Flow cytometry and thromboelastography to assess platelet counts and coagulation in patients with haematological malignancies
Alex Gatt1,2, Fabian Bonello1, Raphael Buttigieg1, Samuel Debono1, Patricia Brincat2, Charlie Grima3, Peter Gatt3, Thomas Lofaro2,4, Stefan Laspina2,5
1Pathology Department, University of Malta Medical School, Msida, Malta; 2Haematology-Oncology Department, Mater Dei Hospital, Msida, Malta; 3Perfusion Department, Department of Surgery, Mater Dei Hospital,Msida, Malta; 4Haematology Department, Guy's & St Thomas' Hospital, London, United Kingdom; 5Blood Transfusion Department, Mater Dei Hospital, Msida, Malta
IntroductionSurvival rates in patients with haematological
malignancies have improved over the last decades not only due to better cancer-directed therapies but also due to better supportive therapy1. This supportive therapy includes the early use of antibiotics as well as adequate blood component transfusion. This practice has been encouraged through the formulation of guidelines which shape local protocols. Whereas there is still considerable variance in the use of red blood cell transfusions, there seems to be a more homogeneous acceptance of proposed triggers for prophylactic platelet transfusion. This stems from the observation, by Gaydos et al., that bleeding episodes are most frequent in patients with platelet counts below 20×109/L as assayed by the "gold
standard" manual method of platelet counting2. This notion has been challenged by very few groups over the years. However, Gmur et al. showed that a much lower threshold of around 5×109/L for routine platelet transfusions might be acceptable. In their study they used an early automated analyser, incorporating a laser-based optical system, to count the platelets and confirmed platelet counts <50×109/L using the manual method3. They also noted that the majority of cases of major bleeding occurred in patients with platelet counts <10×109/L. These studies might not be comparable since the method of platelet counting was different: one used the manual method exclusively whereas the other used an automated analyser combined with confirmatory manual counts. Another point to note is
Background. Accurate platelet counts (PC) are necessary in order to follow recommendations for prophylactic platelet transfusion. We carried out a study comparing the standard way of counting platelets using a routine analyser and compared it with PC determined by flow cytometry (FC) and haemostatic data obtained with thromboelastography (TEG).
Materials and methods. The study was carried out on 24 patients with haematological malignancies, all given one adult dose of platelets. The PC was determined before and after transfusion using an automated blood cell counter and FC. Citrated, "native" whole blood TEG was carried out before and after platelet transfusion to assess global haemostasis.
Results. No bleeding was observed in any of the subjects. Thirty-one assessments were performed in the 24 patients. The mean pre-transfusion PC were 9.8 and 13×109/L with the automated counter and FC, respectively with a difference of 3.7 (p=0.0011). Excellent correlation was observed between the two counts (r=0.89; p<0.0001). Mean post-transfusion increments were 23 and 29×109/L for the routine counter and FC, respectively. Using the immunological PC, patients would not have qualified for transfusion in 18.2% of cases since their PC was >20×109/L. TEG showed a shortened reaction time in 69.6% of cases and a normal mean K time of 6.7 min. Only 9% had a low α angle signifying hypocoagulability. The maximum amplitude was reduced in the majority of cases but normal in 25% despite PC <20×109/L. Mean activated partial thromboplastin time, prothrombin time and fibrinogen were normal prior to transfusion.
Discussion. Although higher PC as assessed by FC could potentially have an impact on platelet transfusion practices, TEG was sensitive enough to detect PC <10×109/L and some between 10-20×109/L. Whether patients with the latter PC are more prone to bleeding remains to be verified in larger studies.
that the coefficient of variation of the manual method is very high, being over 30% in one study4. A recent Cochrane review5 concluded that although a platelet count of <10×109/L is still acceptable as the trigger for platelet transfusions, there is still doubt whether adopting this lower threshold would result in the same number of bleeding episodes as a threshold of 20×109/L with a net reduction in platelet transfusions. In fact, some guidelines still recommend the latter trigger in the case of fever and sepsis6.
The best method of counting platelets in samples from thrombocytopenic patients is still a matter of debate. Due to its high imprecision and laboriousness, the manual method has fallen out of favour and has been replaced by immunological platelet counting using flow cytometry (FC). This latter technique has been adopted as the international reference7. There are, however, appreciable differences between the different technologies for platelet counting using automated analysers, with some showing better results using optical vs impedance technology and vice versa8. Furthermore, it has been appreciated that, as with the inherited platelet disorders, haemostasis depends not only on platelet count but also on platelet function. This is especially important in patients suffering from haematological malignancies9. Apart from the platelet component, there is also an important interplay between other blood cells and coagulation proteins, which cannot be assessed by routine analysers. Thromboelastography (TEG), performed with a simple, point-of-care device, is a whole blood coagulation assay with the capability of assessing all these components. Its use has already been successfully incorporated in blood transfusion guidelines for cardiac and liver surgery where it has been shown to reduce transfusion rates in patients10-12.
The local policy for platelet transfusions in our institution is based on the British Committee for Standards in Haematology guidelines6 which recommend a 10×109/L trigger for platelet transfusion in non-febrile patients, with the trigger being doubled in patients with sepsis or other high-risk features for bleeding. We hypothesized that using FC and TEG might reduce platelet transfusion requirements in patients with haematological malignancies.
Materials and methodsThe study was conducted at Mater Dei Hospital,
Malta between May 2011 and February 2012 after approval by the local research ethics committee. The patients enrolled gave their informed consent. This was a proof-of-concept study and the results of TEG and FC were not used to change standard patient care. Twenty-four patients with various haematological malignancies were enrolled and a total of 31 assessments
were performed. The majority were patients diagnosed with acute leukaemia (70.8%) undergoing intensive chemotherapy. There were 16 males and 8 females and their mean age was 60 years. Pre-transfusion samples were taken on the morning of and within 6 hours of the transfusion. The post-transfusion complete blood count was performed 1 hour after the transfusion of one unit of platelets. Platelet transfusions were requested by the patient's physician in accordance with current guidelines13,14. Accordingly, a decision to transfuse an adult dose of pooled or single donor platelets, depending on availability, was automatically taken if the platelet count was <10×109/L. In case of counts <20×109/L but ≥10×109/L, one unit of platelets was transfused on the basis of the physician's judgement of a perceived increase in risk of bleeding, prior bleeding or fever. All units transfused were leucodepleted. Except for the platelet increment analysis, seven assessments were excluded since the platelet count on the day was higher than 19×109/L and did not, therefore, fulfil the criteria for transfusion.
Blood count and coagulation screeningFor the remaining 24 assessments the platelet count
was assayed using our routine Sysmex XT2000i analyser (impedance method) in blood collected into an EDTA-containing test-tube vacutainer (Becton Dickinson, Plymouth, United Kingdom). A complete blood count was performed, the activated partial thromboplastin time (APTT) and prothrombin time (PT) were determined and the fibrinogen level was assayed using the Clauss method15 as per routine practice. The coagulation assays were performed on a Sysmex 1500 coagulometer, with Innovin (Sysmex Europe GmbH, Norderstedt, Germany) as the thromboplastin source for the PT test and actin FS for the determination of the APTT.
Flow cytometryEDTA samples were also used to evaluate the platelet
count by FC. Whole blood was incubated with FITC-conjugated CD41a (HIP8). Following incubation the cells were washed using Cell Wash (BD Biosciences, San Jose, CA, USA) and analysed on a FACSCalibur FC (BD Biosciences). For each patient a control was included (a sample from a patient with a platelet count of 50-90×109/L) and 50,000 events were acquired for each sample. The staining and gating protocol followed was that proposed by the London Laboratory Service Group and included the following gating strategy7. Two histograms were created for each sample, histogram 1 (side scatter vs forward scatter) and histogram 2 (forward scatter vs CD41a). Two gates were formed within histogram 1, with R1 representing the red and white blood cells and R2 including the platelet events. On histogram 2, the following gates were produced:
All rights reserved - For personal use only No other uses without permission
TEG and fl ow cytometry to guide platelet transfusions
R3 representing the red blood cells, R4 representing the platelets/red blood cells coinciding, R5 including the platelet events and a final gate, R6, composed of debris. The first step in order to calculate the immunological platelet count was to calculate the red blood cell (RBC): platelet ratio using the following formula7:
RBC: Platelet ratio = Events in R3 + Events in R4
Events in R5 + Events in R4
The immunological platelet count could then be calculated as follows:
ThromboelastographyTEG was performed on a citrated blood sample (Becton
Dickinson Vacutainer, containing 9 parts blood to 1 part citrate) after the blood had been left to rest for 15 minutes. A "native" TEG was performed using no activator except for calcium to start blood clotting. This assay was performed on a TEG® 5000 Thromboelastograph Haemostasis System (Hemodyne Inc, Bethesda, Maryland, USA). This whole blood assay measures the reaction (R) time, clot formation (K) time, alpha angle and maximum amplitude (MA)15. The R time is mainly affected by differences in concentrations of the clotting factors, the K time is dependent on the clotting factors and to a certain extent on platelets, whereas the alpha angle and the MA are affected by the platelets and the fibrinogen concentrations15. The normal ranges for these parameters are shown in Table I.
StatisticsResults were initially checked for normality, using
GraphPad Prism version 4.0.3 (GraphPad Software Inc, La Jolla, California, USA). If the data did not pass the normality test and a normal Gaussian distribution was not seen, then non-parametric analysis with the Mann-Whitney test was used. Pearson's correlation was determined for normally distributed correlations whereas Spearman's rank test was used for non-parametric correlations. A paired-t test was used in the case of paired samples. A P value less than 0.05 was taken to be statistically significant. GraphPad Prism version 4.0.3 (GraphPad Software Inc) was used for all analyses.
Results General
The mean pre-transfusion haemoglobin and total white cell count were 9.6 g/dL and 3.23×109/L, respectively, whilst the mean post-transfusion values were 9.3 g/dL and 3.39×109/L, respectively. The mean APTT, PT and fibrinogen were normal at 26.5 seconds (normal range, 23-29 seconds), 11.1 seconds (normal range, 9.3-13.9 seconds) and 3.5 g/L (normal range, 0.9-8.5 g/L), respectively. No statistically significant changes in these three parameters were noted post-transfusion.
Routine blood analyser vs flow cytometryNo bleeding episodes were recorded in our cohort
of patients and all the platelet transfusions were given prophylactically.
Using all the 31 measurements to assess platelet increment after transfusion of one adult unit of platelets, the mean increase in platelet count was 23×109/L (95% CI: 18-27×109/L) according to the automated analyser, whereas the increase was greater, at 28×109/L (95% CI: 23-34×109/L) when measured by FC (P=0.001). The pre-transfusion platelet counts determined by flow cytometry were also higher than those measured by the routine analyser (mean difference 4×109/L, P=0.0011). There was an excellent correlation between the counts determined by the routine analyser and FC (r=0.89, P<0.0001). Using FC, in 18.2% of cases the platelet counts were 20×109/L or greater and the patients would not have required a prophylactic platelet transfusion (Figure 1).
ThromboelastographyPrior to transfusion, TEG showed shortened R time
in 69.6% of cases and a normal mean K time of 6.6 minutes. Only 9% of cases had a low α angle signifying hypocoagulability. The MA showed an excellent correlation with the EDTA platelet count (r=0.91, P<0.0001). The MA was reduced in the majority of cases, although 25% still had normal values despite platelet counts <20×109/L.
After transfusion of the prophylactic platelet unit, the R and K times shortened while the α- angle and the MA increased (Table I). All these changes were statistically significant.
The results were also subdivided on the basis of whether the pre-transfusion platelet count was
Table I - TEG results pre and post-platelet transfusion. The normal ranges shown are those provided by the manufacturer.Pre-transfusion(Mean 95%CI)
Post-transfusion(Mean 95%CI)
Normal range P
R time (min) 7.8(6.6-8.9) 5.7(4.6-6.9) 9-27 0.0008
<10×109/L or >10 but <20×109/L using the routine analyser. There were 12 cases in each group. Of note, there were no statistical differences in the pre-transfusion white blood cell count, haemoglobin, fibrinogen, PT and APTT values between these two groups. Among the patients in the former group, with platelet counts <10×109/L, only one case started with a normal MA. However, the R-time for all of the cases was normal to reduced. Two-thirds of these patients had a normal K-time and half had normal α-angle. This shows that secondary haemostasis is brisk but the platelet dependent part is deficient. The group with a higher platelet count had a normal or reduced R-time (100%), with only one case having an increased K-time together with a reduced α-angle. This case had a platelet count of 21×109/L determined by FC and of 15×109/L according to the routine analyser, and so would not have been revealed as abnormal on FC. Only 6/12 cases (50%) in this group had a low MA (Figure 2). Interestingly, two out of these six patients with a low MA also had a platelet count of <10×109/L determined by FC.
DiscussionThis study was intended as a proof-of-concept study
in order to see whether FC and/or TEG could have an impact on the use of prophylactic platelet transfusions in patients with haematological malignancies. We chose to evaluate FC along with the platelet counting using a standard analyser since it has been shown that FC counting is more sensitive than the normal analysers at low platelet counts. We also performed TEG since, unlike FC, this assesses clot formation and strength. TEG has the advantage of evaluating all components of primary and secondary haemostasis rather than merely counting platelets. It is also an established point-of-care test which can easily be incorporated into use on a haematology ward.
Our study shows that, applying current platelet transfusion cut-offs, using platelet counts determined by FC would have around 18% of platelet transfusions spared on the day. It should be appreciated that this does not necessarily equate to a reduction in the total number of platelets transfused per hospitalisation period. Our study did not try to establish this a priori. Although FC proved to be more sensitive in the enumeration of platelets, one must keep in mind that it is not universally available, is labour-intensive and requires expertise for the identification of the platelet population. One major drawback of the technique is that the sample must be analysed within 4 hours of collection. The samples in our study were brought to the laboratory by hand immediately after collection, which might not be feasible in a routine clinical setting.
TEG showed a shortened R time together with a more obtuse α angle, features seen in hypercoagulable profiles. However, traditionally the MA is the parameter of choice that correlates with platelets especially when fibrinogen levels are normal and in fact the majority of the cases showed a lower than normal MA. Nevertheless, 25% of patients had a normal or increased MA despite a platelet count of <20×109/L. Unsurprisingly, most of these fell in the group with a pre-transfusion platelet count of ≥10×109/L with only one patient in the group with platelet count <10×109/L showing a normal MA. Some would argue that this could be due to the lack of sensitivity of TEG to low platelet counts16, whereas others, including our group, propose that this is mainly due to the fact that since TEG is a global assay of coagulation it is affected by all other blood components that could compensate for the low platelet counts, such as fibrinogen. This is corroborated by studies showing that TEG values are more specific markers of coagulopathy and clinical bleeding than platelet counts per se17. Considered as a whole group, the TEG data could have been the basis for avoiding transfusion of a quarter of the units administered. However, patients with platelet counts <10×109/L would "require" platelet transfusion with or without the TEG information. This means that the utility of such a test might be confined to those patients with platelet counts ≥10×109/L. This is corroborated by a recent trial showing the utility of platelet transfusions in patients with haematological malignancies and platelet counts <10×109/L18.
There are several advantages from using TEG in this scenario. It is a point-of-care test that has proven capabilities of guiding and reducing transfusion requirements in surgery19,20. The test uses whole blood and does not require any centrifugation, thus making it more convenient for integration in a ward setting. The test employed in this study is relatively inexpensive with the main cost being the TEG test cups and calcium chloride,
Figure 1 - Platelet counts determined using the routine analyser (RA)(Sysmex ST2000i) and flow cytometry flow cytometry (FC).
All rights reserved - For personal use only No other uses without permission
TEG and fl ow cytometry to guide platelet transfusions
since "native" TEG does not involve any other extra reagents. Being a whole blood assay, it also incorporates all the components of haemostasis including platelets (number and function21,22), fibrinogen, factor VIII and von Willebrand factor23. Fibrinogen levels were normal in our subjects. We did not measure factor VIII or von Willebrand factor levels since we tried to minimise the blood letting in our patients. However, surrogate markers for factor VIII levels are the APTT and the TEG R time. The APTT in our patients was normal while the R-time was reduced. The latter finding was probably due to elevated factor VIII levels24 although we could not prove this in our study.
There are also certain post-platelet transfusion implications since our results clearly show an increase in MA after the transfusion. In fact, in this period some patients had MA values higher than the normal range.
This, coupled with the heightened coagulation factor response as shown by the low R times and increased α angle, is a clear prothrombotic profile.
Some limitations of our study should be acknowledged. One was the small number of patients assessed. However, this was intended to be an exploratory study to collect the information necessary to estimate the sample size for a larger follow-up study. It would be useful to perform an outcome study to prospectively evaluate the TEG parameters and bleeding and thrombosis in a similar group of patients. Furthermore, it would be important to perform cost/benefit analyses prior to the introduction of similar newer ways of assessing coagulation in haematology-oncology patients. We did not seek correlation with clinical outcomes due to the small sample. Indeed, there were no bleeding episodes in the patients in our study. It is, however, very important
Figure 2 - Pre- and Post- platelet transfusion TEG parameters R, K, alpha angle and MA. Column A shows the cases with a pre-transfusion count <10×109/L and column B those with platelets between 10 and 19×109/L.
The lighter columns denote the pre-transfusion results whereas the darker columns show the post-transfusion values. The horizontal lines show the normal ranges.
All rights reserved - For personal use only No other uses without permission
to correlate in vitro results with outcomes in specific clinical scenarios since this evidence remains lacking25.
Finally, it has been demonstrated that, despite prophylactic platelet transfusions and improvements in platelet counts, patients with haematological malignancies are a vulnerable group of subjects who still experience bleeding20 which highlights the need for further studies on novel ways of assessing coagulation in this group of patients rather than just merely counting platelets.
The Authors declare no conflicts of interest.
References1) Shah A, Andersson TM, Rachet B, et al. Survival and cure of
acute myeloid leukaemia in England, 1971-2006: a population-based study. Br J Haematol 2013; 162: 509-16.
2) Gaydos LA, Freireich EJ, Mantel N. The quantitative relation between platelet count and hemorrhage in patients with acute leukemia. N Engl J Med 1962; 266: 905-9.
3) Gmur J, Burger J, Schanz U, et al. Safety of stringent prophylactic platelet transfusion policy for patients with acute leukaemia. Lancet 1991; 338: 1223-6.
4) Lawrence JB, Yomtovian RA, Dillman C, et al. Reliability of automated platelet counts: comparison with manual method and utility for prediction of clinical bleeding. Am J Hematol 1995; 48: 244-50.
5) Estcourt L, Stanworth S, Doree C, et al. Prophylactic platelet transfusion for prevention of bleeding in patients with haematological disorders after chemotherapy and stem cell transplantation. Cochrane Database Syst Rev 2012; 5: CD004269.
6) British Committee for Standards in Haematology, Blood Transfusion Task Force. Guidelines for the use of platelet transfusions. Br J Haematol 2003; 122: 10-23.
7) International Council for Standardization in Haematology Expert Panel on Cytometry, International Society of Laboratory Hematology Task Force on Platelet Counting. Platelet counting by the RBC/platelet ratio method. A reference method. Am J Clin Pathol 2001; 115: 460-4.
8) Sandhaus LM, Osei ES, Agrawal NN, et al. Platelet counting by the Coulter LH 750, Sysmex XE 2100, and Advia 120: a comparative analysis using the RBC/platelet ratio reference method. Am J Clin Pathol 2002; 118: 235-41.
9) Manoharan A, Brighton T, Gemmell R, et al. Platelet dysfunction in myelodysplastic syndromes: a clinicopathological study. Int J Hematol 2002; 76: 272-8.
10) Shore-Lesserson L, Manspeizer HE, DePerio M, et al. Thromboelastography-guided transfusion algorithm reduces transfusions in complex cardiac surgery. Anesth Analg 1999; 88: 312-9.
11) Westbrook AJ, Olsen J, Bailey M, et al. Protocol based on thromboelastograph (TEG) out-performs physician preference using laboratory coagulation tests to guide blood replacement during and after cardiac surgery: a pilot study. Heart Lung Circ 2009; 18: 277-88.
12) Kang YG, Martin DJ, Marquez J, et al. Intraoperative changes in blood coagulation and thrombelastographic monitoring in liver transplantation. Anesth Analg 1985; 64: 888-96.
13) Schiffer CA, Anderson KC, Bennett CL, et al. Platelet transfusion for patients with cancer: clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol 2001; 19: 1519-38.
14) Clauss A. Rapid physiological coagulation method in determination of fibrinogen. Acta Haematol 1957; 17: 237-46.
16) A g r e n A , Wi k m a n AT, H o l m s t r o m M , e t a l . Thromboelastography (TEG(R)) compared to conventional coagulation tests in surgical patients-a laboratory evaluation. Scand J Clin Lab Invest 2013; 73: 214-20.
17) Essell JH, Martin TJ, Salinas J, et al. Comparison of thromboelastography to bleeding time and standard coagulation tests in patients after cardiopulmonary bypass. J Cardiothorac Vasc Anesth 1993; 7: 410-5.
18) Stanworth SJ, Estcourt LJ, Powter G, et al. A no-prophylaxis platelet-transfusion strategy for hematologic cancers. N Engl J Med 2013; 368: 1771-80.
19) Aoki K, Sugimoto A, Nagasawa A, et al. Optimization of thromboelastography-guided platelet transfusion in cardiovascular surgery. Gen Thorac Cardiovasc Surg 2012; 60: 411-6.
20) Johansson PI, Solbeck S, Genet G, et al. Coagulopathy and hemostatic monitoring in cardiac surgery: an update. Scand Cardiovasc J 2012; 46: 194-202.
21) Sheikh AY, Hill CC, Goodnough LT, et al. Open aortic valve replacement in a patient with Glanzmann's thrombasthenia: a multidisciplinary strategy to minimize perioperative bleeding. Transfusion 2014; 54: 300-5.
22) Apelseth TO, Bruserud O, Wentzel-Larsen T, Hervig T. Therapeutic efficacy of platelet transfusion in patients with acute leukemia: an evaluation of methods. Transfusion 2010; 50: 766-75.
23) Topf HG, Weiss D, Lischetzki G, et al. Evaluation of a modified thromboelastography assay for the screening of von Willebrand disease. Thromb Haemost 2011; 105: 1091-9.
24) Al Hawaj MA, Martin EJ, Venitz J, et al. Monitoring rFVIII prophylaxis dosing using global haemostasis assays. Haemophilia 2013; 19: 409-14.
25) Panzer S, Jilma P. Methods for testing platelet function for transfusion medicine. Vox Sang 2011; 101: 1-9.
Arrived: 18 October 2013 - Revision accepted: 2 December 2013Correspondence: Alex GattHaematology-Oncology DepartmentMater Dei HospitalMsida, Maltae-mail: [email protected]
All rights reserved - For personal use only No other uses without permission
