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Flow cytometric analysis of platelets from children with the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure.

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP-induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.

Actins↗

Does maternal platelet-associated or platelet-bindable IgG correlate with levels in umbilical cord blood or colostrum during normal pregnancy?

Existing data regarding the ability to predict neonatal thrombocytopenia during maternal immune thrombocytopenia are confusing. We studied normal pregnancies (n = 20) to define normal values and the correlation between maternal and umbilical cord platelet counts, platelet-associated immunoglobulin G (IgG), and platelet-bindable IgG. The postpartum serum platelet-bindable IgG level was measured to evaluate peripartum changes and the correlation with colostrum platelet-bindable IgG (n = 6). The mean maternal platelet count was 181,500 cells/cm3 mm and the mean umbilical cord platelet count was 293,500 cells/mm3. The median maternal platelet-associated IgG was 803 molecules per platelet, umbilical cord platelet-associated IgG was 791 molecules per platelet, maternal platelet-bindable IgG was 92 molecules per platelet, and umbilical cord platelet-bindable IgG was 256 molecules per platelet. The postpartum median maternal platelet-bindable IgG was 333 molecules per platelet and for colostrum it was 297 molecules per platelet. No clinically useful correlations for predicting the neonatal platelet count during normal pregnancy were found. Normal pregnancies may have high levels of maternal- or umbilical cord platelet-associated IgG, perhaps due to nonspecific binding.

Blood Platelets↗

Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures.

Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P-selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re-infusion of autologous platelets on platelet function. Autologous platelet-rich plasma (PRP) contained a significantly lower (P < 0.05) percentage of P-selectin-positive and fibrinogen-positive platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P < 0.05) percentage of responsive platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P-selectin or binding fibrinogen increased significantly after CPB (P < 0.05), while the percentage of platelets responsive to in vitro agonists was decreased (P < 0.05 in autologous transfusion patients), consistent with platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re-infusion of autologous PRP. P-selectin expression had returned to pre-CPB levels 24 h post-operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.

Blood Transfusion, Autologous↗

Platelet aggregation induced by type IIb platelet von Willebrand factor.

Platelet lysates from five patients with a form of type IIb von Willebrand's disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWd's platelet-rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard-Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmann's thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate-induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.

Antibodies, Monoclonal↗

Quality control of platelets during storage by the PFA-100: a comparison to platelet aggregation.

BACKGROUND AND OBJECTIVES: The PFA-100 examines platelet function by measuring closure time (CT) when whole blood is passing through a capillary and a filter membrane coated with a collagen/platelet agonist. Termination of the CT is achieved when adhered and aggregated platelets of the passing blood flow occlude the membrane. Two PFA-100 test cartridges are used to measure the CT of citrated whole blood; either the collagen/ADP (PCA) or the collagen/ epinephrine (PCE). We investigated the applicability of the PFA-100 test system for quality control of platelet concentrates in comparison with platelet aggregometry. MATERIALS AND METHODS: For platelet aggregation, the combination of ADP (100 microM/l) and collagen (20 microg/ml) were used as agonists (ADP/COL). In our test system, 25 microl epinephrine (10(-3) M) were added to the PCA cartridge (CT: PCAE) and 25 microl ADP (100 microM/l) to the PCE cartridge (CT: PCEA), respectively. Red blood cells from a blood group 0 donor were adjusted to a hematocrit of 43% using platelet rich plasma (8 x 10(5)/microl) of the respective platelet concentrate. Fourteen irradiated and non-irradiated platelet concentrates were examined on days 0, 3 and 5 after platelet preparation. Swirling without a scoring system and mean platelet volume (MPV) were also tested. Statistical analyses were performed by Pearson's range coefficient, the Mann and Whitney test, and the Wilcoxon test. RESULTS: Swirling was seen in all platelet concentrates. Mean platelet volume was normal during the whole observation period like the PCAE in non-irradiated products. Only the PCAE showed finite CTs during the whole storage time. Consequently, further testing was performed exclusively with the PCAE. Weak but significant correlations could be found between the PCAE and ADP/COL (r = -0.53 for non-irradiated platelet products, r = -0.62 for irradiated platelet products, p < or = 0.0001). Platelet function significantly decreased to around 60% of the initial value in the PCAE on day 5 of storage, ADP/COL decreased to 35% of maximum amplitude. A further decrease of approximately 10% was observed in the irradiated products, a fact which was significant in the PCAE. Only in the irradiated platelet products did MPV show a weak but significant correlation with the PCAE (r = 0.45, p = 0.002). CONCLUSION: Functional features of platelets from platelet concentrates characterized by ADP/COL induced aggregation can also be measured by the PFA-100. Yet, it has a higher sensitivity to platelet lesions induced by gamma-radiation. Further clinical studies will be necessary to determine whether PFA-measurements meet the quality criteria of MPV or swirling, or is superior in predicting in vivo viability. The relatively high cost of the test cartridge remains an obstacle for routine use.

Adenosine Diphosphate↗

Platelet function, size and yield in whole blood and in platelet-rich plasma prepared using differing centrifugation force and time in domestic and food-producing animals.

The effects of centrifugation force and time upon platelets function, mean platelet volume and platelet yield were compared with whole blood platelet counts and size in citrated blood samples from the bovine, canine, caprine, equine, feline, ovine and porcine species. The results were similar, for a given species, irregardless of sample volume. Bovine, caprine, feline and ovine platelet yields and mean platelet volumes were maximal when platelet-rich plasma was prepared using longer centrifugation times and lower gravitational forces. Canine, equine and porcine platelet yields and mean platelet volumes were maximal when platelet-rich plasma was prepared using shorter centrifugation times and higher gravitational forces. Platelet aggregation to adenosine diphosphate or arachidonic acid was not effected by the method of platelet-rich plasma preparation in bovine, caprine, feline, ovine or porcine platelets. Equine platelet aggregation was maximal when platelet-rich plasma was prepared using longer centrifugation times and lower gravitational forces. Canine platelet aggregation, particularly arachidonic acid-induced aggregation, was maximal when platelet-rich plasma was prepared using short centrifugation times and higher gravitational forces. It appeared that the effects of centrifugation parameters upon platelet yield depended upon the relative difference between platelet and red blood cell volumes.

Animals↗

Testing of platelet deposition on polystyrene surface under flow conditions by the cone and plate(let) analyzer: role of platelet activation, fibrinogen and von Willebrand factor.

Recently, we described a method of testing platelet deposition on extracellular matrix under flow conditions. The method was used for assessment of platelet function in various platelet disorders, for monitoring of replacement and anti-platelet therapy. In the present study, we investigated platelet deposition on a polystyrene surface compared with that on extracellular matrix, under defined shear rates, using the original Cone and Plate(let) Analyzer. A correlation of adhesion rate (surface coverage) and aggregate formation (average size) of platelets from normal citrated blood between polystyrene and extracellular matrix was observed. Blocking of von Willebrand factor binding to glycoprotein Ib by a recombinant von Willebrand factor fragment substantially decreased platelet adhesion to both surfaces. Blocking of GPIIb-IIIa by Arg-Gly-Asp-Ser peptide prevented platelet adhesion to the polystyrene while an extensive adhesion of single platelets to extracellular matrix was observed. Furthermore, platelet adhesion to polystyrene but not to extracellular matrix was completely inhibited by platelet inactivation with prostaglandin E(1). Platelets from patients with severe von Willebrand disease yielded very low adhesion to both polystyrene and extracellular matrix. The addition of von Willebrand factor to the blood of these patients or pre-coating of polystyrene surface with von Willebrand factor restored the ability of platelets to adhere and aggregate on the surface. Platelets from patients with Glanzmann's thrombasthenia and afibrinogenemia adhered to extracellular matrix (with defective aggregate formation), while they failed to adhere to the polystyrene. Fibrinogen added to afibrinogenemia blood or pre-coating of the polystyrene with fibrinogen restored the ability of platelets to adhere and aggregate on the surface. In conclusion, the polystyrene surface, like extracellular matrix, can be used to assess platelet function disorders taking in account that platelet deposition on polystyrene under flow is absolutely dependent on platelet activation and on the presence of fibrinogen, von Willebrand factor, and their receptors.

Afibrinogenemia↗

Detection of activated platelets and platelet-leukocyte aggregates in horses.

OBJECTIVE: To determine the potential usefulness of tests for detection of platelet activation and platelet-leukocyte aggregates in horses. SAMPLES: Blood from 3 healthy Thoroughbreds. PROCEDURES: Microscopic and flow cytometric assays were used to evaluate spontaneous platelet aggregation, platelet activation, and platelet-leukocyte aggregates. Platelet activation was detected by evaluation of binding of anti-human fibrinogen to unactivated and ADP-, thrombin-, thrombin agonist receptor peptide-, and platelet activating factor-activated platelets. Platelet-leukocyte aggregates were evaluated microscopically and by flow cytometric determination of leukocyte fluorescence that resulted from binding of fluorescently labeled platelets to leukocytes. RESULTS: Equine platelets readily aggregated spontaneously when blood was stirred at low, medium, and high speeds. Compared with unactivated platelets, activated platelets had a marked increase in the percentage of cells with increased fluorescence intensity and in mean fluorescence intensity. Unactivated platelets formed aggregates with neutrophils and monocytes, but not with lymphocytes. Activation of platelets resulted in a calcium-dependent increase in platelet-leukocyte aggregates. CONCLUSIONS: Flow cytometric techniques can be used to detect in vitro platelet activation and platelet-leukocyte aggregates in horses. CLINICAL RELEVANCE: Flow cytometric techniques may be useful for detection of prothrombotic disorders in horses.

Adenosine Diphosphate↗

Development of optimal techniques for cryopreservation of human platelets. I. Platelet activation during cold storage (at 22 and 8 degrees C) and cryopreservation.

Using the current blood bank storage conditions at 22 degrees C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets needed to treat patients afflicted with thrombocytopenia, a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8 degrees C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at -1 degrees C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8 degrees C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22 degrees C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to -10 degrees C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me2SO solution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me2SO toxicity to platelets was not noted until Me2SO concentrations exceeded 2 M. Finally, a concentration of 1 M Me2SO proved to be the most effective at all cryopreservation ending temperatures tested (-10, -30, -60, and -196 degrees C). In conclusion, under the present experimental conditions, a storage temperature of 8 degrees C appeared to be much better than 22 degrees C. Although the potential chemical toxicity of 1 M Me2SO, EG, or PG is negligible, 1 M Me2SO was found to be optimum for cryopreservation of human platelets. PG has the least cryoprotective function for low-temperature platelet survival.

Blood Platelets↗

Detection of platelet activation with monoclonal antibodies and flow cytometry. Changes during platelet storage.

To investigate whether changes in platelet condition during platelet storage correlate with an altered expression of platelet membrane proteins, the binding of monoclonal antibodies (MoAbs) to fresh platelets was compared with MoAbs' binding to thrombin-activated platelets and to platelets stored as platelet concentrates. The MoAbs included antibodies against the platelet glycoprotein (GP) IIb/IIIa complex and against two activation-dependent antigens, one of which was a component of the internal platelet alpha-granule membrane (GMP 140) and the other of which was a 53-kD protein derived from platelet lysosomes. The binding of MoAbs to platelets fixed with 1 percent paraformaldehyde was measured by flow cytometry. In thrombin-activated platelets, a threefold increase was found in the expression of GP IIb/IIIa over that in fresh platelets. The binding of the activation-dependent MoAbs increased from 2 to 3 percent to 70 to 80 percent of the platelets. Storage of platelet concentrates for 5 days resulted in a 60 percent increase in GP IIb/IIIa expression compared to Day 0 and increased binding of the MoAbs directed against GMP-140 from 3 to 16 percent and against the 53-kD protein from 2 to 8 percent of the platelets, respectively. These changes correlated with modifications in platelet morphology (decrease in swirling), leakage of lactate dehydrogenase, and release of beta-thromboglobulin. These data indicate that platelets become activated and are damaged during the storage of platelet concentrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

Frequency and characterization of platelet-specific antibodies in patients who received multiple platelet transfusions.

BACKGROUND AND PURPOSE: Multiple platelet transfusions may induce alloimmunization. Platelet alloimmunization involves human leukocyte antigen (HLA) antibodies and platelet-specific antibodies, which recognize platelet glycoproteins. No data are available about the frequency and characteristics of platelet-specific antibodies in patients who have received multiple platelet transfusions in Taiwan. METHODS: From June through December 1998, blood samples from 103 patients who had received multiple platelet transfusions were submitted to our laboratory for examination of platelet antibodies. The samples were first screened for HLA antibodies or platelet-specific antibodies using a solid phase method. Positive sera were further tested using an enzyme-linked immunosorbent assay (ELISA) to identify the specificities of the alloantibodies. RESULTS: Forty of the 103 patients who had received multiple platelet transfusions were positive for platelet-reactive antibodies. Further study using ELISA showed that 22 patients had both HLA and platelet-specific antibodies, 12 patients had HLA antibodies alone, and five patients produced platelet-specific antibodies in the absence of HLA antibodies. Most platelet-specific antibodies were found among patients who had HLA antibodies: 81% of platelet-specific antibodies were found in HLA-immunized patients. The most frequently involved platelet glycoprotein antigens were Ia/IIa and IIb/IIIa. CONCLUSIONS: HLA antibodies were the most frequently found platelet-reactive antibodies in Taiwanese patients who had received multiple transfusions, although platelet-specific antibodies were also common. The frequency of platelet-specific antibodies was not as low as previously reported.

Adolescent↗

The effect of transfusion of fresh whole blood versus platelet concentrates after cardiac operations. A scanning electron microscope study of platelet aggregation on extracellular matrix.

To evaluate the effect of fresh whole blood transfusion versus platelet concentrates transfusion on platelet aggregation after cardiac operations, 24 patients were randomized to receive either one unit of fresh whole blood (12 patients) or 10 platelet units (12 patients) after cardiopulmonary bypass. Platelet aggregation on extracellular matrix, platelet count, and mean platelet volume were studied preoperatively, at termination of cardiopulmonary bypass, after protamine administration, and after the transfusion of fresh whole blood or after transfusion of each two platelet units. Extracellular matrix produced by cultured bovine corneal cells closely resembles the vascular subendothelial basal lamina, and is an ideal in vitro model in the study of platelet interaction with the subendothelium. Platelet aggregation on extracellular matrix, studied by a scanning electron microscope, was graded from 1 to 4, wherein grade 1 represents nonactivated platelets and grade 4 a mature platelet aggregate. With this grading system, the two groups were similar in preoperative values (3.3 +/- 0.9 versus 3.7 +/- 0.4) and values after cardiopulmonary bypass (1.5 +/- 1.0 in both groups). One unit of fresh whole blood restored platelet aggregation on extracellular matrix to preoperative status (3.0 +/- 1.0), whereas eight platelet units were needed for the same result (3.2 +/- 0.8). One unit of fresh whole blood increased platelet count in a manner similar to that achieved by six platelet units and increased mean platelet volume to a level higher than that achieved by 10 platelet units. These results suggest that the effect of one unit of fresh whole blood on platelet aggregation after cardiopulmonary bypass is at least equal, if not superior, to the effect of 8 to 10 platelet units.

Adult↗

Loss of high-affinity thrombin receptors during platelet concentrate storage impairs the reactivity of platelets to thrombin.

BACKGROUND: The storage of platelet concentrates (PCs) induces a reduction in the platelet surface expression of glycoprotein (GP) Ib alpha. The location of the platelets' high-affinity binding site for thrombin has been postulated as being located on GPIb alpha. This study attempts to determine whether loss or alteration of GPIb alpha during storage of PCs is related to impairment in the reactivity of platelets to thrombin. STUDY DESIGN AND METHODS: In this study, platelet surface expression of GPIb alpha was monitored by means of flow cytometry, throughout standard storage of PCs for up to 10 days. Two thrombin-induced platelet responses, the binding of radiolabeled fibrinogen and the platelet surface expression of P-selectin, were evaluated. Thrombin-binding assays were also performed to assess the number of thrombin receptors in platelets. RESULTS: The surface expression of the GPIb/IX complex declines during storage of PCs. The thrombin-induced maximal binding of fibrinogen in platelets stored for 3, 7, and 10 days was 77 +/- 7 percent, 60 +/- 20 percent, and 34 +/- 25 percent, respectively, of that found in fresh platelets. Moreover, the concentration of thrombin needed for 50 percent of platelets to express the CD62 antigen P-selectin at the surface increased from 0.05 U per mL in fresh platelets to 0.11, 0.56, and 1.2 U per mL in platelets stored for 3, 7, and 10 days, respectively. Thrombin-binding experiments demonstrated a significant reduction in the number of high-affinity binding sites throughout storage of PCs (55 +/- 21 sites/platelet in 10-day-stored platelets vs. 73 +/- 25 in fresh platelets). A significant correlation was also observed between the number of high-affinity thrombin-binding sites and surface expression of GPIb alpha. Selective blockage of the thrombin-binding site on GPIb alpha with monoclonal antibody LJ-Ib10 also inhibited the response of fresh platelets to thrombin, up to a level equivalent to that found in 3-day-stored platelets. CONCLUSION: The loss of the GPIb alpha-located high-affinity thrombin-binding site may impair the ability of platelets to become activated by thrombin as storage time increases.

Antibodies, Monoclonal↗

Crossmatch-compatible platelets improve corrected count increments in patients who are refractory to randomly selected platelets.

BACKGROUND: HLA-matched platelets and crossmatch-compatible platelets are used to support thrombocytopenic patients who are refractory to randomly selected platelets. Data supporting the effectiveness of crossmatch-compatible platelets are limited, being essentially restricted to the subset of refractory patients previously shown to be alloimmunized. The authors' hospital does not test for alloimmunization. To determine the effectiveness of crossmatch-compatible platelets in an unselected group of refractory patients, the use of such platelets for all patients who are refractory to random-donor platelets was reviewed. STUDY DESIGN AND METHODS: All patients who received crossmatch-compatible platelets between January 1991 and May 1994 were retrospectively reviewed. All study patients were refractory to random-donor platelets, having two consecutive corrected count increments (CCIs) of < 10,000. A solid-phase red cell adherence method was used for platelet crossmatching, and CCI was used to monitor the effectiveness of each platelet transfusion. RESULTS: A total of 475 crossmatch-compatible platelet components were administered to 66 evaluable patients who were refractory to random-donor platelets. A significant improvement was found in the mean CCI when crossmatch-compatible platelets were compared with randomly selected platelets (p < 0.0001): an increase of 8000 +/- 6100 (mean +/- SD). In 59 percent (39/ 66) of the patients, the mean CCI improved to at least 7,500 and in 41 percent (27/66) to at least 10,000. If the 10 patients for whom crossmatch-compatible platelets were not identified are included, the mean CCI in 51 percent (37/76) of the refractory patients improved to at least 7,500; in 36 percent (27/76), it improved to at least 10,000. The effectiveness of crossmatch-compatible platelets did not decline with continued use. CONCLUSION: Crossmatch-compatible platelet components significantly improve the mean CCI for approximately one-half of patients who are refractory to random-donor platelets, even when the patients are not preselected for having alloimmunization to explain their refractory state.

Blood Grouping and Crossmatching↗

Improved removal of white cells with minimal platelet loss by filtration of apheresis platelets during collection.

BACKGROUND: Filtration of apheresis platelets to remove white cells (WBCs) requires operator intervention after the collection procedure (postcollection filtration), which may cause variable and unsatisfactory filter performance (WBC removal and platelet loss). The MCS+ LN9000 apheresis system filters platelets through a WBC-reduction filter during each collection cycle (continuous filtration) at a flow rate of 15 to 25 mL per minute. Apheresis platelets obtained by continuous filtration were evaluated in terms of platelet loss, WBC removal, and platelet storage properties and then were compared to unfiltered apheresis platelets and to apheresis platelets that underwent postcollection filtration. Two WBC-reduction filters were tested (LRF6 and LRFXL). STUDY DESIGN AND METHODS: In 70 apheresis platelets, postcollection filtration was performed by using the LRF6 at flow rates of 80 mL per minute (n = 30) and 50 mL per minute (n = 30) and the LRFXL at 50 mL per minute (n = 10). One hundred fifty-eight apheresis platelets underwent continuous filtration through the LRF6 (n = 58) or the LRFXL (n = 100). Unfiltered apheresis platelets (controls) (n = 30) were obtained by the same collection protocol. RESULTS: Estimated platelet loss with continuous filtration was 7 percent for the LRFXL and 3 percent for the LRF6. A reduction in the filtration flow rate from 80 to 50 mL per minute with postcollection filtration through the LRF6 resulted in markedly lower WBC levels, with 10 percent versus 57 percent of the apheresis platelets having WBC counts <1 x 10(5), respectively. Additional improvements in WBC removal were found with continuous filtration; 85 percent of the apheresis platelets filtered with the LRF6 and 100 percent of the apheresis platelets filtered with the LRFXL had WBC counts <1 x 10(5). CONCLUSIONS: Continuous or postcollection filtration of freshly collected apheresis platelets resulted in minimal platelet loss. Better WBC removal from apheresis platelets was obtained with continuous filtration than with postcollection filtration, likely because of the slower flow rate. Platelet storage quality was not affected by filtration.

Blood Preservation↗

The effects of heparin and extracorporeal circulation on platelet counts and platelet microaggregation during cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass is associated with platelet activation and reduced platelet counts. Platelet activation may artifactually lower platelet counts by causing aggregation. In vivo platelet activation may increase existent platelet microaggregation ex vivo. We studied platelet counts and existent platelet microaggregation at different stages of cardiopulmonary bypass. METHODS: Twenty-one patients were studied before and after heparinization (300 U. kg(-1)) and at the end of cardiopulmonary bypass. Unaggregated (or single) platelets were counted in hirudin-anticoagulated blood, and total platelets were counted in ethylenediaminetetraacetic acid-anticoagulated blood. RESULTS: The total platelet count, 198 +/- 61 x 10(9). L(-1), was unaffected by heparin and stayed at 197 +/- 60 x 10(9). L(-1) (P =.7) but fell during extracorporeal circulation; the hemodilution-corrected count was 163 +/- 52 x 10(9). L(-1) (P =.0004). Heparinization reduced the unaggregated platelet count from (mean +/- 1 SD) 178 +/- 62 x 10(9). L(-1) to 155 +/- 60 x 10(9). L(-1) (P =.0001). Extracorporeal circulation had little additional effect. The hemodilution-corrected count was 142 +/- 48 x 10(9). L(-1) (P =.6). CONCLUSIONS: Heparinization caused platelet activation and increased existent platelet microaggregation ex vivo. During extracorporeal circulation, there was a reduction in total platelets that was greater than could be explained by hemodilution alone, but the unaggregated platelet count did not change significantly when corrected for hemodilution. Furthermore, the increased platelet microaggregation observed after heparinization was no longer evident after this loss. These findings suggest that during extracorporeal circulation, the platelets that formed into microaggregates after heparinization were lost from the circulation in preference to single platelets.

Adult↗

von Willebrand factor binds to platelets and induces aggregation in platelet-type but not type IIB von Willebrand disease.

Platelet-type von Willebrand disease (vWD) and pseudo-vWD are two recently described intrinsic platelet defects characterized by enhanced ristocetin-induced agglutination in platelet-rich plasma. A similar finding is also typical of type IIB vWD, where it has been related to a von Willebrand factor (vWF) rather than a platelet abnormality. Platelet aggregation induced by unmodified human vWF in the absence of other stimuli has been reported in pseudo-vWD. In this study we demonstrate that vWF induces aggregation in platelet-type but not type IIB vWD. Aggregation is observed when normal plasma cryoprecipitate or purified vWF are added to platelet-rich plasma. Cryoprecipitate also aggregates washed platelets, although at higher concentrations than required for platelet-rich plasma. Purified vWF, however, induces significant aggregation of washed platelets only when plasma is added. EDTA inhibits vWF-induced aggregation. Its effect can be overcome by calcium but much less effectively by magnesium ions. Unstimulated platelets in platelet-rich plasma from patients with platelet-type but not type IIB vWD bind 125I-vWF in a specific and saturable manner. All different sized multimers of vWF become associated with platelets. Both aggregation and binding exhibit a similar vWF concentration dependence, suggesting that a correlation exists between these two events. Removal of ADP by appropriate consuming systems is without effect upon such binding or upon vWF-induced aggregation. Thrombin-induced 125I-vWF binding to washed platelets is normal in platelet-type as well as type IIB vWD. These results demonstrate that a specific binding site for unmodified human vWF is exposed on unstimulated platelets in platelet-type vWD. The relatively high vWF concentrations required for aggregation and binding may explain the lack of significant in vivo aggregation and thrombocytopenia in these patients. Moreover, these studies provide additional evidence that platelet-type and type IIB vWD are different diseases with distinct pathogeneses.

Adenosine Diphosphate↗

Prophylactic platelet transfusions from healthy apheresis platelet donors undergoing treatment with thrombopoietin.

Many patients receiving dose-intensive chemotherapy acquire thrombocytopenia and need platelet transfusions. A study was conducted to determine whether platelets harvested from healthy donors treated with thrombopoietin could provide larger increases in platelet counts and thereby delay time to next platelet transfusion compared to routinely available platelets given to thrombocytopenic patients. Community platelet donors received either 1 or 3 microg/kg pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo and then donated platelets 10 to 15 days later. One hundred sixty-six of these platelet concentrates were then transfused to 120 patients with platelets counts 25 x 10(9)/L or lower. Pretransfusion platelet counts (11 x 10(9)/L) were similar for recipients of placebo-derived and PEG-rHuMGDF-derived platelets. Early after transfusion, the median platelet count increment was higher in patients receiving PEG-rHuMGDF-derived platelets: 19 (range, -12-66) x 10(9)/L, 41 (range, 5-133) x 10(9)/L, and 82 (range, -4-188) x 10(9)/L for placebo-, 1-microg/kg-, and 3-micro/kg-derived platelets, respectively. This difference was maintained 18 to 24 hours after transfusion. Transfusion-free intervals were 1.72, 2.64, and 3.80 days for the recipients of the placebo-, 1-microg/kg-, and 3-micro/kg-derived platelets, respectively. The rate of transfusion-related adverse events was not different in recipients of placebo-derived and PEG-rHuMGDF-derived platelets. Therefore, when transfused into patients with thrombocytopenia, platelets collected from healthy donors undergoing thrombopoietin therapy were safe and resulted in significantly greater platelet count increments and longer transfusion-free intervals than platelets obtained from donors treated with placebo.

Adolescent↗