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Experiences with frozen blood products in the Netherlands military
Transfusion and Apheresis Science 34 (2006) 289–298 intl.elsevierhealth.com/journals/tras
Experiences with frozen blood products in the Netherlands military C.C.M. Lelkens ¤, J.G. Koning, B. de Kort, I.B.G. Floot, F. Noorman Military Blood Bank, Plesmanlaan 1C, 2333 BZ, Leiden, The Netherlands Received 22 November 2005; accepted 25 November 2005
Abstract For peacekeeping and peace enforcing missions abroad the Netherlands Armed Forces decided to use universal donor frozen blood products in addition to liquid products. This article describes our experiences with the frozen blood inventory, with special attention to quality control. It is shown that all thawed (washed) blood products are in compliance with international regulations and guidelines. By means of the ¡80 °C frozen stock of red cells, plasma and platelets readily available after thaw (and wash), we can now safely reduce shipments and abandon the backup ‘walking’ blood bank, without compromising the availability of blood products in theatre. © 2006 Elsevier Ltd. All rights reserved.
1. Introduction Blood supply in the Netherlands is a responsibility of the Sanquin Blood Foundation. From the four regional, main blood banks – Northwest, Northeast, Southwest and Southeast – all Dutch hospitals are provided with the necessary blood products, derived from volunteer, unpaid donors. Sanquin operates nationwide in an environment in which demand and supply are more or less balanced. Furthermore, logistical problems are almost non-existent, given the size of the country and its infrastructure. Therefore, the usual shelf lives of particularly red cells and platelets hardly create a problem for the civilian community. The military
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1473-0502/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.transci.2005.11.008
system by contrast, operates in a totally diVerent environment. The Military Blood Bank (MBB), the Wrst link in the NL military blood supply chain, is dependent upon the civilian population in that its donors provide indirectly – through Sanquin – the blood products needed during deployments. All blood products are tested and processed by Sanquin, including those used for military purposes abroad. The MBB is responsible for providing the necessary blood products to deployed military expeditionary units abroad. Finding a balance between demand and supply as observed in the civilian community is virtually impossible, given the current nature and risks of military deployments. In general, transfusing blood in the military situation will be related to trauma, because of massive blood loss and hypovolemic shock [1]. In addition to volume replacement, casualties who have lost more than 25–30% of their original blood volume also
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require restoration of their oxygen-carrying capacity, which calls for transfusing red cells. At the same time, concurring coagulation defects will have to be countered by administering plasma and/or platelets [2,3]. To exclude inadvertent clerical errors as much as possible and to reduce the number of units to be stockpiled, the use of universal donor products is imperative, i.e. O Rh (D) positive and negative red cells, O Rh (D) positive and negative platelets and AB plasma. Blood products have a Wnite life span; the shelf life of the usual, standard liquid stored red cells is measured in weeks and for platelets even in days. If those products were to be readily available in theatre, it would require shipments of platelets every week and red cells preferably every two weeks, but at least every month. This limits their use and creates a substantial logistical burden, particularly, if increased numbers of blood units are needed at short notice. Newly developed additive solutions that signiWcantly prolong storage times of red cells [4], would diminish the frequency of shipments to a certain extent, but these solutions are not available on the market yet. Newly developed storage solutions for platelets do not extend storage times beyond 10 days [5]. Products like hemoglobin based oxygen carriers [6] and artiWcial platelets [5] are not yet available as an alternative. In 1991, during the Wrst Gulf War, the Netherlands Military Blood Bank shipped universal O (Rh D positive and negative) liquid RBC from Amsterdam to the United Arab Emirates, in support of a land-based Royal Netherlands Naval surgical team. Later on, in 1992, we shipped universal liquid RBC and fresh frozen plasma (FFP) to our medical treatment facilities (MTF’s) deployed in Cambodia and Bosnia. Platelets were provided by a so-called “walking blood bank”. The number of new deployments grew steadily and so did the pressure on the blood supply system. Providing standard blood products proved to be cost-ineVective and, moreover, could not guarantee the availability of blood products at all times. To date, freezing is the only technique available to substantially extend the shelf lives of the products we need and to reduce shipments to an absolute minimum. Deep frozen (¡80 °C) red cells can be stored for 10 years at least [7,8], deep frozen plasma for 7 years [9] and deep frozen platelets for 2 years [10,11]. This report describes the experiences of the Netherlands military with these frozen blood products,
with special attention to quality control and compliance to (inter)national regulations. We show that it is now possible to deploy a self-suYcient military blood bank facility, based on an inventory of deep frozen red cells, plasma and platelets. 2. Materials and methods 2.1. Red cells We procured leukodepleted, Wltered whole blood (O, Rh D positive and negative) units from Sanquin Southwest (Rotterdam, The Netherlands), as raw material to produce the frozen red cells from. The unit was transferred to a 1 L PVC bag (Macopharma, Tourcing, France) and centrifuged (Hettich Roto Silenta) at 1615G, 4 min, brake 0. The supernatant plasma was removed in a 600 ml PVC bag (Terumo, Tokyo, Japan) and a volume of 57% w/v glycerol (‘Glycerolite’ Baxter, Toronto, Canada) was sterilely added via the ACP215 (Haemonetics, Braintree, MA, USA) in about 11 min to a Wnal concentration of around 40% (w/v). Subsequently the unit was centrifuged (Hettich Roto Silenta) at 1248G, 10 min, no brake, and the supernatant glycerol was removed in a 600 ml PVC Terumo bag. The Wnal product bag was then folded and vacuumsealed in a plastic overwrap bag. The bag was packed into a rigid cardboard box (Cekumed, Ooltgensplaat, The Netherlands) and frozen to a temperature of ¡80 °C on the bottom of a mechanical freezer (Revco, PolyTemp ScientiWc, Bolsward, The Netherlands) resulting in a freezing rate of 1– 3 °C per minute. After a minimum of 24 h, the product was placed in a quarantine inventory at ¡80 °C (Revco). All units were frozen within 24 h after donation. Prior to transfusion, the deep frozen erythrocyte concentrates (DEC) were thawed in a temperature controlled water bath (Forma ScientiWc, De Meern, The Netherlands), maintained at 42 °C. When the unit reached a temperature of 30–35 °C the unit was deglycerolized in the ACP 215, a semi-automated, functionally closed washing system. In a number of washing steps, the cells were sterilely washed with NaCl 12% (Baxter, DeerWeld, IL, USA), a mixture of normal saline and 0.2 glucose (Baxter, DeerWeld, IL, USA) and Wnally suspended in the storage solution AS3 (Haemonetics, Braintree, MA, USA). The thawed, washed red cells were stored for 14 days at 2–6 °C. Total processing time was 100–120 min after removal from the freezer.
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2.2. Plasma We procured leukodepleted FFP (AB-Rh D positive and negative), isolated by apheresis, from Sanquin Southwest after §1 year of quarantine storage at ¡30 °C, and release from quarantine. Units were thawed in a temperature controlled water bath (Forma ScientiWc), maintained at 37 °C, and warmed to a temperature of 20–30 °C. The thawed plasma was then transferred to a 600 ml PVC bag (Macopharma), folded and vacuum-sealed in a plastic overwrap bag. The unit was packed into a rigid cardboard box (Cekumed) and frozen to a temperature of ¡80 °C on the bottom of a mechanical freezer (Revco) resulting in a freezing rate of 1–3 °C/ min. Before transfusion, the deep frozen plasma (DFP) units were thawed in 25–35 min in a temperature controlled water bath (Forma ScientiWc), maintained at 37 °C, to 30–35 °C. Total processing time was 25–35 min after removal from the freezer. 2.3. Platelets We procured fresh, leukodepleted platelet concentrates, isolated by apheresis (Amicus), from Sanquin Southwest. Units were stored at 22 °C with agitation for 12–20 h, until the freezing procedure was started. A transfer set (Baxter) was sterilely connected to the unit to be frozen and the unit was then put on a Xatbed shaker (GFL, Burgwedel, Germany). A bottle, containing 27% (w/v) DMSO in 0.9% saline (Pharmacy Department, Central Military Hospital, Utrecht, The Netherlands) was hung at 7 in. in a Laminar Air Flow cabinet (PMV, Woerden, The Netherlands). The TeXon cap of the bottle was spiked with the transfer set. While the unit was rotating at 180 rotations per minute on a Xatbed shaker (Beun DeRonde, Abcoude, The Netherlands), 75 ml DMSO was added by means of gravity (§10 ml/min) in §7 min. The unit was subsequently transferred to a 400 ml PVC bag (Baxter) and centrifuged at 1250G for 10 min, brake 0. Supernatant DMSO-plasma was removed and the remaining 10– 20 ml platelet concentrate was carefully mixed in the 400-ml bag by gently rubbing the bag with a nylon gauze. The platelet bag was folded, vacuum-sealed in a plastic overwrap bag, packed into a rigid cardboard box (Cekumed) and frozen to a temperature of ¡80 °C on the bottom of a mechanical freezer (Harris) resulting in a freezing rate of 3–5 °C/min. All units were frozen within 24 h of donation. Prior to transfusion, the DTC were warmed to 30–35 °C in
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§5 min in a temperature controlled water bath (Forma ScientiWc) maintained at 37 °C. After gently mixing the platelet unit by means of gently rubbing the bag with a nylon gauze, a thawed DFP unit was sterilely connected (TSCD, Terumo Europe, Leuven, Belgium) to the platelet bag. The thawed plasma was added to the thawed platelets and the platelets were easily suspended in the plasma by transferring the platelets in plasma back and forth to the platelet-freezing bag. Hereafter, the platelets are ready for transfusion. Total processing time after removal from the freezer is 30–40 min. 2.4. Labels and logistics All blood products used were labelled conforming to ISBT guidelines. For frozen red cells and platelets an extra label for the thawed product was enclosed in the vacuum-sealed plastic overwrap bag. The frozen products were stored at ¡80 °C in mechanical freezers, equipped with on-line alarm systems and CO2 backup, to minimise the consequences of unforeseen power outages, possibly leading to out-of-range inside temperatures. Units were transported in dry ice (ca. ¡80 °C) in insulated small shipping containers with a maximum of 10 units each (Dometic, Alphen a.d. Rijn, The Netherlands) or bigger versions with a maximum capacity of 192 units (Olivo, Roche-la-Moliere, France). Temperature during transportation was monitored continuously by a TempTale® device (TDS, Sassenheim, The Netherlands) in each container and never exceeded the upper limit of ¡65 °C. Products were transferred to a ¡80 °C freezer (Revco) in a room temperature controlled container. The freezer was connected to an audible and visible alarm system and CO2 backup. Temperature was monitored continuously with a 6-in. chart recorder on the freezer. All in-theatre storage, thawing and washing procedures were performed in a room temperature controlled blood bank container, designed by the Dutch Army. 2.5. Quality control and analyses Sanquin Southwest performed the quality control of donor, donation and leukodepletion. To check the Wnal content of frozen/thawed products, a clinical haematology analyser (Sysmex KX-21, GoYn Meyvis, Etten-Leur, The Netherlands) was used. For additional quality control of the RBC, capillary hematocrit and supernatant free hemoglobin (after
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Table 1 Characteristics of deep frozen red cells before, during and after freezing to ¡80 °C Mean § SD
Limit(s)
% OK
Filtered whole blood (N D 1360) Thrombocytes (109/U) White blood cells (106/U) Volume (ml/U) Hb content (g/U)
2§2 0.3 § 0.2 519 § 13 63 § 5
<15c <1a 470–570b >55b
99.8 99.6 99.6 93.3
Deep frozen red cells (N D 1360) Hematocrit (l/l) Glycerol concentration (g/dl) Volume (ml/U) Hb content (g/U)
0.60 § 0.03 40.0 § 0.5 326 § 33 60 § 5
0.50–0.70b 38–42b 250–450b >50b
98.5 99.6 99.6 98.8
Thawed washed red cells (N D 147) Hematocrit (l/l) Glycerol concentration (g/dl) Volume (ml/U) Hb content (g/U) Hemolysis at day 14 (%) Hemolysis at day 14 (%)
0.56 § 0.03 0.18 § 0.03 294 § 4 45 § 3 0.6 § 0.2 0.6 § 0.2
0.50–0.65c <1.0d >245c >40a <1.0d <0.8a
95.2 100 100 95.2 96.3 87.7
a b c d
CE recommendations leukodepleted red cells [10]. MBB internal guidelines. Sanquin internal guidelines leukodepleted red cells. FDA/AABB guidelines deglycerolized red cells [4,12,13].
centrifugation at 2200G, 10 min) was measured on a Plasma low hemoglobin device (Hemocue, Oisterwijk, The Netherlands). The percentage of hemolysis was determined by the ratio of free Hb to total Hb. We used a ReXotron® triglyceride test to measure the residual glycerol concentration in the supernatant after a 1:10 dilution in 0.9% saline. The KC4A (Amelung Coagulometer, Germany) was used to determine the concentration of coagulation factors V and VIII, APTT and PT, according to the manufacturer’s instructions. For these tests standard ¡80 °C plasma, factor VIII deWcient plasma and factor V deWcient plasma from Cryocheck (Precision Biologic, Darmouth, NS, Canada) were used. pH was measured by means of an electrode (Delta OHM, Caselle di Sevazzano, Italy). All data were stored and analysed in Excel 97. The diVerences were studied with the Student’s t-test, a P < 0.05 was considered statistically signiWcant. 3. Results Leukodepleted RBC should contain less than 1 £ 106 WBC and less than 15 £ 109 platelets. Liquid stored red cells should contain at least 40 g of Hb and hemolysis at the end of the storage period should not exceed 0.8% or 1% [6,10,12]. After deglycerolization, the Wnal glycerol concentration
should be less than 1% in order to prevent hemolysis in the recipient [13]. For deglycerolized, stored red cells hemolysis should not exceed 1% at the end of the storage period [12]. From March 2003 to July 2005, a total of 1360 units of Wltered, leukodepleted units of whole blood have been processed to frozen red cells. Table 1 shows an overview of the characteristics of these units. From unit hematocrit prior to freezing (Wxed on 80%) and the unit weight (determined for each unit) the ACP 215 accurately calculates the amount of glycerol needed for each individual unit to reach a Wnal concentration of 40% w/v (40 § 0.5%). Throughout the freezing procedure, only 3 § 1 g Hb was lost due to sampling and removal of supernatant plasma and glycerol. Units were deglycerolized, stored at 2–6 °C for 14 days, after which the product quality was assessed. The process was fully validated in the Netherlands prior to in-theatre validation and implementation. In April 2004, the thaw-wash process was validated in Iraq in the operational, military environment. For two days during 12 h each day, two skilled lab technicians, using two ACP 215, thawed and washed 22 units of DEC. There was no diVerence between the units processed at the MBB in Leiden and those in Iraq. At the end of April 2004, we therefore decided to implement the ACP 215 for deglycerolization of
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85 80 75 70 65 60 55 50 45 40 40
50
60
70
80
90
power failure (2 units), bowl damage during deglycerolization (2 units), ACP215 technical problems (1 unit) and leakage of the sterile connection (1 unit). Another unit was lost due to wrongfully connecting the washing Xuids on the ACP215. In conclusion, out of 1360 units, 1298 units (95.4%) met all the criteria to be stored at ¡80 °C for a maximum of 10 years, ready to be used on deployments. We found that Wltered, leukodepleted whole blood, can eVectively be processed with the ACP215 to produce DEC. The thawed washed products contained at least 40 g Hb and can be stored for 14 days at 4 °C with low hemolysis after deglycerolization and suspension in AS3. Table 2 shows that AB plasma has a high factor VIII and factor V content, even after storage for almost 1 year at ¡30 °C. Compared to FFP a slight reduction of factor VIII concentration (P > 0.05) and factor V (P < 0.05) was observed in DFP. The concentration of factors V and VIII did not decrease to below the minimum of 70%. The volume of plasma is reduced by §6 ml because of sampling, without reducing the plasma volume below the minimum of 225 ml. Thus, thawing and subsequent freezing of AB plasma to ¡80 °C can be performed, without deviation of the product quality from the CE recommendations [10]. Until now a total of 435 DFP units were produced and only 16 units (3.7%) were discarded. The majority (12 units) was discarded because the units were thawed in a defective water bath, which resulted in too slow warming of the units. Furthermore 2 units were discarded due to a high RBC content and another 2 units due to the Hb content of thawed, washed product (gram Hb)
Hb content of frozen product (gram Hb)
DEC in Iraq. The backup system proved to be very useful when during the Iraq election period, logistics were hampered and no liquid red cells could be transported. Twenty units were thawed, washed and stored at 2–6 °C successfully to replace the outdated liquid inventory without further delay. An overview of the quality of thawed, deglycerolized and subsequently stored units (including the data from the 42 units processed in Iraq) is shown in Table 1. The ACP215 eVectively removed glycerol from the thawed product from 40% w/v to below the 1% w/v upper limit [13]. It appeared that the Hb content of the frozen unit was primarily dependent upon the Hb content of the leukodepleted product (Fig 1A). The Hb content of the washed unit was primarily dependent upon the bowl size (275 ml), since hemolysis during washing (19 § 5%) was independent of the Hb content of the frozen product, whereas cell spillage was not (results not shown). As shown in Fig 1B, the Hb content of a unit before freezing should at least be 50 g in order to obtain the minimum required product content of 40 g Hb after deglycerolization. Only 16 of 1360 units (1.2%) did not meet this minimum requirement of 50 g Hb prior to freezing and these units were quarantined. Of these low Hb units, 14 units were washed and Hb content was below 40 g Hb in all cases (Fig 1B). Various other reasons, such as training and education purposes (1.1%) and bad seals of the sterile connection device (1%), accounted for another 3.6% that were not released for transfusion. During the validation period 168 units were deglycerolized and 7 units (4.2%) were discarded. Product loss was caused by
52 50 48 46 44 42 40 38 36 34 32 30 28 26 40
45
50
55
60
65
70
75
80
Hb content of frozen product (gram Hb)
Hb content of leukodepleted whole blood (gram Hb) (A)
293
(B)
Fig. 1. The relation between Hb content of ground substance and Hb content of Wnal product. (A) The relation between Hb content of leukodepleted whole blood and Hb content of frozen red cells (N D 1360). (B) The relation between Hb content of frozen red cells and Hb content of thawed, washed red cells (N D 161).
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Table 2 Characteristics of deep frozen plasma before, during and after freezing to ¡80 °C
Fresh frozen plasma apheresis, leukodepleted (FFP) Thrombocytes concentration (109/l) (N D 68) White blood cells (106/U) Volume (ml/U) (N D 188)
Mean § SD
Limit(s)
% OK
5§7 No datad 290 § 15
<50a <1c 225–315c
100 >90 99.5
Coagulation after FFP storage, 0.9 § 0.01 year at ¡30 °C APTT (s) (N D 12) PT (s) (N D 12) Factor VIII (U/ml) (N D 12) Factor V (U/ml) (N D 12)
30.0 § 2.2 12.2 § 0.5 1.4 § 0.5 1.0 § 0.2
<36b <14b >0.70a >0.70b
100 100 100 100
Deep frozen plasma (DFP) Thrombocytes concentration (109/l) (N D 27) Volume (ml/U) (N D 188)
10 § 8 284 § 14
<50b 225–315b
100 99.5
<36b <14b >0.70b >0.70b
100 100 100 100
Coagulation after DFP storage, 0.76 § 0.02 year at ¡30 °C followed by 1.31 § 0.02 year at ¡80 °C APTT (s) (N D 12) 31.0 § 2.3 PT (s) (N D 12) 13.0 § 0.5 Factor VIII (U/ml) (N D 12) 1.2 § 0.3 Factor V (U/ml) (N D 12) 0.9 § 0.1 a b c d
CE recommendations fresh frozen plasma (apheresis) [10]. MBB internal guidelines. Sanquin internal guidelines fresh frozen plasma apheresis, leukodepleted. Product is manufactured by Sanquin Southwest according to Sanquin internal guidelines.
use of spiking because of short tubing on the original FFP bag. An additional advantage of the DFP procedure is that the folding of the product bag and packaging in a vacuum-sealed overwrap bag, in combination with the rigid cardboard box prevents breakage of the PVC bag. We found 8.2% breakage when thawing ¡30 °C stored FFP (N D 486), and no breakage at all with DFP (N D 82). Similarly, the above mentioned thawed DEC (N D 167) also did not show any breakage, nor did the DTC (N D 27) described below. Thawed DTC units were suspended in thawed DFP and the amount of platelets was determined. As shown in Table 3, the units contained more than the minimum of 200 £ 109 platelets/unit (CE recommendations [10] for fresh liquid platelets) and showed a higher than 40% recovery (CE recommendations [10] for frozen platelets). The pH varied was well above 6.8, varying between 7.6 and 7.8. In the Council of Europe (CE) recommendations [10] a limit is set to the volume of product that can be transfused without washing. We concluded from this recommendation that no more than 10 g of DMSO (200 ml 5% w/v) should be present in one platelet unit. As shown in Table 3 the DMSO content of the unit is very low and did not exceed this limit, in fact 94% of all units have a DMSO content
below 1 g/unit. Out of the 217 units frozen, 13 units (6%) were discarded. This was due to recall because of a positive BacTalert® measurement (2 units), clerical or technical errors (4 units), presence of aggregates prior to freezing (2 units), and failure of a sterile connection device (4 units). Only 1 unit was discarded due to a large starting volume, resulting in a Wnal DMSO concentration below 4%. 4. Discussion It has been shown that the high glycerol method can be used to freeze red cells with an acceptable post-thaw and post-wash recovery. By means of the ACP215 it is now possible to store the red cells after washing for a period of 14 days at 4 °C with an acceptable post-storage hemolysis and in vivo survival [8,14–16]. Platelets can be frozen in 5% DMSO at ¡80 °C [17,18]. Although in vivo survival is reduced [17–21], the frozen platelets are more eVective in reducing blood loss compared to 3-day stored liquid platelets [21]. Fresh frozen plasma (¡30 °C) has been used worldwide, the eVect of refreezing them to ¡80 °C has not been studied before. To ensure readily available blood products on missions all over the world, the NL Ministry of Defence decided to build a state of the art frozen inventory of the blood products most needed: red cells, plasma and platelets.
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Table 3 Characteristics of deep frozen platelets before, during and after freezing to ¡80 °C
Platelets apheresis, leukodepleted (N D 217) Thrombocytes concentration (109/l) White blood cells (106/U) Volume (ml/U) Thrombocytes (109/U) pH
Mean § SD
Limit(s)
% OK
1221 § 220 0.1 § 0.1 312 § 19 381 § 68 7.1 § 0.1
<1500a <1a 150–400c >200a >6.8c
90.2 100 99.5 100 100
Frozen platelets (N D 217) Volume (ml/U) Thrombocytes (109/U) platelet recovery start product–frozen product (%) DMSO concentration (g/dl) DMSO content (gm)
14 § 4 349 § 58 93 § 13 5.1 § 0.4 0.7 § 0.2
<20b >220b >80b 4–6b <1b
Thawed resuspended platelets in DFP (N D 27) Platelet recovery start product–frozen product (%) Platelet recovery freeze-thaw Platelet recovery start product–end product Thrombocytes concentration (109/l) Volume (ml/U) Thrombocytes (109/U) DMSO content (gm) pH
94 § 15 83 § 17 77 § 15 977 § 226 279 § 28 269 § 45 0.7 § 0.2 7.7 § 0.1
>80b >40d >40b <1500a 225–315b >200a <10b (d) >6.8c
a b c d
92.6 98.2 97.2 97.7 94.0 92.6 100 100 92.6 96.3 100 100 100
CE recommendations platelets apheresis, leukodepleted [10]. MBB internal guidelines. Sanquin internal guidelines platelets apheresis, leukodepleted. CE recommendations cryopreserved platelets [10].
Red cells can be frozen in either a high (40% w/v) glycerol or a low (20% w/v) glycerol concentration. The 40% glycerol allows for storage at ¡80 °C in a mechanical freezer, whereas the 20% glycerol requires liquid nitrogen (¡196 °C). We have shown that the ¡80 °C frozen cells are more stable during post-wash storage [22]. Furthermore, introducing ¡80 °C to freeze red cells Wtted well into the concept of a deployable, integrated liquid-frozen blood bank [23]. The feasibility of the concept itself had been shown before as well as the use of previously frozen red cells in the treatment of combat casualties [24– 26]. Using the ¡80 °C frozen RBC allows for 10 years storage, although much longer periods have been shown to be feasible [7,8]. To deglycerolize the thawed red cells in theatre, prior to transfusion, we previously have used the M 115 (Haemonetics, Braintree, MA, USA), a nonclosed washing device, permitting a 24 h post-wash storage period only. Our frozen red cells thus could not serve as a primary source to meet our red cell needs “in the Weld”, but were used as a back-up for the liquid stock, which was supplied every 2 weeks. We used this concept during missions in Bosnia (1993–2005), Afghanistan (2002–2003) and Liberia
(2003). Both in Afghanistan and Liberia the frozen red cells have proven to be essential when sudden demand exceeded the available liquid stock of red cells. With the introduction of the ACP215, a semiautomated, closed washing device, post-thaw, postwash storage of frozen red cells, became possible for at least 14 days at 2–6 °C [8,14–16]. We show that the ACP215 in its current, FDA approved, conWguration can be used eVectively to produce a stock of DEC and to produce on demand liquid red cells in theatre with suYcient product yield and low hemolysis after storage. In addition, we show that leukodepleted whole blood collected in CPD can be used as a source for DEC. Furthermore, in 2005 it became clear in Iraq that a military hospital blood bank facility can be deployed without regular shipments of liquid red cells. It is able to meet the needs of a surgical team by thawing and washing a certain number of frozen units once a week. This creates a new concept of an integrated liquid-frozen blood bank. We have successfully used this new concept in the current mission in Afghanistan. Our conclusion is that the ACP215 enables a more eYcient and Xexible use of
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RBC for military deployments, still meeting the standard quality requirements of the regulatory civilian authorities. It is well known that following massive blood replacement, plasma and platelet transfusion is required to correct coagulation defects. It has been shown that among survivors of massive blood transfusion more platelets were transfused [27]. Fresh whole blood or stored platelet concentrates are required to correct the coagulation defect [3,27–29]. During several missions in the early nineties, a socalled “walking blood bank” consisting of voluntary military personnel in theatre, was to provide whole blood, primarily as a source of platelets. In 1996, however, we encountered a case of hepatitis B transmission in Bosnia, derived from a “walking blood bank” donor. Although under speciWc conditions a single individual may very well beneWt from this source, the risk of transmitting a disease is high even when donors are used that have been tested recently. Most expeditionary missions are land-based operations in third world countries. This means that infections like malaria and Chagas’disease, even in previously healthy personnel, can and will be transmitted inadvertently from the donor pool to patients. Moreover, since blood is needed at unpredictable moments, the listed donors may not be available at the right moment for lots of reasons [29]. The main reason to use fresh whole blood in theatre is to treat major bleeding, due to lack of platelets in the patient, since liquid stored platelets are not available. In order to abandon the walking blood bank, an alternative for fresh whole blood was required. DMSO frozen platelets have been transfused successfully since the 1970s [19,20]. Despite the fact that their functional recovery is less than that of fresh liquid platelets (around 50%), the frozen platelets are more eVective in stopping non-surgical bleeding compared to 3-day liquid stored platelets [21]. Frozen platelets also show a higher capacity to bind factor V [30] and a higher thromboxane A2 production after ADP stimulation [31]. In addition, baboon frozen platelets have a higher in vivo survival and a stronger correction of aspirin induced prolonged bleeding time compared to 5-day liquid stored platelets [32]. With or without leukodepletion, platelets frozen with 4–6% DMSO may be stored at ¡80 °C for at least 2 years [11]. The possibility of adding frozen platelets to our line of products was therefore very logical to investigate.
Washing became part of the original thawing procedure of frozen platelets to reduce all possible adverse side eVects of DMSO. When we visited Dr. Valeri to learn the freezing and thawing procedure of ¡80 °C frozen platelets he had modiWed the procedure in that post-thaw washing was no more required, because supernatant plasma containing the majority of the DMSO was removed prior to freezing. This modiWed procedure saves valuable time (§1 h), technical training and equipment (centrifuge) in theatre. Since this was a major improvement (time-saving is life-saving), we used this modiWed freezing protocol. Whereas Dr. Valeri uses saline, we use AB plasma to suspend the platelets after thaw, reasoning that a trauma patient needing red cells and platelets almost certainly will also need plasma. Autologous plasma has been used previously for the suspension of thawed washed platelets [20]. We chose not to use this option because when using AB plasma and O platelets the product can be used in every patient regardless of the ABO blood type. In our experience, the thawed platelets show clearly visible swirling in plasma and the product looks like a regular platelet unit obtained by apheresis. We found an in vitro recovery of §77% similar to the in vitro recovery reported by others [11,17–19]. By the end of 2001 we implemented the use of frozen platelets in Bosnia, and abandoned the walking blood bank concept. Within half a year two patients were treated that required platelet transfusion. One elderly woman with gunshot wounds in the pelvic region and one young soldier with acute ITP (viral). Both patients experienced unstoppable bleeding due to a low platelet concentration. Although the platelet count barely rose after transfusion, the bleeding of each patient stopped within 20 min after transfusion of one thawed platelet concentrate in AB plasma. We have thus experienced that frozen platelets can be lifesaving and that the use of a walking blood bank can be abolished when this product is available in theatre. From that time onward, frozen platelets and frozen blood bank facilities always have been part of the standard equipment of Dutch deployed military hospitals. Fresh frozen plasma (FFP) is normally kept at temperatures of around ¡20 °C to ¡30 °C with a shelf life from 1 to 2 years. Lower temperatures of at least ¡65 °C even permit a storage period up to 7 years [9,10]. Apheresis, leukodepleted plasma is frozen by Sanquin, according to the guidelines,
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and meets the requirements for FFP. The units are still frozen upon arrival at the MBB, not folded and do not Wt into our standard rigid cardboard boxes. Since we wanted to maintain uniformity in our packing methods and ¡80 °C transportation and storage temperature, we repacked the plasma units. Refrozen (¡30 °C), previously thawed FFP and even liquid stored plasma, have been shown to be safe and eVective, although factor V and FVIII:C levels are signiWcantly decreased with 10–30% [33– 37]. We observed a slight decrease in factor V and VIII levels due to the extra thaw-freeze cycle, however since we only have used AB plasma which has the advantage of high factor VIII levels to start with, factor V and VIII levels are still well above the 0.7 U/ml. It is likely, but still unproven, that these refrozen DFP units can be stored for 7 years at ¡80 °C as described for fresh frozen plasma at temperatures below ¡65 °C [9,10]. In literature it is suggested that breakage of PVC at ¡80 °C is due to freezing and cannot be prevented, instead new plastics are studied to avoid breakage [38]. Strong reduction of PVC breakage by means of folding the blood bag to protect vulnerable parts and by putting it in an overwrap bag and a rigid cardboard box, has been described by Valeri et al. [11,39]. The overwrap bag prevents contact of the product bag with the cardboard box which reduces breakage and is also used during thaw to protect the water bath against contamination in case the product bag is broken. Using the same method in combination with a vacuum-sealed overwrap bag we also could strongly reduce breakage of standard PVC bags stored at ¡80 °C; breakage was reduced to 0.0% even after transportation on dry ice. To date, the (inter)national guidelines advocate the use of frozen cellular blood products for rare blood types and patients with multiple alloantibodies only. We have shown that the frozen blood bank concept can be eVectively used in a military environment and are convinced that it can also be very useful in the civilian community. A frozen blood bank facility with a stock of frozen universal donor products can eVectively be used in remote areas, to compensate for periods when no donors are available (holiday periods) and when suddenly many patients are in need for blood products. In addition a quarantine period similar to that currently used for plasma can also be used for frozen cellular blood products to reduce risks of transmitting diseases.
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5. Conclusions The quality of our stock of frozen blood products is in compliance with the European and US standards. The products can easily, eVectively and safely be used in deployed blood bank units. Deployed military hospitals become thus less dependent on their home countries or on a walking blood bank. Products can be produced on demand and blood ‘spillage’ due to expiration can be reduced to the absolute minimum. The logistic burden can be reduced since less or no shipments are required to support for the blood product inventory abroad. Acknowledgements We like to thank Dr. C.R. Valeri for providing instructions, protocols, advice and technical support, Sanquin Blood Bank Southwest for providing liquid blood products and leukodepletion data. Furthermore we thank the Pharmacists S.M.G.E. van Grinsven, J. Chin and H.M. Meinen, for their leading role in operating the deployed blood bank in Iraq and S.F. Bus, M. Hakvoort, D. van Zandwijk and R. Nieuwenhuizen for their technical assistance. The Netherlands Ministry of Defence Wnancially supported this study. The opinions or assertions contained herein are those of the authors and are not to be construed as oYcial or reXecting the views of the Ministry of Defence. References [1] Champion HR, Bellamy RF, Roberts P, Leppaniemi A. A proWle of combat injury. J Trauma 2003;54:S13–9. [2] Armand R, Hess JR. Treating coagulopathy in trauma patients. Transfus Med Rev 2003;17:223–31. [3] Hardy JF, Moerloose P, Samana CM. The coagulopathy of massive transfusion. Vox Sang 2005;89:123–7. [4] Scott KL, Lecak J, Acker JP. Biopreservation of red blood cells: past, present and future. Transfus Med Rev 2005;19:127–42. [5] Blajchman MA. Novel platelet products, substitutes and alternatives. Transfus Clin Biol 2001;8:267–71. [6] Buehler PW, Alayash AI. Toxicities of hemoglobin solutions: in search of in-vitro and in-vivo model systems. Transfusion 2004;44:1516–30. [7] Valeri CR, Pivacek LE, Gray AD, Cassidy GP, Leavy ME, Dennis RC, et al. The safety and therapeutic eVectiveness of human red cells stored at ¡80 degrees C for as long as 21 years. Transfusion 1989;29:429–37. [8] Valeri CR, Srey R, Tilahun D, Ragno G. The in vitro quality of red blood cells frozen with 40 percent (wt/vol) glycerol at ¡80 degrees C for 14 years, deglycerolized with the Haemonetics ACP 215, and stored at 4 degrees C in additive solution-1
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[9] [10] [11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
C.C.M. Lelkens et al. / Transfusion and Apheresis Science 34 (2006) 289–298 or additive solution-3 for up to 3 weeks. Transfusion 2004;44:990–5. Standards for blood banks and transfusion services, 23rd ed. AABB, 2004. Guide to the preparation, use and quality assurance of blood components, 11th ed. Council of Europe, 2005. Valeri CR, Srey R, Lane JP, Ragno G. EVect of WBC reduction and storage temperature on PLTs frozen with 6 percent DMSO for as long as 3 years. Transfusion 2003;43:1162–6. Sowemimo-Coker S. Red blood cell hemolysis during processing. Transfus Med Rev 2002;16:46–60. Mollison PL, Engelfriet CP, Contreras M. 10th ed. Blood transfusion in clinical medicine Blackwell Science; 1997. p. 302. Valeri CR, Ragno G, Pivacek LE, Srey R, Hess JR, Lippert LE, et al. A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4 degrees C in AS-3: assessment of RBC processing in the ACP 215. Transfusion 2001;41:933–9. Valeri CR, Ragno G, Pivacek L, O’Neill EM. In vivo survival of apheresis RBCs, frozen with 40-percent (wt/vol) glycerol, deglycerolized in the ACP 215, and stored at 4 degrees C in AS-3 for up to 21 days. Transfusion 2001;41:928–32. Bandarenko N, Hay SN, Holmberg J, Whitley P, Taylor HL, MoroV G, et al. Extended storage of AS-1 and AS-3 leukoreduced red blood cells for 15 days after deglycerolization and resuspension in AS-3 using an automated closed system. Transfusion 2004;44:1656–62. Melaragno AJ, Abdu WA, Katchis RJ, Vecchione JJ, Valeri CR. Cryopreservation of platelets isolated with the IBM 2997 blood cell separator: a rapid and simpliWed approach. Vox Sang 1982;43:321–6. Vecchione JJ, Chomicz SM, Emerson CP, Valeri CR. Cryopreservation of human platelets isolated by discontinuousXow centrifugation using the Haemonetics Model 30 Blood Processor. Transfusion 1980;20:393–400. SchiVer CA, Aisner J, Wiernik PH. Clinical experience with transfusion of cryopreserved platelets. Br J Haematol 1976;34:377–85. SchiVer CA, Aisner J, Dutcher JP, Daly PA, Wiernik PH. A clinical program of platelet cryopreservation. Prog Clin Biol Res 1982;88:165–80. Khuri SF, Healey N, MacGregor H, Barnard MR, Szymanski IO, Birjiniuk V, et al. Comparison of the eVects of transfusions of cryopreserved and liquid-preserved platelets on hemostasis and blood loss after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1999;117:172–83. Lelkens CCM, Noorman F, Koning JG, Truijens-de Lange R, Stekkinger P, Bakker JC, et al. Stability after thawing of RBCs frozen with the high and low glycerol method. Transfusion 2003;43:157–64. Valeri CR, Sims KL, Bates JF, Reichman D, Lindberg JR, Wilson AC. An integrated liquid-frozen blood banking system. Vox Sang 1983;45:25–39.
[24] Moss GS, Valeri CR, Brodine CE. Clinical experience with the use of frozen blood in combat casualties. N Engl J Med 1968;278:747–52. [25] Valeri CR, Brodine CE, Moss GE. Use of frozen blood in Vietnam. Bibl Haematol 1968;29:735–8. [26] Rosenblatt MS, Hirsch EF, Valeri CR. Frozen red blood cells in combat casualty care: clinical and logistical considerations. Mil Med 1994;159:392–7. [27] Cinat ME, Wallace WC, Nastanski F, West J, Sloan S, Ocariz J, et al. Improved survival following massive transfusion in patients who have undergone trauma. Arch Surg 1999;124:964–70. [28] Ho AM, Karmakar MK, Dion PW. Are we giving enough coagulation factors during major trauma resuscitation? Am J Surg 2005;190:479–84. [29] Reade MC. Blood products on operational deployments. ADF Health 2001;2:65–70. [30] Barnard MR, MacGregor H, Ragno G, Pivacek LE, Khuri SF, Michelson AD, et al. Fresh, liquid-preserved, and cryopreserved platelets: adhesive surface receptors and membrane procoagulant activity. Transfusion 1999;39:880–8. [31] Valeri CR, MacGregor H, Ragno G. Correlation between in vitro aggregation and thromboxane A2 production in fresh, liquid-preserved, and cryopreserved human platelets: eVect of agonists, pH, and plasma and saline resuspension. Transfusion 2005;45:596–603. [32] Valeri CR, MacGregor H, Giorgio A, Ragno G. Circulation and hemostatic function of autologous fresh, liquid-preserved, and cryopreserved baboon platelets transfused to correct an aspirin-induced thrombocytopathy. Transfusion 2002;42:1206–16. [33] Milam JD, Buzzurro SF, Austin SF, Stansberry SW. Stability of factors V and VIII in thawed fresh frozen plasma units. Transfusion 1980;20:546–8. [34] Smak Gregoor PHJ, Harvey MS, Briet E, Brand A. Coagulation parameters of CPD fresh-frozen plasma and CPD cryoprecipitate-poor plasma after storage at 4 °C for 28 days. Transfusion 1993;33:735–8. [35] Dzik WH, Rubner MA, Linehan SK. Refreezing previously thawed fresh-frozen plasma Stability of coagulation factors V and VIII:C. Transfusion 1989;29:600–4. [36] Ben-Tal O, Zwang E, Eichel R, Badalbev T, Hareuveni M. Vitamin K-dependent coagulation factors and Wbrinogen levels in FFP remain stable upon repeated freezing and thawing. Transfusion 2003;43:873–7. [37] Cardugan R, Lawrie AS, Maackie IJ, Williamson LM. The quality of fresh-frozen plasma produced from whole blood stored at 4 °C overnight. Transfusion 2005;45:1342–8. [38] Hmel PI, Kennedy A, Quiles JG, Gorogias M, Seelbaugh JP, Morrissette CR, et al. Physical and thermal properties of blood storage bags: implications for shipping frozen components on dry ice. Transfusion 2002;42:836–46. [39] Valeri CR, Ragno G. Breakage rate for red blood cells frozen with 40 percent (wt/vol) glycerol in 800-ml polyvinylchloride plastic bags stored in rigid cardboard boxes at ¡80 degrees C. Transfusion 2005;45:822–3.
Experiences with frozen blood products in the Netherlands military C.C.M. Lelkens ¤, J.G. Koning, B. de Kort, I.B.G. Floot, F. Noorman Military Blood Bank, Plesmanlaan 1C, 2333 BZ, Leiden, The Netherlands Received 22 November 2005; accepted 25 November 2005
Abstract For peacekeeping and peace enforcing missions abroad the Netherlands Armed Forces decided to use universal donor frozen blood products in addition to liquid products. This article describes our experiences with the frozen blood inventory, with special attention to quality control. It is shown that all thawed (washed) blood products are in compliance with international regulations and guidelines. By means of the ¡80 °C frozen stock of red cells, plasma and platelets readily available after thaw (and wash), we can now safely reduce shipments and abandon the backup ‘walking’ blood bank, without compromising the availability of blood products in theatre. © 2006 Elsevier Ltd. All rights reserved.
1. Introduction Blood supply in the Netherlands is a responsibility of the Sanquin Blood Foundation. From the four regional, main blood banks – Northwest, Northeast, Southwest and Southeast – all Dutch hospitals are provided with the necessary blood products, derived from volunteer, unpaid donors. Sanquin operates nationwide in an environment in which demand and supply are more or less balanced. Furthermore, logistical problems are almost non-existent, given the size of the country and its infrastructure. Therefore, the usual shelf lives of particularly red cells and platelets hardly create a problem for the civilian community. The military
* Corresponding author. Tel.: +31 71 568 5218; fax: +31 71 568 5297. E-mail address: [email protected] (C.C.M. Lelkens).
1473-0502/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.transci.2005.11.008
system by contrast, operates in a totally diVerent environment. The Military Blood Bank (MBB), the Wrst link in the NL military blood supply chain, is dependent upon the civilian population in that its donors provide indirectly – through Sanquin – the blood products needed during deployments. All blood products are tested and processed by Sanquin, including those used for military purposes abroad. The MBB is responsible for providing the necessary blood products to deployed military expeditionary units abroad. Finding a balance between demand and supply as observed in the civilian community is virtually impossible, given the current nature and risks of military deployments. In general, transfusing blood in the military situation will be related to trauma, because of massive blood loss and hypovolemic shock [1]. In addition to volume replacement, casualties who have lost more than 25–30% of their original blood volume also
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require restoration of their oxygen-carrying capacity, which calls for transfusing red cells. At the same time, concurring coagulation defects will have to be countered by administering plasma and/or platelets [2,3]. To exclude inadvertent clerical errors as much as possible and to reduce the number of units to be stockpiled, the use of universal donor products is imperative, i.e. O Rh (D) positive and negative red cells, O Rh (D) positive and negative platelets and AB plasma. Blood products have a Wnite life span; the shelf life of the usual, standard liquid stored red cells is measured in weeks and for platelets even in days. If those products were to be readily available in theatre, it would require shipments of platelets every week and red cells preferably every two weeks, but at least every month. This limits their use and creates a substantial logistical burden, particularly, if increased numbers of blood units are needed at short notice. Newly developed additive solutions that signiWcantly prolong storage times of red cells [4], would diminish the frequency of shipments to a certain extent, but these solutions are not available on the market yet. Newly developed storage solutions for platelets do not extend storage times beyond 10 days [5]. Products like hemoglobin based oxygen carriers [6] and artiWcial platelets [5] are not yet available as an alternative. In 1991, during the Wrst Gulf War, the Netherlands Military Blood Bank shipped universal O (Rh D positive and negative) liquid RBC from Amsterdam to the United Arab Emirates, in support of a land-based Royal Netherlands Naval surgical team. Later on, in 1992, we shipped universal liquid RBC and fresh frozen plasma (FFP) to our medical treatment facilities (MTF’s) deployed in Cambodia and Bosnia. Platelets were provided by a so-called “walking blood bank”. The number of new deployments grew steadily and so did the pressure on the blood supply system. Providing standard blood products proved to be cost-ineVective and, moreover, could not guarantee the availability of blood products at all times. To date, freezing is the only technique available to substantially extend the shelf lives of the products we need and to reduce shipments to an absolute minimum. Deep frozen (¡80 °C) red cells can be stored for 10 years at least [7,8], deep frozen plasma for 7 years [9] and deep frozen platelets for 2 years [10,11]. This report describes the experiences of the Netherlands military with these frozen blood products,
with special attention to quality control and compliance to (inter)national regulations. We show that it is now possible to deploy a self-suYcient military blood bank facility, based on an inventory of deep frozen red cells, plasma and platelets. 2. Materials and methods 2.1. Red cells We procured leukodepleted, Wltered whole blood (O, Rh D positive and negative) units from Sanquin Southwest (Rotterdam, The Netherlands), as raw material to produce the frozen red cells from. The unit was transferred to a 1 L PVC bag (Macopharma, Tourcing, France) and centrifuged (Hettich Roto Silenta) at 1615G, 4 min, brake 0. The supernatant plasma was removed in a 600 ml PVC bag (Terumo, Tokyo, Japan) and a volume of 57% w/v glycerol (‘Glycerolite’ Baxter, Toronto, Canada) was sterilely added via the ACP215 (Haemonetics, Braintree, MA, USA) in about 11 min to a Wnal concentration of around 40% (w/v). Subsequently the unit was centrifuged (Hettich Roto Silenta) at 1248G, 10 min, no brake, and the supernatant glycerol was removed in a 600 ml PVC Terumo bag. The Wnal product bag was then folded and vacuumsealed in a plastic overwrap bag. The bag was packed into a rigid cardboard box (Cekumed, Ooltgensplaat, The Netherlands) and frozen to a temperature of ¡80 °C on the bottom of a mechanical freezer (Revco, PolyTemp ScientiWc, Bolsward, The Netherlands) resulting in a freezing rate of 1– 3 °C per minute. After a minimum of 24 h, the product was placed in a quarantine inventory at ¡80 °C (Revco). All units were frozen within 24 h after donation. Prior to transfusion, the deep frozen erythrocyte concentrates (DEC) were thawed in a temperature controlled water bath (Forma ScientiWc, De Meern, The Netherlands), maintained at 42 °C. When the unit reached a temperature of 30–35 °C the unit was deglycerolized in the ACP 215, a semi-automated, functionally closed washing system. In a number of washing steps, the cells were sterilely washed with NaCl 12% (Baxter, DeerWeld, IL, USA), a mixture of normal saline and 0.2 glucose (Baxter, DeerWeld, IL, USA) and Wnally suspended in the storage solution AS3 (Haemonetics, Braintree, MA, USA). The thawed, washed red cells were stored for 14 days at 2–6 °C. Total processing time was 100–120 min after removal from the freezer.
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2.2. Plasma We procured leukodepleted FFP (AB-Rh D positive and negative), isolated by apheresis, from Sanquin Southwest after §1 year of quarantine storage at ¡30 °C, and release from quarantine. Units were thawed in a temperature controlled water bath (Forma ScientiWc), maintained at 37 °C, and warmed to a temperature of 20–30 °C. The thawed plasma was then transferred to a 600 ml PVC bag (Macopharma), folded and vacuum-sealed in a plastic overwrap bag. The unit was packed into a rigid cardboard box (Cekumed) and frozen to a temperature of ¡80 °C on the bottom of a mechanical freezer (Revco) resulting in a freezing rate of 1–3 °C/ min. Before transfusion, the deep frozen plasma (DFP) units were thawed in 25–35 min in a temperature controlled water bath (Forma ScientiWc), maintained at 37 °C, to 30–35 °C. Total processing time was 25–35 min after removal from the freezer. 2.3. Platelets We procured fresh, leukodepleted platelet concentrates, isolated by apheresis (Amicus), from Sanquin Southwest. Units were stored at 22 °C with agitation for 12–20 h, until the freezing procedure was started. A transfer set (Baxter) was sterilely connected to the unit to be frozen and the unit was then put on a Xatbed shaker (GFL, Burgwedel, Germany). A bottle, containing 27% (w/v) DMSO in 0.9% saline (Pharmacy Department, Central Military Hospital, Utrecht, The Netherlands) was hung at 7 in. in a Laminar Air Flow cabinet (PMV, Woerden, The Netherlands). The TeXon cap of the bottle was spiked with the transfer set. While the unit was rotating at 180 rotations per minute on a Xatbed shaker (Beun DeRonde, Abcoude, The Netherlands), 75 ml DMSO was added by means of gravity (§10 ml/min) in §7 min. The unit was subsequently transferred to a 400 ml PVC bag (Baxter) and centrifuged at 1250G for 10 min, brake 0. Supernatant DMSO-plasma was removed and the remaining 10– 20 ml platelet concentrate was carefully mixed in the 400-ml bag by gently rubbing the bag with a nylon gauze. The platelet bag was folded, vacuum-sealed in a plastic overwrap bag, packed into a rigid cardboard box (Cekumed) and frozen to a temperature of ¡80 °C on the bottom of a mechanical freezer (Harris) resulting in a freezing rate of 3–5 °C/min. All units were frozen within 24 h of donation. Prior to transfusion, the DTC were warmed to 30–35 °C in
291
§5 min in a temperature controlled water bath (Forma ScientiWc) maintained at 37 °C. After gently mixing the platelet unit by means of gently rubbing the bag with a nylon gauze, a thawed DFP unit was sterilely connected (TSCD, Terumo Europe, Leuven, Belgium) to the platelet bag. The thawed plasma was added to the thawed platelets and the platelets were easily suspended in the plasma by transferring the platelets in plasma back and forth to the platelet-freezing bag. Hereafter, the platelets are ready for transfusion. Total processing time after removal from the freezer is 30–40 min. 2.4. Labels and logistics All blood products used were labelled conforming to ISBT guidelines. For frozen red cells and platelets an extra label for the thawed product was enclosed in the vacuum-sealed plastic overwrap bag. The frozen products were stored at ¡80 °C in mechanical freezers, equipped with on-line alarm systems and CO2 backup, to minimise the consequences of unforeseen power outages, possibly leading to out-of-range inside temperatures. Units were transported in dry ice (ca. ¡80 °C) in insulated small shipping containers with a maximum of 10 units each (Dometic, Alphen a.d. Rijn, The Netherlands) or bigger versions with a maximum capacity of 192 units (Olivo, Roche-la-Moliere, France). Temperature during transportation was monitored continuously by a TempTale® device (TDS, Sassenheim, The Netherlands) in each container and never exceeded the upper limit of ¡65 °C. Products were transferred to a ¡80 °C freezer (Revco) in a room temperature controlled container. The freezer was connected to an audible and visible alarm system and CO2 backup. Temperature was monitored continuously with a 6-in. chart recorder on the freezer. All in-theatre storage, thawing and washing procedures were performed in a room temperature controlled blood bank container, designed by the Dutch Army. 2.5. Quality control and analyses Sanquin Southwest performed the quality control of donor, donation and leukodepletion. To check the Wnal content of frozen/thawed products, a clinical haematology analyser (Sysmex KX-21, GoYn Meyvis, Etten-Leur, The Netherlands) was used. For additional quality control of the RBC, capillary hematocrit and supernatant free hemoglobin (after
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Table 1 Characteristics of deep frozen red cells before, during and after freezing to ¡80 °C Mean § SD
Limit(s)
% OK
Filtered whole blood (N D 1360) Thrombocytes (109/U) White blood cells (106/U) Volume (ml/U) Hb content (g/U)
2§2 0.3 § 0.2 519 § 13 63 § 5
<15c <1a 470–570b >55b
99.8 99.6 99.6 93.3
Deep frozen red cells (N D 1360) Hematocrit (l/l) Glycerol concentration (g/dl) Volume (ml/U) Hb content (g/U)
0.60 § 0.03 40.0 § 0.5 326 § 33 60 § 5
0.50–0.70b 38–42b 250–450b >50b
98.5 99.6 99.6 98.8
Thawed washed red cells (N D 147) Hematocrit (l/l) Glycerol concentration (g/dl) Volume (ml/U) Hb content (g/U) Hemolysis at day 14 (%) Hemolysis at day 14 (%)
0.56 § 0.03 0.18 § 0.03 294 § 4 45 § 3 0.6 § 0.2 0.6 § 0.2
0.50–0.65c <1.0d >245c >40a <1.0d <0.8a
95.2 100 100 95.2 96.3 87.7
a b c d
CE recommendations leukodepleted red cells [10]. MBB internal guidelines. Sanquin internal guidelines leukodepleted red cells. FDA/AABB guidelines deglycerolized red cells [4,12,13].
centrifugation at 2200G, 10 min) was measured on a Plasma low hemoglobin device (Hemocue, Oisterwijk, The Netherlands). The percentage of hemolysis was determined by the ratio of free Hb to total Hb. We used a ReXotron® triglyceride test to measure the residual glycerol concentration in the supernatant after a 1:10 dilution in 0.9% saline. The KC4A (Amelung Coagulometer, Germany) was used to determine the concentration of coagulation factors V and VIII, APTT and PT, according to the manufacturer’s instructions. For these tests standard ¡80 °C plasma, factor VIII deWcient plasma and factor V deWcient plasma from Cryocheck (Precision Biologic, Darmouth, NS, Canada) were used. pH was measured by means of an electrode (Delta OHM, Caselle di Sevazzano, Italy). All data were stored and analysed in Excel 97. The diVerences were studied with the Student’s t-test, a P < 0.05 was considered statistically signiWcant. 3. Results Leukodepleted RBC should contain less than 1 £ 106 WBC and less than 15 £ 109 platelets. Liquid stored red cells should contain at least 40 g of Hb and hemolysis at the end of the storage period should not exceed 0.8% or 1% [6,10,12]. After deglycerolization, the Wnal glycerol concentration
should be less than 1% in order to prevent hemolysis in the recipient [13]. For deglycerolized, stored red cells hemolysis should not exceed 1% at the end of the storage period [12]. From March 2003 to July 2005, a total of 1360 units of Wltered, leukodepleted units of whole blood have been processed to frozen red cells. Table 1 shows an overview of the characteristics of these units. From unit hematocrit prior to freezing (Wxed on 80%) and the unit weight (determined for each unit) the ACP 215 accurately calculates the amount of glycerol needed for each individual unit to reach a Wnal concentration of 40% w/v (40 § 0.5%). Throughout the freezing procedure, only 3 § 1 g Hb was lost due to sampling and removal of supernatant plasma and glycerol. Units were deglycerolized, stored at 2–6 °C for 14 days, after which the product quality was assessed. The process was fully validated in the Netherlands prior to in-theatre validation and implementation. In April 2004, the thaw-wash process was validated in Iraq in the operational, military environment. For two days during 12 h each day, two skilled lab technicians, using two ACP 215, thawed and washed 22 units of DEC. There was no diVerence between the units processed at the MBB in Leiden and those in Iraq. At the end of April 2004, we therefore decided to implement the ACP 215 for deglycerolization of
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85 80 75 70 65 60 55 50 45 40 40
50
60
70
80
90
power failure (2 units), bowl damage during deglycerolization (2 units), ACP215 technical problems (1 unit) and leakage of the sterile connection (1 unit). Another unit was lost due to wrongfully connecting the washing Xuids on the ACP215. In conclusion, out of 1360 units, 1298 units (95.4%) met all the criteria to be stored at ¡80 °C for a maximum of 10 years, ready to be used on deployments. We found that Wltered, leukodepleted whole blood, can eVectively be processed with the ACP215 to produce DEC. The thawed washed products contained at least 40 g Hb and can be stored for 14 days at 4 °C with low hemolysis after deglycerolization and suspension in AS3. Table 2 shows that AB plasma has a high factor VIII and factor V content, even after storage for almost 1 year at ¡30 °C. Compared to FFP a slight reduction of factor VIII concentration (P > 0.05) and factor V (P < 0.05) was observed in DFP. The concentration of factors V and VIII did not decrease to below the minimum of 70%. The volume of plasma is reduced by §6 ml because of sampling, without reducing the plasma volume below the minimum of 225 ml. Thus, thawing and subsequent freezing of AB plasma to ¡80 °C can be performed, without deviation of the product quality from the CE recommendations [10]. Until now a total of 435 DFP units were produced and only 16 units (3.7%) were discarded. The majority (12 units) was discarded because the units were thawed in a defective water bath, which resulted in too slow warming of the units. Furthermore 2 units were discarded due to a high RBC content and another 2 units due to the Hb content of thawed, washed product (gram Hb)
Hb content of frozen product (gram Hb)
DEC in Iraq. The backup system proved to be very useful when during the Iraq election period, logistics were hampered and no liquid red cells could be transported. Twenty units were thawed, washed and stored at 2–6 °C successfully to replace the outdated liquid inventory without further delay. An overview of the quality of thawed, deglycerolized and subsequently stored units (including the data from the 42 units processed in Iraq) is shown in Table 1. The ACP215 eVectively removed glycerol from the thawed product from 40% w/v to below the 1% w/v upper limit [13]. It appeared that the Hb content of the frozen unit was primarily dependent upon the Hb content of the leukodepleted product (Fig 1A). The Hb content of the washed unit was primarily dependent upon the bowl size (275 ml), since hemolysis during washing (19 § 5%) was independent of the Hb content of the frozen product, whereas cell spillage was not (results not shown). As shown in Fig 1B, the Hb content of a unit before freezing should at least be 50 g in order to obtain the minimum required product content of 40 g Hb after deglycerolization. Only 16 of 1360 units (1.2%) did not meet this minimum requirement of 50 g Hb prior to freezing and these units were quarantined. Of these low Hb units, 14 units were washed and Hb content was below 40 g Hb in all cases (Fig 1B). Various other reasons, such as training and education purposes (1.1%) and bad seals of the sterile connection device (1%), accounted for another 3.6% that were not released for transfusion. During the validation period 168 units were deglycerolized and 7 units (4.2%) were discarded. Product loss was caused by
52 50 48 46 44 42 40 38 36 34 32 30 28 26 40
45
50
55
60
65
70
75
80
Hb content of frozen product (gram Hb)
Hb content of leukodepleted whole blood (gram Hb) (A)
293
(B)
Fig. 1. The relation between Hb content of ground substance and Hb content of Wnal product. (A) The relation between Hb content of leukodepleted whole blood and Hb content of frozen red cells (N D 1360). (B) The relation between Hb content of frozen red cells and Hb content of thawed, washed red cells (N D 161).
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Table 2 Characteristics of deep frozen plasma before, during and after freezing to ¡80 °C
Fresh frozen plasma apheresis, leukodepleted (FFP) Thrombocytes concentration (109/l) (N D 68) White blood cells (106/U) Volume (ml/U) (N D 188)
Mean § SD
Limit(s)
% OK
5§7 No datad 290 § 15
<50a <1c 225–315c
100 >90 99.5
Coagulation after FFP storage, 0.9 § 0.01 year at ¡30 °C APTT (s) (N D 12) PT (s) (N D 12) Factor VIII (U/ml) (N D 12) Factor V (U/ml) (N D 12)
30.0 § 2.2 12.2 § 0.5 1.4 § 0.5 1.0 § 0.2
<36b <14b >0.70a >0.70b
100 100 100 100
Deep frozen plasma (DFP) Thrombocytes concentration (109/l) (N D 27) Volume (ml/U) (N D 188)
10 § 8 284 § 14
<50b 225–315b
100 99.5
<36b <14b >0.70b >0.70b
100 100 100 100
Coagulation after DFP storage, 0.76 § 0.02 year at ¡30 °C followed by 1.31 § 0.02 year at ¡80 °C APTT (s) (N D 12) 31.0 § 2.3 PT (s) (N D 12) 13.0 § 0.5 Factor VIII (U/ml) (N D 12) 1.2 § 0.3 Factor V (U/ml) (N D 12) 0.9 § 0.1 a b c d
CE recommendations fresh frozen plasma (apheresis) [10]. MBB internal guidelines. Sanquin internal guidelines fresh frozen plasma apheresis, leukodepleted. Product is manufactured by Sanquin Southwest according to Sanquin internal guidelines.
use of spiking because of short tubing on the original FFP bag. An additional advantage of the DFP procedure is that the folding of the product bag and packaging in a vacuum-sealed overwrap bag, in combination with the rigid cardboard box prevents breakage of the PVC bag. We found 8.2% breakage when thawing ¡30 °C stored FFP (N D 486), and no breakage at all with DFP (N D 82). Similarly, the above mentioned thawed DEC (N D 167) also did not show any breakage, nor did the DTC (N D 27) described below. Thawed DTC units were suspended in thawed DFP and the amount of platelets was determined. As shown in Table 3, the units contained more than the minimum of 200 £ 109 platelets/unit (CE recommendations [10] for fresh liquid platelets) and showed a higher than 40% recovery (CE recommendations [10] for frozen platelets). The pH varied was well above 6.8, varying between 7.6 and 7.8. In the Council of Europe (CE) recommendations [10] a limit is set to the volume of product that can be transfused without washing. We concluded from this recommendation that no more than 10 g of DMSO (200 ml 5% w/v) should be present in one platelet unit. As shown in Table 3 the DMSO content of the unit is very low and did not exceed this limit, in fact 94% of all units have a DMSO content
below 1 g/unit. Out of the 217 units frozen, 13 units (6%) were discarded. This was due to recall because of a positive BacTalert® measurement (2 units), clerical or technical errors (4 units), presence of aggregates prior to freezing (2 units), and failure of a sterile connection device (4 units). Only 1 unit was discarded due to a large starting volume, resulting in a Wnal DMSO concentration below 4%. 4. Discussion It has been shown that the high glycerol method can be used to freeze red cells with an acceptable post-thaw and post-wash recovery. By means of the ACP215 it is now possible to store the red cells after washing for a period of 14 days at 4 °C with an acceptable post-storage hemolysis and in vivo survival [8,14–16]. Platelets can be frozen in 5% DMSO at ¡80 °C [17,18]. Although in vivo survival is reduced [17–21], the frozen platelets are more eVective in reducing blood loss compared to 3-day stored liquid platelets [21]. Fresh frozen plasma (¡30 °C) has been used worldwide, the eVect of refreezing them to ¡80 °C has not been studied before. To ensure readily available blood products on missions all over the world, the NL Ministry of Defence decided to build a state of the art frozen inventory of the blood products most needed: red cells, plasma and platelets.
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295
Table 3 Characteristics of deep frozen platelets before, during and after freezing to ¡80 °C
Platelets apheresis, leukodepleted (N D 217) Thrombocytes concentration (109/l) White blood cells (106/U) Volume (ml/U) Thrombocytes (109/U) pH
Mean § SD
Limit(s)
% OK
1221 § 220 0.1 § 0.1 312 § 19 381 § 68 7.1 § 0.1
<1500a <1a 150–400c >200a >6.8c
90.2 100 99.5 100 100
Frozen platelets (N D 217) Volume (ml/U) Thrombocytes (109/U) platelet recovery start product–frozen product (%) DMSO concentration (g/dl) DMSO content (gm)
14 § 4 349 § 58 93 § 13 5.1 § 0.4 0.7 § 0.2
<20b >220b >80b 4–6b <1b
Thawed resuspended platelets in DFP (N D 27) Platelet recovery start product–frozen product (%) Platelet recovery freeze-thaw Platelet recovery start product–end product Thrombocytes concentration (109/l) Volume (ml/U) Thrombocytes (109/U) DMSO content (gm) pH
94 § 15 83 § 17 77 § 15 977 § 226 279 § 28 269 § 45 0.7 § 0.2 7.7 § 0.1
>80b >40d >40b <1500a 225–315b >200a <10b (d) >6.8c
a b c d
92.6 98.2 97.2 97.7 94.0 92.6 100 100 92.6 96.3 100 100 100
CE recommendations platelets apheresis, leukodepleted [10]. MBB internal guidelines. Sanquin internal guidelines platelets apheresis, leukodepleted. CE recommendations cryopreserved platelets [10].
Red cells can be frozen in either a high (40% w/v) glycerol or a low (20% w/v) glycerol concentration. The 40% glycerol allows for storage at ¡80 °C in a mechanical freezer, whereas the 20% glycerol requires liquid nitrogen (¡196 °C). We have shown that the ¡80 °C frozen cells are more stable during post-wash storage [22]. Furthermore, introducing ¡80 °C to freeze red cells Wtted well into the concept of a deployable, integrated liquid-frozen blood bank [23]. The feasibility of the concept itself had been shown before as well as the use of previously frozen red cells in the treatment of combat casualties [24– 26]. Using the ¡80 °C frozen RBC allows for 10 years storage, although much longer periods have been shown to be feasible [7,8]. To deglycerolize the thawed red cells in theatre, prior to transfusion, we previously have used the M 115 (Haemonetics, Braintree, MA, USA), a nonclosed washing device, permitting a 24 h post-wash storage period only. Our frozen red cells thus could not serve as a primary source to meet our red cell needs “in the Weld”, but were used as a back-up for the liquid stock, which was supplied every 2 weeks. We used this concept during missions in Bosnia (1993–2005), Afghanistan (2002–2003) and Liberia
(2003). Both in Afghanistan and Liberia the frozen red cells have proven to be essential when sudden demand exceeded the available liquid stock of red cells. With the introduction of the ACP215, a semiautomated, closed washing device, post-thaw, postwash storage of frozen red cells, became possible for at least 14 days at 2–6 °C [8,14–16]. We show that the ACP215 in its current, FDA approved, conWguration can be used eVectively to produce a stock of DEC and to produce on demand liquid red cells in theatre with suYcient product yield and low hemolysis after storage. In addition, we show that leukodepleted whole blood collected in CPD can be used as a source for DEC. Furthermore, in 2005 it became clear in Iraq that a military hospital blood bank facility can be deployed without regular shipments of liquid red cells. It is able to meet the needs of a surgical team by thawing and washing a certain number of frozen units once a week. This creates a new concept of an integrated liquid-frozen blood bank. We have successfully used this new concept in the current mission in Afghanistan. Our conclusion is that the ACP215 enables a more eYcient and Xexible use of
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RBC for military deployments, still meeting the standard quality requirements of the regulatory civilian authorities. It is well known that following massive blood replacement, plasma and platelet transfusion is required to correct coagulation defects. It has been shown that among survivors of massive blood transfusion more platelets were transfused [27]. Fresh whole blood or stored platelet concentrates are required to correct the coagulation defect [3,27–29]. During several missions in the early nineties, a socalled “walking blood bank” consisting of voluntary military personnel in theatre, was to provide whole blood, primarily as a source of platelets. In 1996, however, we encountered a case of hepatitis B transmission in Bosnia, derived from a “walking blood bank” donor. Although under speciWc conditions a single individual may very well beneWt from this source, the risk of transmitting a disease is high even when donors are used that have been tested recently. Most expeditionary missions are land-based operations in third world countries. This means that infections like malaria and Chagas’disease, even in previously healthy personnel, can and will be transmitted inadvertently from the donor pool to patients. Moreover, since blood is needed at unpredictable moments, the listed donors may not be available at the right moment for lots of reasons [29]. The main reason to use fresh whole blood in theatre is to treat major bleeding, due to lack of platelets in the patient, since liquid stored platelets are not available. In order to abandon the walking blood bank, an alternative for fresh whole blood was required. DMSO frozen platelets have been transfused successfully since the 1970s [19,20]. Despite the fact that their functional recovery is less than that of fresh liquid platelets (around 50%), the frozen platelets are more eVective in stopping non-surgical bleeding compared to 3-day liquid stored platelets [21]. Frozen platelets also show a higher capacity to bind factor V [30] and a higher thromboxane A2 production after ADP stimulation [31]. In addition, baboon frozen platelets have a higher in vivo survival and a stronger correction of aspirin induced prolonged bleeding time compared to 5-day liquid stored platelets [32]. With or without leukodepletion, platelets frozen with 4–6% DMSO may be stored at ¡80 °C for at least 2 years [11]. The possibility of adding frozen platelets to our line of products was therefore very logical to investigate.
Washing became part of the original thawing procedure of frozen platelets to reduce all possible adverse side eVects of DMSO. When we visited Dr. Valeri to learn the freezing and thawing procedure of ¡80 °C frozen platelets he had modiWed the procedure in that post-thaw washing was no more required, because supernatant plasma containing the majority of the DMSO was removed prior to freezing. This modiWed procedure saves valuable time (§1 h), technical training and equipment (centrifuge) in theatre. Since this was a major improvement (time-saving is life-saving), we used this modiWed freezing protocol. Whereas Dr. Valeri uses saline, we use AB plasma to suspend the platelets after thaw, reasoning that a trauma patient needing red cells and platelets almost certainly will also need plasma. Autologous plasma has been used previously for the suspension of thawed washed platelets [20]. We chose not to use this option because when using AB plasma and O platelets the product can be used in every patient regardless of the ABO blood type. In our experience, the thawed platelets show clearly visible swirling in plasma and the product looks like a regular platelet unit obtained by apheresis. We found an in vitro recovery of §77% similar to the in vitro recovery reported by others [11,17–19]. By the end of 2001 we implemented the use of frozen platelets in Bosnia, and abandoned the walking blood bank concept. Within half a year two patients were treated that required platelet transfusion. One elderly woman with gunshot wounds in the pelvic region and one young soldier with acute ITP (viral). Both patients experienced unstoppable bleeding due to a low platelet concentration. Although the platelet count barely rose after transfusion, the bleeding of each patient stopped within 20 min after transfusion of one thawed platelet concentrate in AB plasma. We have thus experienced that frozen platelets can be lifesaving and that the use of a walking blood bank can be abolished when this product is available in theatre. From that time onward, frozen platelets and frozen blood bank facilities always have been part of the standard equipment of Dutch deployed military hospitals. Fresh frozen plasma (FFP) is normally kept at temperatures of around ¡20 °C to ¡30 °C with a shelf life from 1 to 2 years. Lower temperatures of at least ¡65 °C even permit a storage period up to 7 years [9,10]. Apheresis, leukodepleted plasma is frozen by Sanquin, according to the guidelines,
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and meets the requirements for FFP. The units are still frozen upon arrival at the MBB, not folded and do not Wt into our standard rigid cardboard boxes. Since we wanted to maintain uniformity in our packing methods and ¡80 °C transportation and storage temperature, we repacked the plasma units. Refrozen (¡30 °C), previously thawed FFP and even liquid stored plasma, have been shown to be safe and eVective, although factor V and FVIII:C levels are signiWcantly decreased with 10–30% [33– 37]. We observed a slight decrease in factor V and VIII levels due to the extra thaw-freeze cycle, however since we only have used AB plasma which has the advantage of high factor VIII levels to start with, factor V and VIII levels are still well above the 0.7 U/ml. It is likely, but still unproven, that these refrozen DFP units can be stored for 7 years at ¡80 °C as described for fresh frozen plasma at temperatures below ¡65 °C [9,10]. In literature it is suggested that breakage of PVC at ¡80 °C is due to freezing and cannot be prevented, instead new plastics are studied to avoid breakage [38]. Strong reduction of PVC breakage by means of folding the blood bag to protect vulnerable parts and by putting it in an overwrap bag and a rigid cardboard box, has been described by Valeri et al. [11,39]. The overwrap bag prevents contact of the product bag with the cardboard box which reduces breakage and is also used during thaw to protect the water bath against contamination in case the product bag is broken. Using the same method in combination with a vacuum-sealed overwrap bag we also could strongly reduce breakage of standard PVC bags stored at ¡80 °C; breakage was reduced to 0.0% even after transportation on dry ice. To date, the (inter)national guidelines advocate the use of frozen cellular blood products for rare blood types and patients with multiple alloantibodies only. We have shown that the frozen blood bank concept can be eVectively used in a military environment and are convinced that it can also be very useful in the civilian community. A frozen blood bank facility with a stock of frozen universal donor products can eVectively be used in remote areas, to compensate for periods when no donors are available (holiday periods) and when suddenly many patients are in need for blood products. In addition a quarantine period similar to that currently used for plasma can also be used for frozen cellular blood products to reduce risks of transmitting diseases.
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5. Conclusions The quality of our stock of frozen blood products is in compliance with the European and US standards. The products can easily, eVectively and safely be used in deployed blood bank units. Deployed military hospitals become thus less dependent on their home countries or on a walking blood bank. Products can be produced on demand and blood ‘spillage’ due to expiration can be reduced to the absolute minimum. The logistic burden can be reduced since less or no shipments are required to support for the blood product inventory abroad. Acknowledgements We like to thank Dr. C.R. Valeri for providing instructions, protocols, advice and technical support, Sanquin Blood Bank Southwest for providing liquid blood products and leukodepletion data. Furthermore we thank the Pharmacists S.M.G.E. van Grinsven, J. Chin and H.M. Meinen, for their leading role in operating the deployed blood bank in Iraq and S.F. Bus, M. Hakvoort, D. van Zandwijk and R. Nieuwenhuizen for their technical assistance. The Netherlands Ministry of Defence Wnancially supported this study. The opinions or assertions contained herein are those of the authors and are not to be construed as oYcial or reXecting the views of the Ministry of Defence. References [1] Champion HR, Bellamy RF, Roberts P, Leppaniemi A. A proWle of combat injury. J Trauma 2003;54:S13–9. [2] Armand R, Hess JR. Treating coagulopathy in trauma patients. Transfus Med Rev 2003;17:223–31. [3] Hardy JF, Moerloose P, Samana CM. The coagulopathy of massive transfusion. Vox Sang 2005;89:123–7. [4] Scott KL, Lecak J, Acker JP. Biopreservation of red blood cells: past, present and future. Transfus Med Rev 2005;19:127–42. [5] Blajchman MA. Novel platelet products, substitutes and alternatives. Transfus Clin Biol 2001;8:267–71. [6] Buehler PW, Alayash AI. Toxicities of hemoglobin solutions: in search of in-vitro and in-vivo model systems. Transfusion 2004;44:1516–30. [7] Valeri CR, Pivacek LE, Gray AD, Cassidy GP, Leavy ME, Dennis RC, et al. The safety and therapeutic eVectiveness of human red cells stored at ¡80 degrees C for as long as 21 years. Transfusion 1989;29:429–37. [8] Valeri CR, Srey R, Tilahun D, Ragno G. The in vitro quality of red blood cells frozen with 40 percent (wt/vol) glycerol at ¡80 degrees C for 14 years, deglycerolized with the Haemonetics ACP 215, and stored at 4 degrees C in additive solution-1
298
[9] [10] [11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
C.C.M. Lelkens et al. / Transfusion and Apheresis Science 34 (2006) 289–298 or additive solution-3 for up to 3 weeks. Transfusion 2004;44:990–5. Standards for blood banks and transfusion services, 23rd ed. AABB, 2004. Guide to the preparation, use and quality assurance of blood components, 11th ed. Council of Europe, 2005. Valeri CR, Srey R, Lane JP, Ragno G. EVect of WBC reduction and storage temperature on PLTs frozen with 6 percent DMSO for as long as 3 years. Transfusion 2003;43:1162–6. Sowemimo-Coker S. Red blood cell hemolysis during processing. Transfus Med Rev 2002;16:46–60. Mollison PL, Engelfriet CP, Contreras M. 10th ed. Blood transfusion in clinical medicine Blackwell Science; 1997. p. 302. Valeri CR, Ragno G, Pivacek LE, Srey R, Hess JR, Lippert LE, et al. A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4 degrees C in AS-3: assessment of RBC processing in the ACP 215. Transfusion 2001;41:933–9. Valeri CR, Ragno G, Pivacek L, O’Neill EM. In vivo survival of apheresis RBCs, frozen with 40-percent (wt/vol) glycerol, deglycerolized in the ACP 215, and stored at 4 degrees C in AS-3 for up to 21 days. Transfusion 2001;41:928–32. Bandarenko N, Hay SN, Holmberg J, Whitley P, Taylor HL, MoroV G, et al. Extended storage of AS-1 and AS-3 leukoreduced red blood cells for 15 days after deglycerolization and resuspension in AS-3 using an automated closed system. Transfusion 2004;44:1656–62. Melaragno AJ, Abdu WA, Katchis RJ, Vecchione JJ, Valeri CR. Cryopreservation of platelets isolated with the IBM 2997 blood cell separator: a rapid and simpliWed approach. Vox Sang 1982;43:321–6. Vecchione JJ, Chomicz SM, Emerson CP, Valeri CR. Cryopreservation of human platelets isolated by discontinuousXow centrifugation using the Haemonetics Model 30 Blood Processor. Transfusion 1980;20:393–400. SchiVer CA, Aisner J, Wiernik PH. Clinical experience with transfusion of cryopreserved platelets. Br J Haematol 1976;34:377–85. SchiVer CA, Aisner J, Dutcher JP, Daly PA, Wiernik PH. A clinical program of platelet cryopreservation. Prog Clin Biol Res 1982;88:165–80. Khuri SF, Healey N, MacGregor H, Barnard MR, Szymanski IO, Birjiniuk V, et al. Comparison of the eVects of transfusions of cryopreserved and liquid-preserved platelets on hemostasis and blood loss after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1999;117:172–83. Lelkens CCM, Noorman F, Koning JG, Truijens-de Lange R, Stekkinger P, Bakker JC, et al. Stability after thawing of RBCs frozen with the high and low glycerol method. Transfusion 2003;43:157–64. Valeri CR, Sims KL, Bates JF, Reichman D, Lindberg JR, Wilson AC. An integrated liquid-frozen blood banking system. Vox Sang 1983;45:25–39.
[24] Moss GS, Valeri CR, Brodine CE. Clinical experience with the use of frozen blood in combat casualties. N Engl J Med 1968;278:747–52. [25] Valeri CR, Brodine CE, Moss GE. Use of frozen blood in Vietnam. Bibl Haematol 1968;29:735–8. [26] Rosenblatt MS, Hirsch EF, Valeri CR. Frozen red blood cells in combat casualty care: clinical and logistical considerations. Mil Med 1994;159:392–7. [27] Cinat ME, Wallace WC, Nastanski F, West J, Sloan S, Ocariz J, et al. Improved survival following massive transfusion in patients who have undergone trauma. Arch Surg 1999;124:964–70. [28] Ho AM, Karmakar MK, Dion PW. Are we giving enough coagulation factors during major trauma resuscitation? Am J Surg 2005;190:479–84. [29] Reade MC. Blood products on operational deployments. ADF Health 2001;2:65–70. [30] Barnard MR, MacGregor H, Ragno G, Pivacek LE, Khuri SF, Michelson AD, et al. Fresh, liquid-preserved, and cryopreserved platelets: adhesive surface receptors and membrane procoagulant activity. Transfusion 1999;39:880–8. [31] Valeri CR, MacGregor H, Ragno G. Correlation between in vitro aggregation and thromboxane A2 production in fresh, liquid-preserved, and cryopreserved human platelets: eVect of agonists, pH, and plasma and saline resuspension. Transfusion 2005;45:596–603. [32] Valeri CR, MacGregor H, Giorgio A, Ragno G. Circulation and hemostatic function of autologous fresh, liquid-preserved, and cryopreserved baboon platelets transfused to correct an aspirin-induced thrombocytopathy. Transfusion 2002;42:1206–16. [33] Milam JD, Buzzurro SF, Austin SF, Stansberry SW. Stability of factors V and VIII in thawed fresh frozen plasma units. Transfusion 1980;20:546–8. [34] Smak Gregoor PHJ, Harvey MS, Briet E, Brand A. Coagulation parameters of CPD fresh-frozen plasma and CPD cryoprecipitate-poor plasma after storage at 4 °C for 28 days. Transfusion 1993;33:735–8. [35] Dzik WH, Rubner MA, Linehan SK. Refreezing previously thawed fresh-frozen plasma Stability of coagulation factors V and VIII:C. Transfusion 1989;29:600–4. [36] Ben-Tal O, Zwang E, Eichel R, Badalbev T, Hareuveni M. Vitamin K-dependent coagulation factors and Wbrinogen levels in FFP remain stable upon repeated freezing and thawing. Transfusion 2003;43:873–7. [37] Cardugan R, Lawrie AS, Maackie IJ, Williamson LM. The quality of fresh-frozen plasma produced from whole blood stored at 4 °C overnight. Transfusion 2005;45:1342–8. [38] Hmel PI, Kennedy A, Quiles JG, Gorogias M, Seelbaugh JP, Morrissette CR, et al. Physical and thermal properties of blood storage bags: implications for shipping frozen components on dry ice. Transfusion 2002;42:836–46. [39] Valeri CR, Ragno G. Breakage rate for red blood cells frozen with 40 percent (wt/vol) glycerol in 800-ml polyvinylchloride plastic bags stored in rigid cardboard boxes at ¡80 degrees C. Transfusion 2005;45:822–3.