Laboratory Evaluation of Platelet Function

INTRODUCTION

Platelet aggregation has been the method of choice for assessing platelet function since the early 1970s. The instrumentation for measurement of platelet aggregation has changed very little during that time, although the advent of lumiaggregometry has made the assessment of platelet nucleotide content and release considerably less difficult. When asked to evaluate a patient for a platelet function defect, it is recommended that you examine a peripheral smear and conduct a platelet count in addition to measuring platelet aggregation. Lumiaggregometry or another assay of granule release can be used to distinguish storage pool disease (SPD) and release defects. Flow cytometric analysis of GPIb and GPIIb-IIIa surface density can be used to aid in the diagnosis of Bernard-Soulier syndrome or Glanzmann's thrombasthenia. When performing aggregation, as with any other laboratory procedure, it is essential to control variables that might affect the test results. Some of the variables encountered in performing platelet aggregation will be covered in the following sections.

VARIABLES

Anticoagulants

Citrate. Both 0.1 and 0.129 M citrate (buffered and nonbuffered) at a ratio of 9 parts blood to 1 part anticoagulant are traditionally used for platelet aggregation testing. A correction formula for abnormal hematocrits can be found in the section on Drawing and Processing Blood for Platelet Aggregation. For typical agonists such as ADP, collagen, and epinephrine, at moderate concentrations there is little or no difference seen in the test results between blood drawn into 0.1 or 0.129 M citrate. A discrepancy may be seen when testing in the lowest concentration ranges of agonists such as ADP, where higher concentrations of citrate can result in lower aggregation responses. This can be exacerbated if the patient or donor has a high hematocrit that is not corrected for, thus increasing the citrate-to-plasma ratio. Also, if a situation arises where the specimen will sit on the bench for more than 1-2 hr, it is definitely preferable to use a buffered anticoagulant to help maintain the pH of the PRP. Figure 2.1 demonstrates the aggregation response to ADP in three anticoagulants: 0.1 M buffered citrate, 0.129 M sodium citrate, and ACD (acid-citrate-dextrose). The latter is only used for preparation of washed platelets and not for aggregation assays (see below). The anticoagulant of choice for aggregation studies is 0.1 M buffered citrate.

Heparin. Heparin can be used for platelet testing, but in many donors the PRP platelet count will be significantly lower when drawn into heparin as compared to citrate. In addition, a small percentage of the donor population will exhibit “spontaneous” aggregation in the presence of heparin (Figure 2.2). Heparin is not an anticoagulant of choice for aggregation testing.

EDTA. EDTA is not suitable for use in aggregation testing since it does not leave sufficient Ca2 + available for aggregation to occur.

PPACK. D-Phenylalanine-proline-arginine chloromethyl ketone (PPACK) is an antithrombin. It has the benefit of not chelating calcium and therefore not exerting an effect on platelet function based on available plasma calcium. It also does not have the proaggregating effects of heparin. It has been demonstrated that the plasma free calcium concentration has a direct effect on the IC50 of inhibition of platelet aggregation by some GPIIb-IIIa antagonists currently being evaluated in the treatment of acute coronary syndromes. In order to more accurately reflect the in vivo situation, PPACK-anticoagulated blood is being used in the evaluation of the extent of inhibition of aggregation by these drugs. Unfortunately, PPACK is very expensive relative to the other anticoagulants listed, and its anticoagulant effect is finite. Using a final concentration of 1.6 mg per 10 ml whole blood (0.3 mM final concentration), one can anticipate that the specimen will not clot for several hours. To provide an anticoagulant effect for more than 24 hr, we recommend a final concentration of 12.8 mg per 10 ml (2.4 mM final) whole blood.

ACD. Because this anticoagulant brings the pH of the PRP to 6.5, it is not suitable for use in aggregation experiments (Figure 2.3). It is an excellent choice if the purpose is to obtain platelets for washing or gel-filtration. Use at a ratio of 6 parts blood to 1 part anticoagulant.

ACD-A. This formulation of ACD keeps the pH of the PRP at 7.2 and has been reported as acceptable for aggregation testing.

At this point, it should be noted that anticoagulants such as PPACK and heparin cannot be used in aggregation testing if simultaneous measurements of release are being made (Figure 2.4). The release reaction seen in citrated blood, and which is used in the diagnosis of SPD and release defects, is not seen with the agonist ADP in blood anticoagulated with these antithrombins. Aggregation responses to ADP are generally lower in PPACK- or heparin-anticoagulated blood compared to citrate-anticoagulated blood (see Figure 3.1, Chapter 3).

pH

Platelet aggregation should be performed at pH 7.2-7A. At lower pH, platelet responsiveness is diminished to the point of total inhibition of aggregation at pH 6.5. On the other hand, as the pH rises, so does the response of the platelets. At pH 8.1 and higher, spontaneous aggregation can occur. It is best to store platelets in tubes that are capped and with very little air space relative to PRP volume (Figure 2.5A vs. 2.5B).