Preventing platelet-derived microparticle formation--and possible side effects--with prestorage leukofiltration of whole blood.
Abstract: * Context.--Platelet-derived microparticles (PDMPs) probably function in hemostasis, thrombosis, inflammation, and transfusion-related immunomodulation.

Objectives.--To compare PDMP levels of leukocyte-filtered and unfiltered whole blood during storage.

Design.--Ten whole blood donations were collected and processed. Half of each collection was filtered, half remained unfiltered, and both halves were measured for red cell, white cell, and platelet (PLT) content before storage. Samples were drawn on days 0, 1, 2, 3, 5, 7, 14, 21, 28, and 35 and analyzed by flow cytometry.

Results.--Leukocyte filtration lowered prestorage PDMP and PLT counts by an average of 72% and 99%, respectively. Prestorage PDMP counts were 123 [+ or -] 51/[micro]L in unfiltered whole blood supernatant versus 34 [+ or -] 18/[micro]L after filtration. Prestorage PLT counts were 190 [+ or -] 49/[micro]L in unfiltered whole blood supernatant versus 2 [+ or -] 4/[micro]L after filtration. Moreover, PDMP and PLT counts in filtered whole blood remained low throughout storage, typically below 100/[micro]L . In contrast, unfiltered whole blood PDMP-and PLT-gated events increased approximately 2 log during storage, with the peak number of PLT-gated events tending to coincide with the peak number of PDMP-gated events (4 donors) or to come after the peak number of PDMP-gated events (6 donors).

Conclusion.--Leukocyte filtration of whole blood lowers prestorage PDMP and PLT counts. Platelet-derived micro-particle and PLT counts remain low throughout 35 days of storage. In contrast, PDMP- and PLT-gated events increase significantly in unfiltered whole blood. The nature of PLT-gated events in stored blood warrants further investigation.

(Arch Pathol Lab Med. 2010;134:771-775)
Article Type: Report
Subject: Blood clot (Development and progression)
Blood clot (Physiological aspects)
Thrombosis (Development and progression)
Thrombosis (Physiological aspects)
Flow cytometry (Usage)
Hemostasis (Physiological aspects)
Hemostasis (Research)
Leukocytes (Physiological aspects)
Leukocytes (Research)
Authors: Sugawara, Akiko
Nollet, Kenneth E.
Yajima, Kentaro
Saito, Shunnichi
Ohto, Hitoshi
Pub Date: 05/01/2010
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 College of American Pathologists ISSN: 1543-2165
Issue: Date: May, 2010 Source Volume: 134 Source Issue: 5
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Japan Geographic Code: 9JAPA Japan
Accession Number: 230246565
Full Text: Microparticles were first characterized by electron microscopy, (4) a technique unlikely to be available in a blood center. Optical microscopy is not suited to MP detection, because visible light is scattered by particles close in size or smaller than its wavelength (0.4-0.7 mm). However, light scattering is a useful characteristic in flow cytometry, the principal tool of this investigation, and modern flow cytometry lends itself to quality assurance in other areas. (5-8)

Platelet-derived microparticles (PDMPs), insofar as they display membrane surface antigens characteristic of activated PLTs, may participate in coagulation and thrombogenesis. (3,9,10) One such antigen, CD42b (GPIb[alpha]), is part of the CD42a-d complex that serves as a receptor for von Willebrand factor and thrombin. Platelet activation occurs during cold storage, so residual PLTs in stored blood components may give rise to MPs displaying the CD42b antigen. By itself, fluorescent antibody binding to CD42b cannot distinguish PDMPs from PLTs or other particles to which CD42b might be transferred, but "events" (cells or particles) detected by flow cytometry can be characterized by their fluorescence and lights-cattering properties.

MATERIALS AND METHODS

Donor Selection, Informed Consent, and Conflict of Interest Management

Blood donors were healthy adult volunteers not taking medications known to affect PLT function. Their participation conformed to institutional guidelines for human subjects, and they gave informed consent for the use of their blood in research. Qualified members of the Division of Blood Transfusion and Transplantation Immunology performed all collection, processing, and testing.

[FIGURE 1 OMITTED]

Equipment

Flow cytometry was performed on a Cytomics FC 500 (Beckman Coulter, Fullerton, California), using standard software and reagents supplied by the manufacturer (eg, CXP Cytometer and CXP Analysis software, Flow-Check and Flow-Count fluorospheres). Day 0 blood counts were performed with a KX-21 hematology analyzer (Sysmex, Kobe, Japan). All samples from blood bags were withdrawn through single-use 18-gauge needle and syringe sets and subsequently handled in single-use 12 3 75-mm polypropylene test tubes.

Collection and Processing

Ten healthy adults (5 females, 5 males; age 26-56 years, mean 36 years) consented to donate 200 [micro]L of whole blood (WB), which was collected into a Sepacell Integra CA system designed for that volume (Asahi Kasei Medical, Tokyo, Japan). After thorough mixing of WB with 28 [micro]L of citrate-phosphate-dextrose-adenine anticoagulant in the collection bag, half of the volume was passed through the system's inline leukoreduction filter. Filter and transfer tubing were sealed off, and the bags were separated. Under sterile hood conditions, each bag was spiked with a TC-MP adapter (Terumo, Tokyo, Japan) for periodic sampling. Immediate (day 0) prefiltration and postfiltration samples were evaluated for PLT and PDMP content. Platelet concentration was measured with a Sysmex KX-21 hematology analyzer. Except for periodic mixing and sampling under a room-temperature hood, blood bags were kept at 5[degrees]C.

Samples (4 mL) withdrawn at 0, 1, 2, 3, 5, 7, 14, 21, 28, and 35 days were centrifuged at 2000 g for 20 minutes, followed by soft deceleration. Supernatant aliquots (250 [micro]L) were transferred to new test tubes, from which 50 [micro]L aliquots were withdrawn and added to test tubes freshly loaded with 10 [micro]L of either phycoerythrin-conjugated anti-CD42b or mouse immunoglobulin G (Immunotech, Marseille, France). Sample supernatant and conjugated antibody were incubated at room temperature for 20 minutes. Thereafter, a fixative of 2 [micro]L of 1% paraformaldehyde in phospate-buffered saline was added, followed by incubation at 5[degrees]C for 30 minutes. Flow-Count fluorospheres, 10 [micro]L , were added and thoroughly mixed immediately before the day's flow cytometry run.

Fluorescence, forward scatter, and side scatter gates to distinguish PDMPs and PLTs were developed according to the methods of Ozeki and Nomura,11 from a prior study of platelet-rich plasma from 5 healthy adults (3 females, 2 males; age 26-56 years, mean, 30 years) (A.S., unpublished data, 2006). Figure 1, A, shows fluorescence threshold gating. Figure 1, B, shows forward scatter and side scatter gating.

RESULTS

The Sepacell Integra CA filter, designed to achieve a residual leukocyte count below 1 x [10.sup.6]/unit, also reduces PDMP and PLT counts, as shown in Table 1. Average prestorage PLT concentration of 190 x [10.sup.3]/[micro]L was reduced 99% by filtration to 2 x [10.sup.3]/[micro]L . Average prestorage PDMP concentration of 123/[micro]L was reduced 72% by filtration to 34/[micro]L . Moreover, PDMP- and PLT-gated events remained low in filtered WB during storage, in contrast to unfiltered WB, as shown in Figures 2 and 3.

Platelet-derived microparticle- and PLT-gated events detected in filtered WB supernatant were consistently lower than 100/[micro]L throughout 35 days of storage, with the exception of samples from donor 10 (Figure 2, line F10), in which PDMP-gated events were 21/[micro]L on day 0, but rose to 301/[micro]L on day 28 and were 170/[micro]L on day 35.

In contrast to filtered WB supernatant, PDMP-gated events in unfiltered WB supernatant increased during cold storage by as much as 2 log or more, for example, from 189/[micro]L on day 0 to 36 913/[micro]L on day 7 (Figure 2, donor 1, line U1). In absolute terms, this was also the highest PDMP-gated event count obtained from any of the 10 donors.

Platelet-gated events detected in unfiltered WB supernatant also increased during cold storage by as much as 2 log or more, for example, from 125/[micro]L on day 0 to 19 937/ [micro]L on day 35 (Figure 3, donor 10, line U10). In absolute terms, the highest PLT-gated event count was 35 650/[micro]L on day 35 (Figure 3, donor 9, line U9). The peak number of PLT-gated events tended to coincide with the peak number of PDMP-gated events (4 donors) or come after the peak number of PDMP-gated events (6 donors).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Table 2 shows PDMP- and PLT-gated events during storage as mean [+ or -] standard deviation for all 10 donors. On average, for unfiltered WB supernatant, PDMP-gated events peak on day 7, whereas PLT-gated events continue to increase through 35 days of storage. Platelet-derived microparticle- and PLT-gated events in filtered WB supernatant show no trend and remain low throughout storage.

DISCUSSION

Prestorage leukocyte filtration of blood components has gained favor as a beneficial intervention for at least some patient populations and is recognized as a standard of care in many countries. (12) However, the history of leukocyte filtration includes reports of adverse effects such as the red eye syndrome, (13) back pain, (14) and hypotensive reactions. (15,16) It might be argued that modern leukofiltration is a mature technology for which extraordinary vigilance is no longer required, but this perspective ignores 2 points: (1) Filtration technology is evolving to remove not only leukocytes, but also other contaminants such as prions (17); (2) Interest in storage lesion suggests new areas of concern, for example, MPs. (3,18,19) Thus, quality assessment and quality assurance of filter performance should include more than just the target parameters of contaminant reduction. New performance metrics should be applied to both old and new filter technology. This investigation addressed MP formation during storage of blood components, with and without leukocyte filtration using existing technology. These results have prompted further investigations of combined prion and leukocyte filtration technology. (20)

Microparticles, including PDMPs, are observed in vivo and their functional significance is a matter of substantial investigation. Elevated PDMP levels have been associated with various medical conditions, including diabetes mellitus, inflammation, and thrombosis. (9,21,22) Platelet-derived microparticles arising in vitro as a storage lesion may differ qualitatively from PDMPs arising in vivo. In any case, their presence in stored blood warrants concern, especially in view of recent associations of longer storage with higher morbidity and mortality. (1) Procoagulant properties of PDMPs may contribute to deep vein thrombosis or coronary occlusion in vulnerable patients. In this clinical context, factors that could affect PDMP levels in stored blood should be investigated with the ultimate goal of improving patient outcomes.

A randomized controlled trial (23) recently indicated that prestorage leukofiltration of autologous WB does not improve postoperative outcomes such as infection rate or length of hospital stay. However, the incidence of postoperative deep vein thrombosis, observed in 1 of 488 patients receiving filtered WB versus 2 of 463 receiving unfiltered WB, is still to be elucidated with a cohort size of sufficient power.

Platelet-derived microparticle- and PLT-gated events in unfiltered WB can increase by 2 log or more during storage, with the peak number of PLT-gated events occurring on or after the peak number of PDMP-gated events. It is plausible that PLT-gated events arise from aggregation of PDMPs, association of PDMPs with other formed elements of blood, or from intracellular trafficking of CD42b. Red blood cells (RBCs) and RBC microparticles might acquire CD42b and other platelet antigens in this way. Thus, "RBC-derived microparticles" in WB might be thrombogenic, depending on manufacturing and storage conditions. With or without PLT antigens, RBC microparticles themselves, and RBC microparticles in the context of filtration, warrant further investigation.

Prestorage WB leukofiltration was shown to substantially reduce initial PDMP and PLT counts, consistent with other blood product and filter combinations previously described. (24,25) The observation that filtered WB maintains low PDMP- and PLT-gated event counts throughout storage should not be surprising, but neither should it be taken for granted. Quality assurance concerns itself with surveillance of expected outcomes, and investigation of unexpected outcomes. In this study, unfiltered WB provided an unexpected outcome, namely, that both PDMP- and PLT-gated events increased by as much as 2 log or more during storage. The true nature of these flow cytometric events is a matter of ongoing investigation in this laboratory, not only for current filtration technology, but also for next-generation filters being developed. It is hoped that this report will be one of many from around the world: first, to call attention to epiphenomena associated with filtration technology; second, to compare new and existing technologies with respect to such epiphenomena; and third, to forge new paradigms for quality assurance that are timely, cost-effective, and beneficial to better patient outcomes.

We hereby confirm that Asahi Kasei Medical Co, Ltd, provided grant support and supplies to Fukushima Medical University for an investigation of its filtration technology.

References

(1.) Koch C, Li L, Sessler D, et al. Duration of red-cell storage and complications after cardiac surgery. N Engl J Med. 2008;358(12):1229-1239.

(2.) Lynch SF, Ludlam CA. Plasma microparticles and vascular disorders. Br J Haematol. 2007;137(1):36-48.

(3.) Keuren JF, Magdeleyns EJ, Govers-Riemslag JW, Lindhout T, Curvers J. Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation. Br J Haematol. 2006;134(3):307-313.

(4.) George JN, Thoi LL, McManus LM, Reimann TA. Isolation of human platelet membrane microparticles from plasma and serum. Blood. 1982;60(4):834-840.

(5.) Levering WH, van Wieringen WN, Kraan J, et al. Flow cytometric lymphocyte subset enumeration: 10 years of external quality assessment in the Benelux countries. Cytometry B Clin Cytom. 2008;74(2):79-90.

(6.) Dijkstra-Tiekstra MJ, van der Meer PF, Pietersz RN, de Wildt-Eggen J. Multicenter evaluation of two flow cytometric methods for counting low levels of white blood cells. Transfusion. 2004;44(9):1319-1324.

(7.) van der Meer PF, Gratama JW, van Delden CJ, et al. Comparison of five platforms for enumeration of residual leucocytes in leucoreduced blood components. Br J Haematol. 2001;115(4):953-962.

(8.) Deneys V, Mazzon AM, Robert A, Duvillier H, De Bruyere M. Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates. Vox Sang. 1994;67(2):172-177.

(9.) Nomura S. Function and significance of platelet-derived microparticles. Int J Hematol. 2001;74(4):397-404.

(10.) Keuren JF, Magdeleyns EJ, Bennaghmouch A, Bevers EM, Curvers J, Lindhout T. Microparticles adhere to collagen type I, fibrinogen, von Willebrand factor and surface immobilised platelets at physiological shear rates. Br J Haematol. 2007;138(4):527-533.

(11.) Ozeki Y, Nomura S. Assay methods for platelet-derived microparticle: merit and problem. Kessen Shiketsu Shi [Jpn J Thrombosis Hemostasis]. 2004; 15(3):286-292.

(12.) Beckman N, Sher G, Masse M, et al. Review of the quality monitoring methods used by countries using or implementing universal leukoreduction. Transfus Med Rev. 2004;18(1):25-35.

(13.) Alonso-Echanove J, Sippy BD, Chin AE, et al. Nationwide outbreak of red eye syndrome associated with transfusion of leukocyte-reduced red blood cell units. Infect Control Hosp Epidemiol. 2006;27(11):1146-1152.

(14.) Alvarado-Ramy F, Kuehnert MJ, Alonso-Echanove J, et al. A multistate cluster of red blood cell transfusion reactions associated with use of a leucocyte reduction filter. Transfus Med. 2006;16(1):41-48.

(15.) Abe H, Ikebuchi K, Shimbo M, Sekiguchi S. Hypotensive reactions with a white cell-reduction filter: activation of kallikrein-kinin cascade in a patient [author reply in Transfusion. 1998;38(4):413-415]. Transfusion. 1998;38(4):411412.

(16.) Takahashi TA, Abe H, Hosoda M, Nakai K, Sekiguchi S. Bradykinin generation during filtration of platelet concentrates with a white cell-reduction filter. Transfusion. 1995;35(11):967.

(17.) Cervia JS, Sowemimo-Coker SO, Ortolano GA, Wilkins K, Schaffer J, Wortham ST. An overview of prion biology and the role of blood filtration in reducing the risk of transfusion-transmitted variant Creutzfeldt-Jakob disease. Transfus Med Rev. 2006;20(3):190-206.

(18.) Cauwenberghs S, Feijge MA, Harper AG, Sage SO, Curvers J, Heemskerk JW. Shedding of procoagulant microparticles from unstimulated platelets by integrin-mediated destabilization of actin cytoskeleton. FEBS Lett. 2006;580(22): 5313-5320.

(19.) Simak J, Gelderman MP. Cell membrane microparticles in blood and blood products: potentially pathogenic agents and diagnostic markers. Transfus Med Rev. 2006;20(1):1-26.

(20.) Nollet KE, Ohto H, Sugawara A, Yajima K, Saito S. Evolution of microparticles during storage: testing a new prion-leukocyte filter against current technology. Transfusion. 2008;48(2S):229A.

(21.) Tan KT, Tayebjee MH, Lim HS, Lip GYH. Clinically apparent atherosclerotic disease in diabetes is associated with an increase in platelet microparticle levels. Diabet Med. 2005;22(12):1657-1662.

(22.) Perez-Pujol S, Marker PH, Key NS. Platelet microparticles are heterogeneous and highly dependent on the activation mechanism: studies using a new digital flow cytometer. Cytometry. 2007;71A:38-45.

(23.) Frietsch T, Karger R, Scholer M, et al. Leukodepletion of autologous whole blood has no impact on perioperative infection rate and length of hospital stay. Transfusion. 2008;48(10):2133-2142.

(24.) Krailadsiri P, Seghatchian J, Williamson LM. Platelet storage lesion of WBC-reduced, pooled, buffy coat-derived platelet concentrates prepared in three in-process filter/storage bag combinations. Transfusion. 2001;41(2):243- 250.

(25.) Krailadsiri P, Seghatchian J, Macgregor I, et al. The effects of leukodepletion on the generation and removal of microvesicles and prion protein in blood components. Transfusion. 2006;46(3):407-417.

Akiko Sugawara, AMLT; Kenneth E. Nollet, MD, PhD; Kentaro Yajima, BA; Shunnichi Saito, MT; Hitoshi Ohto, MD, PhD

Accepted for publication July 1, 2009.

From the Division of Blood Transfusion and Transplantation Immunology, Fukushima Medical University, Fukushima City, Japan (Ms Sugawara, Drs Nollet and Ohto, and Mr Saito); and Sepacell Division, Asahi Kasei Medical Co, Ltd, Chiyoda-ku, Tokyo, Japan (Mr Yajima).

Mr Yajima has a financial interest in Asahi Kasei Medical Co, Ltd. The other authors have no relevant financial interest in the products or companies described in this article.

Reprints: Kenneth E. Nollet, MD, PhD, Division of Blood Transfusion and Transplantation Immunology, Fukushima Medical University, 1 Hikariga-oka, Fukushima City, Fukushima 960-1295 Japan (e-mail: nollet@fmu.ac.jp or abo24x7@yahoo.com).
Table 1. Prestorage Platelet-Derived Microparticle

(PDMP) and Platelet (PLT) Concentrations in Whole

Blood Supernatant, With and Without Filtration

                            Unfiltered,          Filtered,
Analyte                  Mean [+ or -] SD    Mean [+ or -] SD

PDMP count,/[micro]L      123 [+ or -] 51     34 [+ or -] 18
PLT count, x
  [10.sup.3]/[micro]L     190 [+ or -] 49      2 [+ or -] 4

                             Removal
Analyte                      Rate, %

PDMP count,/[micro]L      72 [+ or -] 8
PLT count, x
  [10.sup.3]/[micro]L     99 [+ or -] 3

Table 2. Platelet-Derived Microparticle (PDMP)-Gated and Platelet
(PLT)-Gated Events During Storage of Unfiltered and Filtered Whole
Blood (WB) From 10 Healthy Adult Donors

          Unfiltered WB Supernatant, /[micro]L

            PDMP Count,               PLT Count,
Day       Mean [+ or -] SD         Mean [+ or -] SD

 0        123 [+ or -] 48          656 [+ or -] 292
 1        391 [+ or -] 583        1179 [+ or -] 647
 2       1945 [+ or -] 4273       1332 [+ or -] 789
 3       2671 [+ or -] 4197       1602 [+ or -] 948
 5       5816 [+ or -] 9012       4246 [+ or -] 4470
 7      6466 [+ or -] 10392       6185 [+ or -] 4964
 14      5692 [+ or -] 4363      13 012 [+ or -] 5511
 21      3500 [+ or -] 2238      13 062 [+ or -] 4483
 28      2636 [+ or -] 1501      14 510 [+ or -] 6190
 35      2419 [+ or -] 1682      17 180 [+ or -] 9366

              Filtered WB Supernatant, /[micro]L

           PDMP Count            PLT Count,
Day     Mean [+ or -] SD      Mean [+ or -] SD

 0       34 [+ or -] 17        26 [+ or -] 18
 1       31 [+ or -] 12         24 [+ or -] 7
 2        27 [+ or -] 7        20 [+ or -] 11
 3       26 [+ or -] 13        28 [+ or -] 11
 5       23 [+ or -] 13        27 [+ or -] 13
 7        20 [+ or -] 9        26 [+ or -] 15
 14      19 [+ or -] 13        24 [+ or -] 12
 21       15 [+ or -] 7         21 [+ or -] 6
 28      42 [+ or -] 87         22 [+ or -] 6
 35      27 [+ or -] 48         21 [+ or -] 6
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