Platelet Count In Platelet-rich Plasma Research Articles

Effect of platelet concentration in platelet-rich plasma on peri-implant bone regeneration

This study analyzed the effect of the platelet count in platelet-rich plasma (PRP) on bone regeneration in vivo.Twenty male New Zealand white rabbits were used. PRP was produced using the Platelet Concentrate Collection System (PCCS) (3i, Miami, FL, USA). After inducing ketamine–xylazine anaesthesia, a self-tapping titanium screw (Branemark MK III TiUnite, 3.75 × 7 mm) was inserted in each distal femur; the femurs were randomized so that one side was treated with PRP while the other (control) was not. Intravital fluorochrome staining was performed on days 1, 7 (1.5 ml of 2% doxycycline/kg bodyweight), 14 (6% xylenol orange, 1.5 ml/kg), and 21 (1% calcein green, 5 ml/kg). Animals were euthanized on day 28 (n = 20). Specimens were prepared for histomorphological evaluation according to Donath and Breuner [J. Oral Pathol. 11 (1982) 318].Comparing the bone regeneration (fluorochrome staining) in the 4-week implants (n = 19), the only significant difference (sign test, P = 0.004) was seen with intermediate platelet concentrations (n = 9,503,000–1,729,000 platelets/μl PRP).There were no differences in the bone/implant contact rates between the test and the control side among the three groups.The platelet concentration required for a positive PRP effect on bone regeneration seems to span a very limited range. Advantageous biological effects seem to occur when PRP with a platelet concentration of approximately 1,000,000/μl is used. At lower concentrations, the effect is suboptimal, while higher concentrations might have a paradoxically inhibitory effect. On the other hand, the effect of this type of platelet concentrate was not beneficial to accelerate the osseointegration of enosseous dental implants.

The impact of peripheral arterial disease on circulating platelets

Introduction: To test the hypothesis that circulating platelets display evidence of reversible interactions with atherosclerotic lesions, platelet α-granule content and propensity for microaggregate formation were measured in samples from normal donors ( n=65) and from patients with either peripheral arterial disease ( n=47) or renovascular hypertension ( n=22). To measure the effect of a defined arterial injury on platelet function, platelet samples were compared before and 30 min after elective angioplasty. Materials and methods: P-selectin was measured after strong stimulation of ultra-dilute platelets with thrombin (10 nM). Microaggregation was measured as a platelet count deficit in citrate-anticoagulated platelet-rich plasma (PRP) relative to that predicted from the count in EDTA-anticoagulated blood. Results: Platelet α-granule P-selectin was significantly lower from platelets of patients compared to normal donors. In addition, platelets from patients have a significantly greater propensity to form microaggregates in citrate anticoagulant. In contrast to atherosclerotic renovascular hypertension, platelets from patients with fibromuscular dysplasia, a distinct non-inflammatory cause of arterial stenosis, do not differ significantly from normal donors. Other than the PRP platelet count, which rose transiently following angioplasty, other platelet measures were unchanged by the injury. Conclusions: Atherosclerotic arterial disease is associated with an increased share of platelets unable to express P-selectin and an increased fraction of platelets that microaggregate in citrate anticoagulant. These platelet alterations are not completely explained by either focal arterial injury or abnormal rheology associated with arterial stenosis but appear to be an effect of the atherosclerotic process.

Platelet rich plasma and fresh frozen bone allograft as enhancement of implant fixation: An experimental study in dogs

Platelet rich plasma (PRP) is an autologous source of growth factors. By application of PRP around cementless implants alone or in combination with bone allograft chips, early implant fixation and gap healing could be improved. We inserted two porous HA coated titanium implants extraarticularly in each proximal humerus of eight dogs. Each implant was surrounded by a 2.5 mm gap. Four treatments were block randomized to the four gaps in each dog: Treatment 1: empty gap, treatment 2: PRP, treatment 3: fresh frozen bone allograft, treatment 4: fresh frozen bone allograft + PRP. PRP was prepared from each dog prior to operation by isolating the buffycoat from centrifuged blood samples. Platelet count in PRP was increased 670% compared to baseline level. Calcium/thrombin was added to degranulate platelets and form a gel. Three weeks after surgery, push-out test and histomorphometri was performed. After three weeks, the non-allografted implants had poor mechanical properties. Bone grafting significantly increased implant fixation, bone formation in the gap and bone growth on the implant surface. We found no significant effect of PRP alone or mixed with bone allograft on implant fixation or bone formation. In conclusion, we showed the importance of bone allografting on early implant fixation and bone incorporation but we found no effect of PRP. More studies are needed to investigate the effect and possible clinical applications of platelet concentrates which are now being commercialised.

Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count

Introduction: Platelet-rich plasma contains autologous thrombocyte growth factors and might be promising for acceleration of dentoalveolar bone regeneration. In this study, it was analysed for platelet counts and growth factor concentrations. Material and method: Platelet-rich plasma was isolated by discontinuous cell separation from 158 healthy men and 55 women aged 17–62 years. One hundred and fifteen specimens (stratified for age and gender of the donor) were analysed for growth factor concentrations and platelet count. Results: The platelet count in platelet-rich plasma (1,407,640±320,100/μl) was 5 times higher than in donor blood (266,040±60,530/μl). Platelet-derived growth factor AB (117±63 ng/ml), transforming growth factor (TGF) β -1 (169±84 ng/ml), and insulin-like growth factor (IGF) I (84±23 ng/ml) were found in large amounts, while platelet-derived growth factor (PDGF) BB (10±8 ng/ml) and transforming growth factor β -2 (0.4±0.3 ng/ml) were found in small amounts only. The growth factor content was not well correlated with the platelet count in whole blood nor with the platelet-rich plasma (rp=0.35). No influence of gender or age on platelet count or growth factor concentrations was discovered (except IGF-I). Conclusions: While there was substantial variation in the growth factor content of platelet-rich plasma, the factors influencing this are still worthy of further investigation. Furthermore, a technique whereby the growth factor content could be rapidly assessed in platelet-rich plasma may be of therapeutic benefit. Copyright 2002 European Association for Cranio-Maxillofacial Surgery. Published by Elsevier Science Ltd. All rights reserved.

Endotoxin binding to platelets in blood from patients with a sepsis syndrome

Endotoxin, the lipopolysaccharide cell wall constituent of Gram-negative bacteria, produces symptoms of the Gram-negative sepsis syndrome. By measuring endotoxin in blood from septic patients it may be possible to select a subpopulation of patients in which mortality can be prevented by treatment with anti-endotoxin antibodies. We evaluated the performance of an endotoxin-free blood-collection tube. Within-run and between-run CVs of our endotoxin assay were 4-18% and 8-20%, respectively. In endotoxin-positive samples (LPS > or = 6 ng/L), the concentration of endotoxin in platelet-rich plasma was significantly higher (P < 0.001) than in platelet-poor plasma. Apparent binding of endotoxin to platelets ranged from 0% to 92%. The correlation between the apparent percentage binding of LPS to platelets and the platelet count in platelet-rich plasma is linear and positive, but LPS is not bound solely to platelets. We conclude that endotoxin must be measured in platelet-rich plasma.

Laboratory and clinical evaluation of a chromogenic endotoxin assay for horses with acute intestinal disorders

Summary In this study the laboratory and clinical performance of a chromogenic endotoxin assay for equine plasma was evaluated. The assay was sensitive (detection limit 3 ng LPS/L plasma), reproducible (within and between-assay CV at 50 ng LPS/L E.coli 0111:B4 LPS standard addition was 5% and 7.5%, respectively), and not substantially affected by enhancement or inhibition phenomena (recovery of an in vitro spike was 75-125% in 80% of the samples). LPS added to whole blood was rapidly inactivated upon incubation at 37 °C but not at 0 °C. A recently developed blood collection tube for LPS testing was found suitable, i.e. LPS-free and providing non-contaminated samples. In 48 horses suffering from acute abdominal diseases requiring surgical treatment, LPS levels were significantly higher in platelet-rich plasma (PRP) than in platelet-poor plasma (PPP), and the proportional difference was related to the PRP platelet count (r=0.52, p<0.001, mean difference 48%, range 8-77%). LPS levels were also significantly higher in horses that died or were euthanized than in surviving horses (mean 16.5 and 7.1 ng/L PRP, respectively, p<0.05). We conclude that LPS can be measured in equine plasma with picogram sensitivity and recommend the use of PRP instead of PPP for clinical LPS testing. For clinical use a decision limit for endotoxaemia of 5 ng LPS/L PRP appeared to be inadequate. Analysis at a higher cut-off level for endotoxaemia and the evaluation of clinical, pathological, and laboratory parameters would be more meaningful.

The Association of Platelet and Red Cell Count with Platelet Impedance Changes in Whole Blood and Light-Scattering Changes in Platelet Rich Plasma: Evidence from the Caerphilly Collaborative Heart Disease Study

This epidemiological study was undertaken to explore possible relationships among various haematological indices, prevalent ischaemic heart disease and platelet "function" as measured by two rather different methods. ADP-induced platelet impedance changes in whole blood were strongly associated with prevalent ischaemic heart disease in a general population of 49-66 year men at increased risk. Adenosine diphosphate (ADP) induced platelet aggregation in platelet rich plasma (PRP) at a constant platelet count and also the whole blood platelet count and red cell (RBC) count were strongly and independently related to ADP-induced platelet impedance changes. Both platelet count and platelet aggregation in PRP assessed by changes in optical density were directly related to increasing platelet "sensitivity" as measured by impedance changes in whole blood but RBC count was inversely related. Positive independent relationships between platelet impedance changes and plasma viscosity and fibrinogen were markedly attenuated when platelet count was taken into account, but this finding does not discount a role for these factors in platelet aggregation. No relationship was noted between white blood cell (WBC) count and platelet impedance changes; however, a significant inverse relationship was noted with platelet aggregation in PRP. These findings indicate that laboratory-based experimental findings can be observed in population based studies, and that these haematological factors may be important indicators of ischaemic disease in the population.

Elevated Levels of PA IgG in Thrombocytopenic Patients Are Not Related to the Presence of Microthrombocytes or Megathrombocytes

Platelet-associated (PA) IgG levels were measured on peripheral blood samples from 63 thrombocytopenic patients with the use of fluorescein isothiocyanate (FITC)-conjugated anti-IgG antibody. Test specificity was investigated by examining the influence of variables such as the platelet count in platelet-rich plasma (PRP), platelet loss after the assay procedure, and platelet size distribution (including the percentage of microthrombocytes and megathrombocytes) on the PA IgG levels. Additionally, PA IgG levels were compared with PA albumin levels and with levels of nonspecific adherence of FITC anti-IgG by the platelets to ascertain if elevated levels were specific. Elevated PA IgG levels showed only significant correlations (P less than 0.05) with a mean platelet size below normal range and to increased PA albumin levels but not to PRP count, platelet loss after assay procedure, the percentage of microthrombocytes and megathrombocytes, and nonspecific adherence of FITC anti-IgG.

The effects of citalopram (Lu 10-171) on the serotonin (5-HT) uptake kinetics in platelets from endogenously depressed patients.

The uptake kinetics of serotonin (5-HT) in platelets from eight patients with endogenous depression was determined in platelet rich plasma (PRP). From the uptake data the Scatchard correlation coefficient (r[S] ), Sips' index of heterogeneity (alpha), Km and Vmax (Lineweaver-Burk) were calculated. The measurements were performed before and after two weeks treatment with Citalopram, 1-(3-dimethylaminopropyl)-1-(p-fluorophenyl)-5-phtalan carbonitrile (Lu 10-171), a selective 5-HT uptake inhibitor. After two weeks treatment with Citalopram (5, 25 or 50 mg/day) a lowered platelet count in whole blood and PRP was observed and none of the patients had a measurable 5-HT uptake for which kinetic parameters (Km and Vmax, Lineweaver-Burk) could be calculated. However, all the patients but one improved significantly in Global score.

原発性血小板血症の自然凝集におけるアラキドン酸代謝の解析

The amounts of TXB2 and PGD2 which were produced during spontaneous platelet aggregation (SPA) in 3 patients with primary thrombocythemia (PTH) were measured. After platelet counts in platelet-rich plasma (PRP) were adjusted to 120×104μl with autologous platelet-poor plasma, SPA was induced by stirring at 1100 r. p. m. in a Lumi-aggregometer and the lag time was measured. The amounts of TXB2 and PGD2 produced in the aliquots of PRP were measured by HPLC, using 9-anthryl-diazomethane. As a control, the amounts of TXB2 and PGD2 which were produced by the reaction of platelets with arachidonate (0.2mM, f. c.) were measured by similar method, using the platelets from 4 normal subjects (platelet count: 30×104μl).The amounts of TXB2 and PGD2 in normal subjects were 153.0±18.9, and 2.88±0.36ng/108 platelets (mean±S. D.), respectively (TXB2/D2=43.4 to 68.3). The amounts of TXB2 and PGD2 in PTH were as follows: case 1, 48.5 and 0.37; case 2, 56.2 and 1.14; case 3, 53.6 and 0.93 (ng/108 platelets), respectively (TXB2/D2=131.1, 49.3, 57.6). In case 1, the amount of PGD2 was decreased, and TXB2/D2 was significantly increased. In this case, it was also found that SPA occured in a shorter lag time than in the other cases. It was suggested that the increased TXB2/D2 may play some role in the mechanism of SPA in patients with PTH.

Do Patients With Thromboembolic Disease Have Circulating Platelet Aggregates?

Reports of circulating platelet aggregates (ie, microemboli) in thromboembolism and other vascular disorders are based on a method (Wu and Hoak , 1974) in which venous blood is collected via scalp vein needle and tubing into either formaldehyde, which fixes aggregates, or EDTA, which disperses them. The ratio of platelet counts in platelet-rich plasma (PRP) from the two blood samples after centrifugation is interpreted as a measure of platelet aggregates in the circulation in vivo. We compared this standard Wu and Hoak technique with a modified one, in which blood was drawn directly into a syringe, and with a third method that avoided centrifugation by counting single platelets in whole blood. Both modified techniques could detect aggregates generated in vitro with adenosine diphosphate (ADP). In 12 normal subjects, the three methods were equivalent, but in 37 patients with thromboembolic disorders, the standard Wu and Hoak method gave a lower ratio than the other methods. Similar results were found in a subset of eight patients with myocardial infarction. Heparin treatment of patients did not influence the results. The data suggest that formation of platelet aggregates occurred during venipuncture. Platelets may be hyperactive in patients with thromboembolic disease and may form aggregates in vitro during collection, but the concept of chronic microembolism in such patients should be reassessed.

Stimulated Platelet Aggregation, Thromboxane B2 Formation and Platelet Sensitivity to Prostacyclin - A Critical Evaluation

Platelets have been implicated in the development of atherosclerotic and thrombotic vascular diseases. Evaluation of platelet aggregation in relation to endogenously formed compounds which affect platelet function may provide information of clinical and pharmacological relevance. We describe a method in which thromboxane B2 (TXB2) formation was analyzed following stimulation of platelet-rich plasma (PRP) with ADP, 1-epinephrine, collagen, and arachidonic acid. In addition, we determined platelet sensitivity to prostacyclin following ADP- and collagen-induced platelet aggregation. The parameters under study were found to depend on the platelet count in PRP, on the type and dose of the aggregating agent used, and on the test time after blood sampling. By standardization of these variables, a reliable method was established which can be used in clinical and pharmacological trials.

Evaluation of automated whole-blood platelet counts and particle sizing.

The Coulter Counter model S-Plus measures the erythrocyte, leukocyte, and platelet counts, and the mean cell volume of erythrocytes and platelets. Two additional features are novel to the instrument: erythrocyte distribution width (RDW), an index of erythrocyte volume heterogeneity, and mean platelet volume. For 677 subjects, we compared these measurements with complete blood counts obtained by a standard instrument, platelet counts from platelet-rich plasma (PRP), and histograms of erythrocyte and platelet volumes generated by free-standing equipment. The two methods correlated well (r = 0.97) for the complete blood count and the mean platelet volume. The whole-blood platelet count correlated well (r = 0.97) with the PRP platelet count, with whole-blood counts on average 5% higher than PRP counts. The whole-blood index of erythrocyte volume heterogeneity, RDW, correlated less well (r = 0.81) with the erythrocyte coefficient of variation measured by histogram. RDW was abnormal for three of 100 normal subjects with normal histograms. RDW was less sensitive (78% vs. 97%) and less specific (94% vs. 97%) than erythrocyte histograms in distinguishing iron deficiency from heterozygous alpha- or beta-thalassemia. We conclude that the Coulter Counter Model S-Plus reliably measures complete blood cell count, platelet count, and mean cell volumes, but analyzes erythrocyte volume distribution less well than does a histogram.

Platelet aggregation. Adult-onset diabetes mellitus and coronary artery disease.

Using a turbidimetric technique, we determined 1.7 micrometer adenosine diphosphate--induced platelet aggregation and disaggregation at 37 degrees C in the platelet-rich plasmas of two groups of men with coronary artery disease. Eleven men were nondiabetic and 11 had adult-onset diabetes mellitus without retinopathy. There were no significant differences (P greater than .05) between diabetics and nondiabetics of the following variables: age, platelet count in platelet-rich plasma, first and second phases of platelet aggregation, maximum extent of aggregation, and percent disaggregation at three minutes after maximum aggregation occurred. Although the mean adenosine diphosphate--induced platelet aggregation in the platelet-rich plasmas of adult-onset diabetic men with coronary artery disease and no retinopathy was not enhanced, and the mean rate of disaggregation was not reduced, when compared with nondiabetic men with coronary artery disease or with healthy men; a slow rate of platelet disaggregation (less than 10%) occurred more frequently in the platelet-rich plasmas of men with coronary artery disease.

Electronic platelet counting with particular reference to thrombocytopenias (author's transl)

Platelet counts in platelet-rich plasma without hematocrit dependent correction were performed by following rapid and simple steps: 1. pre-dilution of 20 microliter of whole blood by an isotonic solution 1:25; 2. stabilized low-speed centrifugation with 55 g for 5 minutes; 3. final dilution 1 : 5000; 4. enumeration by use of a TOA platelet counter PL-100 which has been technically improved in comparison to similar machines. Erroneously high results were obtained after a too short or too low centrifugation. As reason for this artifical small pulses due to disturbances of the flow patterns around the aperture (so-called vortex-effect) can be assumed having been caused by large-volumed erythrocytes and leukocytes in the suspension. The routinely used procedure was reliable for all platelet ranges, especially in thrombocytopenias between 100 X 10(9)/l and 25 X 10(9)l. In lower ranges comparisons with visual counts are essential.

On the Measurement of Spontaneous Platelet Aggregation. The Platelet Aggregation Test III.

A new measuring device was developed for the study of "spontaneous" aggregating activity of thrombocytes. In the photometric platelet aggregation test (PAT III) 0.6 ml of platelet-rich plasma (PRP) are rotated in a disc-shaped cuvette at 20 rpm and 37 degrees C. Changes in optical density of PRP which are induced by the formation of platelet aggregates are continuously registered using a chart recorder. PAT III was developed for the detection of enhanced platelet aggregation, indicating a risk of thrombosis and thromboembolic complications. In 146 healthy individuals a certain percentage showed slight primary aggregation (alpha1) which in some cases was followed by marked aggregation (alpha2) at a certain time (Tr) after the beginning of rotation. The percentage of individuals showing alpha2 increased with age. An increase of plasma pH in the rotating sample, which was caused by diffusion of CO2, was an important conditioning factor for aggregation. The test results depended on the platelet count in PRP. Aggregation curves were suppressed by admixture of erythrocytes and lipid turbidity. The tendency of platelets to aggregate increased within 60-90 min following blood sampling. During this period the interval to the onset of aggregation (Tr) became shorter and the maximum aggregation speed (alpha 2) increased with time. PAT III yielded reproducible results when it was carried out more than 60 min after blood drawing. In a group of 327 diabetic patients "spontaneous" aggregation occurred more frequently in all age groups as compared with the controls. Additional equipment was available for the registration of ADP-, collagen-, or epinephrine-induced aggregation similar to Born's and O'Brien's method. The device can easily be mounted on an Eppendorf photometer without further alterations.

Second Phase Platelet Aggregation Induced by Adenosine Diphosphate in Patients with Cerebral Vascular Disease and in Control Subjects

SummaryAdenosine diphosphate-induced second phase platelet aggregation was studied in 70 patients with established cerebral vascular disease, 27 patients with chronic cerebral vascular disease taking dipyridamole, and 53 control subjects. The appearances of aggregation graphs relating changes in optical density in platelet-rich plasma to time after addition of adenosine diphosphate are described. An intermediate rate change in optical density was observed between first and second phase aggregation.Induction of second phase aggregation depends on the concentration of adenosine diphosphate and the platelet count of the plasma. Individual rseponses vary greatly, and there is appreciable variability in repeated estimations, but in the same plasma responses to adenosine diphosphate are accurately reproducible.In both patients and controls the mean logarithm of the adenosine diphosphate concentration that just induces second phase aggregation is inversely proportional to the mean platelet count of platelet-rich plasma. At all platelet concentrations studied, the smallest concentration of adenosine diphosphate that brought about second phase aggregation was significantly less in the patients than in controls. There was no significant correlation between the means of the two variables in patients taking dipyridamole. The findings in this group of patients accord with the assumption that dipyridamole favourably affects the abnormal reaction of platelets to adenosine diphosphate in some of the patients with cerebral vascular disease.

The Effect of Warfarin Sodium on the Duration of Platelet Aggregation

SummaryThe effect of the administration of warfarin sodium (Coumadin®) on the duration of platelet aggregation in vitro was studied. Coumadin was given for 4 consecutive days to 10 healthy adults who were followed over a period of 9 days. The duration of adenosine diphosphate-induced platelet aggregation in platelet-rich plasma, the prothrombin time, and the platelet count of platelet-rich plasma were measured. Four other healthy adults received placebos and participated in a double-blind study with those receiving Coumadin.Although administration of Coumadin caused a prolongation of the prothrombin time to 2 or 21/2 times the normal value, a decrease in the duration of platelet aggregation was not observed. In most individuals who received Coumadin an increase in the duration of platelet aggregation occurred. The effect of Coumadin on platelet aggregation was not consistently related to the prothrombin time or to the platelet count. In the placebo group there was a distinct relation between the duration of platelet aggregation and the platelet count in platelet-rich plasma.The mean increase in the duration of platelet aggregation when compared to the control value before medication with Coumadin was 37.7%. In the placebo group there was a mean increase of 8.4%. The difference between the two groups is significant (p &lt;0.001). Increased duration of platelet aggregation also occurred in two individuals who received Coumadin over a period of 10 and 16 days respectively.