Methods for testing platelet function for transfusion medicine.

BACKGROUND: Currently only indirect measures are required for monitoring the function of platelets in platelet concentrates (PC). METHODS: This is an overview on currently available commercialized methods that have been used to determine platelet function in donors, concentrates and after transfusion. We show examples for the application of the no/low shear methods light-transmission aggregometry, flow cytometry, multiple electrode aggregometry, thrombelastography and dynamic light scattering, and those applying high shear, the platelet function analyzer-100, and the cone and plate analyzer. Advantages and disadvantages of the various methods to screen donors, evaluate the haemostatic properties maintained in the PC and after transfusion are discussed, based on considerations of platelet physiology, and the feasibility of the various procedures. This survey focuses on reports from the last 10 years, as the technology for the production of PCs has advanced significantly during the last few years. CONCLUSION: Specific aspects of platelet function can be assessed by the no/low shear methods, while the high shear methods provide more general analysis of platelet haemostatic competence. Yet, there is no strong evidence that the in vitro data correspond with the clinical outcome.

Multiple electrode whole blood aggregometry, PFA-100, and in vivo bleeding time for the point-of-care assessment of aspirin-induced platelet dysfunction in the preoperative setting.

BACKGROUND: Acquired platelet dysfunction due to aspirin ingestion may increase bleeding tendency during surgery. Thus, we examined the diagnostic accuracy of in vivo bleeding time (BT) and 2 platelet function assays for the preoperative assessment of a residual antiplatelet effect in patients treated with aspirin. METHODS: Consecutive patients scheduled for surgery were prospectively enrolled in this study. The patients' last aspirin ingestion had occurred within the previous 48 hours before blood sampling in the "full aspirin effect" group, between 48 and 96 hours before in the "variable aspirin effect" group, and >96 hours before in the "recovered aspirin effect" group. The control group had not taken any aspirin. Multiple electrode aggregometry, platelet function analyzer (PFA)-100, and in vivo BT were performed to assess the effects of aspirin. One-way analysis of variance on ranks with a post hoc multiple-comparison procedure (Dunn) was used to detect differences among the groups. Categorical data were compared using the z test. Receiver operating characteristic (ROC) curves were created to determine the diagnostic accuracy of the platelet function assays investigated. The area under the ROC curve (AUC), sensitivity, and specificity of the assays were calculated. The level of statistical significance was set at P < 0.05. RESULTS: Three hundred ninety-four patients were included in the analysis (133 control and 261 aspirin-treated patients). All 3 methods were able to detect the antiplatelet effect of aspirin in the full aspirin effect group. Furthermore, no difference in the measurement values between the recovered aspirin effect and control group was found, irrespective of the assay performed. Measurement values in the variable aspirin effect group were different from those of the control group in the ASPItest using multiple electrode aggregometry and COL-EPI using PFA-100 but not in BT. ROC analysis showed the highest diagnostic accuracy in excluding the residual aspirin effect in the ASPItest (AUC 0.81, P < 0.001), followed by COL-EPI (AUC 0.78, P < 0.001) and BT (AUC 0.56, P = 0.05). The cutoff value of 53 U in the ASPItest excluded the effect of aspirin with a sensitivity of 88% and specificity of 71%. CONCLUSIONS: The full therapeutic antiplatelet effects of aspirin can be expected within 48 hours of the patient's last aspirin ingestion. Platelet function recovered in our study if aspirin cessation occurred >96 hours (4 days) before; thus, in these patients, preoperative platelet function testing is not useful. To quantify any residual aspirin effect in patients who ceased their intake of aspirin between 48 and 96 hours before surgery, the ASPItest might have the highest diagnostic accuracy.

Automated registration of tail bleeding in rats.

An automated system for registration of tail bleeding in rats using a camera and a user-designed PC-based software program has been developed. The live and processed images are displayed on the screen and are exported together with a text file for later statistical processing of the data allowing calculation of e.g. number of bleeding episodes, bleeding times and bleeding areas. Proof-of-principle was achieved when the camera captured the blood stream after infusion of rat whole blood into saline. Suitability was assessed by recording of bleeding profiles in heparin-treated rats, demonstrating that the system was able to capture on/off bleedings and that the data transfer and analysis were conducted successfully. Then, bleeding profiles were visually recorded by two independent observers simultaneously with the automated recordings after tail transection in untreated rats. Linear relationships were found in the number of bleedings, demonstrating, however, a statistically significant difference in the recording of bleeding episodes between observers. Also, the bleeding time was longer for visual compared to automated recording. No correlation was found between blood loss and bleeding time in untreated rats, but in heparinized rats a correlation was suggested. Finally, the blood loss correlated with the automated recording of bleeding area. In conclusion, the automated system has proven suitable for replacing visual recordings of tail bleedings in rats. Inter-observer differences can be eliminated, monotonous repetitive work avoided, and a higher through-put of animals in less time achieved. The automated system will lead to an increased understanding of the nature of bleeding following tail transection in different rodent models.

Clopidogrel-induced platelet inhibition cannot be detected by the platelet function analyzer-100 system in stroke patients.

The administration of an adenosine diphosphate (ADP) receptor antagonist, such as clopidogrel, is recommended for recurrent stroke patients under aspirin treatment. However, up to 25% of vascular patients have an inadequate response to clopidogrel treatment, which could be associated with increased reinfarction rates. This study investigated whether the platelet function analyzer (PFA-100) system represents an appropriate tool for monitoring clopidogrel's antiplatelet effects in stroke patients. Sixteen stroke patients on clopidogrel therapy (75 mg/day) were included in a prospective analyst-blinded, cross-sectional study. Platelet function was assayed by collagen/epinephrine (CEPI)- and collagen/ADP (CADP)-induced closure times (CTs) using the PFA-100 system. von Willebrand factor antigen (vWF-Ag) levels were measured by enzyme immunoassay. CEPI-CT and CADP-CT values averaged 160 +/- 15 seconds and 102 +/- 10 seconds, respectively, and were in the normal range. vWF-Ag concentrations averaged 153 +/- 17% and correlated inversely with CTs (r = .71; P < .002 for CEPI-CT, r = .54; P < .04 for CADP-CT). Our data indicate that the current PFA-100 cartridges are not sufficiently sensitive to detect clopidogrel-induced platelet inhibition in stroke patients.

Assessment of PFA-100 system for the measurement of bleeding time in oral surgery.

UNLABELLED: The common diagnostic methods to know primary hemostasis have been classified as invasive, depending on the operator, difficult to reproduce and at times not very reliable. Thus, different systems have been proposed to assess bleeding time, one of them being the PFA-100 device, which we present in this paper. OBJECTIVE: Compare specificity between the traditional Ivy method with the PFA-100 system to measure bleeding time. MATERIAL AND METHOD: We obtained a sample of 33 patients between the age of 24-80 years receiving anti-platelet treatment who needed to undergo oral surgery. Bleeding time was obtained by the Ivy method, an INR by an analysis done on the same day and a Coagucheck one hour before surgery as well as measurement of bleeding time with the PFA-100 system. RESULTS: Mean value of bleeding time through the Ivy method was 406.36 sec.. Mean bleeding time with the PFA-100 system for the collagen/epinephrine cartridge was 226.91 sec. and for the collagen/ADP cartridge was 110.27 sec.. All these values were within normality. We observed very high standard deviations with the Ivy method and more regular ones for the PFA-100 system, indicating its greater specificity. We also obtained a large correlation between collagen/epinephrine cartridge and acetylsalicylic acid. CONCLUSIONS: We found greater specificity of the analyzer of PFA-100 platelet function for the measurement of bleeding time in relationship with the traditional Ivy method.

Assessment of PFA-100 system for the measurement of bleeding time in oral surgery

Los métodos diagnósticos habituales para conocer la hemostasia primaria han sido calificados como cruentos, dependientesdel operador, de difícil reproducción y en ocasiones no muy fiables. Es por ello que se han propuesto diferentes sistemas para valorar el tiempo de hemorragia, siendo uno de ellos el dispositivo PFA-100, el cual presentamos en este trabajo.Objetivo: Comparar la especificidad entre el método tradicional Ivy con el sistema PFA-100 para la determinación del tiempo de hemorragia.Material y método: Obtuvimos una muestra de 33 pacientes de entre 24-80 años en tratamiento antiplaquetario a los cuales se debía realizar una cirugía oral. Se les realizó un tiempo de hemorragia mediante el método Ivy , un INR medianteuna analítica realizada el mismo día y un Coagucheck una hora antes de la cirugía así como la determinación del tiempo de sangrado mediante el dispositivo PFA-100. Resultados: El valor medio del tiempo de hemorragia mediante el método Ivy fue de 406.36 sg. El tiempo de hemorragia medio con el sistema PFA-100 para el cartucho de colágeno/epinefrina fue de 226.91 sg. y para el cartucho de colágeno/ADP fue de 110.27 sg. Todos estos valores se encuentran dentro de la normalidad. Observamos desviaciones típicas muy altas con el método Ivy y más regulares para el sistema PFA-100 indicando una mayor especificidad del mismo. Obtuvimos también una gran correlación entre el cartucho colágeno/epinefrina y el ácido acetil salicílico.Conclusiones: Encontramos una mayor especificidad del analizador de función plaquetaria PFA-100 para la medición del tiempo de hemorragia en relación con el método tradicional Ivy

The common diagnostic methods to know primary hemostasis have been classified as invasive, depending on the operator, difficult to reproduce and at times not very reliable. Thus, different systems have been proposed to assess bleeding time, one of them being the PFA-100 device, which we present in this paper.Objective: Compare specificity between the traditional Ivy method with the PFA-100 system to measure bleeding time.Material and method: We obtained a sample of 33 patients between the age of 24-80 years receiving anti-platelet treatment who needed to undergo oral surgery. Bleeding time was obtained by the Ivy method, an INR by an analysis done on the same day and a Coagucheck one hour before surgery as well as measurement of bleeding time with the PFA-100 system.Results: Mean value of bleeding time through the Ivy method was 406.36 sec.. Mean bleeding time with the PFA-100 system for the collagen/epinephrine cartridge was 226.91 sec. and for the collagen/ADP cartridge was 110.27 sec.. All these values were within normality. We observed very high standard deviations with the Ivy method and more regular ones for the PFA-100 system, indicating its greater specificity. We also obtained a large correlation between collagen/epinephrine cartridge and acetylsalicylic acid.Conclusions: We found greater specificity of the analyzer of PFA-100 platelet function for the measurement of bleeding time in relationship with the traditional Ivy method

Correlação entre a recuperação da frequência cardíaca no 1ºminuto após o esforço físico e os marcadores de risco obtidos no teste ergométrico e na tomografia miocárdica de perfusão / Correlation between abnormal heart rate recovery immediately after exercise and the risk markers obtained from exercise treadmill test and myocardial perfusion tomography

Objetivos: Determinar a associação entre os marcadores de mortalidade cardiovascular indicados no teste ergométrico (TE) e na tomografia miocárdica de perfusão (SPECT) e a variável recuperação da FC no 1ºminuto após o esforço físicoMétodos: estudou-se, prospectivamente, um grupo de 2189 pacientes (1294 homens, idade de 56,7 anos), encaminhados para a realização da tomografia miocárdica de perfusão com Tc-99m sestamibi ou tetrofosmin pela técnica tomográfica (SPECT). Foram excluídos todos os pacientes em uso de medicamentos betabloqueadores, antagonistas dos canais de cálcio e antiarrítmicos. O esforço físico foi realizado pelo TE, segundo o protocolo de Bruce, limitado por sintoma. O valor da RFC 1ºminuto da fase de recuperação, sendo considerado como anormal quando menorigual a 12 bpm. A incompetência cronotrópica durante o esforço físico foi definida como a incapacidade de atingir 85 pr cento da FC máxima prevista para a idade. o ventrículo esquerdo (VE) foi dividido em 17 segmentos e a interpretação das imagens do SPECT do miocárdio foi realizada de forma visual semiquantitativa.Resultados: A maioria dos pacientes apresentou RFC 1ºminuto (87 por cento) e SPECT do miocárdio (75 por cento) normais. Os pacientes com RFC 1ºminuto anormal, comparados aos pacientes com RFC 1º minuto normal, apresentaram FC e pressão arterial no repouso, mais elevadas, mais incompetência cronotrópica durante o exercício físico e maiores escores de quantificação do defeito de perfusão. A análise multivariada, após ajuste das variáveis clínicas, ergométricas e cintilográficas, identificou a idade (p menor 0,0001), a FC no repouso (p menor 0,001), a incompetência cronotrópica (p menor 0,005), a duração do exercício físico (p menor 0,0001) e a extensão do defeito de perfusão na fase de repouso no SPECT(p igual 0,001), como preditores da RFC 1º minuto anormal, com valor independente.Conclusão:Neste estudo, a RFC 1ºminuto anormal após o esforço fpisico não se associou aos marcadores de isquemia miocárdica, como o escore de exercício de Duke e o escore somado da diferença (SDS). Entretanto, houve uma significativa associação com os marcadores reconhecidos de maior mortalidade cardiovascular indicados no TE e na tomografia miocárdica de perfusão, representado pelo escore somado de repouso (SRS), um marcador de dano miocárdico

Measurement of primary haemostasis using a pressure clamp technique.

UNLABELLED: With the PFA-100 a sensitive and specific screening test for primary haemostasis has recently become available. An important part of the device is a capillary, providing a defined haemodynamic resistance for the perfusion of the aperture. A modified method to measure platelet function (VCP2) is presented in which the capillary essentially is replaced with an 'electronic capillary' by clamping the pressure/flow relationship. RESULTS AND CONCLUSION: Closure time (CT) and blood volume (BV) as determined by PFA-100 and VCP2 correlated well within (r = 0.922 - 0.952) and between the two methods (r = 0.86). The test variability (CV) of CT could be significantly reduced in the VCP2 method (collagen/epi 3.9 vs. 5.9%, p<0.05; collagen/ADP 3.3 vs. 6.9%, p<0.001), thus considerably increasing test reliability and reducing test variance. In preliminary clinical studies the VCP2 system showed comparable sensitivity for vWD and slightly less sensitivity regarding ASA ingestion. The test spectrum of VCP2 could be extended to more thrombocytopenic samples (< or =20 000/microl) even in combination with low haematocrit levels (20%), thus perhaps permitting the determination of the bleeding risk in bone marrow hypoplasia. Additionally, the sensitivity and applicability can easily be adapted to the desired need only by software modifications.

Evaluation of platelet function with the PFA-100 system in patients with congenital defects of platelet secretion.

The template bleeding time is still the screening test for defects of platelet function, although it is an invasive and poorly reproducible technique. The PFA-100 measures platelet function at high shear. Whole blood is aspirated through a capillary to an aperture of a membrane coated with platelet agonists. The system measures the time required to obtain occlusion of the aperture by a platelet plug (closure time). We measured the closure times in the PFA-100 system and the bleeding time in seven patients with delta-storage pool deficiency, 10 patients with "primary secretion defect" (not due to abnormalities of platelet granules or the arachidonate pathway), and 40 controls. Measurements were repeated I and 4 hours after intravenous infusion of desmopressin in six delta-storage pool deficiency and eight primary secretion defect patients. Baseline bleeding time and closure times with the collagen/epinephrine cartridge were longer in delta-storage pool deficiency and primary secretion defect patients than in controls. In contrast, closure times with the collagen/adenosine diphosphate cartridge were normal in both delta-storage pool deficiency and primary secretion defect patients. Treatment with desmopressin increased the plasma von Willebrand Factor levels, shortened the prolonged bleeding time, shortened the closure times with the collagen/adenosine diphosphate cartridge, and normalized the closure times with the collagen/ epinephrine cartridge. Therefore, the PFA-100 test may be a less invasive alternative to the bleeding time in the diagnosis and therapeutic monitoring of patients with platelet secretion defects. The collagen/epinephrine cartridge is more sensitive than the collagen/adenosine diphosphate cartridge to defects of platelet secretion.

Development and practical use of the new quantitative bleeding time test apparatus.

We have developed a new computerized system for measurement of quantitative bleeding time (QBT) to detect subtle abnormalities of primary hemostasis that are difficult to detect with the standard bleeding time determination. This new apparatus can simultaneously measure the bleeding time (BT; sec), amount of total blood loss (Tv; microl), maximum bleeding rate (Rmax; microl/sec) and bleeding pattern from the bleeding time incision. We have also developed a new holder for the Simplate that enables more consistent incisions and thus improves the reproducibility of the BT test. In this study, the newly developed QBT test was performed in 137 normal healthy volunteers and 10 patients having defined abnormalities of either primary or secondary hemostasis. Comparisons of the standard BT test and our QBT were made in 5 normal subjects and 7 thrombocytopenic patients. Additionally, 6 normal subjects were examined with both tests before and after administration of aspirin. Those results show that our QBT appears to be a more sensitive indicator of primary hemostasis than the standard BT method.

[Clinical evaluation of platelet function analyzer PFA-100 in dogs]. / Untersuchungen zur klinischen Anwendung des Plättchenfunktionsanalysengerätes PFA-100 beim Hund.

The platelet-function-analyzer PFA-100 (Fa. Dade Behring, Liederbach) proofs the capillary in vitro bleeding time by the blood flow through a capillary. The membrane at the end of the capillary is a membrane with a layer of an inductor of the platelet aggregation (collagen/ADP or collagen/epinephrine). The closure time and optional the blood volume flowing out of the capillary can be determined. To find out the applicability of the analyzer for canine blood samples several examinations were carried out in this study. Based on 45 blood samples of clinically healthy dogs a reference range (2.5%- and 97.5% quantile) was defined. Using the collagen/ADP test cartridge the reference range for the closure time was 47-81 s and for the blood volume 223-303 microliters. Using the collagen/epinephrine test cartridge the reference range for the closure time was 67-210 s and for the blood volume 269-611 microliters. In addition, blood samples (n = 19) of dogs with different degrees of thrombocytopenia were examined. Hereby, in contrast to the collagen/epinephrine cartridge, the collagen/ADP cartridge showed a high sensitivity regarding the closure time (n = 18) as well as the blood volume (n = 17). Regarding the results of the healthy dogs and dogs with thrombocytopenia a moderate close correlation was calculated between the in vitro bleeding time and the capillary in vivo bleeding time (rs = 0.590). After intravenous injection of 20 mg/kg acetylsalicylic acid the closure time measured with the collagen/ADP cartridge was 1.3 fold prolonged. The reference range was not exceeded in every case. With artificial variation of the haematocrit a clear prolongation of the closure time at haematocrit values < or = 25% was demonstrated. These results indicate that clinical usability of the platelet function analyzer in dogs is restricted despite its sensitivity to disorders of primary haemostasis.

An assessment of human epidermal repair in elderly normal subjects using immunohistochemical methods.

The purpose of this study was to document a timetable for selected events of epidermal repair in standard partial thickness incised wounds on the legs of normal elderly human subjects. A Simplate-II bleeding-time device was used for producing the wounds, and immunohistochemical techniques were employed for evaluation of the wounds. Antibodies to filaggrin and Ulex europeus I demonstrated little or no staining on migrating epithelium, but staining was apparent whenever epidermal closure had occurred. Bullous pemphigoid antigen was present in the basement membrane zone at all time points examined, including beneath migrating epithelium, whereas antibodies to laminin and type IV collagen were found only at the most lateral aspects of 2-, 3-, and 5-day wounds. Staining progressed centrally by day 7 and was present as a complete linear band beneath most 14-day wounds. The Simplate-II device provides a standard, easy to use, commercially available, sterile, relatively safe method of producing wounds for systematic studies in humans.

"Haemostasis time", a modified bleeding time test and its comparison with the Duke and Ivy/template bleeding times. I. Normal values, application in thrombocytopenic patients and evaluation of heparin and aspirin effects.

The occlusion time ("haemostasis time" - HT) of a thin, short cannula inserted into the cubital vein, was compared with the skin bleeding times of the Duke and Ivy/template techniques. 25 male and 25 female volunteers without a history of bleeding were divided into 5 equally large age groups ranging from 10 to over 50 years of age. They exhibited a range of 46 s-6 min 38 s (95% tolerance interval), while the Duke and Ivy/template bleeding times, which were simultaneously determined, corresponded to values given by other authors. HT is different from the skin bleeding times in that endothelium is replaced by a standard foreign surface which allows better standardization of the method. Similar results were obtained with HT compared to the skin bleeding times. These and a similar, non-significant heparin response with all three techniques suggest that HT is not more influenced by clotting factors than the Duke and Ivy/template bleeding times and, indeed, may be regarded as a bleeding time modification. HT, like both of the skin bleeding times, reflected lowered platelet counts and is even more sensitive in this respect. As tested in a group of 20 male and 20 female volunteers, HT showed a significant prolongation two hours after ingestion of 1 g aspirin. While male individuals exhibited longer bleeding times than females with the Ivy/template technique (sex-related difference p = 0.01), no male to female differences were found both with HT and the Duke bleeding time. HT is easy to perform, inexpensive, leaves no scars and is safe even for the patient with severe bleeding. Moreover, compared to the skin bleeding times, it permits a differential evaluation of vessel wall and tissue effects.

Clinical trial of a new disposable bleeding-time device.

A new disposable bleeding time device (Hemalet) was tested in 20 normal individuals and 11 patients with various bleeding disorders. The results were compared with those of Simplate II. The mean bleeding time for normal individuals was 5.4 +/- 1.5 (mean +/- 1 SD) minutes by Hemalet and 5.8 +/- 1.4 (mean +/- 1 SD) minutes by Simplate II, with good correlation between the results by the two devices (r = 0.81). The bleeding time in patients with various bleeding disorders were also comparably prolonged between the two devices. The new disposable bleeding time device with a disposable blade has quick release (penetration) into skin and retraction, and offers an alternative means of a bleeding time test.