Vojnosanit Pregl 2017; 74(11): 1071–1077.
VOJNOSANITETSKI PREGLED
SHORT
COMMUNICATION
Page 1071
UDC:615.38:[576.3:616.419-006-08
https://doi.org/10.2298/ VSP170505090B
Relative frequency of immature CD34+/CD90+ subset in peripheral
blood following mobilization correlates closely and inversely with the
absolute count of harvested stem cells in multiple myeloma patients
Relativna učestalost nezrelog podtipa ćelija CD34+/CD90+ u perifernoj krvi
posle mobilizacije je u tesnoj i obrnutoj korelaciji sa apsolutnim brojem
matičnih ćelija u afereznom produktu kod bolesnika sa multiplim mijelomom
Bela Balint*†‡§, Ivan Stanojević§||, Milena Todorović¶, Dragana Stamatović§**,
Mirjana Pavlovi憆, Danilo Vojvodić§||
Military Medical Academy,*Institute for Transfusiology and Hemobiology, ||Institute for
Medical Research, Belgrade, Serbia; ** Clinic for Hematology, Belgrade, Serbia;
University of Belgrade, †Institute for Medical Research, Belgrade, Serbia; ‡Serbian
Academy of Sciences and Arts; The University of Defence, §Faculty of Medicine of the
Military Medical Academy, Belgrade, Serbia; Clinical Center of Serbia, ¶Clinic for
Hematology, Belgrade, Serbia; ††Department of Computer and Electrical Engineering
and Computer Science, FAU, VL, USA
Abstract
Background/Aim. Stem cells (SCs) guarantee complete/long-term bone marrow (BM) repopulation after SCtransplants. The aim of the study was to evaluate absolute
count of total SCs (determined by ISHAGE-sequential-gating
protocol – SCish) and relative frequency of immature
CD34+/CD90+ (CD90+SCish) subset in peripheral blood (PB)
as predictive factors of mobilization and apheresis product
(AP) quality. Methods. Mobilization included chemotherapy
and granulocyte-growth-factor (G-CSF). Harvesting was performed by Spectra-Optia-IDL-system. The SCsish were determined as a constitutional part of CD34+ cells in the “stemcell-region” using FC-500 flow-cytometer. In this study, the
original ISHAGE-sequential-gating protocol was modified by
introduction of anti-CD90-PE monoclonal-antibody into the
analysis of CD90 expression on SCish (CD90+SCish). The results were presented as a percentage of SCish per nucleatedcell count, absolute SCish count in μL of the PB or the AP,
percentage of the CD90+SCish expressed to SCish and absolute
CD90+SCish count in μL of the PB or the AP. Results. The
absolute count of total SCish and CD90+SCish was significantly
higher (p = 0.0007 and p = 0.0266, respectively) in the AP
than in the PB samples. The CD90+SCish/total SCish indexes
from PB were higher than indexes from the AP (p = 0.039).
Apstrakt
Uvod/Cilj. Matične ćelije (MĆ) obezbeđuju kompletnu/dugotrajnu repopulaciju kostne srži (KS) posle trans-
The relative frequency of CD90+SCish showed a highly significant inverse correlation with the absolute count of total
SCish in both, the PB and AP (p = 0.0003 and p = 0.0013 respectively). The relative frequency of CD90+SCish from the
PB also showed a significant (p = 0.0002) inverse relationship
with total SCish count in the AP. Patients with less than 10%
CD90+SCish in the PB had evidently higher (p = 0.0025) total
SCish count in the AP. Conclusion. We speculate that lower
CD90+SCish yield in the AP is not a consequence of an inferior collection efficacy, but most likely a result of several still
not fully resolved immature SC cytomorphological/biophysical features. Therefore, following the mobilization by chemotherapy G-CSF, some logical questions appear
– whether we should follow the absolute count of total SCish,
or, whether we should test for relative frequency of
CD90+SCish prior to harvesting. To reach the final conclusions, it is essential to conduct further controlled and larger
investigations concerning the correlation of circulating
and harvested SCs with patients' hematopoietic recovery.
Key words:
stem cells; hematopoietic stem cell transplantation; bone
marrow; flow cytometry; multiple myeloma;
antineoplastic combined chemotherapy protocols.
plantacije. Cilj ove studije bila je procena apsolutnog broja
ukupnih MĆ (utvrđena protokolom „ISHAGE-sequentialgating” – Stem-Cellish [SCish]) i relativne učestalosti primitivnih
podipova CD34+/CD90+ (CD90+SCish) u perifernoj krvi (PK)
Correspondence to: Bela Balint, Military Medical Academy, Institute for Transfusiology and Hemobiology, Crnotravska 17, 11 000 Belgrade, Serbia. Phone: +381 11 3609 134. E-mail: balintbela52@yahoo.com
Page 1072
VOJNOSANITETSKI PREGLED
kao prediktora efikasnosti mobilizacije i pokazatelja kvaliteta
afereznog produkta (AP). Metode. Mobilizacija je postignuta
hemioterapijom/faktor-rasta-granulocitopoeze
(G-CSF).
Prikupljanje je izvedeno pomoću sistema Spectra-Optia-IDL.
Ćelije SCsish determinisane su kao konstitutivni deo CD34+ u
regiji-matičnih-ćelija („stem-cell-region“) upotrebom protočnog citometra FC-500. U ovoj studiji, originalni protokol
„ISHAGE-sequential-gating” modifikovan je uvođenjem
monoklonskog antitela anti-CD90-PE radi analize ekspresije
antigena CD90 na ćelijama SCish (CD90+SCish). Rezultati su prikazani kao procenat ćelija SCish u odnosu na broj nukleisanih
ćelija, apsolutni broj SCish u L PK ili AP, procenat ćelija
CD90+SCish izražen u odnosu na SCish i apsolutni broj
CD90+SCish u L PK ili AP. Rezultati. Apsolutni broj ukupnih
ćelija SCish i CD90+SCish bio je značajno (p = 0,0007 i
p = 0,0266) veći u uzorcima AP nasuprot PK. Indeks
CD90+SCish/ukupne SCish u uzorku PK je bio veći od indeksa u
uzorku AP (p = 0,039). Relativna učestalost CD90+SCish pokazala je vrlo značajnu inverznu korelaciju sa apsolutnim brojem
ukupnih SCish u PK i AP (p = 0.0003 i p = 0,0013). Relativna
učestalost ćelija CD90+SCish u PK takođe je pokazala značajnu
Introduction
The “cytopoiesis” – defined as in vivo cell development
and expansion – is a multi-cyclic event in which a spectrum
of mature cells is produced from a small number of stemcells (SCs). SCs could be characterized as cells having wellbalanced self-renewal, differentiation and proliferative
capacity, as well as potential for plasticity, that is an ability
to “switch” into other cell lineages. The SCs guarantee
steady-state homeostasis in various “tissue-generating” (e.g.
hematopoietic) systems 1, 2.
High-dose chemotherapy followed by allogeneic or autologous SC-transplants is considered as standard treatment
for some malignant and few immune-mediated diseases (e.g.
multiple sclerosis) 1–3. The use of SCs for organ repair (damaged myocardium, liver, pancreas, etc) opens new perspectives in regenerative medicine 1, 3. For transplants, bone
marrow (BM) has been the primary SC-source, in which
approximately 2–4% of total nucleated cells (TNCs) express
the CD34 antigen 4, 5. The CD34+ cells were recognized in
peripheral blood (PB), but in extremely low ratio in the
“steady-state” hematopoiesis: 0.01–0.05% of the TNCs 1, 4.
Mobilization by chemotherapy and recombinant human
granulocyte-colony-stimulating factor (rHuG-CSF) radically
increases the count of circulating CD34+ cells numbers in patients and healthy donors 1. However, just a small fraction of
double positive (CD45+CD34+) cells, with typical size and
specific intracellular granularity/complexity – according to
the International Society for Hematotherapy and Graft Engineering (ISHAGE) protocol – represents “true” SCs (or
SCish) 6–10. Moreover, immature or more primitive hematopoietic progenitors (PHPs) bare antigen CD90 (Thy-1), a 25 to
35 kDa molecule, which is also expressed by 1–4% of human fetal liver cells, umbilical cord blood (UCB), BM and
several PB cells. PHPs are responsible for complete and
long-term BM repopulation with durable or late hematopoie-
Vol. 74, No 11
(p = 0,0002) inverznu korelaciju sa apsolutnim brojem ukupnih
ćelija SCish. Bolesnici sa manje od 10% CD90+SCish u PK su
imali značajno (p = 0,0025) veći apsolutni broj ukupnih SCish
ćelija u AP. Zaključak. Smatramo da niži prinos CD90+SCish u
AP nije prouzrokovan manje efikasnim prikupljanjem, već je
najverovatnije posledica različitih, još uvek samo delimično
razjašnjenih, citomorfoloških/biofizičkih osobina manje zrelih
MĆ. Zato, posle mobilizacije hemoterapijom/G-CSF nameću
se logična pitanja - da li bi trebalo pratiti apsolutni broj ukupnih
SCish ćelija ili je celishodnije testirati relativnu učestalost
CD90+SCish pre sprovođenja afereznog prikupljanja MĆ. Za
donošenje definitivnih zaključaka neophodna su buduća kontrolisana i sveobuhvatnija istraživanja MĆ, u vezi sa utvrđivanjem korelacije cirkulišućih i priku-pljenih ćelija sa hematopoetskim oporavkom bolesnika.
Ključne reči:
ćelije, matične; transplantacija hematopoeznih matičnih
ćelija; kostna srž; citometrija, protočna; multipli mijelom;
lečenje kombinovanjem antineoplastika, protokoli.
tic reconstitution – and are limited within the immature
CD34+/CD90+ subset (or CD90+SCish) 7, 10–12. As confirmed,
the CD34+/CD90+ cells infused most appropriately predict
platelet (Plt) recovery after the SC-transplants 11. The
CD34+/CD90+ subset is also heterogeneous: evidently enriched in PHPs, but contains some less primitive lineage committed progenitors (LCPs). However, the majority of LCPs
exist in an additional CD34+/CD90– (or CD90-SCish) cell population 7.
Traditional sources of the SCs are the BM and PB. The
UCB has been used as an alternative source since the late
1980s 1, 13. Damages caused by the chemotherapy (applied
prior to autologous SC-transplants) could be an important
limiting factor of the SC-mobilization. Regularly, the count
of total CD34+ cells in PB of healthy donors is higher than in
mobilized non-Hodgkin lymphoma patients 12. However, after mobilization in PB of these patients the CD34+ cell population is more immature, since they have a higher CD90
expression 12. That could be a significant factor that influenced marrow repopulation, with special impact of late and durable
hematopoietic
reconstitution
following
SCtransplants 11. This preclinical study aimed to evaluate absolute count of total SCish (including the CD90+SCish and
CD90-SCish subsets) and relative frequency of CD90+SCish in
the PB, as predictive factors of the mobilization efficacy and
of the apheresis product (AP) quality.
Methods
In this pilot study the importance of our own novel predictive factors of the efficacy of the SC mobilization (absolute count of total SCish and relative frequency of CD90+SCish)
using apheresis system Spectra-Optia IDL-system (TerumoBCT, USA) were evaluated. Cell harvesting in a
comparatively homogeneous (considering pre-transplant
chemotherapy, mobilization protocol and conditioning regi-
Balint B, et al. Vojnosanit Pregl 2017; 74(11): 1071–1077.
Vol. 74, No 11
VOJNOSANITETSKI PREGLED
men) category of multiple myeloma patients (n = 12) were
performed. Patients were aged 26–62 years; male/female ratio was 1.3 : 1. The study was performed according to the guidelines of the Declaration of Helsinki Principles and Good
Clinical Practice and was approved by the local institutional
Ethic board.
Cell harvesting technology
Standardized apheresis procedures – processing two patients' total blood volumes with equal quantity (200 mL) of
the AP – were performed. The mobilization protocol included chemotherapy (cyclophosphamide 2–4g/m2 and etoposide 400–800 mg/m2) with rHuG-CSF (12–16 g/kgbm/day).
Citrate-containing anticoagulant (Acid-Citrate-Dextrose,
with 2.2% citrate concentration – ACD formula A, USP) was
applied, at the same ACD : whole blood ratio (1 : 10) for all
procedures. Additional patients' systemic or the AP heparinization was not performed. Vascular access was obtained across central venous catheter applied into subclavian or jugular and occasionally femoral veins. In this study, cell collections were performed when the absolute count of CD34+ in
the patient's PB was 19.4 ± 5 / μL and the relative
frequency of CD90+SCish was 9.3 ± 12.2%, respectively.
The patients tolerated performed apheresis procedures well,
without severe adverse effects. The adverse event of apheresis was considered as severe if it was life-threatening or leads
to irreversible consequence with organ failure.
Page 1073
Forward Scatter vs. Side Scatter dot plot, gated on events
with low CD45 expression and low side scatter, the SCish
were identified by their size slightly larger than small
lymphocytes and uniformly low side scatter. Finally, on the
CD90 vs. Side Scatter dot plot, the selected „true“ CD34+
(SCish) cells were analyzed for CD90 expression. Cell
viability was estimated on the basis of the 7-aminoaktinomicin D (7-AAD) flow cytometric assay (Immunotech, France) 5, 7, 9.
The results obtained in this study were presented as a
percentage of the SCish per TNC count, absolute SCish count in
μL of the PB and AP, percentage of the CD90+SCish expressed
in SCish and absolute CD90+SCish count in μL of PB and AP
(calculated on the basis of CD90+SCish percentage).
For autologous SC-transplants, cryopreservation was
performed according to our original five-step controlled-rate
freezing protocol (with compensation of the released fusion
heat), using dimethyl sulfoxide (DMSO; 10% final concentration) by Planer 560-16 equipment (Planer Products Ltd,
UK), as it was earlier described 13–15.
Statistical analysis
Descriptive data of the SC investigations were
expressed as the mean value ± standard error of mean (SEM)
for each of the parameters examined. Statistical analyses
were performed using GraphPad Prism 5 software. Differences were considered as statistically significant if p value was
less than 0.05.
Cell quantifications techniques
The TNC, mononuclear cell (MNC), Plt and red blood
cell (RBC) numbers in the patients' PB and/or AP samples
were quantified using Advia-2120 blood counter (Bayer,
Germany). The following monoclonal antibodies (mAbs)
were used for the flow cytometric determination of CD45,
CD34
and
CD90
antigens/markers:
anti-CD45ECD, Immunotech, France; anti-CD34-FITC (class III
antibody), BD Pharmingen, USA; anti-CD90-PE, Miltenyi
Biotec GmbH, Germany). The samples were analyzed on
FC-500 flow cytometer (Beckman-Coulter, FL, USA).
The SCsish were determined as a constitutional part of
CD34+ cells in the „stem cell-region“ of the ISHAGE
sequential gating protocol 5–9. In our study, the original
ISHAGE protocol was modified by introduction of antiCD90-PE mAb into the analysis of CD90 expression on
SCish. Briefly, the SCish were first gated on CD45 vs Side
Scatter dot plot in order to separate the CD45+ WBC from
RBCs, Plts and other debris. From the primary gate on the
CD45+ events, the CD34+ cells were identified on the CD34
vs Side Scatter dot plot by their expression of CD34 and characteristic light scatter properties. From the second gate on
the CD34+ events, the SCish were back-gated on CD45 vs Side Scatter dot plot in order to separate “true” CD34+ or SCish
with low CD45 fluorescence and low side scatter, from
nonspecifically stained events – lymphocytes (CD45high),
monocytes (CD45high) and higher Side Scatter) and
granulocytes (high Side Scatter). In the next step, on the
Balint B, et al Vojnosanit Pregl 2017; 74(11): 1071–1077.
Results
Enrichment of total SCish (absolute count) and the
CD90+SCish (relative frequency) cells in the apherosis product
In this study, 12 patients subjected to autologous SCtransplant within the treatment of multiple myeloma were included. We initially assessed the quality of AP by comparing
the absolute counts of targeted cells in the PB samples after
mobilization with their yield in the AP. As expected, higher
absolute count of total SCish in the AP compared with the PB
samples with very high statistical significance (2487 ± 2678
vs 137.6 ± 129.7; p = 0.0007; Figure 1A) was found.
Similarly, the absolute count of CD90+SCish was also
significantly higher in the AP (40.7 ± 42.8 vs 14.4 ± 6.7; p =
0.0266; Figure 1B).
The collection efficiency for the CD90+SCish is lower
than for the CD90-SCish cells
Next, we assessed the harvesting efficiency of
CD90+SCish by comparing of the CD90+SCish yield with the
total SCish yield in the AP. With that purpose, we calculated
the CD90+SCish / total SCish index by dividing the absolute
count of CD90+SCish with the absolute count of total SCish
and multiplied with 100, for both, the PB samples, as well as
for the AP samples.
Page 1074
VOJNOSANITETSKI PREGLED
Vol. 74, No 11
Fig. 1 – Comparison of (A) total SCish and (B) CD90+SCish absolute counts between peripheral blood (PB) and apheresis
product (AP) samples showing significantly higher counts in the AP samples (*p < 0.05; *** p < 0.001, respectively).
(mean ± SEM); Mann Whitney test).
SEM – standard error of the mean.
We found that, despite considerable positive correlation
between these two indexes (Spearman r = 0.6503; p =
0.0221; Figure 2A), the CD90+SCish/total SCish indexes from
the PB samples were significantly higher compared to the
indexes from the AP samples (18.4 ± 12.9 vs 9.3 ± 11.7;
p = 0.0392; Figure 2B). This finding indicates much higher
CD90+SCish/total SCish proportion in the PB samples.
The relative frequency of CD90+SCish correlates
inversely with total SCish in mobilized peripheral blood and
apheresis product
The relative frequency of CD90+SCish showed highly
significant inverse relationship with the absolute count of
total SCish in both, the PB (Spearman's r = -0.8652; p =
0.0003; Figure 3A), and in the AP samples (Spearman r =
-0.8140; p = 0.0013; Figure 3B).
Interestingly, the relative frequency of CD90+SCish from
the PB samples also showed highly significant inverse relationship with the absolute counts of total SCish in the AP samples (Spearman's r = -0.8722; p = 0.0002; Figure 4A). In addition, we found that the patients with less than 10% of relative
frequency of the CD90+SCish in PB, had significantly higher
total SCish [since the higher appearance of mature SCish (CD90SCish) subset] in the AP when compared with the patients with
more than 10% of CD90+SCish in their PB samples
(5067 ± 2249 vs 644 ± 513.1; p = 0.0025; Figure 4B).
Discussion
Although significantly progress in conventional medications has improved the prognosis of hematological malignancies, SC-transplant remains the most effective approach
in obtaining disease free long-term survival of patients.
Fig. 2 – (A) Correlation of peripheral blood (PB) and apheresis product (AP) CD90+SCish/total SCish indexes showing significant positive relationship (Spearman's correlation test); (B) Comparison between PB and AP CD90+SCish/total SCish
indexes showing significantly higher values in the PB samples (*p < 0.05; mean ± SEM; Mann Whitney test).
SEM – standard error of the mean.
Balint B, et al. Vojnosanit Pregl 2017; 74(11): 1071–1077.
Vol. 74, No 11
VOJNOSANITETSKI PREGLED
Page 1075
Fig. 3 – Correlation between relative frequency of CD90+SCish and absolute counts of total SCish in (A) peripheral blood
(PB) and (B) apheresis product (AP) samples showing significant inverse relationship (Spearman’s correlation test).
Fig. 4 – (A) Correlation between relative frequency of CD90+SCish and absolute counts of total SCish in apheresis product
(AP) samples showing significant inverse relationship (Spearman’s correlation test). (B) Comparison of the SCish absolute
counts in the AP samples between the patients with less than 10% relative frequency of the CD90+SCish and patients with
more than 10% of the CD90+SCish in their peripheral blood (PB) samples, showing significantly higher SCish absolute
counts in the group of patients with less than 10% of CD90+SCish in PB (**p < 0.01; mean ± SEM; Mann Whitney test).
SEM – standard error of the mean.
However, high-dose treatment incorporates risks of conditioning-regimen related morbidity/mortality; accordingly it is
limited for relatively younger patients in better clinical condition. This age limitation is regrettable since SC-transplant
candidates in multiple myeloma are frequently older than 60
or even 65 years 1.
The earliest SC collections from PB were accomplished
in “steady state hematopoiesis” – by numerous procedures
since late 1970s, and following cryopreservation was needed 1, 2, 16. The cell harvesting is the same for allogeneic donors as for autologous patients. Vascular access, as mentioBalint B, et al Vojnosanit Pregl 2017; 74(11): 1071–1077.
ned, is regularly realized using central/venous (jugular, subclavian or femoral) catheter.
In the course of cell harvesting, selection of the best collection system and determination of optimal timing for apheresis,
are the most critical events. The most recent SC software design
incorporates the Intermediate density layer (IDL) system using
Spectra-Optia device 1.
There are several advantages of PB as a source of the
SCs as compared to the BM: absence of general anesthesia
and multiple bone aspirations, higher CD34+ yield in the AP
and earlier hematopoietic reconstitution, as well as shortened
Page 1076
VOJNOSANITETSKI PREGLED
hospital stays and reduced transplant related morbidity.
Commonly, the SC-engraftment is defined as the first of
three days with neutrophil count greater than 0.5 × 109/L 1
and Plt count exceeding 20 × 109/L (without transfusion support for 7 consecutive days) 17.
Due to the reasons mentioned above, the number of patients treated by PB derived allogeneic, especially autologous
SC-transplants is increasing worldwide 1, 2. Nowadays, the
PB derived SCs are used for approximately 80% of allogeneic and practically for all autologous SC-transplants 1, 4.
Enumerated CD34+ cell dose by flow cytometry is
routinely performed to optimize timing of the PB stem cell
collections and assess engraftment potential of the AP. Moreover, the immature CD34+/CD90+ cells or PHPs have a
capacity to initiate long-term hematopoiesis ex vivo, and according to some data, they are mobilized into blood to a
maximum level a few days earlier than the peak mobilization
of the total CD34+ cells 18. As mentioned, the CD34+/CD90+
cells in the AP were a better predictor of Plt recovery than the
total dose of the CD34+ cells, and 80x104/kg of the
CD34+/CD90+ cells is the minimal essential dose capable of
durable long-term engraftment 11. Therefore, the measurement
of the CD34+/CD90+ cells is helpful for the evaluation of the
grafts quality in the PB autologous SC-transplant.
There is an increasing interest in evaluation of the
responsibility of the CD34+ subsets for complete and longterm repopulation, as a marker of cell harvest quality 1, 10–12. In
our initial clinical study, as a possible and potentially more objective collection predictor, the CD34+/CD90+ subset evaluation was also recommended 19. Precisely, on the basis of
examination of the immature SC antigens and 7-aminoactinomycin D (7-AAD) viability, we suggested that the
CD34+/CD33-, CD34+/CD38-, CD34+/DR-, CD34+/CD90+
cells could be superior predictive factor over circulating the total CD34+ count for an optimized collection-timing and outcome of autologous SC-transplant 19. Other authors also confirmed that engraftment and hematopoietic recovery are not
necessarily associated with cell dose of the total CD34+ cells 10.
Thus, the CD34+/CD90+ cells could be a useful quality marker
for the AP 10, 19, especially for prediction of the Plt recovery
(reduced hazard of the prolonged thrombocytopenia) 11.
Consequently, the CD34+/CD90+ cells could be a practical
predictive factor for complete and durable hematopoietic reconstitution 10, 11. The quantity of the mature CD34+ subsets in
the AP correlates obviously with engraftment rapidity 1, 2.
However, data concerning the potential of the immature
CD34+ subsets (such as CD90+SCish) for long-term marrow repopulation are still not completely clarified.
Our previous preclinical research (using standardized
cell harvesting protocols) confirmed that Spectra-Optia resulted with superior collection efficiency compared to CobeSpectra for the CD34+ cells (CE2[%]CD34+) 20. The
CE2[%]CD34+ was calculated on the basis of the CD34+ count
in the AP versus their number in processed whole blood 20.
We estimated that the “predictive-value” of circulating total
CD34+ cells for the SC harvesting could be enhanced or improved by determination of the relative frequency of
CD90+SCish in patients' PB.
Vol. 74, No 11
In this study the relative frequency of CD90+SCish demonstrated inverse correlation with the absolute count of total SCish in
both, the PB and AP. Accordingly, on the basis of the inverse
correlation between the PB and the AP counts of CD90+SCish
observed in present study, it is possible that the level of the
collection efficiency of both existing (Cobe-Spectra and
Spectra-Optia) apheresis systems is inferior for the immature (CE[%]CD90+SCish) as compared to the mature
(CE2[%]CD34+) cells from mobilized PB. However, we believe that the lower CD90+SCish yield is not a consequence of
inferior CE[%]CD90+SCish, but possibly a result of several still
not fully resolved immature SC features, such as
cytomorphological and biophysical (intracellular granulation,
cell-density, etc) parameters. Using the „dye-efflux“ method
for SC-sorting, Radley et al. 21 defined more precisely ultrastructural characteristics of the most primitive SCs in murine
BM and provided a basis for studying the structural changes
that occur with progressive activation and differentiation of
immature hematopoietic SCs (HSCs) toward their progeny of
lesser proliferative capacity. The structural changes could
likely have effect on the density and consequently “centrifugation-properties” of these cells. Sharma et al. 22 showed that
the CD90+ cells were predominantly found within the subpopulation of the SCs with low CD34 and very low CD45 antigen expression, in the patients with hematological malignancies. They also showed that those CD90+CD34dimCD45very dim
cells were the smallest among all other examined subpopulations (including CD133+ and CD117+ CSs with the different
CD45 antigen expression) with an electronic volume of
87.1–143.7 µm3 corresponding to a diameter of 5.5–6.5 µm.
In the review article, Kucia et al. 23 postulate that the fraction
of the SCs, small in their size, could be easily lost during collection/isolation protocols based on gradient or centrifugation. Our finding of significantly lower enrichment index for
CD90+SCish, compared with enrichment index of the total
SCish, is in accordance with the aforementioned earlier investigations.
In this study an intriguing observation is the inverse relationship between relative frequencies of the CD90+SCish
and absolute counts of the total SCish in PB, suggesting that
continual increase in absolute counts of the total SCish, during the G-CSF-induced mobilization, is not followed by an
appropriate increase in the CD90+ cells. The G-CSF acting –
either as an endogenously produced after chemotherapy or as
exogenous or administered factor (rHuG-CSF), or a combination of both – is considered to be an initial signal for HSC
mobilization, explained by several proposed pathways 24.
Nevertheless, there are data describing the superior effects of
some new agents, such as mozobil or plerixafor (which is an
antagonist of the alpha chemokine receptor CXCR4) in mobilizing the immature HSC, capable for long-term repopulation (LT-HSC) 25.
Conclusion
We speculate that the inferior CD90+SCish yield in the
AP is not a consequence of the inferior collection efficacy –
but most likely a result of several still not fully understoBalint B, et al. Vojnosanit Pregl 2017; 74(11): 1071–1077.
Vol. 74, No 11
VOJNOSANITETSKI PREGLED
od/clarified immature SC cytomorphological and/or
biophysical features. Thus, when the chemotherapy/G-CSF is
used for mobilization, there are some logical questions to
ask- whether we should follow the absolute count of the total
SCish, or, whether we should test for relative frequency of
CD90+SCish prior to apheresis.
To reach the final conclusions, it is essential to conduct
further controlled and larger SC-investigations (our clinical
study has been already initiated) concerning the correlation
Page 1077
between circulating and harvested SCs, and patients'
hematopoietic recovery.
Acknowledgements
This work was supported by the Ministry of Defence of the
Republic of Serbia (Project MF/VMA 9/17-19) and by the Ministry of Education, Science and Technological Development of
the Republic of Serbia (Project „III“ No 41031).
R E F E R E N C E S
1. Pavlović M, Balint B. Stem cells and tissue engineering. New
York, NY: Springer; 2013.
2. Balint B, Stamatović D, Todorović M, Jevtić M, Ostojić G, Pavlović M, et
al. Stem cells in the arrangement of bone marrow repopulation
and regenerative medicine. Vojnosanit Pregl 2007; 64(7): 481ԟ4.
3. Obradović D, Tukić L, Radovinović-Tasić S, Petrović B, Elez M, Ostojić G, et al. Autologous hematopoietic stem cell transplantation
in combination with immunoablative protocol in secondary
progressive multiple sclerosis ԟ A 10-year follow-up of the
first transplanted patient. Vojnosanit Pregl 2016; 73(5): 504ԟ8.
4. Barnett D, Janossy G, Lubenko A, Matutes E, Newland A, Reilly JT.
Guideline for the flow cytometric enumeration of CD34+
haematopoietic stem cells. Prepared by the CD34+ haematopoietic stem cell working party. General Haematology Task
Force of the British Committee for Standards in Haematology.
Clin Lab Haematol 1999; 21(5): 301ԟ8.
5. Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR. Single platform flow cytometric absolute CD34+ cell counts based
on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. Cytometry 1998; 34(2): 61ԟ70.
6. Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The
ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft
Engineering. J Hematother 1996; 5(3): 213ԟ26.
7. Thornley I, Sutherland R, Wynn R, Nayar R, Sung L, Corpus G, et al.
Early hematopoietic reconstitution after clinical stem cell transplantation: evidence for stochastic stem cell behavior and limited
acceleration in telomere loss. Blood 2002; 99(7): 2387ԟ96.
8. Thornley I, Sutherland DR, Nayar R, Sung L, Freedman MH, Messner
HA. Replicative stress after allogeneic bone marrow transplantation: changes in cycling of CD34+CD90+ and CD34+CD90hematopoietic progenitors. Blood 2001; 97(6): 1876ԟ8.
9. Whitby A, Whitby L, Fletcher M, Reilly JT, Sutherland DR, Keeney
M, et al. ISHAGE protocol: are we doing it correctly? Cytometry B Clin Cytom 2012; 82(1): 9ԟ17.
10. Pratt G, Rawstron AC, English AE, Johnson RJ, Jack AS, Morgan
GJ, et al. Analysis of CD34+ cell subsets in stem cell harvests
can more reliably predict rapidity and durability of engraftment
than total CD34+ cell dose, but steady state levels do not correlate with bone marrow reserve. Br J Haematol 2001; 114(4):
937ԟ43.
11. Sumikuma T, Shimazaki C, Inaba T, Ochiai N, Okano A, Hatsuse
M, et al. CD34+/CD90+ cells infused best predict late haematopoietic reconstitution following autologous peripheral blood
stem cell transplantation. Br J Haematol 2002; 117(1): 238ԟ44.
12. Villaron EM, Almeida J, Lopez-Holgado N, Sanchez-Guijo FM, Alberca M, Blanco B, et al. In leukapheresis products from nonHodgkin's lymphoma patients, the immature hematopoietic
progenitors show higher CD90 and CD34 antigenic expression. Transfus Apher Sci 2007; 37(2): 145ԟ56.
13. Skoric D, Balint B, Petakov M, Sindjic M, Rodic P. Collection
strategies and cryopreservation of umbilical cord blood. Transfus Med 2007; 17(2): 107ԟ13.
Balint B, et al Vojnosanit Pregl 2017; 74(11): 1071–1077.
14. Balint B, Ivanović Z, Petakov M, Taseski J, Jovcić G, Stojanović N, et
al. The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating
ability. Bone Marrow Transplant 1999; 23(6): 613ԟ9.
15. Balint B, Ljubenov M, Stamatović D, Todorović M, Pavlović M, Ostojić
G, et al. Stem cell harvesting protocol research in autologous
transplantation setting: large volume vs. conventional cytapheresis. Vojnosanit Pregl 2008; 65(7): 545ԟ51.
16. Goldman JM, Th'ng KH, Park DS, Spiers AS, Lowenthal RM,
Ruutu T. Collection, cryopreservation and subsequent viability
of haemopoietic stem cells intended for treatment of chronic
granulocytic leukaemia in blast-cell transformation. Br J
Haematol 1978; 40(2): 185ԟ95.
17. Chang YJ, Xu LP, Liu DH, Liu KY, Han W, Chen YH, et al.
Platelet engraftment in patients with hematologic malignancies
following unmanipulated haploidentical blood and marrow
transplantation: effects of CD34+ cell dose and disease status.
Biol Blood Marrow Transplant 2009; 15(5): 632ԟ8.
18. Haas R, Möhle R, Pförsich M, Fruehauf S, Witt B, Goldschmidt H, et
al. Blood-derived autografts collected during granulocyte colony-stimulating factor-enhanced recovery are enriched with
early Thy-1+ hematopoietic progenitor cells. Blood 1995;
85(7): 1936ԟ43.
19. Balint B, Stamatovic D, Todorovic M, Elez M, Vojvodic D, Pavlovic
M, et al. Autologous transplant in the treatment of severe
aplastic anemia--a case report. Transfus Apher Sci 2011; 45(2):
137ԟ41.
20. Balint B, Kanjuh V, Todorovic-Balint M, Ostojic G, Stamatovic D,
Obradovic S, et al. Cobe-Spectra vs. Spectra-Optia apheresis
systems – an overview of current status and a comparative research. Bilt Transfuziol 2014; 60(1–2): 1–5.
21. Radley JM, Ellis S, Palatsides M, Williams B, Bertoncello I. Ultrastructure of primitive hematopoietic stem cells isolated using
probes of functional status. Exp Hematol 1999; 27(2): 365ԟ9.
22. Sharma S, Cabana R, Shariatmadar S, Krishan A. Cellular volume
and marker expression in human peripheral blood apheresis
stem cells. Cytometry A 2008; 73(2): 160ԟ7.
23. Kucia M, Reca R, Jala VR, Dawn B, Ratajczak J, Ratajczak MZ.
Bone marrow as a home of heterogenous populations of nonhematopoietic stem cells. Leukemia 2005; 19(7): 1118ԟ27.
24. Angelopoulou MK, Tsirkinidis P, Boutsikas G, Vassilakopoulos TP,
Tsirigotis P. New insights in the mobilization of hematopoietic
stem cells in lymphoma and multiple myeloma patients. Biomed Res Int 2014; 2014: 835138.
25. Lidonnici MR, Aprile A, Frittoli MC, Mandelli G, Paleari Y, Spinelli
A, et al. Plerixafor and G-CSF combination mobilizes hematopoietic stem and progenitors cells with a distinct transcriptional profile and a reduced in vivo homing capacity compared
to plerixafor alone. Haematologica 2017; 102(4): e120ԟ4.
Received on May 05, 2017.
Accepted on May 22, 2017.
Online First May, 2017.