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- MATERIALS AND METHODS
The PFA-100® (PFA) diagnostic system for the detection of platelet dysfunction was evaluated to determine reference ranges in a normal population. The PFA determines the primary haemostasis capacity (PHC) of anticoagulated whole blood, expressed by the system's closure time (CT). In this study the CT reference ranges were determined for blood samples collected in 105 mmol/l (3.2%) buffered citrate and the effect of gender, smoking, and use of oral contraceptives on reference ranges was assessed. Each of the 309 healthy blood donors from five blood centres was confirmed to have normal platelet function before inclusion in the study. Blood samples were tested in duplicate with both the collagen/epinephrine (Col/Epi) and collagen/ADP (Col/ADP) test cartridges.
PFA reference ranges (90% central intervals of measured closure times) for both cartridge types were similar for all groups. Subgroup analysis showed that neither gender nor oral contraceptive usage had any effect on PHC. The 95% cut-off value for the Col/Epi CT was slightly higher for smokers than for non-smokers, an effect more pronounced in female than in male donors. However, the small difference did not justify establishment of specific reference ranges for smokers. Data from all included subjects were pooled to calculate the CT reference ranges for blood samples collected in 105 mmol/l buffered citrate (Col/Epi 82–150 s; Col/ADP 62–100 s). Normal levels of fibrinogen, as well as normal platelet counts and normal haematocrit levels, appeared not to influence the PHC. Because slight but significant differences of the reference ranges were observed between some of the participating sites, in-house confirmation of these reference range guidelines is recommended.
Evaluation of platelet function in a patient undergoing surgery is important for haemostatic management, particularly if the patient has a history of clinically significant bleeding ( George & Shattil, 1991). Traditionally, the most widely used screening test for platelet disorders is the in-vivo bleeding time test, which suffers from inevitable variance, a lack of predictive value for postoperative bleeding ( Rodgers et al, 1990 ; Lind, 1991) and a certain amount of discomfort for both patient and technician. Furthermore, the test set-up of the bleeding time and associated scarring disqualifies the method for multi-testing and follow-up of the patient to assess the effect of haemostatic therapy. As a reference method, platelet aggregometry is generally accepted as the preferred technology to diagnose platelet dysfunction. Drawbacks of aggregometry include the need for a relatively large sample size, suboptimal standardization, and the fact that reaching a final diagnosis takes considerable time, skill and technical expertise.
Recently, the PFA-100® test system was introduced as an aid in the detection of platelet dysfunction ( Kundu et al, 1995 ). The PFA demonstrated good clinical performance as indicated by both high specificity for normal platelet function and high sensitivity for platelet dysfunction ( Mammen et al, 1998 ). In particular, with respect to acetyl salicylic acid (ASA)-induced platelet dysfunction, the PFA showed superior sensitivity when compared to the traditional bleeding time test ( Marshall et al, 1997 ). Recent detailed characterization of the PFA system demonstrated that its mode of action correlates well with known mechanisms of in-vivo primary haemostasis in that the PFA test was dependent on shear, platelet receptors GPIb and GPIIb–IIIa, platelet count, haematocrit, and von Willebrand factor (VWF) (Kundu et al, 1996). The importance of VWF in the PFA system was further illustrated in several recent reports demonstrating the system's high sensitivity for detection of the dysfunctional primary haemostasis in von Willebrand disease ( Fressinaud et al, 1998 ; Carcao et al, 1998 ).
Most of the aforementioned studies were conducted with blood samples collected in 129 mmol/l (3.8%) buffered sodium citrate. Since the potential influence of the collection system was demonstrated earlier ( Heilmann et al, 1997 ), and a global trend toward use of low-citrate collection systems is anticipated ( Wiseman, 1996, 1998), this study aimed to determine the reference ranges of samples collected in 105 mmol/l (3.2%) buffered sodium citrate. Because smoking and oral contraceptives usage are suspected to influence platelet function, this study also assessed the effects of these behavioural parameters on PFA results. Furthermore, the effect of gender, normal levels of platelets, haematocrit, and fibrinogen on PFA results was evaluated. Finally, the multi-centre set-up of the study allowed assessment of within-site variance, to estimate overall reproducibility of PFA testing, and inter-site variance, to determine any local population influences on PFA testing.
- Top of page
- MATERIALS AND METHODS
As with the introduction of any new diagnostic test system, the clinical acceptance of the PFA-100® relies for the major part on clinical performance and standardization. To allow a correct interpretation of the results generated by the test system, establishment of appropriate reference ranges and cut-off values is a prerequisite. Similar to platelet aggregometry and other test systems dependent on functional cellular blood components, the PFA system cannot be calibrated by the use of control blood, since such controls are not available. Instead, the testing of an adequate number of blood samples, in which normal platelet function can be anticipated or has been confirmed, provides a reasonable alternative for calibration. In this study we evaluated blood samples from ostensibly healthy blood donors with confirmed normal platelet function.
A similar study conducted in the U.S.A. ( Mammen et al, 1998 ) characterized the performance of PFA with samples collected in 3.8% (129 mmol/l) buffered sodium citrate. Because more and more haematology laboratories might convert to using 3.2% (105 mmol/l) citrate based collection systems, consistent with NCCLS guideline H3-A4, the aim of the present study was to evaluate PFA performance in normal healthy subjects from the general population and to establish reference ranges in a standard 3.2% collection system.
From the 359 blood donors recruited 50 were excluded for further evaluation because a normal platelet function in the blood sample could not be confirmed, mostly due to subnormal aggregometry results. Retrospectively, it was determined that most of these drop-outs had normal CTs with the PFA, suggesting a higher specificity for PFA compared to platelet aggregometry, when results were compared to the normal clinical histories obtained from these blood donors.
Both smoking and use of oral contraceptives are considered parameters for increased risk of thrombosis ( Frederiksen et al, 1970 ). Uncertain still is their mode of action which might be directed primarily towards the vessel wall endothelium and/or cellular blood components like platelets ( Levine, 1973) and leucocytes ( Lehr et al, 1994 ). Smoking has been demonstrated to enhance TxA2 formation and raise the cellular level of platelet activation, suggesting that PHC might also be increased in people who smoke. Smoking also stimulates platelets in patients with increased blood pressure ( Gleerup & Winther, 1996), and has a somewhat obscure antagonistic behaviour to aspirin in coronary artery patients ( Davis et al, 1985 ). In the present study no effect of smoking on PHC was observed at the lower CT cut-off value for both Col/Epi and Col/ADP. However, the 95% cut-off for Col/Epi was slightly higher with borderline significance for the combined male and female smokers, and was significantly higher in the female smokers when compared to the non-smoking controls. Although an immediate and reversible platelet activating effect during smoking was not investigated, the results in the blood donors suggest that a certain level of desensitization in platelet function occurs in regular smokers. Further evaluations are needed to clarify whether the apparent inhibitory effects of smoking on platelet function in some donors warrants having such donors withdraw from smoking for a certain time prior to donation.
Oral contraceptive use has also been associated with increased risk of thromboembolic events ( Frederiksen et al, 1970 ). Even newer birth control medications are associated with increased risk for venous thrombosis ( Helmerhorst et al, 1997 ; Comp, 1997). The only significant differences observed between the OC and female control group were higher PT levels and shorter aPTT values in the OC group, although a trend for higher FVII:C levels was also seen. No effect on platelet function as measured in the PFA system was seen. This observation suggests that the prothrombotic character of OC medication is more likely to be related to changes in the balance of the plasma coagulation system rather than to an altered platelet function ( Kluft & Lansink, 1997). Moreover, the apparent changes in plasma concentrations of coagulation factors indicate that such factors do not play a major role in the PFA-100 test mechanism, an observation consistent with the findings that patients with haemophilia A or B have normal closure times ( Fressinaud et al, 1998 ; Carcao et al, 1998 ). Whether the PFA-100 test mechanism is only independent of these factors under anticoagulated conditions is still unclear.
Comparison of the closure time mean values and distributions of the participating sites demonstrated in a few cases a small difference, mostly of borderline significance. An explanation for these differences is unclear. Aside from possible ethnic and dietary differences from region to region, the mere fact that in-house logistics and pre-analytical routing of the blood samples differ could attribute to these findings. Therefore the reference ranges determined in this study should be considered general guidelines. Establishing in-house reference ranges is recommended, though expected to agree with these general guidelines.
In a seven-site field trial conducted in U.S.A., the corresponding reference ranges for normal samples collected in 129 mmol/l buffered citrate were 94–193 s for the Col/Epi and 71–118 s for the Col/ADP test cartridge ( Mammen et al, 1998 ). Compared to that study we found a significant shift to lower closure times for the 105 mmol/l citrate, an observation described earlier by Mammen et al (1995 ) in a small number of subjects. This finding is also in agreement with a more recent publication ( Heilmann et al, 1997 ), suggesting again that platelet function is influenced by different citrate concentrations in the collection systems. Therefore the citrate concentration used in the blood collection system has to be considered for correct interpretation of PFA test results.
The overall within-site variation ranging from 3.7% to 5.8% in the duplicate tests was acceptable, considering the complex test matrix in which the test process is taking place.
Moreover, the low number of samples which needed re-testing (1.45%), due to an unacceptable variation of >20% between the duplicates (0.8%) or to test interruptions resulting from flow obstructions in one of the two replicates (0.65%), strongly suggests that in routine usage there is no need to perform duplicate testing. This study does not exclude that the variance in abnormal samples could be higher.
The weak correlation between factor VIII levels and PHC is probably due to underlying VWF levels (not evaluated in this study), which are known to influence PHC ( Fressinaud et al, 1998 ) and, because of the VWF–VIII protein organization, correlate with FVIII levels in normals.
In an earlier report ( Kundu et al, 1995 ) the inverse correlation of platelet count and haematocrit level with PFA-100 closure time was described. These observations could not be confirmed by our data, possibly because in this study only subjects with a normal blood profile were included. Apparently, platelets and haematocrit effect PHC only at pathological levels. A possible role of fibrinogen in the test process could not be found, albeit that again only normal levels were evaluated.
In summary, centre specific reference ranges for Col/ADP and Col/Epi closure times show slight, mostly non-significant, local differences indicating the need for establishment of in-house reference ranges for the PFA-100 test system. OC usage and gender do not significantly influence PHC in normal subjects. The small effect of smoking on an increased Col/Epi upper cut-off value needs further explanation. Both smoking and oral contraceptive usage do not effect the closure time lower cut-off values suggesting that these behavioural variables do not cause a significant change in platelet function. Normal levels of haematocrit and platelet count in normal subjects do not significantly influence PHC. Reference ranges for 3.2% buffered citrate show a significant shift to shorter CTs when compared to the 3.8% reference ranges described in literature.