Journal of Clinical Apheresis 6:143-149 (1991)

Preparation and Storage of “Leukocyte-Depleted Single Donor” Platelet Concentrate: A Step Forward in Effective Haemotherapy Mona K. Elias, Joost Th.M. de Wolf, Nel Blom, Luit Rijskamp, Ruud M. Halie, and Cees Th. Smit Sibinga Red Cross Blood Bank Groningen-Drenthe (M.K.E., L.R., C.T.S.S.) and Department of Haematology, University of Groningen (J.T. M.d. W., N.B., R.M. H.), Groningen, The Netherlands “Leukocyte-depleted” platelet concentrates (LD-SDPC) were prepared by cotton-wool and by polyester filtration of “leukocyte-poor” PC collected by the elutriation technique. The storage characteristics of LD-SDPC were comparable to those of the filtered pools of multiple donor platelet concentrates (LD-MDPC). However, LD-SDPC were less activated and less damaged than LD-MDPC over a 7-day storage period, as evidenced by P-thromboglobin percent release and lactic dehydrogenase percent leakage in both products. LD-SDPC were prophylactically transfused to 12 thrombocytopenic patients; the mean bleeding time was shortened from 12 min 20 sec to 4 min 39 sec. Corrected count increment (CCI) was 22.8 at 1 hr and 14.1 at 24 hr compared to 15.5 and 10.0, respectively, with standard PCs.

Key words: elutriation, leukocyte depletion, filtration, bleeding time correction, corrected count increment


The benefit of leukocyte depletion from blood components for minimising the incidence of alloimmunisation is well recognised [ 1-71. Leukocyte-poor blood products were reported to prevent febrile transfusion reactions [8-201 and more recently to reduce cytomegalovirus infectivity [2 I]. In addition, contaminating leukocytes were found to adversely affect the quality of stored platelets directly [22,23] as well as through pH lowering [24-271. Although the reduced level of leukocyte contamination achieved by any depletion technique that removes 90% of leukocytes seems adequate to prevent febrile non-haemolytic reactions, a leukocyte level below 10 million per transfusion may be necessary to avoid alloimmunisation [28-301. To guarantee a leukocyte contamination below this level, we used a combination of two leukocyte depletion techniques: elutriation followed by filtration in a closed system. We report on the storage characteristics over 7 days, as well as the clinical efficacy of this leukocyte-depleted product.

V50 modified autosurge protocol). This technique uses a discontinuous flow mode and preprogrammed adjustable variables. After separation of the cellular components, platelet collection begins at the platelet-plasma interface which is automatically detected by an optical sensor system. Platelets are surged out of the buffy coat by high speed reinjection of a volume of the collected plasma into the bowl. This results in elutriation of platelets from the red cell mass and increases the resolution of platelets and leukocytes in the buffy coat. A closed apheresis software system (extended storage pack PL-724) including two 1,500 ml PVC-TOTM (Polyvinyl chloride plasticised with trimellitate) containers (CLX-7) was preconnected by sterile connection (DuPont SCD 321) to a pig-tail ACDA anticoagulant bag. One of the two collection bags was disconnected and saved for collection of the filtered end product. Differential Centrifugation

Standard multiple donor platelet concentrates (MDPC) were prepared at room temperature by first spinning at 1,OOOg for 9 min, followed by a second spin



Single donor platelet concentrates (SDPC) were collected by the elutriation technique [311 (Haemonetics

Address reprint requests to Mona K Elias, P 0 Box 1191, 9701 ED Groningen, The Netherlands

0 1991 Wiley-Liss, Inc.


Received for publication Apnl30, 1991, accepted September 8, 1991


Elias et al.

at 1,300g for 30 min. The platelet pellet was resuspended in 50 ml plasma and left undisturbed at room temperature, for 1'/2 hr. Six donor units were pooled, filtered, and stored for 7 days in parallel with elutriated and filtered SDPC.

chamber, 1:lO dilution, whole grid counts, lowest detection limit 10 cells/pl). Throughout storage, the metabolic status was assessed by pH (22°C) maintenance, HCO, depletion, lactate production, and glucose consumption. Platelet function was assessed by aggregation responses to dual agonist (epinephrine 2 p M ADP 10 pm) [32] and recovery from hypotonic shock [33]. Platelet structural integrity was determined by the morphology score [34]. Change in platelet ultrastructure following filtration was observed by a Philips 201 electron microscope. Representative pre- and post-filtration (n = 10) samples were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 2 hr, pelleted, rinsed, and post-fixed in 1% OSO, for 1 hr, dehydrated in a graded series of alcohol, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and examined. Beta thromboglobulin (PTG) release was used as a marker of activation and release reaction. Lactic dehydrogenase (LDH) leakage was used as an index of cell lysis (normal plasma range: 114-235 U/L). The assay uses a modified lactate to pyruvate procedure (Du Pont aca). Both markers were expressed as a percent release [35] according to the formula:



Elutriated SDPC were filtered within 2 hr of collection through a cotton wool filter (Imugard IG 500) or a polyester filter (Pall PL 100s) designed for use with the sterile connection device. MDPC needed longer time to become fully disaggregated, and were preferably filtered on the next day to avoid clogging of the filter. The two upper tubings of the cotton wool filter were sterilely connected to the platelet bag and to a pig-tail saline bag. The two lower tubings were connected to a 400 ml pack serving as waste container and to the empty CLX-7 container. The filter was washed and primed with 150 ml saline (discarded). The PC was positioned 90 cm above the filter and filtration was performed by gravity. A 100 ml of saline was used first to rinse the emptied PC container, then to recover the platelets trapped in the filter. To decrease the saline percentage in the filtrate (25%) either more PC volume was filtered or the first 50 ml of almost platelet free filtrate was discarded in the waste bag. Filtration with the polyester filter was performed at 10 ml/min flow rate, without priming. For comparative reasons, the filter was rinsed with 30-40 ml saline.


level of PTG (LDH)/ml supernatant total PTG (LDH)/ml triton-lysed PC

x 100

Storage Conditions

The filtered PC (LD-PC) was divided over the two sterilely connected CLX-7 containers. Routinely used 300 ml polyolefin containers (PO) were also used for comparison. Filling ratios ranging from 1:4 to 1:lO were compared. PC was stored for 7 days, at 22-24"C, on a flat bed agitator reciprocating at 70 strokedmin.

LDH level was also expressed as units per 10" platelets. Endotoxin was assessed by chromographic substrate (KabiVitrum). The test is based on activation of a proenzyme in the Limulus Amoebocyte Lysate (LAL) by endotoxin. Bacterial culture was done on random samples before and after filtration, as well as during and after 7-day storage.


Samples were drawn before and after filtration and on days 2, 5 , and 7 through a plasma exchange coupler. The ending with the plastic cover spike was welded to the container tubing and a kocher clamp was applied proximal to the spike with cotton that allows exit of air. The sample was allowed to flow in the coupler by slightly releasing the kocher clamp. Both endings were sealed off and thus the system was kept closed. In Vitro Platelet Testing

Cell counts and distribution curves were performed on a Coulter S cell counter (lowest leukocyte detection limit 0.2 X 109/L). Post filtration leukocyte counts were performed manually using a haemocytometer (Biirker



The effect of various filling ratios on the metabolic and functional parameters was assessed by Spearman rank analysis. Multiple regression analysis (Systat) was performed to assess the effect of saline added during filtration and the degree of bag filling on pH. Wilcoxon Mann Whitney test was used to compare the storage-induced changes between both products and between both container types. In Vivo Study

Twelve clinically stable thrombocytopenic leukemia patients received leukocyte-depleted SDPC during the induction remission period.

LD-SDPC Preparation and Storage

Non-immunological causes which accelerate platelet consumption were absent. Post-transfusion platelet recovery at 1 and 24 hr was corrected for the individual recipient and the dose of cells transfused according to the formula: Absolute increment ( l 09) X body surface area (m2) CCI = Total platelets transfused (10") A CCI of 10 or less at 1 hour and 7.5 or less at 24 hr was defined as less than adequate response to the transfused platelets [36]. Increments obtained with leukocytedepleted transfusions were compared with those obtained with non-filtered standard PC. The haemostatic effectiveness of leukocyte depleted PC was determined by measuring the template bleeding time [37,38] before and 1 hr after the completion of the transfusion (upper normal range 9 min).

RESULTS In Vitro Results

Pre- and post-filtration data [means and ( ) standard deviations] of SDPC for each filter type are summarised in Table I. No significant difference in filtration parameters was observed between both filters. Platelet recovery after filtration ranged from 94 to 100% in SDPC with a mean of 98.2 compared to 86% in pooled MDPC ( P < .05). Microscopic platelet aggregates were observed in pooled MDPC, but not in elutriated SDPC, on the day of preparation. Leukocyte depletion was also more efficient in SDPC. The initial leukocyte count in SDPC (168 & 105 X lo6) was significantly different from the leukocyte count in pooled MDPC (615 ? 235 X lo6). In LD-SDPC, 85% of the units contained no detectable leukocytes. In the units where leukocytes were detected, they never exceeded 10 million. In pooled LD-MDPC, residual leukocytes varied between undetectable and 21 million, with only 54% of units without detectable leukocytes. All platelet parameters were preserved following filtration. Following elutriation PTG % release was 632 (k328) kg/L compared to 1,178 ( *1,065) pg/L in the pool, reflecting different grades of activation in both products. Figure 1 shows the ultrastructure of elutriated (A) and filtered platelets by the polyester (B) and the cotton wool (C) filters. Platelet ultrastructure was hardly altered after polyester filtration. Following cotton wool filtration, all shapes were present. Most platelets have acquired a spheroidal configuration but no other signs of platelet activation were observed. The cytoplasmic membrane


TABLE I. Filtration-Induced Changes in Platelet Parameters Pre


Post ~

A. Cotton wool filtration (n = 44)a

Volume (ml) Platelets (10") Leukocytes (lo6) PH HSR 9% BTG % release LDH % leakage Morphology score Total score Disc % ...~. - . ~..

B. Polyester filtration (n Volume (ml) Platelets (10") Leukocytes (lo6) PH HSR % Synergistic aggr. % PTG % release LDH (unitll0" plat.) Morphology score Total score Disc %


379 3.8 270 6.97 78.3 4.6 5.8

(52) (1.0) (120) (0.07) (7.3) (1.7) (1.0)

424 3.7 0.04 6.94 74.4 4.9 6.1

(47) (0.9) (0.06) (0.1) (5.8) (3.1) (1.6)





308 57

(33) (15)

30)" 371 4.7 168 6.96 73.9 88.4 1.1 18.1

(19) (1.2) (105) (0.05) (8.6) (8.9) (0.9) (6.5)

359 4.5 0.077 6.93 69.4 86.1 1.4 20.1

(18) (1.2) (0.27) (0.06) (9.0) (11.8) (1.0) (7.2)

(24) (9)

320 65

(15) (7)

344 71


"NB: In cotton wool filtration study, LDH leakage was expressed as a percentage of the total LDH content. In polyester filtration study, it was calculated per 10" platelets. Values are means and (standard deviations).

was intact with normal aspect, number, and distribution of organelles. Storage Characteristics

Before leukocyte removal, the ratio of PC volume to container volume correlated significantly (P < . O l ) with post storage pH [39]. In LD-PCs the extent of bag filling (range 1: 4- 1:10) on post-storage pH was not significant. A trend to a positive Correlation ( P < . l ) was found between H + production during storage and the saline percentage in PC. Table I1 summarises the ex vivo parameters of both leukocyte-depleted SDPC and MDPC (polyester filtration) throughout 7-day storage, irrespective of the container filling ratio. Data obtained by cotton wool filtration were similar. With the exception of the levels of PTG and LDH, the functional and metabolic parameters were comparable for both products. Both markers were already higher in LD-MDPC ( P < .Ol) than LD-SDPC, and did rise further during storage in LD-PC ( P < .05 and P < .01 for both markers, respectively). Post-storage pH of LD-SDPC was not significantly different from the LD-pool. However, HCO, depletion was significantly higher ( P < . O l ) in LD-MDPC. In both


Elias et al.

Fig. 1. Ultrastructure of platelets following elutriation (A) and subsequent polyester filtration (B) or cotton wool filtration (C). A = a granules (eccentric nucleoids); M = mitochondrion; CS = open canalicular system; T = microtubules; G = glycogen particles.

therefore excluded from the results. The mean bleeding time was shortened from 12 min 20 sec (? 4 min) to 4 min 13 sec (5 1 min 28 sec). Figure 3 shows the corrected count increment (CCI) in 8 patients after excluding 3 patients with HLA antibodies and a fourth lacking data on 24-hr CCI. The mean CCI at 1 hr was 22.8 (? 5.1) and at 24 hr was 14.1 (+ 4.4) (compared to 15.5 and 10.0, respectively, for the controls transfused with standard PC). No transfusion reactions were observed. DISCUSSION AND CONCLUSIONS

single and pooled PCs, HCO, was more rapidly depleted in PO containers ( P < . O l ) . Bacterial cultures and endotoxin tests were negative.

In Vivo Results Figure 2 ihstrates the haemostatic effect of transfusions with the cotton wool filtered fresh SDPC, in 12 thrombocytopenic patients. The only patient showing a 1 hr post-transfusion bleeding time above 9 had a very tough skin which interfered with the test. The data were

Leukocyte depletion of platelet concentrates represented a challenge over the last decade. However, the residual leukocytes in a proportion of PC still exceeded the theoretical threshold for alloimmunisation, which could compromise the therapeutic regimen. In the method reported (elutriation followed by filtration), residual leukocytes never exceeded the immunogenic dose of 10 million. Moreover, the platelet recovery was more favourable for SDPC, presumably due to the initial lack of aggregates in elutriated platelets. This observation was valid for both cotton wool and polyester filters. Further, this approach has the advantage that platelets are not pelleted, a step resulting in platelet damage, activation, and aggregate formation. The difference in PTG and LDH levels between both LD-PCs reflect different


LD-SDPC Preparation and Storage TABLE 11. Storage Characteristics of Leukocyte-DepletedPC (Polyester Filtration) in TOTM Containers*


plat (10")


HCO, PO, PCO, LDH (mmol') (rnmol) (rnmol) (U) - -~ ~-




Elutriated PC (n = 13) 0 1.9 6.93 0.9 0.06 1.8 7.04 2 0.9 0.09 5 1.9 6.90 1.0 0.12 7 1.8 6.76 0.9 0.14 _ _ _ _ . ~

14.0 2.5 11.3 1.7 7.1 2.4 4.9 2.1. ~

Pooled PC (n 0 2.5 0.8 2 2.3 1.0 5 2.4 0.9 7 2.3 0.8

16.0 2.4 10.7 2.8 8.0 2.8 5.9 3.0


13) 7.09 0.04 7.08 0.08 6.87 0.15 6.78 0.24


16.2 2.7 13.5 2.6 14.5 2.9 14.3 3. I -

8.9 0.7 6.1 1.7 5.2 2.1 4.6 1.5 ~~





Syn. aggr

Morph Score tot --disc, % ~

20 1 72 22 4 69 26 7 83 310 96 ~

13 10 2 1 12 30 14 34 14

68 8 90 65 3 75 62 0 69 610 94

40.1 19.5 53.9 7.0 59.5 34.8 67.9 40.6

3.5 1.1 3.3 0.9 4.5 1.1 6.5 1.7

54.2 10.3 56.5 4.5 54.7 7.8 53.6 10.4

86 I 64 6 231 50 7 198 478 345

320 15 302 19 275 14 253 19

65 7 58 8 50 7 45 6~

67.3 16.6 53.6 12.2 49.7 13.6 46.1 18.8

262 12 258 34 246 22 226 19

42 4 43




14.1 2.3 14.5 3.7 13.0 2.8 13.8 3.1

7. I 1.1 5.1 1.1 5.7 1.2 5.0 1.2


36 8 31 8

*Means and (standard deviation). LDH is expressed as units per 10" platelets.

Bleeding time before and after transfusion of LD-SDPC

Corrected count increments (CCI) by LD-SDPC

Bleeding time (minutes)

CCI at 1 and 24 hours




20 16


12 8 4

0 1












Patients Before












1-hour CCI


24-hour CCI

Fig. 2. Bleeding in 12 thrombocytopenic patients before and after transfusion with leukocyte-depleted single donor platelet concentrates.

Fig. 3. Corrected count increments in 8 stable thrombocytopenic patients after transfusion of leukocyte-depleted single donor platelet concentrates.

grades of platelet activation and damage by the two processing techniques. It is unlikely that the overnight storage of MDPC with leukocytes would account for the higher levels of LDH and PTG during storage. When SDPC were also filtered after 1 or 2 days of storage (unpublished observations) they did not show the extent of storage lesion observed in the pelleted and pooled MDPC . The polyester filter is time saving and does not require saline priming or rinsing, thus allowing platelets to be stored in their physiological medium.

The introduction of closed apheresis systems, the sterile connection device and filters with a blind tubing (pig tails), render the storage of LD-PC possible. This would eliminate the concern about the transfusion of leukocyte fragments in case filtration is performed after storage. Elutriation of platelets followed by filtration through an appropriate leukocyte depleting filter combines several advantages in view of the product quality, the depletion capacity, in addition to the advantages of single donation. The results from the present study indicate that elutriated and filtered PC ensure a leukocyte depletion


Elias et al.

below the immunogenic dose in all the units, have good in vitro and in vivo function, and can be sterilely stored for 7 days. The pH could be satisfactorily maintained provided less than 20% v/v of saline is added during filtration and a reasonable filling ratio is considered. Prevention of primary alloimmunisation by leukocyte-free products is within reach. Whether these products can reverse HLA alloimmunisation in already sensitised patients needs yet to be answered.





The authors thank R.L. McShine, M . Weggemans, and H.K.A. Carper from the Red Cross Blood Bank Groningen-Drenthe and J. Medema from the Nuclear Medicine Department of the University Hospital Groningen for their expert assistance, and Maya ten Cate for preparing this manuscript.

16. 17. 18.


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Preparation and storage of "leukocyte-depleted single donor" platelet concentrate: a step forward in effective haemotherapy.

"Leukocyte-depleted" platelet concentrates (LD-SDPC) were prepared by cotton-wool and by polyester filtration of "leukocyte-poor" PC collected by the ...
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