intl.elsevierhealth.com/journals/tras

Transfusion and Apheresis Science 33 (2005) 37–45

Matrix metalloproteinase-9 and cell kinetics during thecollection of peripheral blood stem cells by leukapheresis

Dragoslav Domanovic a,*, Gordana Wozniak a, Peter Cernelc b,Marina Samardzija c, Sanja Balen-Marunic d, Primoz Rozman a

a Blood Transfusion Centre of Slovenia, Slajmerjeva 6, 1000 Ljubljana, Sloveniab Department of Haematology, University Medical Centre, Zaloska 7, 1000 Ljubljana, Slovenia

c Department of Transfusion Medicine, Osijek University Hospital, Huttlerova 4, 31000 Osijek, Croatiad Department of Transfusion Medicine, Rijeka University Hospital Centre, T. Strizica 3, 51000 Rijeka, Croatia

Received 27 July 2004; received in revised form 15 September 2004; accepted 19 September 2004

Abstract

The plasma levels of matrix metalloproteinase-9 (MMP-9) were determined during 41 collections of peripheral blood

stem cells (PBSC) by standard volume (two blood volumes) leukaphereses (SVL) in 29 donors (7 allogeneic and 22

autologous) mobilized with the granulocyte-colony stimulating factor (G-CSF). The association between MMP-9 levels

and cell counts in donor�s blood was explored. During the processing of the first blood volume (BV), MMP-9 levels

declined on average by 31% and persisted at the same level during the processing of the second BV. During the collec-

tion, a slight decline of white blood cells (WBC), polymorphonuclear neutrophils (PMN) and platelets (PLT) in donor�sblood was accompanied by a significant drop of CD34+ cells by 37% after 1 BV and by 44% after 2 BV had been pro-

cessed (p = 0.001). The drop of MMP-9 plasma levels showed a loose correlation with the decrease of WBC (r = 0.68,

p = 0.002) and PMN counts (r = 0.67, p = 0.001). We conclude that the levels of MMP-9 that have been elevated by the

mobilization of donors with G-CSF, decrease during the collection of PBSC by 4 h SVL. The observed decrease was

indirectly related to the drop of WBC and PMN counts, suggesting that certain other factors have an influence on

MMP-9 kinetics during PBSC collection.

� 2005 Elsevier Ltd. All rights reserved.

Keywords: MMP-9; Leukapheresis; PBSC; CD34+ cells; G-CSF

1473-0502/$ - see front matter � 2005 Elsevier Ltd. All rights reserv

doi:10.1016/j.transci.2004.09.009

* Corresponding author. Tel.: +386 1 54 38 123; fax: +386 1

54 38 135.

E-mail address: dragoslav.domanovic@ztm.si (D. Domano-

vic).

1. Introduction

Peripheral blood stem cells (PBSC) have be-come the standard source of haematopoietic stem

cells (HSC) for bone marrow reconstitution after

ed.

Table 1

Demographics and diagnosis

Autologous

donors

Allogeneic

donors

Total

Number of

aphereses (N tested)

33 8 41

Number of cases 22 7 29

Females 8 4 12

Males 14 3 17

Average age 47 34 44

Average number

of aphereses/case

1.5 1.1 1.4

1 Aphereses/case 12 (54%) 6 (86%) 18 (62%)

2 Aphereses/case 9 (41%) 1 (14%) 10 (34%)

3 Aphereses/case 1 (5%) 0 1 (3%)

Diagnosis

MM 9 (31%) – –

ALL 4 (14%) – –

NHL 3 (10%) – –

AML 6 (21%) – –

CML 6 (21%) – –

Amyloidosis 1 (3%) – –

MM = multiple myeloma, ALL = acute lymphoblastic leukae-

mia; NHL = non-Hodgkin lymphoma; AML = acute myeloid

leukaemia; CML = chronic myeloid leukaemia.

38 D. Domanovic et al. / Transfusion and Apheresis Science 33 (2005) 37–45

myeloablative therapy [1–3]. Rapid and sustained

engraftment correlates with the number of infused

HSC usually quantified by immunophenotyping

for CD34+ cells [4–6]. Although the recommended

doses for safe engraftment range from 1.0 to5.0 · 106 CD34+ cells/kg [7–9], the greater

CD34+ cell doses may accelerate haematopoietic

recovery and significantly impact the clinical out-

come after allogeneic transplantation [10–12].

Thus, the factors that influence the quantity of

CD34+ cells collected by leukapheresis have been

investigated in order to ensure an adequate cell

dose for transplantation or to obtain greater cellyields for the compensation of cell loss during fur-

ther processing of the harvest. The quantity of

CD34+ cells collected can be increased either by

increasing the number of mobilized cells in the

circulation, by the greater number of collections,

or by increasing the volume of blood processed

during each collection [13–17].

To increase the concentration of circulatingCD34+ cells, various mobilization regimens with

chemotherapeutic agents, haematopoietic growth

factors or their combination, have been developed.

Among the growth factors, the predominant mobi-

lizing agent is the granulocyte colony-stimulating

factor G-CSF [18–22]. Although the mechanism

of G-CSF mobilization is not fully understood, it

has been proposed that it may initiate mobilizationby down-regulation of CD114 expression on poly-

morphonuclear neutrophils (PMN) consequent

degranulation and the release of matrix metallo-

proteinase-9 (MMP-9). Secreted MMP-9 cleaves

the extracellular matrix and weakens adhesive cell

interactions within marrow that promotes the re-

lease of HSC from marrow. MMP-9 also causes

disorganization of endothelial junctions facili-tating HSC transmigration and egress into the

circulation [23–26]. MMP-9 is a zinc-dependent

endopeptidase which is secreted as zymogen (pro-

MMP-9) by various cells. Pro-MMP-9 is activated

by a variety of proteinases or by reaction with

organic mercurials and inactivated by endogenous

inhibitors. The substrate for active MMP-9 are the

components of an extra cellular matrix [25].Previous studies have shown that the MMP-9

levels are increased during the G-CSF mobiliza-

tion of donors [21,22]. However, it is unknown

whether the MMP-9 levels elevated by mobiliza-

tion change during the collection of PBSC by leuk-

apheresis. Therefore, we have wanted to elucidate

the kinetics of MMP-9 and its relationship with

the peripheral blood cell counts during the collec-tion of PBSC by SVL in G-CSF-mobilized donors,

which could give us supplemental data about

the mechanism of G-CSF-induced mobilization

through the release of MMP-9.

2. Materials and methods

2.1. Population and study design

Twenty-two autologous and seven allogeneic

donors who underwent 41 consecutive PBSC col-

lections were included in the study (see Table 1

for the demographic data). Procedures were per-

formed between April and October 2003. The con-

centrations and activities of MMP-9, blood cell

D. Domanovic et al. / Transfusion and Apheresis Science 33 (2005) 37–45 39

counts and CD34+ cell counts were determined in

the peripheral blood of donors at the beginning of

collection and after each of the two blood volumes

(BV) processed by standard volume leukapheresis

(SVL). The concentration of MMP-9 was alsodetermined in 10 healthy blood donors that served

as a control group. The blood cell counts and

CD34+ cell counts were also determined in the col-

lected product after the first and the second BV

had been processed. The Medical Ethics Commit-

tee of the University Medical Centre in Ljubljana

approved the study. Informed consent for the col-

lection of PBSC and additional testing required forthis study was obtained from all donors.

2.2. Mobilization regimen

Autologous donors were mobilized by a combi-

nation of chemotherapy and recombinant human

G-CSF (rHG-CSF) Filgrastim (Neupogen�,

Amgen Inc. Thousand Oaks, CA, USA) 10 lg/kgs.c. divided into two doses daily until a desired

CD34+ cell count was achieved. Before rHG-

CSF application, multiple myeloma patients

underwent mobilization with cyclophosphamide

at 4 g/m2, B-NHL patients received the same dose

of cyclophosphamide and MabThera and ALL pa-

tients received high-dose methothrexate. The pa-

tient with amiloydosis was mobilized only withrHG-CSF. The allogeneic donors received the

same dose of rHG-CSF (10 lg/kg s.c. divided in

two doses daily) for five consecutive days before

collection. A level of >20 · 106 l�1 of CD34+ cells

in peripheral blood was the trigger to initiate the

collection in both groups of donors.

2.3. Collection of PBSC and sampling

The Amicus cell separator (Baxter Healthcare,

IL, USA) software version 2.42 was used for the

collection of PBSC by SVL according to the man-

ufacturer�s instructions. The total BV required to

be processed was determined in each procedure

as the sum of two BV of each donor calculated

from a standard equation [27].Blood samples were taken from donors at the

beginning of collection and sequentially after pro-

cessing 1 BV and 2 BV, as well as from the final

product. For MMP-9 determination, blood sam-

ples were collected in heparin and centrifuged at

1000g for 10 min within 30 min of collection. The

decanted plasma was then centrifuged at 10000g

for 10 min at 4 �C. Plasma samples were then fro-zen at �80 �C within 6 h after collection. After

thawing, the samples were analysed for total

MMP-9 concentration and enzyme activity. Sam-

ples for cell counts and CD34+ cells were collected

in the K3EDTA tubes and counted within 2 h after

collection.

2.4. Concentration and activity of MMP-9 in

plasma

For the quantitative determination of the total

(active and pro) MMP-9 in plasma, a commercial

test kit (Quantikine�, human MMP-9 total Immu-

noassay, R&D Systems) that employs the sand-

wich enzyme-linked immunoassay was used.

Active MMP-9 was quantitatively measured byfluorometric assay (Fluorokine�E, human active

MMP-9 total Fluorescent Assay, R&D Systems)

without the addition of AMPA (4-aminophenyl-

mercuric acetate) that can activate MMP-9 proen-

zyme. Both tests were performed according to the

manufacturer�s instructions.Proteolytic activity of MMP-9 in plasma

samples was determined under non-reducing con-ditions using the modified sodium dodecyl sul-

phate (SDS)–polyacrylamide gel electrophoresis

through 10% SDS–polyacrilamide gel containing

1 mg/ml gelatin (Sigma–Aldrich, St. Luis, MO,

USA) as an enzyme substrate [28]. Two microli-

ters of plasma were mixed with 44 ll of loading

buffer (0.075 mol/l Tris–HCl, 11.25% glycerol,

2.25% SDS and 0.002% bromophenol blue, pH6.8) and 4 ll of the mixture were applied onto

the gel. After electrophoresis, SDS was removed

from the gel by washing with 2.5% Triton X-100

3· for 20 min and then incubated in a zymogra-

phy buffer (50 mmol/l Tris–HCl, 150 mmol/l

NaCl, 5 mmol/l CaCl2, pH 7.4) for 24 at 37 �C.The gels were then stained with 0.05% Coomassie

brilliant blue G-250 (Sigma–Aldrich, St. Luis,MO, USA). Proteolytic activities were evidenced

as clear bands against the blue background of

the stained gelatin.

40 D. Domanovic et al. / Transfusion and Apheresis Science 33 (2005) 37–45

2.5. Cell counts

Automated blood cell counts were performed

with an electronic cell counter CELL-DYN 3200

(Abbott Laboratories, IL, USA). Cell differentialswere performed microscopically on May-Grun-

wald-Giemsa-stained specimens. The number of

mononuclear cells (MNC) was calculated as the

sum of lymphocytes and monocytes.

Collection efficiency, number of cells released in

to the peripheral blood during the apheresis and

the cell release rate were calculated using the previ-

ously described formulas [16,29].

2.6. Flow cytometry

CD34+ cell counts were performed on a flow

cytometer, the Coulter Epics XL-MCL (Beck-

man-Coulter/Immunotech, Hialeah, FL, USA)

according to the ISHAGE guidelines [30]. Stem-

KIT (Beckman-Coulter/Immunotech, Hialeah,FL, USA), which utilizes CD45FITC/CD34PE

for specific and CD45FITC/Isotype control PE

non-specific staining, was used for determining

absolute CD34+ counts.

2.7. Statistical analysis

A commercial computer software program Stat-istica (StatSoft, OK, USA) was used for the statis-

tical analysis. The paired and unpaired Wilcoxon�stest was used to compare changes in MMP-9, as

well as the cell counts determined before, during

Table 2

The characteristics of collection procedures on the Amicus cell separa

N tested = 41 Autologous collections

WB/cycle (ml) 1400 (1000–1400)a

No. of cycles 9.0 (6.0–11.0)

WB flow rate (ml/min) 55 (42–60)

ACD used (ml) 990 (772–1437)

WB processed (ml) 11,970 (8311–17,268)

Saline (ml) 614 (596–654)

Collection time (min) 239 (135–292)

There was a constant setting of ACD:WB ratio at 1:12 and the cit

collection phase was set at 0.6, the red blood cells offset volume at 6.3

whole blood processed in each cycle was set at 1000 ml (allogeneic don

>35 · 103 ll�1) or 1400–1600 ml (allogeneic donors with WBC <50 ·a Shown are medians (range). WB = whole blood.

and after apheresis. Spearman�s rank correlation

was used to evaluate the association between the

MMP-9 levels and cell counts.

3. Results

We evaluated a total of 41 leukapheresis proce-

dures in the 29 donors of PBSC (22 autologous

and 7 allogeneic) that were aimed at obtaining

the yield target of >2 · 106 CD34+ cells/kg of the

recipients body weight (BW). The autologous do-

nors underwent 33 leukapheresis procedures (1.5leukaphaereses per case) whereas allogeneic do-

nors underwent eight procedures (1.1 leukaphaere-

ses per case). On average, a single leukapheresis

was sufficient to collect the targeted yield in 18

(62%) collections: in 6 (86%) out of 7 allogeneic

donors and in 12 (54%) out of 22 in autologous

donors.

There were no statistically significant differencesbetween autologous and allogeneic donors in the

duration of collection, volume of whole blood pro-

cessed and in other characteristics of leukapheresis

procedures using the Amicus cell separator (see

Table 2).

The zymographic analysis demonstrated that

the MMP-9 related proteolytic activities were

higher in the plasma of mobilized donors as com-pared to healthy controls (Fig. 1).

The median concentration of MMP-9 at the

beginning of the 41 collections was 474 ng/ml

(range 45–1933 ng/ml) which was significantly

tor

Allogeneic collections Total

1200 (1000–1600) 1400 (1000–1600)

7.0 (5.0–12.0) 7.0 (5.0–12.0)

55 (30–70) 55 (30–70)

839 (750–1184) 860 (550–1437)

9905 (7658–14,236) 10,801 (7310–17,268)

632 (846–1072) 630 (546–1072)

231 (138–325) 232 (132–325)

rate infusion rate of 1.25 mg/kg/min. The interface during the

and the platelet poor plasma set point at 0.35. The volume of

ors with WBC >50 · 103 ll�1 and autologous donors with WBC

103 ll�1 and autologous donors with WBC <35 · 103 ll�1).

Fig. 1. Zymographic analysis. The representative zymogram

shows the presence of enzyme activity at the band 92 kDa

(MMP-9) that is markedly higher in the mobilized donors than

in the control healthy blood donors in the two left lanes. MMP-

2 (an enzyme with similar but not identical activity to MMP-9)

that runs at the band 72 kDa was not significantly different in

the samples from the mobilized donors and un-mobilized

healthy blood donors. This finding is consistent with the

previous reports in humans during G-CSF mobilization [25].

D. Domanovic et al. / Transfusion and Apheresis Science 33 (2005) 37–45 41

higher (p = 0.0001) than the median values of

78 ng/ml (range 44–116 ng/ml) in 10 healthy con-trols, which is comparable to the data of others

[21,22]. The starting median MMP-9 concentra-

tion in allogeneic donors of 881 ng/ml (range

146–1933 ng/ml) was significantly higher than in

autologous donors (428 ng/ml, range 45–1897 ng/

ml) (p = 0.001). After processing 1 BV, we ob-

served a significant decline of MMP-9 concentra-

tion from the starting value to the median levelof 327 ng/ml (range 38–1569 ng/ml; p = 0.0027).

A similar degree of decline was observed in both

groups. After processing the second BV, the con-

Table 3

The concentration of MMP-9 in plasma during PBSC collection by a

N tested = 41 Start

MMP-9 (ng/ml) autologous collections 428 (45–189

MMP-9 (ng/ml) allogeneic collections 881 (146–19

MMP-9 (ng/ml) autol. and allog. collections 474 (45–193

The median level of MMP-9 in a control group of 10 healthy blood do

than the starting values in donors (p < 0.001).a Shown are medians (range).

centration of MMP-9 fell insignificantly to the

median level of 314 ng/ml (range 61–1704 ng/ml)

(p = 0.949). The changes in MMP-9 concentration

are presented in Table 3.

The major part of MMP-9 found in plasma wasas a latent precursor (pro-MMP-9) and only low

levels of active MMP-9 were observed during the

collections with a total median value of 0.585 ng/

ml (range 0.07–4.12 ng/ml) (data not shown).

The counts of the cell populations in peripheral

blood during leukapheresis, as well as the number

of released cells and cell release rates are presented

in Table 4. The white blood cell (WBC), polymor-phonuclear neutrophil (PMN) and mononuclear

cell (MNC) counts declined during the whole pro-

cedure with an average drop of 9% after 2 BV had

been processed. The platelet (PLT) count dropped

after processing the 1 BV by an average of 12%

and 13% (±11%) after the second BV. A greater

decline of an average of 21% after 1 BV and of

19% at the completion of the procedure was ob-served for MNC and for the CD34+ cells that

dropped by an average of 37% after processing 1

BV and by 44% after the second BV. The relative

drops of cell populations and MMP-9 levels are

presented in Fig. 2.

The cell yields in the collected products (see

Table 5) were in accordance with the results

previously reported [31,32].The correlation analysis showed an association

between MMP-9 levels and WBC as well as PMN

counts in allogeneic donors (r = 0.82; p = 0.011

for WBC and r = 0.83; p = 0.010 for MNC),

whereas correlation coefficients were lower in

autologous donors, consequently giving a loose

total correlation (r = 0.68; p = 0.002 for WBC

and r = 0.67; p = 0.001 for PMN). Low correlation

pheresis

1 BV 2 BV

7)a 253 (38–1569) 277 (61–1704)

33) 625 (87–848) 760 (86–1432)

3) 327 (38–1569) 314 (61–1704)

nors was 78 ng/ml (range 44–116 ng/ml) and significantly lower

Table 4

Blood cell concentrations, number of cells released and cell release rates in donors

N tested = 41 Start 1 BV 2 BV

WBC count (·109 l�1) 27 (6.41–85.9)a 24.4 (4.8–80.0) 22.1 (5.52–79.6)

WBC released (·1010) – 0.4 (�5.3–5.0) 1.5 (�6.2–7.6)

WBC release rate (·107 min�1) – 3.9 (�77.5–41.8) 7.1 (�26.4–25.9)

PMN count (·109 l�1) 22.5 (3.87–68.1) 21.5 (3.2–68.4) 19.7 (3.82–72.8)

PMN released (·1010) – �0.2 (�7.8–2.2) �0.3 (�7.1–4.7)

PMN release rate (·107 min�1) – �1.8 (�84.2–32.0) �1.2 (�30.5–17.9)

MNC count (·109 l�1) 3.29 (1.01–10.4) 2.8 (0.20–10.0) 2.6 (1.02–11.7)

MNC released (·1010) – 1.1 (�0.4–3.8) 1.5 (0.4–5.5)

MNC release rate (·107 min�1) – 10.1 (�3.5–42.5) 6.6 (1.4–24.7)

PLT count (·109 l�1) 129 (37.4–272.0) 113 (31.8–274.0) 119 (27.9–263.0)

PLT released (·1010) – �4.5 (�48.8–10.8) �6.5 (�50.8–3.5)

PLT release rate (·107 min�1) – �37.8 (�650.7–164.0) �28.2 (�376.6–14.4)

CD34+ count (·106 l�1) 51.8 (0.7–220.7) 34.58 (2.62–182.7) 25.44 (1.42–113.5)

CD34+ released (·108) – 2.7 (�20.7–35.5) 6.1 (�25.6–45.2)

CD34+ release rate (·106 min�1) – 21.9 (�157.0–495.1) 27.3 (�109.4–342.8)

The number of cells released from other compartments was calculated from the difference between the number in the peripheral

circulation at the completion of the procedure plus what was present in the collection bag and the number in the peripheral circulation

at the start of the procedure. The cell release rate was determined by dividing that number by the duration of the collection procedure

[29].a Shown are medians (range). WBC = white blood cells; PMN = polymorphonuclear leukocytes; MNC = mononuclear cells;

PLT = platelets.

WBCPMN

MNC

PLT

CD34+

MMP-9

50%

60%

70%

80%

90%

100%

110%

Start 1BV 2BVTime points

Perc

ents

of t

he s

tarti

ng v

alue

s

Fig. 2. Relative drops of blood cell counts and MMP-9 concen-

trations. Shown are the average percents of peripheral blood cell

counts and MMP-9 concentrations measured at various time

points during the apheresis procedure. The starting values were

taken as 100%. The time points refer to the amount of donor�sblood volume (BV) processed. Collections were completed after

processing 2 BV. The abbreviations are the same as in the text.

Table 5

The characteristics of collected product

N tested = 41 1 BV 2 BV

WBC (·109 l�1) 354 (120–738)a 219 (92–416)

WBC total (·1010) 1.6 (0.5–4.1) 2.7 (1.3–6.1)

PMN (·109 l�1) 110 (12–381) 59 (3–160)

PMN total (·1010) 0.5 (0.05–0.16) 0.9 (0.1–1.9)

MNC (·109 l�1) 240 (75–516) 161 (51.2–253)

MNC total (·1010) 1.1 (0.3–3.3) 1.8 (0.7–5.3)

PLT (·109 l�1) 380 (80–1350) 398 (107–1160)

PLT total (·1010) 1.7 (0.4–6.1) 4.8 (1.3–14.5)

CD34+ (·106 l�1) 2388 (186–8965) 1135 (92–4730)

CD34+ total (·108) 1.14 (0.109–4.893) 1.69 (0.125–5.993)

Total number of cells in the collected product were calculated

from the number of cells per liter and the volume of collected

product.

Median collection efficiencies on the Amicus apheresis device

were 62% for MNC and 53% for CD34+ cells. Through one

leukapheresis we collected a median of 2.6 · 108 MNC/kg

recipients body weight (BW) (range, 0.8–7.0 · 108) and

2.1 · 106 CD34+ cells/kg recipients BW (range, 0.2–6.7 · 106).a Shown are medians (range).

42 D. Domanovic et al. / Transfusion and Apheresis Science 33 (2005) 37–45

coefficients were found between MMP-9 concen-

trations and MNC and CD34+ cell counts (r =

0.48, p = 0.03 for MNC and r = 0.47; p = 0.02 for

CD34+ cells). There was no correlation between

the MMP-9 levels and the calculated number of

cells released into the circulation from the other

compartments or their calculated release rates.

D. Domanovic et al. / Transfusion and Apheresis Science 33 (2005) 37–45 43

4. Discussion

Mobilization of PBSC into the peripheral blood

after G-CSF administration encompasses several

processes. Disruption of cytoadhesive interactionsof HSC with the bone marrow stroma is among

the leading hypothesis that was initially demon-

strated by the mobilization of HSC into the

peripheral blood after the inhibition of the very

late activation antigen 4 (VLA-4) by a specific

antibody [33]. This hypothesis was also supported

by the inhibition of IL-8-induced mobilization of

HSC in rhesus monkeys using antibodies againstMMP-9 [23], although the recent study in MMP-

9 knockout mice demonstrates that MMP-9 is

not absolutely required for G-CSF or Flt-3 mobi-

lization [24].

In our study, we demonstrated a decline of

MMP-9 levels during the collection of PBSC by

SVL in G-CSF-mobilized donors. After processing

the 1 BV we observed a significant decline inMMP-9 concentration by 31% from the starting

value. The same drop was observed in both groups

of donors. After processing the second BV, the

concentration of MMP-9 dropped by an addi-

tional 3% to a final 34% of the initial value.

The cell populations in the peripheral blood de-

clined steadily during the apheresis procedures.

The drop was most evident after processing thefirst BV, which was in accordance with the previ-

ously reported results [34]. It is presumably a con-

sequence of the initial rapid removal of the cells

and only partially due to the dilution effect after

packing the blood into the apheresis device and

the return of the priming solution or due to the

cell adherence to the surface of the tubing [35].

In the second part of the procedure, the cell de-cline was smaller, implying the release of cells

from other compartments into the peripheral

blood over the 4 h period during which apheresis

was performed. Although a mild to moderate

thrombocytopenia has been reported after alloge-

neic and autologous collection of PBSC [31], in

our study the end-procedure platelet count de-

creased by only 13% in average. This lower plate-let loss was related to the specific separation

method employed using the Amicus apheresis

machine.

Analysis showed a loose but statistically signifi-

cant correlation between the MMP-9 levels and

WBC and PMN counts during PBSC collection by

apheresis. This finding confirms the previously de-

scribed role of PMN that release MMP-9 in theG-CSF-induced mobilization [26] and is in accor-

dance with the findings that the elevated MMP-9

levels during theG-CSFmobilization correlate with

the increased number of PMN [21,22]. The low cor-

relation coefficients, however, indicate that the

other factors are important in explaining the rela-

tionship between the decrease of the MMP-9 levels

and the cell populations. The declinedMMP-9 con-centration coincides with a diminished stimulation

of donors by G-CSF given 2–3 h before collection,

which corresponds to the half-life of G-CSF that

is 3.5 h after subcutane injection [34] and by the nor-

mal renal clearance ofMMP-9. The role of the anti-

coagulant used (ACD) in the collection procedure

should be evaluated, because Na citrate as a weak

metal chelator can inhibit MMP-9 by binding thezinc and calcium that are required forMMP-9 enzy-

matic activity. Additionally, theMMP-9 levels were

not in correlation with the quantities of collected

cells and the calculated number of released cells or

with the release rates of cells during the apheresis.

This confirmed the previous findings that the num-

ber of collected CD34+ cells could not be predicted

by MMP-9 levels at the onset of collection and alsosuggests that other factors are involved in the trans-

migration of cells fromother compartments into the

blood to replenish those removed. We think that a

study of MMP-9 levels during the collection of

PBSC by large volume leukapheresis, where the

greater blood volumes are processed, higher cell

yields are collected and the intraprocedure recruit-

ment of CD34+ cells is eventually taking place,could give us additional data about MMP-9 levels

during apheresis.

In conclusion, we have demonstrated that the

elevated MMP-9 levels in the plasma of donors

mobilized with G-CSF, decrease during the 4 h col-

lection of PBSC by SVL, which implies that SVL

itself does not exert a stimulatory effect on the re-

lease MMP-9 into the blood. Changes in MMP-9concentration were indirectly related to the changes

of WBC and PMN counts and were not asso-

ciated with the number of collected CD 34+ cells,

44 D. Domanovic et al. / Transfusion and Apheresis Science 33 (2005) 37–45

suggesting that other factors have an influence on

the kinetics of MMP-9 during PBSC collection.

References

[1] Bensinger WI, Clift R, Martin P, et al. Allogeneic periph-

eral blood stem cell transplantation in patients with

advanced hematologic malignances: a retrospective

comparison with marrow transplantation. Blood 1996;88:

2794–800.

[2] Ottinger HD, Beelen DW, Scheulen B, et al. Improved

immune reconstitution after allotransplantation of periph-

eral blood stem cells instead of bone marrow. Blood

1996;88:2775–9.

[3] Couban S, Simpson DR, Barnett MJ, et al. A randomized

multicenter comparison of bone marrow and peripheral

blood in recipients of matched sibling allogeneic trans-

plants for myeloid malignancies. Blood 2002;100:

1525–31.

[4] 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;86:3961–9.

[5] Bensinger W, Appelbaum F, Rowley S, et al. Factors that

influence collection and engraftment of autologous periph-

eral-blood stem cells. J Clin Oncol 1995;13:2547–55.

[6] 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;77:400–9.

[7] Singhal S, Powels R, Treleaven J, et al. A low CD34+ cell

dose results in higher mortality and poorer survival after

blood or marrow stem cell transplantation from HLA-

identical siblings: should 2 · 106 CD34+ cells/kg be con-

sidered the minimum threshold? Bone Marrow Transplant

2000;26:489–96.

[8] Weisneth M, Schreiner T, Friedrich W, et al. Mobilization

and collection of allogeneic peripheral blood progenitor

cells for transplantation. Bone Marrow Transplant

1998;21:S21–4.

[9] Heimfeld S. HLA-identical stem cell transplantation: is

there an optimal CD34 cell dose? Bone Marrow Transplant

2003;31:839–45.

[10] Bensinger W, Martin P, Storer B, et al. Transplantation of

bone marrow as compared with peripheral-blood cells

from HLA-identical relatives in patients with hematologic

cancers. N Engl J Med 2001;344:175–81.

[11] Champlin RE, Schmitz N, Horowitz MM, et al. Blood

stem cells compared with bone marrow as a source of

hematopoietic cells for allogeneic transplantation. Blood

2000;95:3702–9.

[12] Ketterer N, Salles G, Raba M, et al. High CD34+ cell

counts decrease hematologic toxicity of autologous periph-

eral blood progenitor cell transplantation. Blood 1998;91:

3148–55.

[13] Mohle R, Murea S, Pforsich M, et al. Estimation of

progenitor cell yield in a leukapheresis product by previous

measurement of CD34+ cells in the peripheral blood. Vox

Sang 1996;71:90–6.

[14] Perez-Simon JA, Cabalero MD, Corral M, et al. Minimal

number of circulating CD34+ cells to ensure successful

leukapheresis and engraftment in autologous peripheral

blood progenitor cell transplantation. Transfusion 1998;38:

385–91.

[15] Malachowski ME, Comenzo RL, Hillyer CD, et al. Large-

volume leukapheresis for peripheral blood stem cell

collection in patients with hematologic malignancies.

Transfusion 1992;32:732–5.

[16] Stroncek DF, Clay ME, Smith J, et al. Comparison of two

blood cell separators in collecting peripheral blood stem

cell components. Transfus Med 1997;7:95–9.

[17] Brown RA, Adkins D, Goodnough LT, et al. Factors that

influence the collection and engraftment of allogeneic

peripheral-blood stem cells in patients with hematologic

malignancies. J Clin Onc 1997;15(9):3067–74.

[18] Bensinger W, Singer J, Appelbaum FR, et al. Autologous

transplantation with peripheral blood mononuclear cells

collected after administration of recombinant granulocyte

stimulating factor. Blood 1993;81:3158–63.

[19] Blume KG, Thomas ED. A review of autologous hema-

topoietic cell transplantation. Biol Bone Marrow Trans-

plant 2000;6:1–12.

[20] Welte K, Gabrilove J, Bronchud MH, Platzer E, Morstyn

G. Filgrastim (r-metHuG-CSF): the first 10 years. Blood

1996;88:1907–29.

[21] Carstanjen D, Ulbricht N, Iacone A, Regenfus M, Salama

A. Matrix metalloproteinase-9 (gelatinase B) is elevated

during mobilization of peripheral blood progenitor cells by

G-CSF. Transfusion 2002;42:588–96.

[22] Van Os R, van Schie M, Willemze R, Fibbe WE.

Proteolitic enzyme levels are increased during granulo-

cyte-colony-stimulating-factor-induced hematopoietic stem

cell mobilization in human donors but not predict the

number mobilized stem cells. J Hematother Stem Cell Res

2002;11(3):513–21.

[23] Pruijt JFM, Fibbe WE, Laterveer L, et al. Prevention of

interleukin-8-induced mobilization of hematopoietic pro-

genitor cells in rhesus monkeys by inhibitory antibodies

against the metalloproteinase gelatinase B (MMP-9). Proc

Nat Acad Sci 1999;96(19):10863–8.

[24] Robinson SN, Pisarev VM, Chavez JM, et al. Use of

matrix metalloproteinse (MMP)-9 knockout mice demon-

strates that MMP-9 activity is not absolutely required for

G-CSF or Flt-3 ligand-induced hematopoietic progenitor

cell mobilization or engraftment. Stem Cells 2003;21:

417–27.

[25] Janowska-Wieczorek A, Marquez LA, Nabholtz J, et al.

Growth factors and cytokines upregulate gelatinase expres-

sion in bone marrow CD34+ cells and their transmigration

D. Domanovic et al. / Transfusion and Apheresis Science 33 (2005) 37–45 45

trough reconstituted basement membrane. Blood 1999;

93(10):3379–90.

[26] Jilma B, Hergovich N, Homoncik M, et al. Granulocyte

colony-stimulating factor (G-CSF) downregulates its

receptor (CD114) on neutrophils and induces gelatinase

B release in humans. Br J Haematol 2000;111:314–20.

[27] Nadler SB, Hidalgo JU, Bloch T. Prediction of blood

volume in normal human adults. Surgery 1962;51:224–

323.

[28] Heussen C, Dowdle EB. Electrophoretic analysis of plas-

minogen activators in polyacrylamide gels containing

sodium dodecyl sulfate and copolymerized substrates.

Anal Biochem 1980;102:196–202.

[29] Rowley SD, Prather K, Bui KT, et al. Collection of

peripheral blood progenitor cells with an automated

leukapheresis system. Transfusion 1999;39:1200–6.

[30] Sutherland DR, Andreson L, Keeney M, Nayar R, Chin-

Yee I. The ISHAGE guidelines for CD34+ cell determi-

nation by flow cytometry. J Hematother 1996;5:213–26.

[31] Kozuka T, Ikeda K, Teshima T, et al. Predictive value of

circulating immature cell counts in peripheral blood for

timing of peripheral blood progenitor cell collection after

G-CSF plus chemotherapy-induced mobilization. Transfu-

sion 2002;42:1514–22.

[32] Schreiner T, Wiesneth M, Krug E, et al. Collection of

allogeneic peripheral blood progenitor cells by two proto-

cols on an apheresis system. Transfusion 1998;38:1051–5.

[33] Papayannopoulou L, Nakamoto B. Peripheralization of

hematopoietic progenitors in primates treated with anti-

VLA-4 integrin. Proc Nat Acad Sci, USA 1993;90:9374–8.

[34] Lord BI, Bronchud NH, Owens S, et al. The kinetics of

human granulopoiesis following treatment with granulo-

cyte colony—stimulating factor in vivo. Proc Nat Acad Sci

1989;86:9499–503.

[35] Humpe A, Riggert J, Meineke I, et al. A cell-kinetic model

of CD34+ cell mobilization and harvest: development of

predictive algorithm for CD34+ cell yield in PBPC

collections. Transfusion 2000;40:1363–70.