different erythrocyte-lysing procedures on the concentration of HPCs from unselected and selected apheresis products was investigated. The flow cytometric analysis and gating strategy are exemplarily explained using the measurement of an unselected stem cell product (Fig. 1). The following parameters were analyzed and compared.

Enumeration of CD34  Cells in the 2-PF Mode+ b# d" [  U' G& V* a" p6 A
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For the unselected samples, a high consistency between the two devices was demonstrated, with a correlation coefficient of r = 0.998 for the range 334¨C7,712 CD34  cells/µl (Fig. 2A). The same high consistency was shown for the selected samples (r = 0.999) for the range 2,959¨C17,425 CD34  cells/µl (Fig. 2B).' U3 u4 V  i2 l  j9 X
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Figure 2. Dual platform (2-PF) determination of CD34  cells in unselected and selected hematopoietic progenitor cell (HPC) products. Cells were counted using the 2-PF standard method (SM) and the 2-PF comparison method (CM) in unselected (A) and selected (B) HPC products. The line of identity is given as a diagonal. Abbreviations: RM, reference method; CM, comparison method; SM, standard method.' U; K4 d  T- `

There was no statistically significant difference between the two determinations in unselected (p = .075) and selected (p = .409) products (Wilcoxon test). In conclusion, both systems resulted in identical values for both groups when running in the 2-PF mode. Therefore, it was concluded that the comparison method (PAS) was comparable with the standard method (FACSCalibur) in the 2-PF mode.3 Q( G/ }0 m& `1 {9 r8 F, M
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Enumeration of CD34  Cells in the 1-PF Mode$ W+ M  L5 \7 L: q: d  L

Comparing the 2-PF results with the 1-PF results, the mean loss of CD34  cells was 59.5% for unselected apheresis products. Exact figures were: lysis I, 47% (standard deviation , 5.8%); lysis II, 45% (SD, 14.8%); lysis III, 46% (SD, 6.3%); lysis IV, 100% of the 2-PF results (Fig. 3A). In selected apheresis products, the mean loss of CD34  cells was 52%. Exact figures were: lysis I, 36% (SD, 10.8%); lysis II, 35% (SD, 8.8%); lysis III, 37% (SD, 1.9%); lysis IV, 100% of 2-PF results (Fig. 3B). The mean absolute numbers of CD34  cells/µl and SDs are given in Table 2.5 k* S8 X1 E$ j$ L- v
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Figure 3. Comparison of 1-PF and 2-PF determination of CD34  cells in unselected and selected hematopoietic progenitor cell (HPC) products. HPCs were counted using the 2-PF SM and the 2-PF CM and using the CM in the 1-PF mode in unselected (A) and selected (B) HPC products using different lysing procedures. The lysis IV sample was not measurable and is not shown in this graphic. Abbreviations: 1-PF, single platform; 2-PF, dual platform; CM, comparison method; SM, standard method.

Table 2. Absolute number (¡Àstandard deviation) of CD34  cells/µl in unselected and selected hematopoietic progenitor cell products

Determination of Leukocytes by Flow Cytometry and by HC

Similar to the kinetics of CD34  cell loss found by comparing the results of the 2-PF and 1-PF enumerations, CD45  cells decreased in a like manner (compared with the reference HC values), with a mean loss of cells in unselected products of 50.75%. Exact figures were: lysis I, 33% (SD, 3.1%); lysis II, 34% (SD, 9.0%); lysis III, 36% (SD, 4.2%); lysis IV, 100% (Fig. 4A). The selected products, with a purity of HPCs of 99.7%, had a mean CD45  cell loss of 49.75%. Exact figures were: lysis I, 33% (SD, 8.8%); lysis II, 32% (SD, 6.3%); lysis III, 34% (SD, 4.6%); lysis IV, 100% (Fig. 4B). The mean absolute numbers of CD45  cells/µl and SDs are given in Table 3.

Figure 4. Comparison of leukocytes counted by the hematology counter (HC) and the 1-PF determination of CD45  cells in unselected and selected hematopoietic progenitor cell (HPC) products. Leukocytes were counted in unselected (A) and selected (B) HPC products using the HC as the RM. The results were compared with those of the 1-PF evaluation of the CM using lysed (columns 3¨C5) and unlysed blood samples (columns 1 and 2). The lysis IV sample was not measurable and is not shown in this graphic. Abbreviations: 1-PF, single platform; 2-PF, dual platform; CM, comparison method; RM, reference method.. P, ]4 w% `2 h' n

Table 3. Absolute number (¡Àstandard deviation) of CD45  cells/µl in unselected and selected hematopoietic progenitor cell products2 ~9 K( V. x; y  t: d6 u
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Determination of Nuclear-Stained Leukocytes by the CM and HC5 ^1 p+ y9 J8 c1 o8 T+ a
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In order to verify the volumetric counting method, aliquots of unselected and selected products were fluorescently labeled with DAPI, and the nucleated cells were counted using the CM. Total numbers of leukocytes were measured in parallel using the HC and CM. A high consistency between the two methods was shown, with a correlation coefficient of r = 0.998 (p = .098) for the unselected products and r = 0.999 (p = .810) for the selected products.

In summary, the following results were obtained:& l# U! p' S2 ~" i3 I' E2 E

If both flow cytometric systems (FACSCalibur and PAS) were used in the 2-PF mode, results for the enumeration of CD34  cells were identical (unselected cell products, r = 0.998, p = .075; selected cell products, r = 0.999, p = .409).

In relation to the standard system (FACSCalibur 2-PF), the 1-PF method demonstrated a mean loss of 59.5% (SD, 1.0%)¡ªincluding one lysing reagent (lysis IV) with a loss of 100%¡ªfor CD34  cells in unselected stem cell products. The same was true for selected products, with a mean loss of 52% (SD, 1.5%)¡ªincluding one lysing reagent (lysis IV) with a loss of 100%.% c* B7 R/ G( d1 j+ H7 M$ {
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If the same unmanipulated (unlysed) samples were counted with the HC in parallel with the CM after nuclear staining with DAPI, the results were identical: r = 0.998 (p = .098) for unselected products and r = 0.999 (p = .810) for selected products.
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DISCUSSION; X& K; ~5 ]# ^/ h

The success of reconstituting the hematopoietic system after myeloablative therapy and subsequent SCT is closely related to the administration of sufficient and functioning HPCs. Traditionally, these parameters have been evaluated using the colony-forming test, but these methods are handicapped by a lack of reproducibility and a long assay time . The last sample treatment, in particular, results in a loss of accuracy that has not been investigated in as much detail as has been done for the precision and sensitivity in the determination of CD34  cells. Furthermore, most studies focus on mobilized whole blood and waive the modified conditions during the HPC purification process. Therefore, this study represents, for the first time, a systematic investigation of pre- and perianalytic cellular loss in unmanipulated apheresis and purified HPC products.

This was done by comparing the results of a volume-based 1-PF flow cytometer (CM).

The CM technique was already used in a previous study to quantify lysing-induced cell loss in G-CSF¨Cmobilized whole blood .2 S7 r- r0 _. s, `( C
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Unselected and selected stem cell products include different fractions and populations. In the latter group, cells are out of their natural environment. This, and the addition of citrate as an anticoagulant in apheresis products, may be responsible for the greater sensitivity of selected CD34  cells to particular components of lysing buffers, and results in a complete loss of immunologic detectability when using detergent-based lysing procedures (lysis IV). The cell destructive properties of citrate in combination with detergents have already been described and used in another context to isolate nuclei from cells or tissues . However, all these studies agree that erythrocyte lysing has a high impact on the enumeration of HPCs, and this is underlined by the results of this article.( l1 R" P$ U' K1 k+ |' ]

We have recently established a reliable protocol to count immunolabeled lymphocyte subsets like CD4  T-cells in unlysed blood samples . We found 10% lower numbers of CD4  T cells in lysed blood samples than in the same no-lyse blood samples. Therefore, we recommend the use of a simplified but reliable no-lyse protocol that may also be suitable for quantifying CD34  cells. Because of the high practical and clinical consequences of lysing effects on the determination of CD34  stem cells in transplants, this protocol is currently under investigation in a larger comparative study with other established protocols.
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In conclusion, both unselected and selected HPC products are strongly influenced by lysing procedures. Moreover, it was found that the change in HPC environment from blood to a buffer-based solution induces a higher susceptibility to single components of lysing reagents. The present results give an explanation for the high interlaboratory variability found in different studies and confirm the accuracy problems of the 2-PF method. This has to be taken into account for flow cytometric analysis and for further guidelines focused on the determination of CD34  cells in different compartments.5 T: I9 t- ~2 b$ r

DISCLOSURES
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS

We thank Gerlind Bellmann, Alexandra Mertens, and Nikola Gözelmann for excellent technical assistance.
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