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Differences in lymphocyte subpopulation count and function in cord, maternal and adult blood / Kordon kani, maternal ve eriskin kanda sayisal ve fonksiyonel lenfosit alt grup farkliliklari.

Abstract

Objective: Phenotypical characterization and functional activity of lymphocytes and natural killer (NK) cells in cord blood (CB) were investigated, and maternal peripheral blood (MPB) values were compared to those of adult peripheral blood (APB) (control).

Materials and Methods: To determine cytotoxic activity target cells (K562) were labeled with carboxy-fluorescein diacetate (CFDA) or fluorescein isothiocyanate (FITC), and propidium iodide (PI) was used to label dead cells. Cell surface expression in CB, APB, and MPB cells were analyzed using flow cytometry.

Results: CB and MPB mononuclear cells had similar CD45, CD34, CD4, and surface molecule for T helper cell expression, but had low-level expression of total T-lymphocyte surface molecules CD3 and CD8. CD19 and HLA-DR expression was higher in CB than in MPB. The same high-level of expression for CD19 and HLA-DR was observed in APB, as compared to MPB. All other cell surface expressions were similar in APB and MPB samples. NK (CD[16.sup.+] and CD[56.sup.+]+) cells in CB was similar to that in MPB and APB, and the level of inhibitory KIR receptors in NK cells was higher in venous CB than in MPB and APB. The only difference between MPB and APB was that the CD158a level was higher in MPB. No difference was observed in NK cells in CB and MPB, in terms of cytotoxicity.

Conclusion: The present results show that there was numerical and proportional variability of lymphocytes and their subgroups in CB and APB, but no cytological difference. (Turk J Hematol 2011; 28: 33-41)

Key words: NK activity, cord blood lymphocytes, flow cytometry

Received: September 10, 2009

Accepted: April 30, 2010

Ozet

Amac: Kordon kanindaki lenfosit ve NK hucrelerinin fonksiyonel aktivitesi ve fenotipik karakterizas-yonu arastirilmis, anne kan degerleri (MPB) kontrol olarak eriskin periferik kan (APB) degerleri ile karsilastirilmistir.

Yontem ve Gerecler: Sitotoksik aktivite icin hedef hucreler (K562) karboksifloresandiasetat (CFDA) veya floresanizotiyosiyanat (FITC) ile isaretlenmis, olu hucreler ise propidiumiyodid (PI) ile saptan-mistir. Kordon, anne ve eriskin periferik kan hucre yuzey ifadeleri flow sitometri ile analiz edilmistir.

Bulgular: Kordon kani CD45, CD34 ve CD4 ifadesi acisindan maternal periferik kana benzerlik gosterirken, CD3 ve CD8 ifadesi dusuk, CD19 ve HLA-DR ifadeleri ise kordon kaninda maternal kana gore yuksek saptanmistir. Maternal kana gore CD19 ve HLA-DR agisindan benzer yuksek ifade eriskin kanda da gozlenmis, diger tum yuzey ifadeleri eriskin ve maternal orneklerde benzer olarak saptanmistir. Kordon kani NK (CD16+CD56+) hucreleri maternal ve eriskin kana benzerlik gosterirken, NK hucre yuzeyinde aktivator CD 161 ve inhibitor anti-Hu-KIR ifadesi hem maternal hem de eriskin kana gore, CD 158a ifadesi ise sadece eriskin kana gore yuksek saptanmistir. Eriskin ve maternal kan karsilastirildiginda sadece CD 158a ifadesi maternal kanda yuksek olarak bulun-mustur. Kordon, eriskin ve maternal kan orneklerinde NK sitotoksisitesi acisindan farklilik gozlen-memistir.

Sonuc: Bulgulanmiz lenfosit ve altgruplari acisindan kordon kani ile yetiskin immun sistem arasinda sayi ve fonksiyonel anlamda farkhhk oldugunu gostermekle birlikte, sitotoksik aktivite acisindan bir farkhhk olmadigim ortaya koymaktadir. (Turk J Hematol 2011; 28: 33-41)

Anahtar kelimeler: NK aktivitesi, kordon kani lenfositleri, flow sitometri

Gelis tarihi: 10 Eylul 2009

Kabul tarihi: 30 Nisan 2010

Introduction

Allogeneic cord blood transplantation (CBT), especially from unrelated donors, has become an extensively used treatment for patients with both malignant and nonmalignant disorders (1). As compared to bone marrow transplantation (BMT), the advantages of CBT include ease and safety of hematopoietic collection, low risk of viral contamination, prompt availability when an unrelated donor is used, and reductions in the incidence and severity of graft-versus-host disease (GVHD) (2), (3).

Several immunologic properties and peculiarities of cord blood lymphocytes (CBL) may be responsible for the reduction of GVHD following CBT (4). In fact, CBLs are naive and characterized by a low number of interleukin-2 (IL-2), interferon (IFN)-[gamma], and tumor necrosis factor (TNF)-[alpha] producing cells, and have been shown to produce lower quantities of proinflammatory cytokines, such as IFN-[gamma] and TNF-[alpha], and to display no or markedly reduced responsiveness to allogeneic stimuli in a secondary mixed lymphocyte reaction (MLR) (5), (6). One of the reasons for the lower incidence of GVHD may be the reduced cytotoxic potential of cord blood (CB)-derived natural killer (NK) and cytotoxic T cells, as well as reduced levels of T helper I (Thl) cytokines, which are known to take part in the GVHD mechanistic cascade (7-10). On the other hand, cytotoxic T and NK cells are the key effector cells that mediate the graft-versus-leukemia (GVL) effect, which are used clinically in adoptive cell-mediated immunotherapy to control minimal residual disease and for re-induction of remission in chronic myelogenous leukemia patients that relapse following allogeneic stem cell transplantation (alloSCT) (11-13).

Most clinical experimental data indicate that the greater the GVL affect after alloSCT, the higher the risk of developing GVHD (14), (15). Reducing the risk of GVHD after alloSCT, either by T cell depletion or by immunosuppression, is known to lead to an increase in leukemic relapse, which may indicate a decrease in the GVL effect (16), (17). As such, one of the main concerns in CBT was that the reduced incidence of GVHD observed following CBT would lead to a decrease in the GVL effect and a subsequent increase in the relapse rate. Nevertheless, it has been shown that CB-derived NK and lymphokine-activated killer cells are able to lyse non-cultured fresh leukemia blasts, readily respond to IL-2 and IL-12, and mediate relatively high levels of apoptosis-mediated cytotoxicity against target cell lines (18), (19).

Furthermore, CB is rich in unique NK cell subsets that may possess greater potential proliferative capacity than adult peripheral blood (APB) NK cells (20). NK cells in CB appear to be similar to those in APB, and these cells may actually have greater proliferative capacity when exposed to alloantigens or exogenous cytokines (8), (18), (21). This suggests that CB may have substantial GVL potential and that CB-derived NK cells may be used effectively, if properly amplified, for adoptive cell-mediated immunotherapy and amplification of the GVL effect.

The function of NK cells is important for the clearance of tumor cells, the removal of immunoglobulin-bound antigens, and the control of viral infections (22). It was reported that NK function is decreased in some patients, including those with primary immunodeficiencies, those with late-stage human immunodeficiency virus (HIV) infection, and pregnant women (23), (24). NK cell function is tightly regulated by a fine balance of inhibitory and activatory signals that are delivered by a diverse array of cell surface receptors. Killer cell Ig-like receptor (KIR) binds to HLA class I molecules on the surface of target cells, and it confers inhibitory signals to NK cells (25), (26). Upon its ligation by HLA class I molecules, KIR can deliver inhibitory signals via the immune-receptor tyrosine-based inhibitory motif. As such, NK cells can recognize cells that do not express HLA class I molecules as cytotoxic target cells, and KIR plays a role in NK cells' cytotoxic target discrimination (27), (28). Among the inhibitory receptors, some are specific for different groups of MHC class I alleles, while others are orphan receptors. In contrast, various activating receptors are involved in the triggering of NK cell-mediated natural cytotoxicity (25).

The present study analyzed activatory KIR: CD 161 (NKR-PIA), and inhibitory KIRs: CD158a (NKAT1, KIR2DL1) and anti-human KIR (NKB1). As more is learned about NK cells and their function, and more simplified and precise means to quantify their numbers and functions become available, analysis of lymphocytes from healthy human controls and patients may become a more routine practical approach in clinical trials. Moreover, low-level NK cell activity may be useful for predicting patient outcome (23); therefore, a suitable clinical assay for NK cell activity is necessary.

The role of the functional and phenotypic characteristics of CB lymphocytes and NK cell activity against K562 in CB were investigated and compared to that in maternal peripheral blood (MPB) and APB (control). Target cells were labeled with fluorescein isothiocyanate (FITC) or carboxyfluorescein diacetate (CFDA) before contact with effector cells. The red fluorescent dye propidium iodide (PI) was applied for the identification of dead cells.

Materials and Methods

Study population

CB samples (n=10) were obtained immediately postpartum from full-term, normally delivered healthy babies via cannulation of the umbilical vein and collected into heparinized tubes. Then, mono-nuclear cell fraction of CBL was obtained via Ficoll-Hypaque (Sigma Chemical Co., St Louis, MO, USA) density-gradient centrifugation. Mononuclear cell fractions of heparinized MPB (derived from healthy maternal donors [n=10]) and APB (derived from adult healthy donors [n=10]) were also separated by the same procedure described above. The viability of separated CB, MPB, and APB lymphocytes was measured using Trypan blue. The study protocol was approved by the Istanbul University Ethics Committee.

Flow cytometric analysis

Assay of cell surface markers

Mononuclear cells (2 x [10.sup.5] cells [mL.sup.-1]) were stained with anti-CD45-FITC/anti-CD14-PE, anti-CD3-FITC, anti-CD19-PE, anti-CD4-FITC, anti-CD8-PE, anti-CD16/56-PE, anti-HLA-DR-PE, anti-CD158a-FITC, anti-CD161-FITC, anti-human KIR (NKB1)-FITC, and anti-CD34-FITC (all obtained from Becton Dickinson, San Jose, USA). Stained cells were fixed in 2% paraformaldehyde. The controls were FITC-and PE-conjugated mouse IgG1 and IgG2A (Becton Dickinson, San Jose, USA). Flow cytometric analysis was performed using FACSCalibur (Becton Dickinson, San Jose, USA).

Cytotoxic activity

Target cell

The cell line K562, an NK-sensitive tumoral human erythroleukemia cell line, was used as target cells. Cells were grown in RPMI-1640 (Gibco-BRL, UK) supplemented with 10% fetal bovine serum (FBS, Dutscher, France) and cultured for 24 h before cytometric analysis.

Cell labeling

Two fluorescent dyes were initially tested for labeling target cells: FITC (Sigma Chemical Co., St Louis, MO, USA) at 50 [micro]g [mL.sup.-1] in PBS and CFDA (Sigma Chemical Co., St Louis, MO, USA) at 30 [micro]g m [L.sup.-1] in PBS. For FITC and CFDA labeling, target cells were adjusted to [10.sup.5] cells m [L.sup.-1] in PBS, incubated for 30 min at 37[degrees]C, and then rinsed 2 times with PBS. Cell viability was assessed using PI (Sigma Chemical Co., St Louis, MO, USA), which permeates only through the membrane of dead cells and emits a red fluorescence (10 [micro]L [mL.sup.-1] is the optimal concentration determined after a calibration assay).

Effector cells

CB, MPB, and APB mononuclear cells were obtained from heparinized blood via density gradient centrifugation over Ficoll (Sigma Chemical Co., St Louis, MO, USA), and were used as the source of NK effector cells. These effector cells were washed twice with RPMI-1640 medium (Sigma Chemical Co., St Louis, MO, USA) and resuspended to a final concentration of 5 x [10.sup.6] cells mL-1 for NK assay.

Cytotoxicity

Effector and target cells were placed in 12 x 75-mm round bottom polystyrene tubes (Falcon, NJ, USA) to yield effector:target ratios of 50:1, 25:1, and 12.5:1. Control tubes that included only target cells were assayed to identify spontaneous cell death. For maximum lysis (100% of death) target cells were incubated with 0.1% Triton X-100 (Sigma Chemical Co., St Louis, MO, USA). To enhance cell contact the mixture was centrifuged at 800 rpm for 1-2 min, and then incubated as a cell pellet in complete medium for 4 h at 37 [degrees]C under 5% [CO.sub.2]. After incubation, 10 44mL-1 of PI was added to each tube for detection of dead cells, and cooled for 5-10 min on ice before acquisition. The samples were gently mixed and analyzed using flow cytometry (FACSCalibur, Becton Dickinson, San Jose, USA). Forward and side scatter parameters were adjusted to accommodate the inclusion of both target and effector cells within the acquisition gate. No cells were excluded from the analysis, and 10,000 cells were counted. Data were analyzed using BD FACSCalibur with CellQuest software (BD Bioscience, San Jose, USA).

Statistical analysis

Data are expressed as mean[+ or -]standard deviation (SD). Statistical analysis was performed by Student's t test using SPSS 11.5 version

Results

Expression of cell surface molecules

To characterize the subpopulations of cells undergoing expansion cells were stained with T and NK cell-associated surface markers, and flow cytometric analysis was performed. Table 1 summarizes the percentage of lymphocyte subsets observed in CB, MPB, and APB with a viability >95%. In MPB and CB mononuclear cells total leukocyte marker CD45, and stem cell marker CD34 and CD4 (marker for helper T cells) expression were similar (13=0.068, p=0.075, p=0.059, respectively); however, CD3, total T cell marker, and CD8 (a marker for cytotoxic T cells) were significantly lower in CB (p=0.046, p=0.012, respectively, Figure 1A). Expression of the B-lymphocyte marker CD19 and activation marker HLA-DR were significantly higher in CB than in MPB (p=0.0089, p=0.0014, respectively, Figure 1A). Expression of CD 19 and HLA-DR were also significantly higher in APB than in MPB; however, there wasn't a significant difference in the expression of CD3 and CD8 between APB and MPB (p=0.019, p=0.0085, respectively).
Table 1 A. Lymphocyte subsets in CB and MPB

Surface molecules             CB            MPB         P

CD[45.sup.+]       97.2 [+ or -]  98.2 [+ or -]  p= 0.068
                             1.5            0.5

CD[3.sup.+]        63.8 [+ or -]  73.2 [+ or -]  p= 0.046
                             3.6            5.2

CD[4.sup.+]        47.0 [+ or -]  41.4 [+ or -]  p= 0.059
                             9.0            5.4

CD[8.sup.+]        23.8 [+ or -]  36.8 [+ or -]  p= 0.012
                             5.2            4.7

CD[19.sup.+]       17.6 [+ or -]   9.6 [+ or -]        p=
                             5.4            0.8    0.0089

HLA-[DR.sup.+]     19.2 [+ or -]   8.2 [+ or -]        p=
                             6.4            4.0    0.0014

CD[34.sup.+]        1.6 [+ or -]   1.0 [+ or -]  p= 0.075
                             1.1            0.4

CD[16.sup.+]       20.0 [+ or -]  18.0 [+ or -]  p= 0.094
[56.sup.+]                   6.6            6.6

CD[161.sup.+]      30.2 [+ or -]  15.6 [+ or -]        p=
                             7.0            3.0    0.0021

CD 158[a.sup.+]     8.2 [+ or -]   4.4 [+ or -]        p=
                             2.5            1.1    0.0068

Hu-KIR              5.6 [+ or -]   2.8 [+ or -]  p= 0.036
                             0.4            1.3

Lymphocyte subsets in CB, MPB, and APB. Phenotypic analysis of
lymphocytes isolated From CB, MPB, and APB. The percentage of
positive cells was determined using FACSCalibur. The results
are given as mean % [+ or -] SD

Table 1 B. Lymphocyte subsets in APB and MPB

Surface molecules            APB            MPB          P

CD[45.sup.+]       98.7 [+ or -]  98.2 [+ or -]   p= 0.073
                             0.6            0.5

CD[3.sup.+]        76.0 [+ or -]  73.2 [+ or -]   p= 0.056
                             0.8            5.2

CD[4.sup.+]        41.0 [+ or -]  41.4 [+ or -]   p= 0.078
                             6.5            5.4

CD[8.sup.+]        36.0 [+ or -]  36.8 [+ or -]   p= 0.076
                             2.0            4.7

CD[19.sup.+]       14.0 [+ or -]   9.6 [+ or -]   p= 0.019
                             4.3            0.8

HLA-[DR.sup.+]     14.7 [+ or -]   8.2 [+ or -]   p=0.0085
                             4.8            4.0

CD[34.sup.+]        1.1 [+ or -]   1.0 [+ or -]   p= 0.095
                             0.4            0.4

CD[16.sup.+]       16.0 [+ or -]  18.0 [+ or -]   p= 0.088
[56.sup.+]                   6.5            6.6

CD[161.sup.+]      14.3 [+ or -]  15.6 [+ or -]  p= 0.0965
                             3.0            3.0

CD158[a.sup.+]      2.7 [+ or -]   4.4 [+ or -]  p= 0.0046
                             0.6            1.1

Hu-KIR              3.3 [+ or -]   2.8 [+ or -]   p= 0.098
                             1.2            1.3

Lymphocyte subsets in CB, MPB, and APB. Phenotypic analysis of
lymphocytes isolated from CB, MPB, and APB. The percentage of
positive cells was determined using FACSCalibur. The results are
given as mean % [+ or -] SD


[FIGURE 1 OMITTED]

CD[16.sup.+] and CD[56.sup.+] NK cell expression was similar in CB, MPB, and APB samples; however, activatory KIR CD 161, and CD 158a, and Hu-KIR cell surface molecules, which are inhibitory KIR receptors in NK cells, were more highly expressed in venous CB than in MPB (p=0.0021, p=0.0068, p=0.036, respectively, Figure 1 B). Compared to MPB, only CD158a expression was significantly lower in APB and there wasn't a difference in the expression of CD161 and anti-Hu-KIR (p=0.0046).

Flow cytometric assay for NK cytotoxicity

The cytotoxic potential of CB, MPB, and APB was analyzed via direct cytotoxic activity in NK-sensitive K562 cells. A flow cytometry-based assay, which correlates well with the standard chromium-release assay, was used (29). Cells from CB, MPB, and APB were used as effector cells against the labeled target cells (K562) with 2 different dyes, namely CFDA and FITC. Dead cells were identified base on PI incorporation. Effector and target cells were mixed at ratios of 12.5:1, 25:1, and 50:1, and co-incubated for 4 h at 37[degrees]C under a 5%-[CO.sub.2] atmosphere; the results are shown in Table 2. Cytotoxic activity of CB, MPB, and APB increased in an effector:target ratio-dependent manner, and cytotoxicity increased as the number of effector cells increased; however, there wasn't a significant difference between CB, MPB, and APB cells, in terms of cytotoxic activity.
Table 2. The cytotoxic effect of effector cells on FITC-labeled and
CFDA-labeled K562 target cells

            12.5:1                 25:1              50:1 +

         FTTC      CFDA      FTTC      CFDA     FTTC       CFDA

APB      52.0      54.7      42.0      42.0      29.0      32.0
     [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
          3.2       6.5      1.5        4.0      3.5        5.0

MPB      54.8      56.0      40.0      42.0      30.4      29.6
     [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
          1.5       3.7       3.8       6.7       3.4       6.0

CB       54.2      52.0      39.2      38.9      27.6      27.0
     [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]  [+ or -]
          3.0       6.6       2.0       5.2       5.1       5.2

The cytotoxic effect of effector cells on FITC-labeled and
CFDA-labeled K562 target cells. NK cytotoxic activity in CB and MPB
at different effector: target ratios (12.5:1,25:1, and 50:1) is shown
as mean %[+ or -]SD


[FIGURE 2 OMITTED]

Discussion

CB T cells have been studied since it was demonstrated that recipients of related and unrelated umbilical CBT experience less acute and chronic GVHD than recipients of BMT (30). The major theory regarding the reduced immunological response of CB lymphocytes is that CB T and NK cells are naive and exhibit decreased cytokine production and are therefore not primed for activation (31).

In the present study immunophenotypic characterization of CB mononuclear cells and cytotoxic activity of NK cells in CB were investigated. The phenotypic subsets of CB lymphocytes differed significantly from those of MPB lymphocytes; In comparison to MPB cells, higher expression of B-lymphocyte surface molecules, CD19, and the activation molecule HLA-DR, and lower expression of CD3 and CD8 were observed in CB cells. Similar to CB, lower expression of CD19 and HLA-DR was also noted in APB than in MPB; however, CD3 and CD8 did not differ significantly. The observed decrease in the ratio of total T-lymphocytes was proportional to the decrease in the ratio of cytotoxic T-lymphocytes, and low CD3 expression may be considered indicative of immature T-lymphocytes during the intermediate phase. There wasn't a statistically significant difference in the expression of CD4 as a surface molecule for T helper cells between CB and MPB. In contrast to the present results, it has been reported that CB contains a higher absolute number of T, NK, and B cells than MPB. The same higher expression of NK cells was observed even in CB obtained during caesarean sections than in CB obtained during vaginal deliveries, indicating variability in NK cell numbers according to the type of delivery (32). In CB a relative excess of naive cells among T cells and in the CD[4.sup.+] and CD[8.sup.+] subsets individually were reported. Similarly, the B-lymphocyte compartment had a smaller fraction of memory IgE receptor (CD23)-positive cells, along with a higher percentage of the so-called B1 ontogenetically more primitive CD19 -E/CD[5.sup.+] B cells (33), (34). A smaller fraction of CB T cells exhibited markers of peripheral activation, such as HLA-DR, or the CCR5 subset performing tissue surveillance in the skin was absent, which may also explain the lower incidence of skin GVHD after CBT (35).

In the present study CB the NK (CD[16.sup.+]/CD[56.sup.+]) cell ratio was similar to the MPB NK cell ratio, as was previously reported for NK cells in APB and CB (36). The nature of CB NK cells remains controversial, despite the similar frequency of NK cells in CB and APB, CB NK cell dysfunction, as compared to APB and MPB was reported. In contrast, NK cells differentiated from CB stem cells exhibited comparable cytotoxicity as those from bone marrow or APB stem cells (37). Although NK cell counts in CB were higher, cytotoxic activity was reported to be lower in some studies, and CB T-lymphocytes were immature and their cytotoxic activity was insufficient. Other possible mechanistic explanations for the reduction in killing by CB NK cells include relatively higher expression of inhibitory receptor complexes, including CD94/NKG2A and/or KIR (38). This KIR is expressed in 10%-30% of CB NK cells and in the present study compared to MPB the observed higher expression of CD 161, CD158a, and anti-human-KIR in CB supports the protective role of CB in GVHD. Some studies suggest that expression of NK cells triggering receptors, including CD94, KIR (CD158a/h and CD158b/j), NKp46, and NKG2D, does not differ between CB and APB NK cells, while others reported that a higher percentage of CB NK cells express the inhibitory receptor complex of CD94/ NKG2A and CD158b/j (38), (39). Considering the role of T-lymphocytes and NK cells in the pathophysiology of GVHD, the reported lower incidence of GVHD after CBT could be due to insufficient T-lymphocyte and NK cell function (40-42).

The sources for bone marrow transplantation are bone marrow, CB, and APB. Cells necessary for short-and long-term reconstitution after transplantation are thought to exist among the mononuclear cell population expressing CD34. CD34 stem cells can be isolated from CB, bone marrow, and APB and all three sources are deemed to have sufficient capacity for reconstitution of the hematopoietic system (43). In the present study the CD34 cell ratio in CB was similar to that in MPB. Although most studies reported similar results, CD34 stem cells obtained from these sources had qualitative and quantitative differences (44), (45).

Many studies reported that CB NK cells have lower-level cytotoxic function against traditional cell lines, such as K562 and Daudi, than MPB NK cells; however, low-level CB NK cell cytotoxic function may be increased by various cytokines, such as IFN-[gamma], IL-2, IL-7, IL-12, and IL-15 individually and in combination. In CBT the existence or absence of several cytokines is considered to lower the incidence of GVHD (46), (47). In the present study NK activity was measured using flow cytometry and FITC-or CFDA-labeled K562 target cells. According to the present results, no difference in cytotoxicity was observed between MPB and CB samples, in contrast to studies that reported suppression of NK cell cytotoxicity in postpartum women (48). The same suppression pattern was observed in NK cells from premature infants, as compared to those from full-term infants (49-51), indicating that not only the number, but also the function differs according to the type of delivery.

The standard method for determining NK activity is the chromium-release assay; however, this assay is not a preferred method for use in clinical laboratories for a variety of reasons, e.g. it requires the use of radioactive chromium, which is very expensive and requires specialized handling and disposal. For these reasons a new flow cytometry-based method was developed for measuring NK cell activity. Flow cytometric assays avoid the problems associated with the use of radioactive methods, and are rapid and easier to standardize. The present results support the notion that flow cytometry could be a suitable alternative for measuring NK activity (23), (52).

In conclusion, the results of the present study suggest that there is numerical and proportional variability between lymphocytes and their sub-groups in CB and APB, although no significant difference was observed between these 2 samples, according to cytotoxic activity. The clinical relevance of these subsets for GVHD and the development of protective immunity in allogeneic CBT is substantial, and we are conducting ongoing prospective research.

Conflict of interest statement

None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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Address for Correspondence: Prof. Gunnur Deniz, Department of Immunology, Institute of Experimental Medicine, Istanbul University, Vakif Gureba Cad. Sehremini 34393 Istanbul, Turkey Phone: +90 212 414 20 97 E-mail: gdeniz@istanbul.edu.tr

Nilgun Akdeniz, Esin Aktas, Gaye Erten, Sema Bilgic, Gunnur Deniz

Department of Immunology, Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey

doi:10.5152/tjh.2011.03
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Title Annotation:Research Article
Author:Akdeniz, Nilgun; Aktas, Esin; Erten, Gaye; Bilgic, Sema; Deniz, Gunnur
Publication:Turkish Journal of Hematology
Date:Mar 1, 2011
Words:5888
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