effect of edta on platelet count and other platelet parameters in blood and blood components...

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Original Papers Vox Sang 1991;61:84-89 0 1991 S. Karger AG. Basel 0042-9007/9l/0612-0084 $2.75/0 Effect of EDTA on Platelet Count and Other Platelet Parameters in Blood and Blood Components Collected with CPDA-1 R. L. McShinea, P.C. Dasa, C. Th. Smit Sibinga", B. BrozoviCb 'Red Cross Blood Bank Groningen-Drenthe, Groningen, The Netherlands; "North London Blood Transfusion Centre, UK Abstract. We have shown in this study that addition of dried KzEDTA (1.5 mg/ml) to blood samples anticoagulated with CPDA-1 increases significantly the platelet count and mean platelet volume (MPV) of whole blood, red cell concentrate (RCC) and platelet concentrate (PC), but not of platelet-rich plasma (PRP) or of platelet-poor plasma (PPP). Transmis- sion and scanning electron microscopy illustrated that platelet aggregates which are present in some components are dispersed on mixing of the sample with EDTA and that this is accompanied by a change in platelet morphology. Determination of the platelet distribution width (PDW) indicated that the platelet populations in whole blood and RCC seem to be more uniform in size than the populations in PRP, PPP and PC. The determination of MPV and PDW changes after addition of EDTA may provide a new approach to quality control of PC. Introduction The application of quality control on blood components prepared in blood transfusion centres (blood banks) is be- coming more widespread. One of the components for which there is an ever-increasing demand for quality con- trol is platelet concentrate (PC). An essential indicator of the quality of PC is the platelet count. However, it has already been shown that some platelet microaggregates develop spontaneously in citrated whole blood, that these aggregates are not recognised and counted as platelets in the modern automated haematology counters and that the platelets in aggregates dissociate on the addition of EDTA salt [ 1-31, CPDA-1 solution (1 part to 7 parts blood) gives a final concentration of 11.02 mM citrate ion, which is consid- erably more than the minimum concentrations of some anticoagulants in general use, necessary to prevent gross evidence of coagulation: EDTA (12-15 mM), oxalate (20- 30 mM) and citrate (50-75 mM) when used as 1 part anti- coagulant to 9 parts blood [4]. However, the figure used for concentration of citrate is only approximate since it does not take into account the facts that (a) a bag of blood is difficult to mix, especially when almost full, (b) citrate salts are poor calcium binders compared to other anticoagu- lants, and (c) there is a great variability in donor haema- tocrit. Inadequate anticoagulation leads to platelet activa- tion (aggregation); however, the phenomenon could also be triggered off in PC and red cell concentrate (RCC) by ADP released from damaged platelets during the centrifu- gation process [5,6]. There are several reports indicating that the platelet population is heterogeneous and can be separated through differences in sedimentation rate, and on a basis of volume and density using density gradients and counter flow cen- trifugation (elutriation) [7-151. These reports also illustrate the relationship between platelet age, density, volume and biological activity. The conclusions in these studies suggest that young platelets are larger, denser and haemostatically more active. Other studies do not support any relationship between size and age [16,17]. Not much is known in the literature about measurements of mean platelet volume (MPV) and platelet distribution width (PDW) in the different fractions obtained during preparation of PC.

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Page 1: Effect of EDTA on Platelet Count and Other Platelet Parameters in Blood and Blood Components Collected with CPDA-1

Original Papers

Vox Sang 1991;61:84-89 0 1991 S. Karger AG. Basel

0042-9007/9l/0612-0084 $2.75/0

Effect of EDTA on Platelet Count and Other Platelet Parameters in Blood and Blood Components Collected with CPDA-1

R. L. McShinea, P.C. Dasa, C. Th. Smit Sibinga", B. BrozoviCb 'Red Cross Blood Bank Groningen-Drenthe, Groningen, The Netherlands; "North London Blood Transfusion Centre, UK

Abstract. We have shown in this study that addition of dried KzEDTA (1.5 mg/ml) to blood samples anticoagulated with CPDA-1 increases significantly the platelet count and mean platelet volume (MPV) of whole blood, red cell concentrate (RCC) and platelet concentrate (PC), but not of platelet-rich plasma (PRP) or of platelet-poor plasma (PPP). Transmis- sion and scanning electron microscopy illustrated that platelet aggregates which are present in some components are dispersed on mixing of the sample with EDTA and that this is accompanied by a change in platelet morphology. Determination of the platelet distribution width (PDW) indicated that the platelet populations in whole blood and RCC seem to be more uniform in size than the populations in PRP, PPP and PC. The determination of MPV and PDW changes after addition of EDTA may provide a new approach to quality control of PC.

Introduction

The application of quality control on blood components prepared in blood transfusion centres (blood banks) is be- coming more widespread. One of the components for which there is an ever-increasing demand for quality con- trol is platelet concentrate (PC). An essential indicator of the quality of PC is the platelet count. However, it has already been shown that some platelet microaggregates develop spontaneously in citrated whole blood, that these aggregates are not recognised and counted as platelets in the modern automated haematology counters and that the platelets in aggregates dissociate on the addition of EDTA salt [ 1-31,

CPDA-1 solution (1 part to 7 parts blood) gives a final concentration of 11.02 mM citrate ion, which is consid- erably more than the minimum concentrations of some anticoagulants in general use, necessary to prevent gross evidence of coagulation: EDTA (12-15 mM), oxalate (20- 30 mM) and citrate (50-75 mM) when used as 1 part anti- coagulant to 9 parts blood [4]. However, the figure used for concentration of citrate is only approximate since it does

not take into account the facts that (a) a bag of blood is difficult to mix, especially when almost full, (b) citrate salts are poor calcium binders compared to other anticoagu- lants, and (c) there is a great variability in donor haema- tocrit. Inadequate anticoagulation leads to platelet activa- tion (aggregation); however, the phenomenon could also be triggered off in PC and red cell concentrate (RCC) by ADP released from damaged platelets during the centrifu- gation process [5,6].

There are several reports indicating that the platelet population is heterogeneous and can be separated through differences in sedimentation rate, and on a basis of volume and density using density gradients and counter flow cen- trifugation (elutriation) [7-151. These reports also illustrate the relationship between platelet age, density, volume and biological activity. The conclusions in these studies suggest that young platelets are larger, denser and haemostatically more active. Other studies do not support any relationship between size and age [16,17]. Not much is known in the literature about measurements of mean platelet volume (MPV) and platelet distribution width (PDW) in the different fractions obtained during preparation of PC.

Page 2: Effect of EDTA on Platelet Count and Other Platelet Parameters in Blood and Blood Components Collected with CPDA-1

EDTA Effect on Platelet Parameters 85

More knowledge of MPV and PDW values in the compo- nents obtained during the preparation of PC may thus be of value in the quality control of this very important compo- nent.

In this study, the platelet parameters in CPDA-1 whole blood and blood components were measured and com- pared, and the effect of EDTA on these parameters is presented.

Materials and Methods

Preparation of Platelet Concentrate Blood from blood donors selected according to Guidelines for the

Selection, Medical Examination and Care of Blood Donors in the UK was collected into triple blood packs with CPDA-1 (FGR 1648, Baxter Healthcare, Thetford, UK). PC was prepared using a 1st centrifugation of 1,611 g for 3 min and a 2nd centrifugation of 1,850 g for 7 min in a Beckman 5-68 centrifuge (Beckman Instruments. High Wycombe. UK) and the platelet pellet with approximately 50 ml of platelet-poor plasma (PPP) was left for 1.5 h before resuspension. Samples for count- ing of platelets were taken immediately after each stage of processing with the exception of PC samples which were taken after resuspension.

Determination of EDTA Concentrations Studies on whole blood, platelet-rich plasma (PRP), PC and PPP

were performed in order to establish the optimal concentration of EDTA required for counting. Samples were mixed with dried K2EDTA in tubes (FBG Trident, Bristol, UK) to obtain final concentrations varying between 0.19 and 4.5 mg/ml. After standing at room temper- ature for 1 h, the samples were mixed and analysed in duplicate on a Technicon H*l system. The Technicon H*l system utilises the principle of flow cytometry and determines the platelet count as well as the platelet size and density by measuring the laser beam light scattering at a low and a high angle. The mean platele volume (MPV) is the mode of the measured volumes and the PDW is the coefficient of variation of the platelet volume distribution histogram [18]. The highest platelet count on each type of component was considered as 100% and the other values were expressed as a percentage.

Platelet Count, MPV und PDW Measurements Twelve units of whole blood and components prepared from these

units were weighed and mixed before sampling (fresh) in duplicate tubes -one plain polystyrene and one tube with K2EDTA. The samples were mixed and left to stand at room temperature for 0.5 h before analysis.

Transniission and Scanning Electron Microscopy Fresh samples of CPDA-1 PRP and resuspended PC prepared from

6 units of blood were prefixed in 0.2% glutaraldehyde in cacodylate buffer (SO mM sodium cacodylate, pH 7.2) for electron microscopy (performed in the Anatomy Department, Royal College of Surgeons, Lincoln's Inn Fields, London).

Sfatistics The Student paired t test was used for comparison of results: p <

0.05 was considered to indicate a significant difference.

Fig. 1 The effect of EDTA concentration on platelet counts in CPDA-1 whole blood (*), platelet concentrate (0) and red cell concen- trate (m) (n=6).

Results

EDTA Concentration and Platelet Counts The effect of EDTA concentration on platelet count

in whole blood, RCC and PC is shown in figure 1. With all three preparations a plateau was reached at 1.5 mg/ml. This effect was not observed in PRP and PPP, and the addition of increasing amounts of EDTA to PRP and PPP caused no change in the count [results not shown]. An EDTA concentration of 1.5 mg/ml sample was used for further studies.

Platelet Counts and Absolute Numbers of Platelets The mean platelet counts in samples of whole blood and

corresponding components are shown in figure 2. Whole blood, RCC and PC showed a significantly (p<O.OOl) high- er platelet count after the addition of EDTA. The range of increase varied for whole blood from 12 to 69%, for RCC from 1 to 33% and for PC from 1 to 57%. The mean absolute numbers in whole blood, RCC and PC with EDTA were significantly higher (p<O.OOl) than those without EDTA (fig. 3). EDTA created a slight but not significant increase in PRP and none in PPP (see also fig.3) The mean percent- age yield (+ 1 SD) of platelets in PC, calculated using the counts with and without EDTA was 66 f 12 and 70 f 12%, respectively. The change of shape of the white blood cell distribution curve which is known to occur in the presence of platelet aggregates [1,19-211 was commonly seen on the printed results for samples without EDTA.

Page 3: Effect of EDTA on Platelet Count and Other Platelet Parameters in Blood and Blood Components Collected with CPDA-1

86 McShine/Das/Smit SibingalBrozoviC

Fig. 2. Platelet counts in CPDA-1 whole blood and components. - =Without EDTA; + =with EDTA.

Fig. 3. Absolute number of platelets in CPDA-1 whole blood (WB) and components. On the left without EDTA, on the right with EDTA. The columns represent the mean of 12 determinations and the vertical bars indicate SD.

Fig. 4. Mean MPV in CPDA-1 whole blood (WB) and components. - =Without EDTA; + =with EDTA. Vertical bars indicate SD (n = 12).

Comparison of MPV The mean MPV values in RCC were higher and in PRP,

PPP and PC lower than those in whole blood, both with and without EDTA (fig. 4, table 1). These differences were statistically significant in all cases (p<0.05). Addition of EDTA (1.5 mg/ml) to the CPDA-1 samples created a highly significant increase of MPV in whole blood (p<O.OOl) and in RCC (p<O.OOl), and a slightly significant increase in PC

(p=0.02); it had no effect on the MPV in PRP and PPP. The addition of higher concentrations of EDTA also failed to alter the MPV in the last two fractions.

Comparison of PDW The mean PDW of the components are shown in figure

5. The values in whole blood and RCC were significantly (p<O.OOl) lower than those in PC and PPP and slightly

Page 4: Effect of EDTA on Platelet Count and Other Platelet Parameters in Blood and Blood Components Collected with CPDA-1

EDTA Effect on Platelet Parameters 87

lower than that in PRP. The addition of EDTA lowered the PDW significantly (p<O.OOl) in whole blood, RCC and PC by 9, 12 and 6%, respectively, and only by 1% in PRP and PPP.

Transmission and Scanning Electron Microscopy The presence of platelet aggregation in CPDA-1 PC and

disaggregation on the addition of EDTA is illustrated in figure 6. Other features induced by EDTA such as sphere forming and increase of MPV can also be seen. Aggrega- tion bridges are formed via pseudopodia (fig. 6c), which decrease considerably in number on the addition of EDTA (fig. 6d).

Table 1. Mean MPV values (+I SD) found in samples of whole blood and components, (n = 12)

Anti- Blood component coagulant

whole RCC PRP PPP PC blood

Discussion

The lower platelet counts observed in samples of CPDA-1 whole blood, RCC and PC without EDTA are probably due to the presence of platelet microaggregates which are not recognised as platelets in the Technicon H*l flow cell cytometer. A similar phenomenon was also ob- served in whole blood analysed by the Coulter S Plus 4 and the Sysmex PL 110 automated cell counters [l-31. The pres- ence of these aggregates was illustrated with the help of transmission and scanning electron microscopy, and was confirmed by the change in shape of the printed white blood cell distribution curve in the automated cell counters. EDTA causes higher platelet counts by disaggregation Of

platelet microaggregates (clumps), It is probable that

Fig. 5. Mean PDW in cpDA-l whole (WB) and components~ bx=Without EDTA; +=with EDTA. Vertical bars indicate SD (n = 12).

a

C

CPDA-1 7.71f0.64 8.25k0.75 7.21f0.68 4.57f0.52 6.88f0.76 CPDA- 1 + EDTA 8.32k0.72 9.21f0.98 7.18k0.76 4.58f0.50 7.05f0.78

b

d

Fig. 6. Transmission (a,b) and scanning (c,d) electron microscopy photographs of fresh CPDA-1PC. On the left without EDTA, on the right with EDTA. X 3,000.

Page 5: Effect of EDTA on Platelet Count and Other Platelet Parameters in Blood and Blood Components Collected with CPDA-1

88 McShinelDaslSrnit SibingalBrozoviC

bound calcium plays a role in the formation of the aggrega- tion bridges and its removal by EDTA causes these bridges to break. An increased concentration of citrate may also bring about the same effect.

The EDTA effect described is predominantly seen in whole blood, RCC and PC. It is present to a lesser extent in PRP and is absent in PPP. However, Slichter and Harker [22] found that the addition of EDTA to PC was not a critical factor in electronic counting of platelet concen- trates. Their study was carried out on PC prepared with an additional amount of ACD added to the PRP, thereby not only decreasing the pH but also increasing the final citrate concentration; both factors and not only the first one may have been beneficial to resuspension.

In our study, significantly lower counts in the samples of CPDA-1-whole blood without EDTA created spuriously higher platelet yields in PC than that obtained with the counts with EDTA.

The lower MPV found in PRP, PPP and PC and the higher MPV in RCC, compared to the values in whole blood, are due to separation of the platelet populations with different properties [23-251 during the first centrifu- gation step. This study clearly demonstrated differences in the MPV of the different fractions produced by routine centrifugation, the highest MPV being measured in RCC. It may be that this lost platelet population (10-36% in RCC) is most important in haemostasis. The highest changes in MPV and PDW observed in whole blood and RCC after the addition of EDTA to CPDA-1 samples seem to suggest that larger and denser platelets, which are present in greater numbers in these two products, are more sensitive to ED- TA changes than smaller and less dense ones. It is of in- terest that MPV and PDW determined in PRP were essen- tially the same in samples without and with EDTA. We believe that an explanation for this observation is found in the population of platelets which will separate into PPP during the subsequent centrifugation. Although these pla- telets represent only one tenth of the total number of plate- lets in PRP, their small MPV, high PDW and absence of response to EDTA are of sufficient magnitude to remove the statistical significance from the observed changes in PRP.

The evidence presented in this study shows clearly that the addition of dried K2EDTA to samples of CPDA-1 whole blood, RCC, PRP, and PC is a necessary step for accurate platelet counts. Without EDTA the counts are bound to be incorrect due to the presence of platelet microaggregates. A question left unanswered is what will be the fate of these microaggregates after transfusion. One report in the litera- ture [26] has suggested that platelet microaggregates may

disaggregate spontaneously in vivo. If that proves to be true, then counting the sample of a fresh PC without EDTA will lead to falsely high calculations of platelet recovery. It is our view that aggregates sufficiently large to be retained by a 180-ym filter in the infusion set are unlikely to be disaggregated by action of EDTA. Platelets in these large aggregates are not counted by cell counters and are exclud- ed when platelet yields are calculated for PC. Therefore, their retention by the filter during transfusion will not affect the calculation of platelet recovery in vivo.

At present work is in progress to assess the significance of the magnitude of change in MPV (delta MPV) caused by EDTA in the blood components.

Acknowledgements

We would like to thank all blood donors and members of the staff, especially those in the donor centre and blood component departments of both Blood Centres.

Thanks also to Maya ten Cate for preparing the manuscript and to Joost Schijfsma for his technical assistance.

References

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2 McShine RL, Das PC, Smit Sibinga CTh, BrozoviC B: Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean platelet volume. Clin Lab Haematol 1990;12:277- 285.

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EDTA Effect on Platelet Parameters 89

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Received: September 6,1990 Revised manuscript received: January 30,1991 Accepted: February 3,1991

R.L. McShine, FIMLS, MPhil Red Cross Blood Bank Groningen-Drenthe PO Box 1191 NL-9701 BD Groningen (The Netherlands)