Laboratory Diagnosis of Qualitative Platelet Disorders

Laboratory analysis of patients with apparent bleeding problems utilizes initial testing of the cellular and fluid-phase components of hemostasis, followed by more specialized testing of the suspected problematic aspect (Fig. 64.2). A platelet count will identify patients with a quantitative platelet defect, although one must remember that some qualitative defects are associated with lower platelet counts as well (Table 64.1). The possibility of coagulation factor inhibitors and deficiencies should be assessed using the prothrombin time (PT) and the activated partial thromboplastin time (aPTT). A prolonged aPTT can occur in vWD and should be explored by confirmatory testing of vWF activity, vWF antigen, factor VIII activity, (see Case 65), or hemophilia A (FVIII), B (FIX), or C (FXI) (see Case 61). The bleeding time (BT) and platelet function screen (PFA-100, Dade-Behring, Deerfield, IL, USA) are screening tests of primary hemostasis that include, but are not limited to, evaluation of platelet function. Platelet function screening tests should be performed on patients with adequate (> 100 x 103/mL) platelet counts, and in the absence of drugs known to cause platelet dysfunction. An abnormal result in the absence of a diagnosis of vWD should be pursued with definitive testing by platelet aggregometry. In some cases where clinical suspicion of platelet dysfunction remains high despite normal BT or PFA-100 results, platelet aggregometry should be considered because of its greater sensitivity for detecting some mild qualitative disorders.

The Ivy bleeding time (BT), or a modification of the technique, is used to assess the integrity of the subendothelium-von Willebrand factor (vWF)-platelet-fibrinogen

Von Willebrand Screen Test

Figure 64.2 Algorithm for evaluation of patient with abnormal bleeding history (PT—prothrombin time; aPTT—activated partial thromboplastin time; vWD—von Willebrand disease; BT—bleeding time; PFA-100—platelet function screen, Dade-Behring, Deerfield, IL, USA; col/epi—collagen/epinephrine cartridge on PFA-100; col/ADP—collagen/ADP cartridge on PFA-100).

Figure 64.2 Algorithm for evaluation of patient with abnormal bleeding history (PT—prothrombin time; aPTT—activated partial thromboplastin time; vWD—von Willebrand disease; BT—bleeding time; PFA-100—platelet function screen, Dade-Behring, Deerfield, IL, USA; col/epi—collagen/epinephrine cartridge on PFA-100; col/ADP—collagen/ADP cartridge on PFA-100).

system. A specially trained laboratory technician performs this bedside test. A blood pressure cuff placed on the upper arm is inflated to 40 mm Hg as a lancet device is used to deliver a standardized incision on the patient's forearm. A stopwatch tracks the time elapsed before incisional bleeding ceases. This time, in minutes, is compared to the normal range for the person's age. While the BT holds the advantage of in vivo testing of the subendothelial interaction with blood components, the test has fallen out of favor in most modern laboratories. The requirement for a specially trained technician to perform the test on the patient limits its availability and increases laboratory costs. Because it involves laceration of the forearm, associated with bleeding and scarring, repetitive testing is relatively contraindicated. Normal test ranges in healthy adults can be generated and validated with effort, but pediatric normal ranges for this invasive test are usually inferred from published reference ranges. Many technical and patient variables affect the precision and accuracy of the bleeding time. For example, variations in skin thickness with age and nutritional status can affect the depth of the laceration, even when age range-specific lancet devices are used. BT results may be prolonged when platelet counts fall below 100 x 103/mL, so verification of a platelet count of at least 100 x 103/mL should always precede BT testing. BT normal ranges are usually given for newborn infants to age 6 months, and for all others >6 months of age. The shortened BT result in early infancy is attributed to the preponderance of larger vWf multimers at this age.

An alternative to the BT for evaluation of vWF-platelet-fibrinogen interactions is the PFA-100 two-cartridge test system. Citrated whole blood is added to test cartridges with a collagen-coated membrane and either epinephrine (col/epi) or adenosine diphosphate (col/ADP). The sample is pulled through a capillary tube and the central aperture of the membrane to generate high shear flow as a physical platelet activator, in addition to the physiologic activators collagen, epinephrine, and ADP. Activated platelets adhere to the membrane and to each other until the aperture is occluded. The interval from sample application to occlusion, in seconds, is designated the closure time (CT). CT is dependent on platelet number; intact platelet adhesion, activation, and aggregation; and interactions of platelets with fibrinogen and vWF. Hematocrit and platelet count should be obtained prior to PFA-100 testing, since values for hematocrit of <25 or >50% and platelet count <100 x 103/mL may cause inaccurate closure times.

The PFA-100 has been validated to differentiate between the effect of aspirin or other nonsteroidal antiinflammatory drugs (NSAIDs), which should prolong the col/epi cartridge results only, and other congenital or acquired qualitative platelet defects, vWD, and dysfibrinogenemia, which should result in prolonged CTs of both col/epi and col/ADP cartridges. However, the PFA-100 test has not been validated for predicting the risk of bleeding in asymptomatic, unselected patients prior to surgery or invasive procedures. Published CT normal ranges for newborns (cord blood) are shorter than those for toddlers through adulthood, consistent with BT results and the prediction that the high-molecular-weight vWf multimers in babies enhance platelet performance in platelet function screening tests. In pediatric patients with suspected bleeding problems, the sensitivity and specificity of the PFA-100 was superior to the BT in identifying patients with qualitative platelet disorders or vWD. The utility of the PFA-100 to detect patients with qualitative platelet defects has been assessed using platelet aggregometry as the gold standard, with favorable results for vWD and for severe platelet dysfunction syndromes such as Glanzmann thrombasthenia and Bernard-Soulier syndrome. Both the BT and the PFA-100 have been shown to be less effective at identifying patients with congenital defects of platelet secretion. Therefore, a patient with significant platelet-type symptoms and an otherwise negative evaluation should be considered for platelet aggregation studies.

Platelet aggregometry remains the gold standard for evaluation of in vitro platelet function in response to physiologic agonists and also to ristocetin. Citrated whole blood is centrifuged at 900 r/min (revolutions per minute) for 10 minutes to pellet red cells and white cells, but not platelets, to obtain platelet-rich plasma (PRP). Platelet-poor plasma (PPP) is prepared by centrifuging the residual cell pellet at 15,000 r/min for 10 minutes. The PRP is diluted, if necessary, with PPP to obtain a platelet count of 200250 103/mL. PRP is added to a glass tube containing a magnetic stir bar to maintain platelets in suspension, and the tube of PRP is placed within the light path of the aggreg-ometer instrument. Platelet agonists are added to separate tubes, and as platelets undergo activation and aggregate, more light passes through the tube and reaches the detector (PRP = 0% light transmission = PPP, 100%).

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