R. N. I. Pietersz

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from platelet‐rich plasma (PRP) and from the other as nonpelleted platelets from the buffy coat (BC). The activation of platelets in these PCs was studied immediately after preparation and during storage for up to 9 days at 22°C with gentle agitation. The binding of monoclonal antibodies (MoAbs) against the GP IIb/IIIa complex and against activation‐dependent antigens (GMP 140 from the alpha granules and a 53‐kDa glycoprotein from the lysosomal granules) was measured. β‐thromboglobulin (β‐TG) release was also determined. Disc‐to‐sphere transformation was quantitated by measuring on an aggregometer the difference in light transmission during stirring at different rates and also by light microscopy. Immediately after preparation, platelets derived from PRP had a more spheric morphology (p < 0.01), had a higher β‐TG release (p < 0.01), bound more MoAbs against GP IIb/IIIa (p < 0.01), and expressed more GMP 140 and 53‐kDa glycoprotein (p < 0.01) than did BC‐ derived platelets. However, these differences had disappeared after 2 days of storage. It was concluded that, immediately after preparation, PRP‐derived platelets are more activated than BC‐derived platelets. This is most likely a result of the pelleting that follows the second high‐speed centrifugation of the PRP.

Preparation of Leukocyte-Poor Platelet Concentrates from Buffy Coats

Abstract. A special insert was developed for centrifuge cups in order to prepare leukocyte‐poor platelet concentrates from buffy coats by using quadruple citrate phosphate dextrose‐saline adenine glucose mannitol systems from different manufacturers. Each centrifuge cup could contain up to 4 sets of double bags allowing the preparation of 24 platelet concentrates per run. Optimal conditions for centrifugation of the buffy coats in the inserts were found to be 6 min at 380 g (2,150 g min). A platelet count of 69 ± 19 × 109 and a leukocyte contamination of 14± 10.5 × 106 per platelet concentrate was thereby obtained in a plasma volume of 63 ± 10.5 ml (mean ±SD). The method described allows large scale production of leukocyte‐poor platelet concentrates from buffy coats in a closed system.

Detection of bacteria in platelet concentrates: comparison of broad-range real-time 16S rDNA polymerase chain reaction and automated culturing

BACKGROUND: Based on real‐time polymerase chain reaction (PCR) technology, a broad‐range 16S rDNA assay was validated and its performance was compared to that of an automated culture system to determine its usefulness for rapid routine screening of platelet concentrates (PCs).

Prevention of Yersinia enterocolitica growth in red-blood-cell concentrates

In response to concern about Yersinia enterocolitica contamination of blood products, we have studied the effects on Y enterocolitica growth of holding whole blood at 22 degrees C for 20 h and then removing leucocytes. Thirty pools of three bags of blood were inoculated with Y enterocolitica (2 x 10(1)-3 x 10(4) colony-forming units/ml). One bag in each pool was processed to red-blood-cell concentrate after 6 h at 4 degrees C (RBC); the other two were held at 22 degrees C for 20 h before processing to buffy-coat-depleted RBC (BCd-RBC). One of these bags was then depleted of leucocytes by filtration (Ld-RBC). All bags were stored at 4 degrees C for 5 weeks. RBC bags showed Y enterocolitica growth after the shortest storage times, followed by BCd-RBC then Ld-RBC (p less than 0.03-0.001). We recommend that whole blood should be held at 22 degrees C to make use of inherent bactericidal activity; leucocytes should then be removed.

Storage of platelets in additive solution for up to 12 days with maintenance of good in-vitro quality.

BACKGROUND:  Storage of PLT concentrates (PCs) may be extended beyond 5 days, provided in‐vitro and in‐vivo variables allow longer storage and bacterial screening is performed. The aim of this study was to examine in‐vitro storage characteristics of PCs in various storage solutions: plasma only, or mixtures of plasma with PAS‐II, PAS‐III, PAS‐IIIM, and Composol.

Detection of bacterial contamination of platelet concentrates.

R. N. I. Pietersz, C. P. Engelfriet, H. W. Reesink, E. M. Wood, S. Winzar, A. J. Keller, J. T. Wilson, G. Henn, W. R. Mayr, S. Ramírez-Arcos, M. Goldman, J. Georgsen, P. Morel, P. Herve, G. Andeu, A. Assal, E. Seifried, M. Schmidt, M. Foley, C. Doherty, P. Coakley, A. Salami, E. Cadden, W. G. Murphy, M. Satake, D. de Korte, V. Bosnes, J. Kjeldsen-Kragh, C. McDonald, M. E. Brecher, R. Yomtovian & J. P. AuBuchon

UPDATE ON LEUCOCYTE DEPLETION OF BLOOD COMPONENTS BY FILTRATION

It has long been recognized that allogenic leucocytes from donor blood are responsible for serious untoward effects in some transfused patients such as alloimmunization, febrile reactions, platelet refractoriness, transfusion associated acute lung injury, immunosuppression as well as transmission or reactivation of viruses such as CMV, HTLV or EBV. Leucocytes are also known to accelerate the rate of storage lesion. The optimal method to remove leucocytes from blood components has been shown to be filtration. However, many variables exist in the properties of leuco-depletion filters (material, composition, surface charge, mechanisms of leucocyte entrapment), the blood components to be filtered (composition, age), and the filtration method (pre- or post-storage, priming and rinsing, temperature, flow rate). In this paper principles of filtration and subsequent logistic consequences will be discussed. It is recommended to carefully select a filter for a specific blood component and to perform leuco-depletion procedures under controlled conditions according to validated methods meeting Good Manufacturing Practice (GMP) and Good Laboratory Practice (GLP).

Correlation between the extent of platelet activation in platelet concentrates and in vitro and in vivo parameters

Background and Objectives  Platelet activation, which is necessary to stop bleeding, also occurs in vitro during the storage of platelet concentrates (PCs). However, it is unknown whether in vitro‐activated platelets are able to reduce blood loss in the patient. We studied correlations between platelet activation in PCs and in vitro parameters (pH, platelet count, swirling effect, storage time). In addition, we studied the correlation between platelet activation and in vivo parameters [the volume of thorax drain fluid as a measure of blood loss, platelet count, international normalized ratio (INR), and activated partial thrombin time (APTT)] in a clinical pilot study.

Bacterial contamination in platelet concentrates

R. N. I. Pietersz, H. W. Reesink, S. Panzer, S. Oknaian, S. Kuperman, C. Gabriel, A. Rapaille, M. Lambermont, V. Deneys,D. Sondag, S. Ramirez-Arcos, M. Goldman, G. Delage, F. Bernier, M. Germain, T. Vuk, J. Georgsen, P. Morel, C. Naegelen,L. Bardiaux, J.-P. Cazenave, J. Dreier, T. Vollmer, C. Knabbe, E. Seifried, K. Hourfar, C. K. Lin, M. Spreafico, L. Raffaele,A. Berzuini, D. Prati, M. Satake, D. de Korte, P. F. van der Meer, J. L. Kerkhoffs, L. Blanco, J. Kjeldsen-Kragh,A.-M. Svard-Nilsson, C. P. McDonald, I. Symonds, R. Moule, S. Brailsford, R. Yomtovian & M. R. JacobsSeptic reactions after transfusion, particularly of plateletconcentrates, still occur and belong to the most serioustransfusion reactions. From a previous InternationalForum [1] on the subject, it could be concluded that inpart of the countries that participated in the forum, plate-let concentrates (PCs) were tested for bacterial contamina-tion and that culture-based methods, particularly theBacT/Alert system, were used.In recent years, several rapid bacterial detection meth-ods, such as surrogate measurements of the pH or glu-cose, the detection of bacteria with a scan system orPCR tests that detect bacterial RNA, have been devel-oped. These tests can either be performed immediatelyprior to transfusion of the PC or at a variety of testmoments at which culture and release tests are com-bined.Pathogen inactivation (PI) methods also affect bacterialcontamination of PCs. In 2007 [1], in some countries, theIntercept method of PI of PCs was implemented insteadof bacterial screening.It seemed of interest to evaluate the present state ofthe art of this subject. In order to obtain the desiredinformation, the following questions were sent to expertsin the field.Question 1: How long do you store PC and is there adifference between whole-blood-derived PC and apheresisPC? Which method of preparation do you use for whole-blood-derived PC? Are PCs leuco-reduced?Question 2: Do you use a culture method to detect bac-terial contamination of PC? If so,

Storage of leukocyte-poor red cell concentrates: filtration in a closed system using a sterile connection device

Abstract. Storage of leukocyte‐poor red cell concentrates (LP‐RCC) was investigated after filtration in a closed system that was assembled using a Sterile Connection Device (SCD). The LP‐RCC were stored for up to 6 weeks following filtration with either 0.9% saline solution (n = 14) or saline‐adenine‐glucose‐mannitol (SAG M) solution (n = 15) to prime and rinse the cellulose acetate filter. The results were compared with the data of nonfiltered buffy‐coat‐poor red cell concentrates (BC‐poor RCC) stored in SAG M solution (n = 10). All LP‐RCC contained less than 106 leukocytes whereas the nonfiltered BC‐poor RCC contained 6751286 × 106 leukocytes at day 1, decreasing to 83±49 × 106 at day 42. Although glucose consumption, lactic acid production and decrease in pH was similar from day 7 through 28 in both groups of LP‐RCC, a significantly steeper decline of ATP values as well as a higher hemolysis and LDH release was observed in the LP‐RCC filtered with saline. During storage of the nonfiltered BC‐poor RCC in SAG M, significantly higher glucose consumption (p<0.01), LDH release (p<0.001), rate of hemolysis (p<0.001) and a lower pH (p<0.001) were found, compared to the filtered units. It is postulated that the leukocytes present in the nonfiltered BC‐poor RCC were responsible for these differences. The ATP values in the SAG‐M‐filtered and nonfiltered BC‐poor RCC in SAG M were comparable. By comparing the ATP levels and values of the filtered RCC and the nonfiltered BC‐poor RCC we conclude that the LP‐RCC can be stored for 35 days if SAG M solution is used to prime and rinse the filter.