Prediction of bleeding and prophylactic platelet transfusions in cancer patients with thrombocytopenia

Studies on markers for bleeding risk among thrombocytopenic cancer patients are lacking. This prospective observational cohort study investigated whether platelet parameters and a standardised bleeding questionnaire predicted bleeding or prophylactic platelet transfusions in patients with cancer and thrombocytopenia. Admitted adult patients with cancer and platelet count <80 × 10⁹/L were enrolled, but excluded if they experienced surgery or trauma within 7 days or platelet transfusion within 14 days. Patients were interviewed, blood samples collected and, subsequently, spontaneous bleeding and prophylactic platelet transfusion within 30 days were registered.

Of 197 patients enrolled, 56 (28%) experienced bleeding. In multivariate analyses, predictors of bleeding were infection (adjusted odds ratio (OR) = 2.65 and 95% confidence interval (95% CI) 1.04–6.74); treatment with platelet inhibitors, heparin or warfarin OR = 2.34, 95% CI 1.23–4.48; urea nitrogen OR = 1.15, 95% CI 1.07–1.25; creatinine OR = 1.01, 95% CI 1.01–1.01; and haemoglobin OR = 0.62, 95% CI 0.41–0.93. Specific information regarding previous gastrointestinal bleeding OR = 3.33, 95% CI 1.19–9.34 and haematuria OR = 3.00, 95% CI 1.20–7.52 predicted bleeding whereas the standardised bleeding questionnaire did not.

Prophylactic platelet transfusions were administered to 97 patients. Predictors of prophylactic platelet transfusions were: platelet count OR = 0.96, 95% CI 0.94–0.97; fibrinogen OR = 0.88, 95% CI 0.83–0.95; mean platelet volume OR = 0.69, 95% CI 0.49–0.97; platelet aggregometry with OR = 2.48, 95% CI 1.09–5.64 for collagen-induced platelet aggregation within the lowest quartile; and albumin OR = 1.07, 95% CI 1.01–1.15.

In conclusion, except for immature platelet fraction (IPF), platelet parameters predicted prophylactic platelet transfusion but not bleeding. Bleeding risk factors were previous haematuria or gastrointestinal bleeding, infection, antiplatelet or anticoagulant treatment, high urea nitrogen, low haemoglobin or high creatinine.     

Prospective observational cohort study of the association between thromboelastometry,coagulation and platelet parameters and bleeding in patients with haematological malignancies‐ The ATHENA study

Previous studies have shown that total platelet count (TPC) inadequately predicts bleeding in thrombocytopenic patients with haematological malignancies. This prospective cohort study evaluated whether rotational thromboelastometry (ROTEM), coagulation or other platelet parameters were more strongly associated with bleeding than TPC. Adults treated at two UK haematology centres for haematological malignancy were enrolled if they had thrombocytopenia (TPC ≤ 50 × 10⁹/l) at beginning of, or during treatment (International Standard Randomized Controlled Trial Number 81226121). TPC and bleeding symptoms were recorded daily for up to 30 d or until platelet count recovery, hospital discharge or death. Blood samples were tested thrice weekly using ROTEM, Platelet Function Analyser (PFA)‐100^®, coagulation and platelet cytometry assays. Bleeding symptoms and TPC from 49/50 enrolled participants who completed the study were recorded on 754/760 study days. Mean platelet volume and PFA‐100^® closure times were frequently inestimatable because of thrombocytopenia. TPC, absolute immature platelet number (AIPN) and ROTEM maximum clot firmness were significantly associated with bleeding on the day after blood sampling. Only AIPN was associated with bleeding after adjustment of test results for TPC (Odds Ratio 0·52, 95% confidence interval 0·28–0·97; P = 0·038). In a predictive model, AIPN was superior to TPC for predicting bleeding. This study indicates that AIPN may be more clinically useful than TPC at predicting bleeding.     

Beyond the platelet count: immature platelet fraction and thromboelastometry correlate with bleeding in patients with immune thrombocytopenia

Platelet counts (PC) estimate bleeding risk in Immune Thrombocytopenia (ITP). We investigated whether measures of thromboelastometry and absolute immature platelet fraction (A‐IPF) would correlate better with acute bleeding score (ABS) than PC or mean platelet volume (MPV). Simultaneous determination of ABS, complete blood count and thromboelastometry was performed in 141 ITP patients; 112 underwent A‐IPF testing. Subgroup analyses were performed for paediatric subjects, PC <60 × 10⁹/l and <30 × 10⁹/l. PC significantly inversely correlated with ABS in all subjects, PC <30 × 10⁹/l and total paediatric cohort. MPV did not correlate with ABS in any subgroup. Thromboelastometry measures of clot firmness, but not PC, significantly correlated with ABS in all subjects with PC <60 × 10⁹/l, and children with PC <60 × 10⁹/l and <30 × 10⁹/l. A‐IPF demonstrated stronger correlation with ABS than did PC among all subjects, those with PC <60 × 10⁹/l, all children and children with PC <30 × 10⁹/l (r = ?0·37; r = ?0·34; r = ?0·44; r = ?0·60) versus ABS with PC (r = ?0·36; ns; r = ?0·32; ns). Stronger correlations of both thromboelastometry measures of clot firmness and A‐IPF than PC with ABS suggest factors beyond PC, i.e. related to platelet function, contribute to ITP bleeding pathophysiology. Thromboelastometry, A‐IPF and ABS can be incorporated into routine or acute visits.     

Continuing developments with the automated platelet count

The four main procedures for platelet counting are: manual phase contrast microscopy, impedance, optical light scatter/fluorescence and flow cytometry. Early methods to enumerate platelets were inaccurate and irreproducible. The manual count is still recognized as the gold standard or reference method, and until very recently the calibration of platelet counts by the manufacturers of automated cell counters and quality control material was performed by this method. However, it is time-consuming and results in high levels of imprecision. The introduction of automated full blood counters using impedance technology resulted in a dramatic improvement in precision. However, impedance counts still have limitations as cell size analysis cannot discriminate platelets from other similar-sized particles. More recently, light scatter or fluorescence methods have been introduced for automated platelet counting, but there are still occasional cases where an accurate platelet count remains a challenge. Thus, there has been interest in the development of an improved reference procedure to enable optimization of automated platelet counting. This method utilizes monoclonal antibodies to platelet cell surface antigens conjugated to a suitable fluorophore. This permits the possible implementation of a new reference method to calibrate cell counters, assign values to calibrators, and to obtain a direct platelet count on a variety of pathological samples. In future, analysers may introduce additional platelet parameters; a reliable method to quantify immature or reticulated platelets would be useful.     

Flow cytometry and thromboelastography to assess platelet counts and coagulation in patients with haematological malignancies

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.     

Cone and platelet analyser (CPA): a new test for the prediction of bleeding among thrombocytopenic patients

The risk of bleeding among thrombocytopenic patients was evaluated using our new cone and platelet analyser (CPA) test. Using this test, adherence of platelets was quantitated on extracellular matrix and expressed as percent of surface coverage (SC) and the average size (AS) of aggregates. 42 thrombocytopenic patients with ITP (n=23), post chemotherapy (n=12) and others (n=7) were tested over a total of 82 visits. On each visit, complete blood count and CPA tests were performed and patients were evaluated for evidence of bleeding (found in 40 visits). Bleeding patients had significantly lower platelet counts (27.4 ± 22.0 v 47.1 ± 21.0 × 10⁹/l), lower haematocrit values (30.2 ± 8.1 v 35.2 ± 6.6%), lower MPV (6.83 ± 1.89 v 8.98 ± 1.13 fl), and lower SC (4.87 ± 3.95 v 10.33 ± 5.48%) and AS (33.99 ± 14.94 v 52.9 ± 24.34 μm²). Univariate analysis yielded platelet count 20.0 × 10⁹/l, MPV 8fl, haematocrit <35%, SC < 5%, AS 40 μm² as significantly associated with bleeding, whereas only MPV and SC were associated with bleeding (OR 6.95, CI 2.25–21.46 and OR 4.27, CI 1.29–14.16, respectively) by multivariate analysis. When taken together, 21/22 of patients (95%) with both low SC (<5%) and low MPV (<8.0 fl) had bleeding symptoms, whereas only 9/43 (21%) patients with both these parameters above these values experienced bleeding symptoms. We conclude that the CPA test and the parameter SC (<5%) together with MPV (8 fl) might be used as independent predictors of bleeding in the management of thrombocytopenic patients.     

Platelet count evaluation using three automated haematology analysers compared with the immunoplatelet reference method, and estimation of possible inadequate platelet transfusion

The accuracy of three automated haematology analysers [Sysmex XE-2100 (both optical and impedance mode), Bayer Advia 120, and Beckman Coulter LH-750] was compared with the immunoplatelet reference method for platelet measurement. A total of 165 blood specimens were obtained from patients and platelet counts were determined using the four-automated haematology analyser methods and the immunoplatelet reference method. The coefficients of determination (R(2)) between the automated haematology analyser methods and the immunoplatelet reference method for the overall platelet range were >0.98. A bias study, however, showed some disagreement. The use of a coincidence correction calculation for the immunoplatelet method did not improve the correlation between the immunoplatelet method and the automated haematology analyser methods. To estimate the possibility of inadequate platelet transfusion, the number of prophylactic platelet transfusion indications determined by the automated haematology analyser platelet counts were compared with the number of transfusion indications according to the platelet counts determined by the immunoplatelet method. An additional 48 blood specimens were included in this analysis. All of the automated haematology analysers showed some disagreement in the transfusion indications when compared with the immunoplatelet method, suggesting the possibility of inadequate platelet transfusion.     

MicroRNAs as potential regulators of platelet function and bleeding diatheses

Although a growing number of studies suggest that microRNAs (miRNAs) play a relevant role in platelet biology, their implications in bleeding diatheses are starting to be investigated. Indeed, several studies have shown that alterations in the intracellular levels of highly expressed platelet miRNAs provoke a thrombotic phenotype. On the other hand, primary immune thrombocytopenia (ITP), which is considered the hallmark of acquired bleeding disorders, has been recently associated with altered levels of miRNAs in peripheral blood mononuclear cells, plasma, and platelets. In this review, we will focus on miRNAs that may affect the hemostatic and thrombotic functions of platelets, and we will discuss the different studies that have attempted to associate miRNAs with regulatory mechanisms of ITP.     

Effect of heparin on platelet count and platelet aggregation

The in vitro effect of heparin on platelet aggregation was studied in three groups: in 26 subjects recently treated with heparin, in 18 subjects on maintenance hemodialysis, and in 20 normal controls. With the aid of Technicon H6000, platelet counts and platelet aggregations were compared in whole blood samples collected in ethylenediaminetetraacetic acid (EDTA) and in heparinized tubes. Although there was no significant difference between platelet count of heparinized and EDTA blood in the control group, the dialysis group and the group recently treated with heparin showed significantly lower platelet counts and more platelet aggregation in heparinized tubes than in EDTA tubes. We speculate that the majority of subjects exposed to heparin develop an antibody or a proaggregator which can aggregate or agglutinate platelets in the presence of heparin and causes destruction of platelets; but only in a small percentage of subjects receiving heparin is this reaction severe enough to cause thrombocytopenia.     

The association between haemorrhage and markers of endothelial insufficiency and inflammation in patients with hypoproliferative thrombocytopenia: a cohort study

In daily haematological practice, predicting bleeding in thrombocytopenic patients is difficult, and clinicians adhere to transfusion triggers to guide patients through the aplastic phase of chemotherapy. Platelet count is not the only determinant of bleeding and additional mechanisms for impending haemostasis are likely. Beside clot formation, platelets are essential for the maintenance of integrity of vascular beds. We therefore prospectively investigated associations between biomarkers for endothelial damage (urine albumin excretion) and inflammation (C-reactive protein) and bleeding (WHO grading) in 88 patients with 116 on-protocol episodes. We found an increase in grade 2 bleeding with a higher urine albumin/creatinine ratio one day after the measurement [odds ratio (OR) 1·24 for every doubling of the ratio, 95% CI 1·05–1·46, P-value 0·01] and a 29% increase in the odds of grade 2 bleeding for every doubling of serum C-reactive protein (CRP) (95% CI 1·04–1·60, P-value 0·02) after correction for morning platelet count. The 24 h post-transfusion corrected count increment (CCI₂₄) showed a significant association with these biomarkers: increasing urine albumin/creatinine ratio and CRP were associated with lower CCI_24. We report two inexpensive and easy-to-apply biomarkers that could be useful in designing a prediction model for bleeding risk in thrombocytopenic patients.     

The frequency of bleeding complications in patients with haematological malignancy following the introduction of a stringent prophylactic platelet transfusion policy

Indications for platelet transfusion remain controversial and are frequently based on arbitrary numerical criteria. In October 2000, we introduced a stringent prophylactic-platelet transfusion policy < 10 x 109/l for stable patients and < 20 x 10(9)/l in the presence of major bleeding or additional risk factors. A trigger of < 50 x 10(9)/l was introduced for patients undergoing invasive procedures. A prospective analysis was performed measuring the frequency of minor and major bleeding events, morbidity, mortality and duration of pancytopenia. Blood product usage was assessed and health care savings measured. A total of 98 patients were evaluated on 2147 patient study days and 271 bleeding episodes were recorded. Major bleeding occurred on 1.39% (30/2147) of the study days when platelet counts were < 10 x 10(9)/l and 2.3% (50/2147) of the study days when platelet counts were 10-20 x 10(9)/l. In patients with platelets > 20 x 10(9)/l, there were 117 major bleeding episodes observed on 5.4% of the study days. In patients with no identified additional risk factors present, major haemorrhages were recorded in 0.51% (11/2147) of the study days in patients with platelet counts > or = 10 x 10(9)/l . There was a 36% reduction in platelet units transfused compared with retrospective data when an arbitrary transfusion trigger of 20 x 10(9)/l was in place (P = < 0.02). Of note, a 16% reduction in red cell transfusions was recorded. These data confirm that the introduction of a transfusion trigger of < 10 x 10(9)/l in the absence of fresh bleeding and sepsis (> 38 degrees C) is safe and has a significant impact on overall hospital transfusion costs.     

An overview of platelet indices and methods for evaluating platelet function in thrombocytopenic patients

Thrombocytopenia is associated with bleeding risk. However, in thrombocytopenic patients, platelet count does not correlate with bleeding risk and other factors are thus likely to contribute to this risk. This review presents currently available platelet‐related markers available on automated haematology analysers and commonly used methods for testing platelet function. The test principles, advantages and disadvantages of each test are described. We also evaluate the current literature regarding the clinical utility of the test for prediction of bleeding in thrombocytopenia in haematological and oncological diseases. We find that several platelet‐related markers are available, but information about the clinical utility in thrombocytopenia is limited. Studies support that mean platelet volume (MPV) can aid diagnosing the cause of thrombocytopenia and low MPV may be associated with bleeding in thrombocytopenia. Flow cytometry, platelet aggregometry and platelet secretion tests are used to diagnose specific platelet function defects. The flow cytometric activation marker P‐selectin and surface coverage by the Cone‐and‐Plate[let] analyser predict bleeding in selected thrombocytopenic populations. To fully uncover the clinical utility of platelet‐related tests, information about the prevalence of platelet function defects in thrombocytopenic conditions is required. Finally, knowledge of the performance in thrombocytopenic samples from patients is essential.     

Platelet transfusion improves clot formation and platelet function in severely thrombocytopenic haematology patients

Prophylactic platelet (PLT) transfusion is a common practice in severely thrombocytopenic patients that reduces mortality, but responses to platelet transfusions are variable and difficult to predict in individual patients. In this prospective study, we evaluated the outcome of PLT transfusions in 40 patients with haematological malignancies, linking corrected count increment (CCI) to clot formation and agonist-induced platelet activation after transfusion. The CCI was highly variable between patients and 34% showed no response (1-h CCI < 7,5). Short time since the last PLT transfusion and extended storage time of the PLT product were linked to poor transfusion response, while patient sex, C-reactive protein or the number of chemotherapy cycles prior to transfusion did not influence transfusion outcome. High CCI and good PLT responsiveness to agonist stimulation predicted efficient clot formation in rotational thromboelastometry, but transfusion did not restore poor PLT function in patients to the level of healthy controls. Our study provides new insights into factors affecting PLT transfusion outcome in haematology patients with severe thrombocytopenia, and suggests that the thrombocytopenic environment, or disease-associated factors, may hamper platelet responsiveness.     

Determinants of platelet count in pediatric patients with congenital cyanotic heart disease: Role of immature platelet fraction

Objectives

Congenital heart defects are common noninfectious causes of mortality in children. Bleeding and thrombosis are both limiting factors in the management of such patients. We assessed the frequency of thrombocytopenia in pediatric patients with congenital cyanotic heart disease (CCHD) and evaluated determinants of platelet count including immature platelet fraction (IPF) and their role in the pathogenesis of thrombocytopenia.

Methods

Forty‐six children and adolescents with CCHD during pre‐catheter visits were studied; median age was 20.5 months. Complete blood count including IPF as a marker of platelet production and reticulated hemoglobin content (RET‐He) as a marker of red cell production and iron status were done on Sysmex XE 2100 (Sysmex, Japan). C‐reactive protein, prothrombin time (PT), Activated partial thromboplastin time (APTT) were also assessed.

Results

Thrombocytopenia was found in 6 patients (13%). PT was prolonged (P = .016) and IPF was significantly higher in patients with thrombocytopenia compared with patients with normal platelet count (14.15 ± 5.2% vs 6.68 ± 3.39%; P = .003). Platelet count was negatively correlated with IPF while significant positive correlations were found between IPF and hemoglobin, red blood cells (RBCs) count, hematocrit (Hct), PT, reticulocytes count, and immature reticulocyte fraction.

Conclusions

We suggest that elevated IPF in CCHD patients with thrombocytopenia may denote peripheral platelets destruction as an underlying mechanism. Hemoglobin level, RBCs count, Hct, and RET‐He were not significant determinants for platelet count in CCHD.