Original Article Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood Paolo Rebulla 1, Simonetta Pupella 2, Michele Santodirocco 3, Noemi Greppi 1, Ida Villanova 4, Marina Buzzi 5, Nicola De Fazio6, Giuliano Grazzini2 for the Italian Cord Blood Platelet Gel Study Group (see Appendix 1) Blood Transfusion Centre, Foundation Ca' Granda Ospedale Maggiore Policlinico, Milan; 2Italian National Blood Centre, National Institute of Health, Rome; 3Puglia Cord Blood Bank, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo; 4Pescara Cord Blood Bank, UOSD Tissue and Biobank, Department of Haematology, Transfusion Medicine and Biotechnologies, Ospedale Civile dello Spirito Santo, Pescara; 5Emilia Romagna Cord Blood Bank, Immunohaematology and Transfusion Medicine, Policlinico S. Orsola-Malpighi, Bologna; 6Organ and Tissue Transplant Unit, Foundation Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy 1
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Background. In addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel. Materials and methods. Cord blood units collected at public banks with total nucleated cell counts 150×109/L and volume >50 mL, underwent soft centrifugation within 48 hours of collection. Platelet-rich plasma was centrifuged at high speed to obtain a CBPC with target platelet concentration of 800-1,200×109/L, which was cryopreserved, without cryoprotectant, below −40 °C. Results. During 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×109/L. Total platelet count per CBPC was 11.3±4.9×109. Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC. Discussion. This study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC. Keywords: platelet concentrate, platelet gel, umbilical cord blood, skin ulcers.
Introduction
Umbilical cord blood (CB) (also termed placental blood) collected by venipuncture of umbilical cord vessels after delivery of healthy term neonates is an established source of haematopoietic stem and progenitor cells that has been used in more than 30,000 haematopoietic stem cell transplants carried out in patients suffering from leukaemia, lymphoma, thalassaemia, immunodeficiencies and metabolic disorders. Since the development of regular CB banking programmes in the USA and in Europe in the early 1990s, about 600,000 CB units voluntarily donated
after informed consent by healthy mothers have been cryopreserved for long-term storage in more than 100 public CB banks worldwide1. Consolidated clinical experience shows that the clinical outcome of CB transplantation is significantly and positively associated with the number of nucleated cells contained in the CB unit (a proxy of haematopoietic stem cell content)2. Moreover, recent reports from organisations in charge of managing CB unit requests by transplant centres show that CB units containing fewer than 1.5×109 nucleated cells are infrequently selected and used for clinical transplants3. Following the
Blood Transfus 2016; 14: 73-9 DOI 10.2450/2015.0122-15 © SIMTI Servizi Srl
All rights reserved - For personal use only No other use without premission
73
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standardising the routine production of cryopreserved CBPC from CB units not fulfilling the criteria for banking for haematopoietic transplant purposes but otherwise potentially usable for other therapeutic applications as compliant with negative donor medical history, infectious markers screening and additional criteria (see below). Eleven additional ITCBN banks later agreed to participate in a national project. After a pilot exercise carried out during January-October 2013, routine CBPC production was started on November 1st, 2013. Table I reports the criteria adopted for the selection of CB units to be processed into CBPC. Meanwhile, an inquiry into the appropriate classification of CBPC and CBPG (as blood components or as blood derivatives) was submitted to the Committee for Experimentation in Phase I Clinical Trials of the Italian National Institute of Health (Istituto Superiore di Sanità) which determined that allogeneic CBPC and CBPG, in analogy to similar products obtained from adult peripheral blood, belong to the category of blood components. The Italian National Health Council, which is the scientific counselling board of the Minister of Health (Consiglio Superiore di Sanità), released the same opinion under the regulatory point of view.
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publication of the above evidences and in consideration of the high cost of cryogenic space, several public banks, including those forming the Italian Cord Blood Network (ITCBN), elected to exclude from banking for haematopoietic transplant purposes those CB units containing less than 1.5×109 nucleated cells, which represent more than 80% of CB collections4,5. Although justified on grounds of clinical evidence and economic evaluations, this decision has the potential to discourage mothers in their generous attitude towards altruistic CB donation for public use. The above considerations and the presence of several potentially valuable biological materials in CB prompted the development of investigations to identify new blood components that could be obtained from routine CB collections. A procedure for the preparation of allogeneic CB platelet gel (CBPG) suitable for tissue regenerative purposes was developed at the Milan Cord Blood Bank and the Blood Transfusion Service of the Policlinico Hospital in Milan, Italy6, and a patent for the preparation of a "Platelet Fraction Deriving From Placental Blood" (US patent n. 8,501,170 B2) was granted to the Policlinico Hospital on August 6th, 2013. This article describes the outcomes of a national programme developed to standardise the production of cryopreserved CB platelet concentrates (CBPC) suitable for future preparation of clinical grade CBPG by the public CB banks belonging to the ITCBN.
Materials and methods
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Preparation and quality control of cord blood platelet concentrates CB units were collected after the mothers' informed consent into plastic bags containing 25-30 mL of citratephosphate-dextrose anticoagulant by trained midwives, before and after placental delivery in natural deliveries and in Caesarean sections respectively, according to locally validated standard operating procedures. After storage and transportation at monitored room temperature to the CB banks, the units were processed within 48 hours of collection. The participating banks were allowed to use locally available centrifuges and bags of convenient size and nominal volume that they considered appropriate for this protocol. Moreover, in agreement with the platelet concentration defined by the Italian Society of
©
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The Italian Cord Blood Network The ITCBN comprises 19 public CB banks accredited by the health authorities of the Italian Regions. Among them, the CB banks located in Bologna, Milan, Pavia and Treviso have also been accredited by the Foundation for the Accreditation of Cellular Therapy (FACT). A detailed report of activities of the ITCBN is available at the website of the Italian National Blood Centre5. Briefly, during 2014 the 19 CB banks belonging to the ITCBN collected 19,459 CB units for allogeneic non-family-related use in 320 delivery rooms. Of them, 1,738 (8.9%) were added to the national inventory of cryopreserved CB units stored for allogeneic haematopoietic transplantation, which totalled 38,437 CB units on December 31, 2014. Starting from 1993 (date of the onset of the first public CB banking programme in Italy) until December 31, 2014, 1,352 CB units from the ITCBN inventory have been distributed for allogeneic non-family-related haematopoietic transplants worldwide. The Italian Cord Blood Platelet Gel project In 2011, the public CB banks located in Milan and Pavia developed a research project aimed at
Table I - Criteria adopted for the selection of cord blood (CB) units to be processed into CB platelet concentrates (CBPC). -
- - - - -
Maternal informed consent to CB use for the preparation of CBPC and CBPG if CB unit does not match the criteria for inclusion in the cryopreserved inventory for haematopoietic transplant. Negative medical history of the CB donor. Total nucleated cell count 50 mL. Platelet count >150×109/L. Negative CB donor screening for blood transmissible infections (hepatitis B virus, hepatitis C virus, human immunodeficiency virus, syphilis, human T-lymphotropic virus I/II).
Blood Transfus 2016; 14: 73-9 DOI 10.2450/2015.0122-15 74
All rights reserved - For personal use only No other use without premission
Allogeneic cord blood platelet concentrate
a mechanical freezer at a temperature below −40 °C in view of future clinical use of the CBPG, which requires thawing at 37 °C in a water-bath before activation. The main characteristics of the disposable bags used for the preparation of CBPC by the 13 CB banks are shown in Table II. PPP was used for the detection of bacteria and fungi, according to standard blood component culture procedures.
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Quality assurance A common Excel database was developed and distributed to the banks to collect detailed information (weight and complete blood counts) on each CB unit before processing, the PRP, the PPP and the CBPC before cryopreservation. Automated internal controls checked the coherence, expressed as percent recovery, between the sums of cell counts (platelets, white blood cells, red blood cells, haemoglobin) and volumes in the recovered fractions and the total cell counts and volume in the original CB unit. A common bleeding list was used by all the participating banks to report information relevant for the validation of clinical grade CBPC and CBPG units and for national haemovigilance purposes to the Italian
Se
Transfusion Medicine for platelet gel obtained from adult peripheral blood7, it was decided to define a mean target platelet concentration in the CBPC of 1,000×109/L (range, 800-1,200×109/L). During pilot optimisation studies at each bank, a common protocol was defined which included an initial centrifugation of CB at 200-210 g×10-15 minutes, followed by transfer of the platelet-rich plasma (PRP) into a secondary bag, centrifugation of the PRP at 1,800-2,600 g×15 minutes and removal of the supernatant platelet-poor plasma (PPP) in excess of the final target volume of the CBPC. The latter was defined by an automated algorithm performed by an Excel spreadsheet used for data collection (Microsoft Corp., Redmond, WA, USA), which takes into account the platelet concentration in the PRP and the minimum (800×109/L) and maximum (1,200×109/L) values of the platelet concentration expected in the final CBPC. The platelet concentrations in the PRP divided by 800×109/L and by 1,200×109/L provided the upper and lower bounds of the target CBPC volume, respectively. The final volume was set as (upper + lower bounds)/2, by determining the net weight of the CBPC on an electronic scale. The CBPC units were finally transferred into a storage bag and cryopreserved without cryoprotectant in
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Table II - Number of CBPC prepared from November 2013 to December 2014 by each CB bank participating in this study (total 1,080), bag manufacturer, code and volume of anticoagulant in the primary bags used for CB collection, manufacturer and code of the bags used for the production of CBPC. Bank (n. of CBPC)
C (129) D (112) E (89)
CBPC preparation
CBPC cryopreservation
FK: P4208
MA: MSC1202PU (29, CPD)
MA: VQX0001XU FK: A3AB0010 FE: R4R2004
FK: A3AB0010 B: PRPS B: PRPS
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B (152)
CB collection (mL of anticoagulant) J: 811-1010 (30, CPDA1)
MA: MSC1201PU (29, CPD) MA: MSC1205DU (29, CPD)
©
A (180)
Type of bag used for
B: PRPS
FK: A3AB0020 FK: A3AB0010 MA: VRT0000XU
B: PRPS
FK: P4208
B: PRPS
F (71)
FK: T4029 (30, CPD) J: 811-1010 (30, CPDA1) J: 811-1010 (30, CPD)
G (70)
FK: T4029 (30, CPD)
J: 814-0132
B: PRPS
H (64)
MA: MSC1201DU (29, CPD)
FK: A3CA0060
I (61)
T: BBT015CM FK: A3AB0030
M (47)
G: 722270 (25, CPD) MA: MSC1206DU (29, CPD) FK: T4029 (30, CPD) FK: T4029 (30, CPD) MA: MSC1201DU (29, CPD) MA: MSC1201DU (29, CPD)
MA: GSR 1000 AU B: PRPS B: PRPS
N (30) O (17)
L (58)
B: PRPS
B: CBB150
B: PRPS
MA: VSE2001XR
B: PRPS
FK: T4029 (30, CPD)
FK: P4208
B: PRPS
J: 811-1010 (30, CPDA1)
T: BBT015CM
B: PRPS
CB: cord blood; CBPC: CB platelet concentrates; B: Biomed Device, Modena, Italy; FK: Fresenius Kabi, Bad Homburg, Germany; FE: Fenwal, Mont Saint Guibert, Belgium; G: Grifols, Sant Cugat del Vallès, Spain; J: JMS, Singapore; MA: MacoPharma, Turcoing, France; T: TerumoBCT, Lakewood, CO, USA.
Blood Transfus 2016; 14: 73-9 DOI 10.2450/2015.0122-15 All rights reserved - For personal use only No other use without premission
75
Rebulla P et al Table III - Volume, total platelet count, total white blood cell (WBC) count, total red blood cell (RBC) count, and haemoglobin (Hb) content of cord blood (CB) units before processing, of platelet rich plasma (PRP) obtained after the first centrifugation and of the final CBPC before cryopreservation. Unit/Fraction
Volume (mL)
Platelets (109)
WBC (106)
RBC (109)
Hb (g)
CB unit
97.6±20.2
23.4±6.8
956.8 (535.9-1430.2)
305.5 (195.1-463.5)
7.4±2.1
PRP % recovery from CB
34.7±9.8 n.d.
14.3±5.8 59.7±19.3
12.7 (2.7-151.5) 1.4 (0.3-13.2)
0.8 (0-3.0) 0.3 (0-0.9)
n.d.
CBPC % recovery from PRP % recovery from CB
11.4±4.4 n.d. n.d.
11.3±4.9 79.2±21.7 47.7±17.8
7.2 (1.0-105.7) 67.5 (10.6-144.8) 0.8 (0.1-12.0)
0.6 (0.2-2.4) n.d. 0.2 (0.1-0.7)
n.d.
Data are given as mean ± standard deviation (volume, platelets, Hb) or median (5th-95th percentiles) (WBC, RBC). CBPC: CB platelet concentrates; n.d.: not determined.
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Discussion
Platelet gel from adult peripheral blood is a standard blood component that has been used for therapeutic purposes for many years, primarily as an autologous biological product for the treatment of skin ulcers and bedsores8-11 and other conditions12,13. Autologous platelet gel is generally considered microbiologically safer than allogeneic donor blood with regard to the risk of acquiring microbial and viral transmissible
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Statistical analysis Data showing a normal distribution are presented as mean±SD, those diverting from a normal distribution as median, 5 th and 95 th percentiles. The statistical significance of differences in volume and platelet content in CB units and in CBPC prepared by the different banks was evaluated by analysis of variance (ANOVA).
An evaluation carried out in one bank over the period from January to June 2014 showed that about one third of CB units originally donated for haematopoietic transplant purposes complied with the selection criteria listed in Table I for the production of CBPC. Table V reports costs incurred at one bank for the production of one CBPC (€ 160.92). On average, banks reported that about 2 hours were needed for one technician to prepare four units of CBPC.
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National Blood Centre, including manufacturer, code and lot number of kits used for microbiological screening tests carried out on blood samples collected from the mothers of neonates from whom the CB units were collected, according to standard CB banking procedures.
Results
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Between November 2013 and December 2014, the 13 banks produced 1,080 CBPC. The mean±SD time from CB collection to the start of processing into CBPC was 31±12 hours. Quality control data from the 1,080 CBPC are shown in Table III. It can be seen that the CB units used for the production of CBPC had a mean±SD volume (including anticoagulant) of 97.6±20.2 mL. The PPP fractions obtained after centrifugation of the PRP had a mean±SD volume of 19.6±9.8 mL and median total platelet count of 0.13×109 platelets (5th-95th percentiles, 0-1.54). The 1,080 CBPC had a mean ± SD volume of 11.4±4.4 mL, platelet concentration of 1,003±229×109/L and total platelet count of 11.3±4.9×109 platelets. Platelet recovery from CB was 47.7±17.8%. The median white cell count per CBPC was 7.2×106 (5th-95th percentiles, 1.0-105.7). The ANOVA showed highly statistically significant differences (p
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Background. In addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel. Materials and methods. Cord blood units collected at public banks with total nucleated cell counts 150×109/L and volume >50 mL, underwent soft centrifugation within 48 hours of collection. Platelet-rich plasma was centrifuged at high speed to obtain a CBPC with target platelet concentration of 800-1,200×109/L, which was cryopreserved, without cryoprotectant, below −40 °C. Results. During 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×109/L. Total platelet count per CBPC was 11.3±4.9×109. Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC. Discussion. This study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC. Keywords: platelet concentrate, platelet gel, umbilical cord blood, skin ulcers.
Introduction
Umbilical cord blood (CB) (also termed placental blood) collected by venipuncture of umbilical cord vessels after delivery of healthy term neonates is an established source of haematopoietic stem and progenitor cells that has been used in more than 30,000 haematopoietic stem cell transplants carried out in patients suffering from leukaemia, lymphoma, thalassaemia, immunodeficiencies and metabolic disorders. Since the development of regular CB banking programmes in the USA and in Europe in the early 1990s, about 600,000 CB units voluntarily donated
after informed consent by healthy mothers have been cryopreserved for long-term storage in more than 100 public CB banks worldwide1. Consolidated clinical experience shows that the clinical outcome of CB transplantation is significantly and positively associated with the number of nucleated cells contained in the CB unit (a proxy of haematopoietic stem cell content)2. Moreover, recent reports from organisations in charge of managing CB unit requests by transplant centres show that CB units containing fewer than 1.5×109 nucleated cells are infrequently selected and used for clinical transplants3. Following the
Blood Transfus 2016; 14: 73-9 DOI 10.2450/2015.0122-15 © SIMTI Servizi Srl
All rights reserved - For personal use only No other use without premission
73
Rebulla P et al
iz
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standardising the routine production of cryopreserved CBPC from CB units not fulfilling the criteria for banking for haematopoietic transplant purposes but otherwise potentially usable for other therapeutic applications as compliant with negative donor medical history, infectious markers screening and additional criteria (see below). Eleven additional ITCBN banks later agreed to participate in a national project. After a pilot exercise carried out during January-October 2013, routine CBPC production was started on November 1st, 2013. Table I reports the criteria adopted for the selection of CB units to be processed into CBPC. Meanwhile, an inquiry into the appropriate classification of CBPC and CBPG (as blood components or as blood derivatives) was submitted to the Committee for Experimentation in Phase I Clinical Trials of the Italian National Institute of Health (Istituto Superiore di Sanità) which determined that allogeneic CBPC and CBPG, in analogy to similar products obtained from adult peripheral blood, belong to the category of blood components. The Italian National Health Council, which is the scientific counselling board of the Minister of Health (Consiglio Superiore di Sanità), released the same opinion under the regulatory point of view.
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publication of the above evidences and in consideration of the high cost of cryogenic space, several public banks, including those forming the Italian Cord Blood Network (ITCBN), elected to exclude from banking for haematopoietic transplant purposes those CB units containing less than 1.5×109 nucleated cells, which represent more than 80% of CB collections4,5. Although justified on grounds of clinical evidence and economic evaluations, this decision has the potential to discourage mothers in their generous attitude towards altruistic CB donation for public use. The above considerations and the presence of several potentially valuable biological materials in CB prompted the development of investigations to identify new blood components that could be obtained from routine CB collections. A procedure for the preparation of allogeneic CB platelet gel (CBPG) suitable for tissue regenerative purposes was developed at the Milan Cord Blood Bank and the Blood Transfusion Service of the Policlinico Hospital in Milan, Italy6, and a patent for the preparation of a "Platelet Fraction Deriving From Placental Blood" (US patent n. 8,501,170 B2) was granted to the Policlinico Hospital on August 6th, 2013. This article describes the outcomes of a national programme developed to standardise the production of cryopreserved CB platelet concentrates (CBPC) suitable for future preparation of clinical grade CBPG by the public CB banks belonging to the ITCBN.
Materials and methods
TI
Se
Preparation and quality control of cord blood platelet concentrates CB units were collected after the mothers' informed consent into plastic bags containing 25-30 mL of citratephosphate-dextrose anticoagulant by trained midwives, before and after placental delivery in natural deliveries and in Caesarean sections respectively, according to locally validated standard operating procedures. After storage and transportation at monitored room temperature to the CB banks, the units were processed within 48 hours of collection. The participating banks were allowed to use locally available centrifuges and bags of convenient size and nominal volume that they considered appropriate for this protocol. Moreover, in agreement with the platelet concentration defined by the Italian Society of
©
SI M
The Italian Cord Blood Network The ITCBN comprises 19 public CB banks accredited by the health authorities of the Italian Regions. Among them, the CB banks located in Bologna, Milan, Pavia and Treviso have also been accredited by the Foundation for the Accreditation of Cellular Therapy (FACT). A detailed report of activities of the ITCBN is available at the website of the Italian National Blood Centre5. Briefly, during 2014 the 19 CB banks belonging to the ITCBN collected 19,459 CB units for allogeneic non-family-related use in 320 delivery rooms. Of them, 1,738 (8.9%) were added to the national inventory of cryopreserved CB units stored for allogeneic haematopoietic transplantation, which totalled 38,437 CB units on December 31, 2014. Starting from 1993 (date of the onset of the first public CB banking programme in Italy) until December 31, 2014, 1,352 CB units from the ITCBN inventory have been distributed for allogeneic non-family-related haematopoietic transplants worldwide. The Italian Cord Blood Platelet Gel project In 2011, the public CB banks located in Milan and Pavia developed a research project aimed at
Table I - Criteria adopted for the selection of cord blood (CB) units to be processed into CB platelet concentrates (CBPC). -
- - - - -
Maternal informed consent to CB use for the preparation of CBPC and CBPG if CB unit does not match the criteria for inclusion in the cryopreserved inventory for haematopoietic transplant. Negative medical history of the CB donor. Total nucleated cell count 50 mL. Platelet count >150×109/L. Negative CB donor screening for blood transmissible infections (hepatitis B virus, hepatitis C virus, human immunodeficiency virus, syphilis, human T-lymphotropic virus I/II).
Blood Transfus 2016; 14: 73-9 DOI 10.2450/2015.0122-15 74
All rights reserved - For personal use only No other use without premission
Allogeneic cord blood platelet concentrate
a mechanical freezer at a temperature below −40 °C in view of future clinical use of the CBPG, which requires thawing at 37 °C in a water-bath before activation. The main characteristics of the disposable bags used for the preparation of CBPC by the 13 CB banks are shown in Table II. PPP was used for the detection of bacteria and fungi, according to standard blood component culture procedures.
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Quality assurance A common Excel database was developed and distributed to the banks to collect detailed information (weight and complete blood counts) on each CB unit before processing, the PRP, the PPP and the CBPC before cryopreservation. Automated internal controls checked the coherence, expressed as percent recovery, between the sums of cell counts (platelets, white blood cells, red blood cells, haemoglobin) and volumes in the recovered fractions and the total cell counts and volume in the original CB unit. A common bleeding list was used by all the participating banks to report information relevant for the validation of clinical grade CBPC and CBPG units and for national haemovigilance purposes to the Italian
Se
Transfusion Medicine for platelet gel obtained from adult peripheral blood7, it was decided to define a mean target platelet concentration in the CBPC of 1,000×109/L (range, 800-1,200×109/L). During pilot optimisation studies at each bank, a common protocol was defined which included an initial centrifugation of CB at 200-210 g×10-15 minutes, followed by transfer of the platelet-rich plasma (PRP) into a secondary bag, centrifugation of the PRP at 1,800-2,600 g×15 minutes and removal of the supernatant platelet-poor plasma (PPP) in excess of the final target volume of the CBPC. The latter was defined by an automated algorithm performed by an Excel spreadsheet used for data collection (Microsoft Corp., Redmond, WA, USA), which takes into account the platelet concentration in the PRP and the minimum (800×109/L) and maximum (1,200×109/L) values of the platelet concentration expected in the final CBPC. The platelet concentrations in the PRP divided by 800×109/L and by 1,200×109/L provided the upper and lower bounds of the target CBPC volume, respectively. The final volume was set as (upper + lower bounds)/2, by determining the net weight of the CBPC on an electronic scale. The CBPC units were finally transferred into a storage bag and cryopreserved without cryoprotectant in
TI
Table II - Number of CBPC prepared from November 2013 to December 2014 by each CB bank participating in this study (total 1,080), bag manufacturer, code and volume of anticoagulant in the primary bags used for CB collection, manufacturer and code of the bags used for the production of CBPC. Bank (n. of CBPC)
C (129) D (112) E (89)
CBPC preparation
CBPC cryopreservation
FK: P4208
MA: MSC1202PU (29, CPD)
MA: VQX0001XU FK: A3AB0010 FE: R4R2004
FK: A3AB0010 B: PRPS B: PRPS
SI M
B (152)
CB collection (mL of anticoagulant) J: 811-1010 (30, CPDA1)
MA: MSC1201PU (29, CPD) MA: MSC1205DU (29, CPD)
©
A (180)
Type of bag used for
B: PRPS
FK: A3AB0020 FK: A3AB0010 MA: VRT0000XU
B: PRPS
FK: P4208
B: PRPS
F (71)
FK: T4029 (30, CPD) J: 811-1010 (30, CPDA1) J: 811-1010 (30, CPD)
G (70)
FK: T4029 (30, CPD)
J: 814-0132
B: PRPS
H (64)
MA: MSC1201DU (29, CPD)
FK: A3CA0060
I (61)
T: BBT015CM FK: A3AB0030
M (47)
G: 722270 (25, CPD) MA: MSC1206DU (29, CPD) FK: T4029 (30, CPD) FK: T4029 (30, CPD) MA: MSC1201DU (29, CPD) MA: MSC1201DU (29, CPD)
MA: GSR 1000 AU B: PRPS B: PRPS
N (30) O (17)
L (58)
B: PRPS
B: CBB150
B: PRPS
MA: VSE2001XR
B: PRPS
FK: T4029 (30, CPD)
FK: P4208
B: PRPS
J: 811-1010 (30, CPDA1)
T: BBT015CM
B: PRPS
CB: cord blood; CBPC: CB platelet concentrates; B: Biomed Device, Modena, Italy; FK: Fresenius Kabi, Bad Homburg, Germany; FE: Fenwal, Mont Saint Guibert, Belgium; G: Grifols, Sant Cugat del Vallès, Spain; J: JMS, Singapore; MA: MacoPharma, Turcoing, France; T: TerumoBCT, Lakewood, CO, USA.
Blood Transfus 2016; 14: 73-9 DOI 10.2450/2015.0122-15 All rights reserved - For personal use only No other use without premission
75
Rebulla P et al Table III - Volume, total platelet count, total white blood cell (WBC) count, total red blood cell (RBC) count, and haemoglobin (Hb) content of cord blood (CB) units before processing, of platelet rich plasma (PRP) obtained after the first centrifugation and of the final CBPC before cryopreservation. Unit/Fraction
Volume (mL)
Platelets (109)
WBC (106)
RBC (109)
Hb (g)
CB unit
97.6±20.2
23.4±6.8
956.8 (535.9-1430.2)
305.5 (195.1-463.5)
7.4±2.1
PRP % recovery from CB
34.7±9.8 n.d.
14.3±5.8 59.7±19.3
12.7 (2.7-151.5) 1.4 (0.3-13.2)
0.8 (0-3.0) 0.3 (0-0.9)
n.d.
CBPC % recovery from PRP % recovery from CB
11.4±4.4 n.d. n.d.
11.3±4.9 79.2±21.7 47.7±17.8
7.2 (1.0-105.7) 67.5 (10.6-144.8) 0.8 (0.1-12.0)
0.6 (0.2-2.4) n.d. 0.2 (0.1-0.7)
n.d.
Data are given as mean ± standard deviation (volume, platelets, Hb) or median (5th-95th percentiles) (WBC, RBC). CBPC: CB platelet concentrates; n.d.: not determined.
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Discussion
Platelet gel from adult peripheral blood is a standard blood component that has been used for therapeutic purposes for many years, primarily as an autologous biological product for the treatment of skin ulcers and bedsores8-11 and other conditions12,13. Autologous platelet gel is generally considered microbiologically safer than allogeneic donor blood with regard to the risk of acquiring microbial and viral transmissible
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Statistical analysis Data showing a normal distribution are presented as mean±SD, those diverting from a normal distribution as median, 5 th and 95 th percentiles. The statistical significance of differences in volume and platelet content in CB units and in CBPC prepared by the different banks was evaluated by analysis of variance (ANOVA).
An evaluation carried out in one bank over the period from January to June 2014 showed that about one third of CB units originally donated for haematopoietic transplant purposes complied with the selection criteria listed in Table I for the production of CBPC. Table V reports costs incurred at one bank for the production of one CBPC (€ 160.92). On average, banks reported that about 2 hours were needed for one technician to prepare four units of CBPC.
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National Blood Centre, including manufacturer, code and lot number of kits used for microbiological screening tests carried out on blood samples collected from the mothers of neonates from whom the CB units were collected, according to standard CB banking procedures.
Results
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Between November 2013 and December 2014, the 13 banks produced 1,080 CBPC. The mean±SD time from CB collection to the start of processing into CBPC was 31±12 hours. Quality control data from the 1,080 CBPC are shown in Table III. It can be seen that the CB units used for the production of CBPC had a mean±SD volume (including anticoagulant) of 97.6±20.2 mL. The PPP fractions obtained after centrifugation of the PRP had a mean±SD volume of 19.6±9.8 mL and median total platelet count of 0.13×109 platelets (5th-95th percentiles, 0-1.54). The 1,080 CBPC had a mean ± SD volume of 11.4±4.4 mL, platelet concentration of 1,003±229×109/L and total platelet count of 11.3±4.9×109 platelets. Platelet recovery from CB was 47.7±17.8%. The median white cell count per CBPC was 7.2×106 (5th-95th percentiles, 1.0-105.7). The ANOVA showed highly statistically significant differences (p
