原 田 智 美
＊１野 田 美保子
＊２齋 藤 久美子
＊２古 川 照 美
＊３北 宮 千 秋
＊３木 田 和 幸
＊３木 立 るり子
＊２對 馬 栄 輝
＊２米内山 千賀子
＊２西 村 美 八
＊３倉 内 静 香
＊３大 津 美 香
＊２北 嶋 結
＊２牧 野 美 里
＊４赤 池 あらた
＊３小 枝 周 平
＊２小 池 祐 士
＊４成 田 句 生
＊５三 田 禮 造
＊６（2012 年 9 月 30 日受付，2012 年 12 月 11 日受理）
要旨：【目的】骨密度低下を予防する方法を検討するために，健常高齢者の骨密度と歩行速度の 関係を調べた。【方法】対象は青森県Ｔ町主催のことぶき大学を受講しており我々の測定に参加 した78名の高齢者（女性60名，男性18名）とし，身長，体重，BMI，骨密度及び新体力テスト の測定を実施した。【結果】骨密度の若年成人比較％の値が70％未満の骨粗鬆症の危険領域にい る者が，女性では女性全体の57％，男性は男性全体の39％を占めていた。新体力テストにおいて，
全体，男女ともにスティフネス指数と「 6 分間歩行」に有意な正の相関がみられた。【結論】こ の結果から骨密度が低い健常高齢者は骨密度が高い者に比べて歩行速度が遅いことが示唆され た。
キーワード：高齢者，骨密度，歩行速度
＊１弘前大学大学院保健学研究科健康支援科学領域 老年保健学分野（院生）
〒036‑8564 青森県弘前市本町66番地 E‑mail：[email protected]‑u.ac.jp
＊２弘前大学大学院保健学研究科健康支援科学領域 老年保健学分野
＊３弘前大学大学院保健学研究科健康支援科学領域 健康増進科学分野
＊４弘前大学大学院保健学研究科健康支援科学領域 障害保健学分野
＊５弘前医療福祉大学作業療法学科
＊６青森中央短期大学看護学科
【Original paper】
Optimum time from collection to cell separation of human
placental/umbilical cord blood for yield of mononuclear and CD34 + cells
Satoko E BINA
＊１, Masaru Y AMAGUCHI
＊２and Ikuo K ASHIWAKURA
＊２♯（Received September 28, 2012 ; Accepted December 11, 2012）
Abstract
: The aim of this study was to clarify the association between time from collection to cell separation of human placental/umbilical cord blood (CB) and the number of mononuclear cells (MNC) and CD34+ cells.
: A total of 182 CB units collected from a single hospital were analyzed in a retrospective study using perinatal data for mothers and babies.
: Statistically signifi cant correlations were observed between time and MNC counts/g of CB. When CB units were classified into <24 h and >24 h groups, the number of MNC detected in the >24 h group was markedly higher than those in the <24 h group. Whereas, the recovery rate of CD34+ cells from MNC obtained in the <24 h group was signifi cantly higher than that in the >24 h group. However, no diff erences were found for CD34+ cells.
When CB units were classifi ed into four groups of 6 h periods, no signifi cant diff erences were observed among groups.
: It is desirable to perform cell separation within 24 h after CB collection. (172 words)
Key words：umbilical cord blood; mononuclear cells; CD34+ cells.
＊１Sapporo Medical University Graduate Course in Midwifery, South2West15, Chou-ku, Sapporo, 060‑0062, JAPAN.
E‑mail: [email protected]
＊２Department of Radiological Life Sciences, Hirosaki University Graduate School of Health Sciences
♯ correspouding author
Placental/umbilical cord blood (CB) contains multipotent hematopoietic stem/progenitor cells１）, which can self-renew and differentiate into all hematopoietic lineages throughout the lifetime of an organism. CB is a promising source of these cells because the collection process is painless and non-invasive, causes no harm to the mother or newborn, and recovers a material that is usually discarded.
Accordingly, CB is being increasingly used as an alternative source of cells used in treating patients with diseases such as hematopoietic malignancies２，３）. Previous reports have shown that the clinical
outcomes of CB transplantation are generally influenced by the number of mononuclear cells (MNC) per CB unit transplanted. A reduction in this n u m b e r o f t e n c a u s e s s e r i o u s i s s u e s d u r i n g transplantation in adult primates, even though the hematopoietic stem cells present in CB have higher proliferative potential than that of bone marrow cells. Therefore, it is important to obtain and use CB units that contain a suffi cient number of MNC３，４−10）. A white paper released by the Japanese Cord Blood Bank recommends that CB processing should start within 24 h after CB collection and be applied only to samples containing >8×108 MNC11）. However, it is difficult to predict the number of nucleated/CD34+
cells per CB unit because of the extremely wide variations in individual samples.
Perinatal factors including birth weight, placental weight, gestational age, neonatal sex, the mode of birth, and the collection and length of the umbilical cord generally infl uence the cellular content of CB and the volume that can be collected12−16）. However, not much has been reported to date on the optimal time from collection of CB to cell separation. The association between time from CB collection to cell separation and the number of MNC and CD34+ cells was analyzed in a retrospective study.
This study was approved by the Committee of Medical Ethics of Hirosaki National Hospital (Hirosaki, Japan) and the Committee of Medical Ethics of Hirosaki University Graduate School of Medicine (Hirosaki, Japan). Informed consent was obtained from the mothers of all patients. During the period between January 2009 and February 2012, CB units were collected at a single hospital (Hirosaki National Hospital, Hirosaki, Japan). The criteria for inclusion were low-risk pregnancies, singleton gestations, vaginal deliveries, and neonates born without requirement of resuscitation or immediate rescue procedures. According to the guidelines of the Tokyo Cord Blood Bank, the segment of CB was double-clamped immediately after neonatal delivery and blood was obtained from the umbilical vein before placental delivery ( collection). CB was collected in a sterile collection bag that contained 28 mL citrate‒phosphate‒dextrose anticoagulant (CBC-20; Nipro, Osaka, Japan) until the fl ow ceased. A total of 301 CB units were available for cell separation within 48 h of CB collection. Relevant perinatal data including maternal age, gestational age, duration of labor, birth weight, Apgar score, and umbilical arterial pH were obtained from the pregnancy charts.
+
MNC were separated using Ficoll-Paque. Twenty milliliters of CB was diluted 2-fold with phosphate-
buff ered saline (PBS) (−) and 5 mM ethylenediamine-tetraacetic acid (EDTA; Wako Pure Chemicals, Tokyo, Japan) and layered onto 15 mL Ficoll-Paque (1.077 g/mL; Amersham Pharmacia Biotech AB, Uppsala, Sweden). The samples weighed 300 g and were centrifuged for 30 min at room temperature.
The buffy coat was harvested and diluted with EDTA-PBS solution. To remove platelets, cells were washed twice with EDTA-PBS and centrifuged at 100×g for 10 min at 4°C. The cells were then resuspended in EDTA-PBS at 4°C and counted using Türk solution. Following the manufacturerʼs instructions, magnetic cell sorting (Miltenyi Biotec, Germany) was used for positive selection of CD34+ cells. Following the procedure, the proportion of C D 3 4+ c e l l s r e c o v e r e d f r o m t h e M N C w a s approximately 0.1%‑0.6% and the purity, as determined by flow cytometry, was 80%‑95%. Cell viability was verifi ed using the trypan blue exclusion method. CD34+ cells were not purifi ed when the CB unit contained an extremely low number of MNC (<8
× 107 cells) because of poor recovery.
After normality tests, data were also analyzed by univariate analysis using the Mann‒Whitney -test, Kruskal‒Wallis test, and Spearman rank correlation coeffi cient depending on the distribution pattern of the data. Descriptive statistics are presented as arithmetic median (range). Statistical analysis was performed using SPSS 16.0 (SPSS Japan, Inc., Tokyo, Japan), and Origin (Origin Lab, Northampton, MA, USA) for Windows. < 0.05 was considered signifi cant.
Of 301 CB units collected, 182 were used for analysis. Cases with gestational ages of <37 and 侒42 weeks, Apgar scores at 1 min of <7, or unknown or missing data for these obstetric factors, caused by delay or no cell separation, were also excluded. The m a t e r n a l a n d n e o n a t a l c h a r a c t e r i s t i c s a r e summarized in Table 1. The characteristics of all CB units and the number of MNC and CD34+ cells are shown in Table 2. The median net weight of CB units was 47.4 g ranging from 10.1 g to 118.7 g. The median values of total MNC and CD34+ cells were
1.48 × 108 and 1.03 × 106 cells, respectively. The median recovery percentage was 0.6% ranging from 0.1% to 5.3%. When a univariate analysis was performed to determine the association between net CB weight and the total number of MNC and CD34+ cells, statistically significant correlations were observed between net CB weight and total MNC count (r = 0.70, < 0.001) and total CD34+ cell count (r = 0.24, < 0.01; data not shown). Given that the total number of MNC and CD34+ cells were aff ected by CB volume as reported previously17，18）, the number of MNC and CD34+ cells per g of CB were also evaluated (Table 2). These values were 3.52 × 106 cells and 2.13 × 104 cells, respectively. However, no significant correlation was observed between these values and the maternal/neonatal factors except for umbilical arterial pH (data not shown).
These findings were consistent with our previous f i n d i n g s1 7 ，1 8 ） a n d t h o s e o f o t h e r g r o u p s1 9 ，2 0 ）, confi rming that the CB samples used in the present study were appropriate samples for investigation.
Next, the time from CB collection to cell separation for 182 CB units was analyzed. The median time was 785 min, ranging from 80 to 2813
min. Significant correlations between the time and the number of MNC contained per g of CB (Fig.
1‑A) were observed. In contrast, no correlations were found for CD34+ cell counts per g of CB and the recovery rate of CD34+ cells from MNC (Fig.
1‑B, C). When CB units were classifi ed by 24 h (284 min) intervals between collection and cell separation, 151 units (83.0%) fell into the <24 h group and 31 units (17.0%) into the >24 h group. The number of MNC detected in the >24 h group was signifi cantly higher, but the recovery rate significantly lower, than those in the <24 h group (Fig. 2). However, no significant differences were found for CD34+ cells.
With respect to these differences, the white blood cell population in CB is composed primarily of mature granulocytes that generally account for approximately 55%‑65% of the total nucleated cell (TNC) content21，22） and the remaining MNC population in CB (35%‑45% of TNC) includes monocytes, lymphocytes, and CD34+ and CD34− hematopoietic stem/progenitor cells23）. In peripheral blood or CB in vivo, granulocytes are “programmed”
to die by apoptosis and generally do so within 12‑24 h after collection in vitro24−26）. In contrast to mature Factors n (%) Median (range)
Maternal
Age(years) 30 (16―43)
Nulliparous 103 (56.6)
Gestational age (week) 39 (37―41) Total duration of labor (min) 459 (46―3011) Neonatal
Birth weight (g) 3064 (2038―4100)
Male 90 (49.5)
Umbilical arterial pH 7.32 (7.02―7.51) Umbilical arterial Base excess ‑3 (‑14―7) Placental weight (g) 550 (385―1200) Cord length (cm) 57.5 (35―90)
Table 1 Maternal and neonatal characteristics of 182 deliveries
Factors N Median (range) Net weight of CB (g) 182 47.4 (10.1―118.7) Total MNC (×106) 182 148.0 (3.15―705.0) MNC / g (×106) 182 3.52 (0.135―11.53) Total CD34＋ cells (×104) 132 102.6 (15.0―10000) CD34＋ cells / g (×104) 132 2.13 (0.36―13.26) Recovery percentage 132 0.59 (0.11―5.26)
Table 2 CB volumes and numbers of total MNC and CD34＋ cells
granulocytes, engrafting MNCs are not programmed for “self-destruction” by apoptosis . Although the precise reason for the differences between the two cell types is not clear from the present results alone, the aforementioned points may provide an explanation. In addition, Solomon et al. recently reported a time- and temperature-dependent decrease in viability of granulocytes, monocytes, lymphocytes, and CD34+ cells in CB at room temperature27）. Further studies are needed to clarify the effect of temperature before cell separation, because we did not consider temperature in the
present analysis. However, all 151 CB units were classifi ed into four groups of 6 h intervals: 48 units (31.8%) in <6 h; 26 units (17.2%) from 6 h to <12 h; 44 units (29.1%) from 12 h to <18 h, and 23 units (15.2%) in >24 h. No significant differences were observed among the four groups (Fig 3).
In conclusion, the present study demonstrated that the number of MNC detected per g of CB is dependent on the time from CB collection to cell separation, indicating that it is desirable to perform cell separation within 24 h after CB collection.
0 720 1440 2160 2880
20 40 60 80 100 120
0 720 1440 2160 2880
2 4 6 8 10 12 14
0 720 1440 2160 2880
0 2 4 6
㼇㻯㼉 㼇㻮㼉
r=0.220 P=0.003
Mononuclear cells/g(x105 )
Time (min) 㼇㻭㼉
r=-0.082 P=0.351
CD34+ cells/g(x104 )
Time (min)
r=-0.176 P=0.043
Recovery percentage (%)
Time (min)
Figure 1 Correlation between time from CB collection to cell separation and the number of MNC and CD34+ cells contained per g of CB. A positive correlation is seen between time and number of MNC (n = 182) [A]. While, no correlations were found for CD34+ cell counts per g of CB (n = 132) [B] and the recovery rate of CD34+ cells from MNC (n = 132) [C].
0 20 40 60 80 100
120 P=0.006
>24 h
<24 h Mononuclear cells/g(x105 )
0 2 4 6 8 10 12 14
16 P=0.357
>24 h
<24 h CD34+ cells/g(x104 )
0 1 2 3 4 5 6 㼇㻮㼉 㼇㻯㼉
㼇㻭㼉
P=0.047
>24 h
<24 h
Recovery percentage(%)
Figure 2 Distribution of the number of MNC and CD34+ cells per g of CB and the recovery rate of CD34+ cells from MNC observed in the <24 and 24‒48 h groups. The number of MNC detected in the >24 h group (n = 31) is signifi cantly higher than that in the <24 h group (n = 151) [A]. However, the recovery rate in the <24 h group (n = 108) is signifi cantly higher than that in the >24 h group (n
= 24) [C]. No signifi cant diff erence was observed for CD34+ cells [B].
We are indebted to Dr. Ayako Tarakida, Dr.
Tomoka Ogasawara, Dr. Mami Manabe, Dr. Sei-ichi Katagiri and Dr. Takashi Ozaki of the Hirosaki National Hospital for collecting the cord blood units.
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