In vitro quality tests for cellular blood products

Project leader: Dirk de Korte PhD

Although the final proof for the quality of cellular blood products is still the result after clinical use, in vitro tests can be of value to predict in vivo behavior. In 2011 a relatively new test was added to the in vitro set of parameters for platelets, the ThromboElastoGraphy or TEG. In 2011 the effect of lipemic plasma on blood products was also investigated with in vitro parameters.

Use of ThromboElastoGraphy in product evaluation
Thromboelastography (TEG) monitors the thrombodynamic properties of blood as it is induced to clot under a low-shear environment resembling sluggish venous flow. The patterns of change in shear-elasticity enable the determination of the kinetics of clot formation and growth as well as the strength and stability of the formed clot. The strength and stability of the clot provide information about the ability of the clot to perform the work of haemostasis, while the kinetics determines the adequacy of quantitative factors available to clot formation. With TEG the quantitative clot formation and the kinetics are measured, followed by measurement of the clot strength and stability, plus the clot resolution (fibrinolysis).

In the citrate-kaolin (CK) test, the intrinsic pathway is activated with kaoline (after addition of calcium, because citrate anti-coagulated blood is used) and a clot is formed by fibrinogen and platelets. In the citrate-functional-fibrinogen (CFF) test the extrinsic pathway is activated by tissue factor addition (also after calcium addition), whereas the platelet aggregation is inhibited, therefore the strength of the formed clot is representative for the amount of functional fibrinogen.

To obtain information on the normal values and the extent of variation in testing with the TEG due to donor variation, whole blood from 100 different donors was tested with the CK and the CFF test. In total the tests from 95 donors were evaluable (tested between 30 and 120 min. after collection, mean 65± 29 min.). For both tests normal values were defined for the various measured parameters (mean with 95% CI). The moment of testing for citrate blood is of importance as well as the concentration of citrate. There are also some differences between men and women. In general, women have a more rapid clot formation and the strength of the clot is higher. Whereas the concentration of fibrinogen showed a clear correlation with the maximal clot strength in the CFF results, the concentration of platelets did not show a correlation with the clot strength. This might be due to the fact that very low and very high platelet counts were not represented in the tested whole blood samples from healthy voluntary blood donors.

The project will have a follow-up in 2012 with measurement of platelet concentrates from different aphaeresis donors, to investigate donor variation in the product and to check whether TEG analyses on either product or whole blood can be used to select donors.

Effect of lipemic plasma on the in vitro quality of erythrocytes or platelets during storage
Dutch guidelines for blood transfusion require that plasma should not be turbid or lipemic (not milky). However, no data on the effect of lipemic plasma on the quality of cellular blood components during storage are available. Therefore the effect of lipemic plasma on the quality of cellular blood components during storage was investigated.

Either whole blood units that were discarded from regular component production because of lipemic plasma, or released units, were used for further processing. Whole blood (500±50 ml) was collected in citrate phosphate dextrose, centrifuged and separated automatically with the Compomat™ G5 into a plasma, buffy coat (BC) and red blood cell (RBC) concentrate. Plasma was sampled for triglyceride analysis. After the addition of SAGM, the RBC concentrates were leukodepleted by filtration. RBC concentrates were stored at 2-6°C for 42 days and sampled at regular intervals for in vitro analysis. BCs were used to make a single donor platelet concentrate (SD-PC) in plasma. SD-BCs were stored on a flatbed shaker at 20-24°C and sampled at days 1, 6 and 8 for in vitro analysis. Cellular components made from lipemic blood (n=8) were compared with those made from regular, non-lipemic blood (n=11).

The triglyceride concentration of lipemic plasma and normal plasma was 6.9±3.0 and 1.5±0.6 mmol/L respectively. The results of SD-PC during storage are shown in table 2. Platelets stored in lipemic plasma showed a stronger decline in pH and swirling as compared to storage in regular plasma. Metabolic activity, as measured by lactate production, and activation (number of CD62 positive cells) were more pronounced during storage in lipemic plasma. The high numbers of Annexin V-positive cells combined with the high oxygen tension and the decline in platelet count, suggest that a vast majority of platelets is apoptotic after 8 days’ storage in lipemic plasma.

Red cells prepared from lipemic whole blood (WB) showed significantly higher levels of hemolysis during storage. The level of hemolysis correlated with the triglyceride concentration in the plasma: in particular, RBC concentrates made from WB with a triglyceride concentration above 10 mmol/L showed high levels of hemolysis. For the other in vitro parameters, including glucose and ATP levels, no significant difference between lipemic RBCs and regular RBCs could be observed.

It can be concluded that lipemic plasma has a negative effect on the in vitro quality of both platelets and red cells during storage, and discard of the whole unit in case of lipemic plasma seems valid. Further research is necessary to determine the mechanism for the effect of lipids on cellular blood products and the maximal acceptable degree of lipemia.

Key publications

Last edited on: 2 October 2012