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Year : 2018  |  Volume : 61  |  Issue : 3  |  Page : 366-370
Platelet function tests: A 5-year audit of platelet function tests done for bleeding disorders in a tertiary care center of a developing country

Department of Hematology and Clinical Pathology, Sir Ganga Ram Hospital, New Delhi, India

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Date of Web Publication13-Jul-2018


Introduction: The platelet function disorders remain largely undiagnosed or incompletely diagnosed in developing nations due to lack of availability of tests like lumiaggregometry, granule release assay or molecular testing. We performed a retrospective analysis of all the platelet function test (PFT) carried out in past 5 years by Light transmission aggregometery (LTA) using a panel of agonist. The indications and the test results were analyzed by two hematopathologist with the aim to look into the present diagnostic facilities or lack of it for correct diagnosis. This is essential for better management and genetic counselling. Materials and Methods: The PFT was performed both on patients and healthy unrelated age specific controls by light transmission aggregometry on Chronolog platelet aggregometer using platelet rich plasma. The panel of agonists included ADP (10μm/l and 2.0 μm/l), epinephrine (10.0 μm/l), collagen (2μg/ml), arachidonic acid (0.75 mM) and ristocetin (1.25 mg/ml & 0.25 mg/l). Results: The 5 years records of 110 cases were audited, 101 of these were tested for clinical bleeding , 35 adults and 66 children. The adults included 29 women and 6 men, 17 to 82 years of age. The children were 16 years to 3 months of age, 30 girls and 36 boys. Platelet function test abnormality was found in 31.6% (32/101) cases ,a majority remained undiagnosed of these about 21% had clinically significant bleeding.The cases diagnosed included Glanzmann Thromboasthenia-11 , von Willebrand Disease-6, Bernard Soulier'syndrome-1, storage pool disorder-6, mild defect of Epinephrine-3, isolated defect with collagen in1. Conclusion: An epidemiologically large proportion of platelet function disorders amongst people living in developing nations remain undiagnosed. This lacunae needs to be highlighted and addressed on larger scale. The options available are to increase the available armamentarium of tests or international collaboration with a specialized laboratory to aid in complete diagnosis.

Keywords: Bleeding disorders, light transmission aggregometry, platelet function test

How to cite this article:
Langer S, Dass J, Saraf A, Kotwal J. Platelet function tests: A 5-year audit of platelet function tests done for bleeding disorders in a tertiary care center of a developing country. Indian J Pathol Microbiol 2018;61:366-70

How to cite this URL:
Langer S, Dass J, Saraf A, Kotwal J. Platelet function tests: A 5-year audit of platelet function tests done for bleeding disorders in a tertiary care center of a developing country. Indian J Pathol Microbiol [serial online] 2018 [cited 2021 Jun 13];61:366-70. Available from: https://www.ijpmonline.org/text.asp?2018/61/3/366/236628

   Introduction Top

Although rare, the bleeding disorders in people living in one of the most populated parts of the world with population reaching 1.3 billion and more in India are found in larger numbers, especially in parts of country where consanguinity is a common practice. Although platelet function defects (PFDs) were found to be less prevalent than coagulation factor defects, they form a major portion of undiagnosed bleeding disorders.[1] Diagnostic algorithms have been laid down for inherited platelet disorders by various study groups.[2] Light transmission aggregometry (LTA) is the gold standard for detecting acquired and inherited PFDs in the nonthrombocytopenic patients. These need to be combined with other tests such as granule release assay, flow cytometric immunophenotypic analysis, and molecular studies.[3] However, a large number of cases of platelet type bleeding remain undiagnosed in developing countries due to marked limitations of resources and availability of tests. Recently, there are efforts of international collaboration with developing nations to support the diagnosis of inherited platelet defects.[4]

Only a handful laboratories provide testing on platelet function testing cases are referred to these from various parts of the country. This study was done to determine the practices and outcomes in our hospital to screen and diagnose platelet disorders in a resource-limited setting. The aim of the study was to highlight the infrastructure or lack of it for an epidemiologically significant population of world.

   Materials and Methods Top

All the records for the platelet function tests done in the past 5 years were retrieved and analyzed retrospectively by two hematopathologists. They independently looked into the indications for ordering platelet function tests and test results. The history taken at the time of testing was obtained from the records. The bleeding symptoms were scored as per international society of thrombosis and haemostasis –bleeding assessment tool (ISTH BAT) into significant and not significant by a total of all the individual scores drawn for final bleeding score (BS). The normal adult BS range was taken 0–3 in males and 0–5 in females. The BS of ≥ 4 for males, ≥6 for females, and ≥ 3 for children were cutoffs for abnormal.[5]

LTA is affected by several preanalytical and analytical variables. In our laboratory, test is carried out by trained personnel according to laid down standard operating procedures as per the recent recommendations for carrying out LTA.[6]

Besides LTA, the other tests performed on all the patients included complete blood counts on automated hematology analyzer and peripheral blood film examination after staining with Wright–Giemsa stain. The bleeding time and clot retraction tests were not done on any patient. Prothrombin time and activated partial thromboplastin time were done in majority of cases. The other tests done to help supplement the diagnosis included serum fibrinogen levels, Factor VIII, Von Willebrand factor Ag, Factor IX, clot solubility test, ristocetin cofactor assays.

Patient preparation

The platelet function tests by LTA tests were carried out only after prior appointment. The patients and controls were instructed to be off medication for 4 days before testing with overnight fasting.

Blood sample collection

The blood samples were collected up to the mark in sky blue capped vacutainer containing trisodium citrate, th us assuring 1:9 ratio of blood is to anticoagulant. The platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared according to standardized procedures in the laboratory. The quality of PRP is regularly monitored. All the platelet function tests were then carried out on the Chrono-log platelet aggregometer based on the principle of LTA.

Preparation of platelet-rich plasma and platelet-poor plasma and ascertaining platelet-rich plasma quality

The quality control measures in our laboratory include stringent controls on timings of collection and performing tests, centrifuge speed and calibration, temperature, and the quality of PRP and PPP made.

The panel of agonists used included adenosine diphosphate (ADP) (10 μm/L and 2.0 μm/L), epinephrine (10.0 μm/L), collagen (2 μg/ml), arachidonic acid (0.75 mM), and ristocetin (1.25 mg/ml and 0.25 mg/L).

Reporting of results

The platelet aggregation tracing was evaluated with respect to shape change, length of the lag phase, slope of aggregation, maximal amplitude, or % aggregation. The laboratory has established its internal reference intervals for platelet aggregation by calculating the mean and standard deviation of the tests performed on healthy controls.

   Results Top

The 5-year records of 110 cases were retrieved. Among these, platelet aggregation studies were done in 101 cases to define the cause for bleeding. There were 35 adults and 66 children. The adults were mainly women (29 women and 6 men) aged 17–82 years of age. The pediatric age group included children from 16 years to 3 months of age with gender breakup of 30 girls and 36 boys.

The bleeding history in children commonly included ecchymosis, petechiae, epistaxis, and gum bleeds with BSs ranging from 3 to 17. In adolescent and young females, the menorrhagia was the most common presenting symptom [Table 1]. As per the defined cutoffs for BSs, the significant bleeding was present in 60.6% (40/66) children, 51.7% (15/29) women, and 66.6% (4/6) men tested. There were about ~ 42% (42/101) cases in which the bleeding history felt into the not significant score.
Table 1: Clinical profile of patients

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Among 101 patients tested, platelet function test defect was present in only 31.6% (32/101) of cases and 68.3% (69/101) of cases exhibited no platelet function test abnormality using aforementioned agonists. Two of the patients falling into the nonsignificant bleeding category displayed platelet function abnormality by LTA. The test results where a well-characterized platelet function test disorder could be ascertained using light transmission platelet aggregometry were even a smaller fraction of total bleeders tested [Table 2]. These cases included 11 cases of Glanzmann's thrombasthenia which characteristically showed markedly reduced response to the agonists such as ADP, collagen, epinephrine, and arachidonic acid and normal or near-normal response to ristocetin [Figure 1]. The diagnosis of von Willebrand disease (vWD) was confirmed in six cases, which showed markedly reduced response to ristocetin and normal responses to all other agonists, one case of Bernard–Soulier syndrome [Figure 2], whereas seven cases were suggestive of storage pool disorder [Figure 3]. Isolated epinephrine defect was found in 3 cases and isolated collagen defect was found in a single case. In four cases, there was reduced aggregation response to arachidonic acid alone, suggesting intake of nonsteroidal anti-inflammatory drugs (NSAIDs). In some cases isolated epinephrine and arachidonic acid defects were found. In these cases patients were advised to come for repeat testing to rule out effect of NSAID or other drugs. However none of the patients came back for repeat testing. Three cases were found to have factor deficiencies of Factor XIII, Factor XI, and Factor VIII.
Table 2: The platelet function defects classified by light transmission aggregometory

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Figure 1: Light transmission aggregometry chromatograph showing markedly reduced platelet aggregation response to adenosine diphosphate, collagen, arachidonic acid, and epinephrine with mildly reduced response to ristocetin suggestive of Glanzmann's thrombasthenia

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Figure 2: Light transmission aggregometry chromatograph showing normal platelet aggregation response to adenosine diphosphate, collagen, arachidonic acid, and epinephrine with markedly reduced response to ristocetin which did not get corrected by mixing with normal plasma suggestive of Bernard–Soulier syndrome

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Figure 3: Light transmission aggregometry chromatograph showing reduced platelet aggregation markedly reduced response to adenosine diphosphate, and epinephrine with normal response to collagen, arachidonic acid ristocetin suggestive of storage pool disorder

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   Discussion Top

In many developing nations where a large proportion of cases of suspected inherited platelet disorders are seen, the laboratories are not adequately equipped to correctly diagnose and classify these disorders. These countries have major financial constraints and majority of the diagnostic laboratories are under resourced, depriving the patients of proper treatment and counseling. There being only a few tertiary care centers which provide testing for bleeding disorders, patients are referred from various parts of the country for diagnosis and workup of. It is therefore important to assess the quality of diagnostic services that we provide and steps that can be taken to improve.

LTA is the only test available with our laboratory to screen and diagnose PFDs. Although labor intensive, it has been well standardized as per the international guidelines. However, our analysis showed that only 31.6% of cases were diagnosed with specific PFD from all tested cases in the past 5 years. This is similar to studies in literature where studies have shown that about 40% of cases could be diagnosed by LTA.[3]

The children had presented most frequently with a history of petechiae, epistaxis, and gum bleeds; in adolescent and young females, the menorrhagia was the most common presenting symptom.

Among the abnormal patterns, 34.3% were diagnostic of Glanzmann's thrombasthenia, having markedly reduced platelet aggregation response to APD, collagen, and epinephrine with near-normal response to ristocetin [Figure 1]. The percentage of cases of Glanzmann's thrombasthenia was higher in our laboratory, as cases get referred from all over the country. In the large worldwide survey, the cases of Glanzmann's thrombasthenia constituted 9.8% of all cases and the majority were Gi signaling defect; these were only 10% in our laboratory.[7] This could be partly due to non-availability for more sensitive assays. The collagen receptor defect in the large survey was 10%; we reported only an occasional case. The cases which had shown markedly reduced response to high dose ristocetin (1.25mg/dl) and normal aggregation with other agonist, corrected after adding normal plasma were diagnosed as vWD. [Figure 2] There was one case in which the platelet aggregation abnormality (reduced response to high dose ristocetin) was not corrected after mixing with normal plasma. This result on platelet aggregometry, presence of large platelets and clinical profile the possibility of Bernard–Soulier Syndrome was suggested. The further subtyping of vWD was done based on the levels of Factor VIII, von Willebrand antigen, and ristocetin cofactor assay. Three cases which were tested and found to have normal platelet function test cases of factor deficiency. One case each of Factor XI, Factor XIII, and Factor VIII deficiency were. In the cases where there was a reduced response to collagen and epinephrine, a storage pool disorder was suggested which was however remained to be confirmed by granule secretion studies or lumi-aggregometry. The audit of the tests done in our laboratory revealed that although LTA can classify the classical bleeding disorders, a major fraction remains unclassified. This was also demonstrated in other studies in literature. Our audit also revealed that in spite of the repeated emphasis laid on the history taking, still many patients underwent the test where the defect was clearly of coagulation factor defect or clinically insignificant bleeding.

A prospective study by Hayward et al. done on diagnostic utility of light transmission aggregometry (LTA) for individuals referred for bleeding disorders showed that abnormal LTA was common in individuals with bleeding disorders, and the likelihood was increased when abnormal aggregation was reduced by 2 or more agonists.[3]

In a study on 280 patients with mucocutaneous bleeding or bleeding of unknown cause, 50 (17.9%) patients had vWD.[8] PFDs and mild clotting factor deficiencies were found in 65 (23.2%) and 11 (3.9%) patients, respectively. 11.5% patients had combined defects, and the remaining 167 (59.6%) patients had bleeding of unknown cause with prolonged bleeding time in 18.6% as their only abnormality. In our study, the vWD was diagnosed in only 5.9% of cases, and in about 25% of cases with a significant BS, the platelet function test with LTA was normal.

In an Indian study by Ahmad et al., 48.3% of patients were found to have unclassified PFDs.[9] In these cases when retested on a separate occasion, a similar abnormal pattern was seen. Inspite of abnormal pattern seen on aggregometry the defect in specific granule or platelet secretion could not identified due to absent facilities to study platelet secretion studies or lumiaggregometry in the laboratory. Therefore a large cases were added to a blanket category of unclassified PFDs. This inability to satisfactorily classify the bleeding disorders are also seen in other published Indian studies. In their study on mild bleeders Kotru et al. were unable to identify the underlying defect in as high as 75% patients by light transmission aggregometry and labeled them as bleeding disorders– Unclassified.[10] In their study in India Ghosh et al. had tested 497 patients on repeat testing they found persistent platelet aggregation abnormality to one or more of the agonists where they could only suggest some form of platelet secretory defect without being able to pinpoint the exact functional or molecular defect.[11]

Patients in which no abnormality was detected on testing by light transmission aggregometry there were about ~15% cases who had significant bleeding (score 7–16) and yet we were unable to determine the defect or explain the cause with presently available tests. This makes us sit up and think that our diagnostic expertise and tests need to be upgraded and researched further to be able to define these cases so as to be able to treat and counsel them. The decisions like what next step to be taken if a bleeder does not response to vasopressin and further genetic counseling will rely on our diagnostic capabilities to define qualitative platelet disorder. Patients must be advised with regard to avoiding aspirin, aspirin-like drugs, and NSAIDs and advised regarding dental hygiene.[12] The inherent low platelet count in some patients such as suspected cases of Bernard–Soulier made it difficult to make PRP (especially in infants) and carry out platelet aggregation studies as facilities for whole blood platelet function tests are not available. In such cases, flow cytometry immunophenotyping can help achieve quick diagnosis. The differences in the incidence of various platelet function disorders could be due to under diagnosis because of unavailability of tests or due to different ethnic backgrounds; this will require a well-planned study with available resources in the laboratory to correctly assess and classify the disorders.

Thus, various studies done on platelet aggregometry have shown that diagnosis is reached in only up to 40% of bleeding cases.[3],[8],[9] This can further be increased by further enhancing and upgrading the facilities in hematology laboratory for diagnosing platelet disorders. Tests such as lumi-aggregometry, flow cytometry, immunophenotyping, and molecular studies are required to decipher the causes in a greater number of patients.[13]

In the recent past, there have been international collaborations of under-resourced laboratories to overcome the limitations and provide access to diagnosis.[4],[14] There has been successful adoption of established algorithms and collaboration with an expert center which has have shown to be effective. These also helped in correcting the incorrect diagnosis and reclassify the disorders; this will prove important to assess the correct prevalence of disorders in the different geographical and ethnic populations. The 5-year audit of our laboratory has brought forth the essential requirement of creating such a network for Third World countries like India.

   Conclusion Top

It is time to provide and or strengthen the existing facilities for diagnosing platelet function disorders in developing nation. Despite a large population of bleeding disorder patients living in developing countries a large majority remain undiagnosed due to high cost of testing and financial constraints.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Rodeghiero F, Tosetto A, Abshire T, Arnold DM, Coller B, James P, et al. ISTH/SSC bleeding assessment tool: A standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. J Thromb Haemost 2010;8:2063-5.  Back to cited text no. 1
Lowe GC, Lordkipanidzé M, Watson SP, UK GAPP study group. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders. J Thromb Haemost 2013;11:1663-8.  Back to cited text no. 2
Hayward CP, Pai M, Liu Y, Moffat KA, Seecharan J, Webert KE, et al. Diagnostic utility of light transmission platelet aggregometry: Results from a prospective study of individuals referred for bleeding disorder assessments. J Thromb Haemost 2009;7:676-84.  Back to cited text no. 3
Glembotsky AC, Marta RF, Pecci A, De Rocco D, Gnan C, Espasandin YR, et al. International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country. J Thromb Haemost 2012;10:1653-61.  Back to cited text no. 4
Elbatarny M, Mollah S, Grabell J, Bae S, Deforest M, Tuttle A, et al. Normal range of bleeding scores for the ISTH-BAT: Adult and pediatric data from the merging project. Haemophilia 2014;20:831-5.  Back to cited text no. 5
Cattaneo M, Cerletti C, Harrison P, Hayward CP, Kenny D, Nugent D, et al. Recommendations for the standardization of light transmission aggregometry: A Consensus of the working party from the platelet physiology subcommittee of SSC/ISTH. J Thromb Haemost 2013. doi: 10.1111/jth.12231. [Epub ahead of print].  Back to cited text no. 6
Gresele P, Harrison P, Bury L, Falcinelli E, Gachet C, Hayward CP, et al. Diagnosis of suspected inherited platelet function disorders: Results of a worldwide survey. J Thromb Haemost 2014;12:1562-9.  Back to cited text no. 7
Sánchez-Guiu I, Antón AI, Padilla J, Quiroga T, Goycoolea M, Oetal P. High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls. Haematologica 2007;92:357-65.  Back to cited text no. 8
Ahmad F, Kannan M, Ranjan R, Bajaj J, Choudhary VP, Saxena R, et al. Inherited platelet function disorders versus other inherited bleeding disorders: An Indian overview. Thromb Res 2008;121:835-41.  Back to cited text no. 9
Kotru M, Mutereja D, Purohit A, Tyagi S, Mahapatra M, Saxena R, et al. Mild bleeders: Diagnosis is elusive in large number of patients. Mediterr J Hematol Infect Dis 2016;8:e2016049.  Back to cited text no. 10
Ghosh K, Nair S, Kulkarni B, Khare A, Shetty S, Mohanty D, et al. Platelet function tests using platelet aggregometry: Need for repetition of the test for diagnosis of defective platelet function. Platelets 2003;14:351-4.  Back to cited text no. 11
Bolton-Maggs PH, Chalmers EA, Collins PW, Harrison P, Kitchen S, Liesner RJ, et al. A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO. Br J Haematol 2006;135:603-33.  Back to cited text no. 12
Pai M, Wang G, Moffat KA, Liu Y, Seecharan J, Webert K, et al. Diagnostic usefulness of a lumi-aggregometer adenosine triphosphate release assay for the assessment of platelet function disorders. Am J Clin Pathol 2011;136:350-8.  Back to cited text no. 13
Sánchez-Guiu I, Antón AI, Padilla J, Velasco F, Lucia JF, Lozano M, et al. Functional and molecular characterization of inherited platelet disorders in the Iberian Peninsula: Results from a collaborative study. Orphanet J Rare Dis 2014;9:213.  Back to cited text no. 14

Correspondence Address:
Sabina Langer
Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi - 110 060
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_758_17

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