Light transmission aggregometry: Useful but difficult diagnostic tool

Introduction We started using light transmission aggregometry (LTA), specifically platelet aggregation profiler (PAP-8E from BioData, Canada) in the National Center of Hematology/Al-Mustansiriya University, Baghdad, Iraq, in December 2011 till the present date, and although it did constitute the basic functional diagnostic technique for hundreds of our patients who presented mainly with some type of bleeding, and hence, we are supposed to acquire some experience with this machine and the efforts paid for standardization of working protocol,[1] but still, we face a lot of challenges when using the platform. Therefore, in this short letter, we will try to define some of the frequent difficulties with using LTA to diagnose patients with bleeding. Diagnostic Approach Bleeding disorders include some rare conditions, therefore, the best way for diagnosis can be by utilizing all the available clinical data within an “integrated report” that starts with a search for personal and family history of bleeding, assessment of severity and type of bleeding preferably using accepted tools like the International Society of Thrombosis and Hemostasis Bleeding Assessment tool,[2] basic screening tests for bleeding including complete blood count, peripheral blood film, template bleeding time or PFA-100, prothrombin time (PT) and PT ratio, activated partial thromboplastin time and if required mixing studies. Sometimes, we ask for fibrinogen assay using Claus method and thrombin time, factor assay, factor XIII assay using clot solubility in 2% acetic acid, von Willebrand’s factor antigen assay using ELISA and ristocetin cofactor (RiCof) assay using LTA, and platelet function analysis using LTA. The final diagnosis should be issued after reviewing the integrated report by a multidisciplinary team meeting that includes at least the operator of LTA, the hematopathologist, and the attending physician. Request Form Writing clear clinical indications for performing LTA can be very helpful as we basically cannot infer conclusions without being aware of the presentation or knowing the reason for sending the patient for LTA testing when looking for very specific indications like response to aspirin and/or plavix. Technical Requirements LTA is described in the literature as the gold-standard test for the diagnosis of heritable platelet disorders, however, it may inaccurately identify the underlying condition in up to 4 out of 10 patients.[3,4] In practice, we learned that using medications even those that are not listed in the literature may affect LTA testing and be confusing as it may have some effect and induce unwanted changes in LTA plots. Therefore, we usually advise our patients to stop using any drugs that are not deemed highly essential such as multivitamins and oral iron. When using LTA to study RiCof, we found that preparing platelet-free plasma may provide superior results to the manufacturer’s recommended platelet-poor plasma, although the difference does not usually affect the diagnosis. In our practice, we use the following agonists: adenosine diphosphate, collagen, epinephrine, and low- and high-dose ristocetin. We faced a period where the response to epinephrine from certain lot numbers was absent/markedly reduced unexpectedly from many patients and even some control subjects, forcing us to omit reading absent/markedly reduced epinephrine as abnormal and recommending not relying on its result. The importance of using a carefully calibrated micropipette may seem trivial, but in practice, it can have significant unwanted effects, such as being unable to draw the standard curve of VWF: RiCoF testing using four dilutions, i.e., incorrectly calibrated micropipette can result in imperfect linearity of the standard curve. In our experience, using LTA in a more general way, i.e. to search for bleeding disorders instead of trying to confirm their cause can prove very disappointing as it usually ends with data and plots that cannot fit with the patient clinical presentation. LTA is a test specific for heritable platelet disorders but is not sensitive to bleeding in general, so it should be used only in patients with the proper clinical context of bleeding. As LTA is basically a functional test, having a control plot from an apparently healthy donor with blood withdrawn on the same day as the patient’s blood and be treated under similar conditions can be essential for proper assessment of findings. Choosing a healthy donor by itself can be somewhat challenging, and in our experience, we prefer to withdraw blood from the same control subject as when trying to have a sample from different donors can provide some less than typical graphs [Figure 1] like absent response to epinephrine or atypical aggregation in response to other agonists.Figure 1: Typical findings of light transmission aggregometry in a healthy donor. LTA: Light transmission aggregometry, ADP: Adenosine diphosphateTrying to carefully select patients who may benefit from LTA can be very useful; as avoiding LTA test in patients with immune thrombocytopenia (ITP) with a typical clinical and hematological presentation, no family history of bleeding and are responding to first-line immune treatment as steroids or intravenous immunoglobulin can be helpful to avoid confusing findings like a reduced response to some or even all agonists apart from usually a normal response to ristocetin, as the autoantibody in patients with ITP is usually against GPIIb/IIIa receptors. Also, it is important to remember that LTA cannot be used to differentiate between ITP and heritable platelet disorders, as the LTA results in ITP can be normal or abnormal, and the abnormal findings in ITP may mimic findings in some heritable platelet disorders [Figure 2 for findings of LTA in a child with a clinical diagnosis of ITP].[5]Figure 2: Light transmission aggregometry findings in two patients with immune thrombocytopenia*, in (a) the patient has a normal response to collagen and high-dose ristocetin with impaired response to adenosine diphosphate (ADP); while in (b) the patient has normal response only to ristocetin with impaired response to both ADP and Collagen. *(a) Is for a 5-year-old female, and (b) is for a 3-year-old male, both presented with petechial hemorrhage and easy bruising, with isolated severe thrombocytopenia <15 × 109/l responding to treatment with oral prednisolone. LTA: Light transmission aggregometry, ADP: Adenosine diphosphate, ITP: Immune thrombocytopeniaConclusions LTA is a functional test for patients with heritable platelet disorders and it results in a specific diagnosis in only certain patients with conditions such as Glanzmann thrombasthenia, Bernard–Soulier syndrome, von Willebrand disease (VWD), and platelet-type VWD. Otherwise, patients can only be labeled as having a heritable platelet disorder, with no specific diagnosis, and usually require confirmatory testing with polymerase chain reaction, next-generation sequencing, or flow cytometry. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

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