Light transmittance aggregometry

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Associate Editors-in-Chief: Davide Capodanno, M.D. [1]


Overview

Light transmittance aggregometry (LTA): This is a laboratory based study that evaluates the aggregation or clumping of platelets in response to aggregating stimuli. For historical reasons, it is broadly accepted as the gold standard in-vitro test of platelet function. The most immediate information for basic diagnostic considerations is obtained by using agents such as adenosine diphosphate (ADP), epinephrine and collagen. Both ADP and epinephrine are contained in storages organelles within the platelets and are released during formation of the primary hemostatic plug thus enhancing further platelet aggregation. Conversely, collagen is found in the supporting connective tissue of the vessels and is considered to be the first proagulant factor that the platelet encounters following vessel’s injury. Other reagents such as arachidonic acid, ristocetin, serotonin, calcium and Factor VIII are also used to study platelet response for more specific purposes. Different aggregating agents stimulate alternative pathways of activation in the platelets and different concentrations of the same agonist are often used to elicit dose-dependent response.

Aside from the detection and diagnosis of acquired or congenital qualitative platelet defects, LTA has an important role to reveal patterns of GPIIb/IIIa-dependent platelet-to-platelet aggregation in response to specific agonists (e.g. arachidonic acid to assess aspirin response; ADP to test thienopyridines response). The chambers of a typical aggregometer are designed so that a beam of infrared light shines through two cuvettes. One of these contains the sample, namely a suspension of platelet rich plasma (PRP) obtained by a relatively low centrifugal force centrifugation. The other cuvette contains a reference sample of platelet poor plasma (PPP) obtained by centrifuging the blood sample at a relatively high force. Silicon photodiodes detect the light able to pass through the samples, with PRP arbitrarily considered to be 0% light transmission (or 0% aggregation) and PPP considered to be 100% light transmission (or 100% aggregation). The optical aggregation output is proportional to the continuously measured difference in light transmission between the PRP and PPP samples. Following the addition of a stimulus to the cuvette containing PRP, changes in light transmission occur as a consequence of platelet response and are recorded over time. In fact, the larger size of activated platelet allows less light to pass through the PRP: this is recorded as less light transmission relative to the PPP. Conversely, when platelets form aggregates, more light is able to pass through the test sample. Aggregation recordings are curves characterized by several features:

  1. Shape changes;
  2. A first wave of aggregation that may reverse (primary aggregation);
  3. A second wave of aggregation that occurs when the granule contents become the stimulus and lead to further aggregation;
  4. Maximum amount of change in light transmission caused by the stimulus (percent aggregation);
  5. Late amount of aggregation recorded after a certain timeframe;
  6. Slope of the aggregation, or percentage of aggregation per minute.

Designed as a measure of defective [[platelet\\ function, the main disadvantage of LTA is that the test is time-consuming, expensive, weakly standardized and need to be performed in specialized lab by specialized personnel. Additionally, several procedural variables may account for poor reproducibility.




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