Human platelets are critically involved in normal haemostasis, pathological bleeding and thrombosis.[1] The inherited and acquired platelet dysfunctions have to be diag¬nosed early, as rapid identification and treatment of patients at risk of bleeding is really necessary. The monitoring of antiplatelet therapy has therefore become increasingly important for the identi-fication of hypo- or hyper responder patients at risk of both thrombosis and hemorrhage.[2][3]

Prevalence of platelet disorders is low, but the percentage of cases of unclassified platelet disorders are high which causes the need for the creation of a network for comprehensive care of these patients.[4] Light transmission (LTA) remains the gold standard for testing platelet function as It provides important information that is essential forthe diagnostic work-up of patients with platelet function defects.[5]

In our country, prevalence of hereditary type platelet disorder is suspected to be higher due to consanguinity, which is much common in India.[6] Many laboratories worldwide are involved in the diagnosis of platelet function defects, but due to high variability in the diagnostic approaches many patients remain undiagnosed. [7]

Some basic clotting tests (e.g. the APTT and PT) should always be included in the laboratory evaluation of platelet dysfunction to exclude any potential coagulation defects that may be the primary or additional cause(s) of bleeding. In primary haemostatic disorders immediate bleeding occurs after injury with a disproportionate amount of bleeding compared to the degree of trauma, however in coagulation defects delayed bleeding is seen associated with deep tissue bleeding, haemarthroses and intramuscular haematomas. [8]

Data of 153 patients from Jan 2012 to April 2021 was collected who came for testing in the Central India Institute of Haematology and Oncology (CIIHO), Nagpur.

LTA is affected by many pre-analytical and analytical variables, also it is very time-consuming and technically challenging technique, so these must be carefully controlled by expert personnel. For this reason, LTA is performed only in specialized laboratories.[5]

Patient preparation and sample collection

 Medications were stopped for 5 days and fasting samples were collected. Blood sample were collected in 3.2 % sodium citrate in the ratio of 1 part anticoagulant to 9 part of blood with a minimum trauma or stasis at the venepuncture site. Samples were collected in plastic tube only so as to reduce the activation of platelet during sample preparation. Collected sample was kept at room temperature and test started within 30min and completed within 3 hours of collection. An EDTA sample was also collected from the patient for platelet count.

Preparation of plasma

Sample was properly mixed by gentle inversion and centrifuged at 100g for 15 minute. Platelet rich plasma (PRP) was taken with a polypropylene transfer pipette in another plastic tube and labeled properly with a patient name and sample type. Sample was then checked on CBC (complete blood count) cell counter to look for the platelet count.

For making platelet poor plasma (PPP) remaining sample was again centrifuged at 2400g rpm for 20 minutes. PPP was then taken in another tube and labeled similarly. While performing test PRP was used as a sample and PPP as a reference.

Different laboratories often use panels of different agonists at different ranges of concentrations.[8] In our laboratory we used adenosine diphosphate (ADP) (5μm/l and 2.5 μm/l), collagen (2μg/ml) and ristocetin (1.25 mg/ml). LTA determines platelet aggregation percentage in platelet-rich plasma by measuring the increase in light transmission in response to platelet agonist to the platelet suspension. So the results were given accordingly by adding agonists to the sample.[9]

Total 153 cases were studied in 8 years, out of which platelet function abnormality was found in 32.02% (49/153) cases. Patients with bleeding history presented with ecchymosis, petechie, epistaxis, purpura, gum bleeding, post surgical bleeding, tooth extraction bleed and few with GI bleed. In addition females mostly presented with menorrhagia. Patients had a variable age of onset of bleeding manifestations (2 months to 98 years) with a mean age of 49 years. Of the total cases 73 were males and 80 females. Inherited platelet function disorders were seen in 49(32.02%) cases. Based on the aggregation pattern seen in LTA, patients were classified into different groups [Table 1]. Glanzmann's thrombasthenia in 13 (8.5%), Bernard–Soulier disease in 1 (0.6%), vonWillebrand disease in 4 (2.6%), and storage pool defect in 17 (4.6%) and defect with drug aspirin 14 (15.2%) were seen. Remaining patients showed normal response with different agonists on aggregometry.

Patients with Glanzmann’s thrombasthenia showed absent response with ADP and normal response with ristocetin confirming diagnosis [figure 1]. The diagnosis of Bernard Soulier syndrome in one and vonWillebrand disease in 4 was confirmed which showed reduced response to ristocetin and normal response to other agonists [figure 2]. Storage pool defect seen in17 patients and 14 patients showed defects with drug aspirin with reduced response to low dose collagen. Out of 153 patients in 2 patients the aggregation pattern did not fall in any of the platelet defect categories, so they were labeled as sample with defective aggregation.

Total 153 cases were studied in 8 years, out of which platelet function abnormality was found in 32.02% (49/153) cases. Patients with bleeding history presented with ecchymosis, petechie, epistaxis, purpura, gum bleeding, post surgical bleeding, tooth extraction bleed and few with GI bleed. In addition females mostly presented with menorrhagia. Patients had a variable age of onset of bleeding manifestations (2 months to 98 years) with a mean age of 49 years. Of the total cases 73 were males and 80 females. Inherited platelet function disorders were seen in 49(32.02%) cases. Based on the aggregation pattern seen in LTA, patients were classified into different groups [Table 1]. Glanzmann's thrombasthenia in 13 (8.5%), Bernard–Soulier disease in 1 (0.6%), vonWillebrand disease in 4 (2.6%), and storage pool defect in 17 (4.6%) and defect with drug aspirin 14 (15.2%) were seen. Remaining patients showed normal response with different agonists on aggregometry.

Patients with Glanzmann’s thrombasthenia showed absent response with ADP and normal response with ristocetin confirming diagnosis [figure 1]. The diagnosis of Bernard Soulier syndrome in one and vonWillebrand disease in 4 was confirmed which showed reduced response to ristocetin and normal response to other agonists [figure 2]. Storage pool defect seen in17 patients and 14 patients showed defects with drug aspirin with reduced response to low dose collagen. Out of 153 patients in 2 patients the aggregation pattern did not fall in any of the platelet defect categories, so they were labeled as sample with defective aggregation.

Total 153 cases were studied in 8 years, out of which platelet function abnormality was found in 32.02% (49/153) cases. Patients with bleeding history presented with ecchymosis, petechie, epistaxis, purpura, gum bleeding, post surgical bleeding, tooth extraction bleed and few with GI bleed. In addition females mostly presented with menorrhagia. Patients had a variable age of onset of bleeding manifestations (2 months to 98 years) with a mean age of 49 years. Of the total cases 73 were males and 80 females. Inherited platelet function disorders were seen in 49(32.02%) cases. Based on the aggregation pattern seen in LTA, patients were classified into different groups [Table 1]. Glanzmann's thrombasthenia in 13 (8.5%), Bernard–Soulier disease in 1 (0.6%), vonWillebrand disease in 4 (2.6%), and storage pool defect in 17 (4.6%) and defect with drug aspirin 14 (15.2%) were seen. Remaining patients showed normal response with different agonists on aggregometry.

Patients with Glanzmann’s thrombasthenia showed absent response with ADP and normal response with ristocetin confirming diagnosis [figure 1]. The diagnosis of Bernard Soulier syndrome in one and vonWillebrand disease in 4 was confirmed which showed reduced response to ristocetin and normal response to other agonists [figure 2]. Storage pool defect seen in17 patients and 14 patients showed defects with drug aspirin with reduced response to low dose collagen. Out of 153 patients in 2 patients the aggregation pattern did not fall in any of the platelet defect categories, so they were labeled as sample with defective aggregation.

Table 1: Platelet Function defect by LTA

Table 2: Gender

Platelet function testing presents many challenges to ensure that accurate and meaningful results should be obtained. While performing test many potential problems with samples are commonly overlooked causing significant problems and artifacts. Unlike coagulation tests, internal or external quality control materials are not available for PFT, so most assays are performed on fresh blood. Many laboratories either establish normal

ranges using control volunteer blood and/or assay known normal samples in parallel to ensure that each test/reagent is viable.[8]

The blood films of all cases were seen as it gives valuable information about platelets including their size, clumps, granule content and number, although one must be aware of potential artifacts (e.g.pseudothrombocytopenia caused by platelet satelletism or cold reacting agglutinins).[5][8]

There are many alternative techniques to measure platelet aggregation in PRP or whole blood such as impedance aggregometry, 96-well plate aggregometry, single platelet counting and flow cytometry enabling faster and user-friendly study of the platelet response to agonists. Out of all these potential advantages, the majority of these techniques have not been widely adopted as they fail to provide important additional diagnostic information that can be given by LTA such as platelet shape change, the occurrence of secondary wave of aggregation or platelet deaggregation.[5]

In Indian study Langer et al., GT was the most common inherited platelet defect found in 34.3% patients, unlike our study where we got 27.1% (13/49). In study done by Ahmad et al., they found that most of the cases of GT belonged to particular community . Prevalence in India is relatively higher compared to Western countries and majority in northern state of India as compared to other states due to consanguineous marriages.[10]

We found only single case of BSS which showed absent response with ristocetin and normal response with other agonists, this failure to agglutinate could not be corrected by the addition of normal plasma, which distinguished BSS from von Willebrand's disease (VWD).[4] Our finding also correlated with the study done by Meenal et al., which proved that BSS is very rare in the Indian population when compared to all platelet function disorders. [11] In the populations of Europe, North America, and Japan, which have been studied most intensively, the prevalence of this disease is less than 1 in 1,000,000 as estimated from the cases reported in the literature. [12]

VWD was confirmed in 4 cases. For further subtyping, patients were advised levels of Factor VIII, von Willebrand antigen, and ristocetin cofactor assay. In Ahmad et al., study they stated that, PFDs were comparatively low in frequency in the Indian population but higher when compared to the West. Comparing platelet function disorder with other factor deficiency bleeding disorders, the occurrence of PFDs in India is lesser than factor deficiency but more when compared alone with rare coagulation disorders. Also the severity of overall PFDs is less as compared to other coagulation factor disorders, but the management in patients is important in cases of very severe type of PFDs.[10]

In our study we confirmed 14 cases of acquired platelet function defect, all of them were due to aspirin defect. Platelet defects due to other acquired causes were not seen in cases studied. Although acquired defects are more common, but less studied as they are more complex and more difficult to classify. Occuring in a wide variety of clinical settings, e.g. autoimmune disorders, drug treatment, systemic diseases (e.g, uremia, liver diseases) or surgery, their severity of bleeding or thrombotic tendency varies greatly.[14] Out of all, the best test of hemostatic function remains detailed history and physical examination performed by an experienced observer, particularly in individuals who have been hemostatically challenged by surgery, trauma, or the vicissitudes of daily life. [13] Although LTA remains the mainstay of the diagnosis for platelet function defects, there are wide range of other methods which are now in use in specialized laboratories worldwide, including flow cytometry and lumiaggregometry, and some sophisticated techniques, such as TEM and molecular tests. [7]

Both Inherited and acquired platelet function disorders represent a significant fraction of all the bleeding diatheses. In our study we found more inherited defects than acquired, although acquired platelet function defects are most com¬mon types of bleeding disorders. Despite the relative frequency of platelet disorders their diagnosis is still cumbersome, but if diagnosed correctly can lead to proper treatment and minimize bleeding risk associated with it. This article will help to make awareness amongst clinicians that suspected platelet function disorders should be promptly investigated to come to conclusion.

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