Comparison of the measurement of surface or total platelet-associated IgG in the diagnosis of immune thrombocytopenia

Platelet-associated IgG (PAIgG) can be measured on intact platelets or following platelet lysis. Measurement of PAIgG following platelet lysis may provide different or additional information compared to PAIgG measured on intact platelets. The PAIgG of lysed platelets represents "total" PAIgG, ie, IgG on the surface of platelets plus any IgG that was inside the platelet. To investigate the clinical relevance of the two types of PAIgG assay we performed a prospective study on washed platelets collected from 47 patients with idiopathic thrombocytopenic purpura (ITP). The PAIgG was measured on intact and lysed platelets using an immunoradiometric assay. Platelet-associated IgG was 2-3 times higher when measured on lysed platelets from healthy controls or patients with ITP compared to PAIgG measured on the same intact platelets. The higher level of PAIgG observed following platelet lysis was not due to the reactions not achieving equilibrium. Using lysed platelets, PAIgG was elevated on 29 of 47 samples from different ITP patients and elevated in 31 samples when measured on intact platelets. The PAIgG is invariably higher when measured following platelet lysis compared measurements made on intact platelets. Neither technique offers a diagnostic advantage over the other.     

Influences of antiplatelet autoantibodies on platelet function in immune thrombocytopenic purpura

We investigated the characteristics of the antiplatelet autoantibodies in 60 patients with ITP. Using flow cytometry, the binding of monoclonal antibodies to the platelet glycoprotein (GP) IIb/IIIa complex and to GPIb was examined in these patients. The extent of binding was decreased in 15 patients (anti-GPIIb/IIIa in 12 patients and both anti-GPIIb/IIIa and anti-GPIb in 3 patients). Western blotting revealed that 10 of these 15 patients had either anti-GPIIb or anti-GPIIIa and 2 had anti-GPIb autoantibodies, ADP-induced aggregation of normal platelets was inhibited by autoantibodies in 12 of 60 patients, and 11 of these had anti-GPIIb/IIIa antibodies. Ristocetin-induced aggregation was inhibited in 4 of these patients, and 2 with prominent inhibition had anti-GPIb antibodies. There was a significant relationship between platelet-associated IgG value and ATP secretion. These results suggest that some antiplatelet autoantibodies can affect platelet function and thus have an influence on the pathophysiology of ITP.     

Reevaluation of platelet function in chronic immune thrombocytopenia: impacts of platelet size,platelet-associated anti-αIIbβ3 antibodies and thrombopoietin receptor agonists

Platelet function of immune thrombocytopenia (ITP) has been controversial because of methodological problems associated with low platelet counts. In this study, we evaluated platelet function in 21 patients with chronic ITP (cITP) using the recently developed flow cytometry (FCM)-based platelet aggregation assay (FCA) along with a PAC1/CD62P assay. Since ITP platelets are larger than controls, whole platelets (whole gating method) and size-adjusted platelets (size-adjusted method) were analysed in the PAC1/CD62P via FCM. We found that: (i) aggregation was equivalent [phorbol myristate acetate (PMA) or adenosine diphosphate (ADP)-induced] or enhanced [protease-activated receptor 1-activating peptide (PAR1AP)-induced] in cITP compared with control by FCA; (ii) PAC1 or CD62P was also equivalent or enhanced in cITP in the whole gating method; and (iii) in sharp contrast, the size-adjusted method revealed that ADP-, PAR1AP-, and collagen synthetic liquid reactive peptide (SRP)-induced PAC1 and ADP-induced CD62P were impaired in cITP. These data suggested that an increase in the number of larger-sized platelets may compensate for the impaired platelet function of cITP, leading to non-inferiority of overall platelet function in cITP. Furthermore, we revealed that ADP-induced aggregation was impaired in the patients with thrombopoietin receptor agonists (TPO-RAs) or platelet-associated anti-αIIbβ3 antibodies compared with the control, suggesting that the presence of anti-αIIbβ3 autoantibodies and/or administration of TPO-RAs may have a negative impact on platelet function.     

Increase in density and accumulation of serotonin by human aging platelets

⁵¹Cr-labeled autologous platelets were infused into splenectomized subjects and the specific radioactivities of high-density (HD) and low-density (LD) platelet subpopulations were determined sequentially in postinfusion samples. A rapid decrease in the specific radioactivity of LD cohorts (T 1/2 = 2.5 days) was observed, but the specific radioactivity of HD platelets remained constant or increased slightly during the first 4 days and then gradually declined for the next 5 days. No experimental artifacts during the platelet-labeling steps that could account for these results were demonstrated. These findings confirm previous observations in eusplenic individuals and support the hypothesis that human LD platelets are, on the average, younger than HD platelets. LD platelets contain 33.8 ± 13.5 ng serotonin (5HT)/10⁸ platelets and HD platelets 76.8 ± 9.5 ng 5HT/10⁸ platelets (P < 0.001). Sequential measurements of 5HT in PRP platelets were performed during the recovery phase of thrombocytopenia following splenectomy in patients with idiopathic thrombocytopenic purpura (ITP), a condition associated with aging of platelets in circulation. Presplenectomy platelet 5HT was 17.7 ng/10⁸ platelets and on days 1, 6, and 12 after surgery it increased to 18.1, 37.8, and 61.0 ng/10⁸ platelets (n = 7). When three healthy volunteers were given aspirin (500 mg/day) for up to 15 days, no significant change in the 5HT content of circulating platelets was observed. If aspirin blocks, at least partially, the secretory process in vivo without interfering the 5HT uptake by the platelets, this finding stands against the possibility that a net depletion of 5HT occurs during the life-span of normal human platelets. The observation that human HD platelets, enriched with older cells, contain more 5HT than LD platelets taken together with the parallel increase in platelet 5HT and age during the recovery from thrombocytopenia in ITP patients and the lack of effect of aspirin on platelet 5HT content, provides initial evidence that human platelets accumulate 5HT during their life-span in circulation.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Platelet aggregability and platelet volume in the postoperative course: problems in the platelet aggregation test derived from the measurement of platelet volume

Platelet count, aggregability and volume in the postoperative course of 20 patients were examined. Platelet count was decreased on the 1st postoperative d, and increased on the 7th and 14th d compared with the preoperative value. The maximal aggregation rate of platelets induced by ADP was decreased on the 3rd postoperative d, and then recovered to the preoperative level. In contrast, platelet volume was only slightly increased on the 3rd postoperative d. In this study, there was no correlation between platelet aggregability and platelet volume in PRP. We have proposed one parameter, 'platelet concentration ratio' (platelet concentration in PRP/platelet concentration in whole blood). In the postoperative course, this concentration ratio changed depending on platelet volume, and possibly on other conditions of blood such as hematocrit, viscosity and specific gravity. The concentration ratio influenced the subpopulations of platelets in PRP. Platelet aggregation tests may be performed using PRP in which platelet subpopulations differ from those in whole blood, especially in the postoperative state.     

Platelet calcium-dependent proteins: Identification and localization of the calcium-dependent regulator,calmodulin, in platelets

The calcium-dependent regulator protein, calmodulin, is a 17,000 molecular weight polypeptide which binds calcium and has been shown to confer calcium sensitivity on contractile and other proteins. In the present study, we have examined the presence and subcellular distribution of this protein in preparations of human platelets. Calmodulin was quantified using a two-stage phosphodiesterase assay. Whole platelets contained 1.33 ± 0.06 units calmodulin per 10⁶ platelets or 26.5 ± 3.4 fg calmodulin per platelet. The distribution of calmodulin in the platelet was predominantly soluble with over 80 percent of calmodulin activity in the soluble fraction of the cell. There was no apparent difference in the distribution of calmodulin between soluble and particulate compartments in recalcified platelet homogenates compared to homogenates in EDTA. Indirect immunofluorescent studies with monospecific antisera to dinitrophenylated calmodulin showed intense staining of platelets in a diffuse pattern. The identification of calmodulin in platelets raises the possibility that this protein may participate in calcium-dependent reactions important in platelet aggregation and release.     

The amount of platelet-bound albumin parallels the amount of IgG on washed platelets from patients with immune thrombocytopenia

The biologic relevance of the increased platelet-associated IgG (PAIgG) on platelets from patients with idiopathic thrombocytopenic purpura (ITP) is unclear. Platelets from ITP patients are often larger than normal, and it is possible that the increased IgG is not specific but passively related to platelet size. The measurement of platelet-bound albumin could provide information concerning the specificity of the platelet-bound IgG, since albumin, like IgG, is a plasma protein, but unlike IgG, is not an active participant in immunologic reactions. Albumin is also a normal constituent of platelet membrane, and increased platelet albumin could indicate an increased platelet mass. Platelet-bound albumin, IgG, and total platelet protein were measured on both intact and disrupted platelets from healthy individuals (n = 25) and patients with ITP (n = 21). Platelet IgG and albumin were measured in an immunoradiometric assay using intact antisera and F(ab')2 fragments prepared from the same antisera. There was no relationship between platelet-bound IgG or albumin, and platelet size measured by either platelet protein or platelet volume, (r less than 0.3 for all interactions). In contrast, there was a significant correlation between platelet-bound albumin and platelet-bound IgG (r = 0.7, n = 21, p less than 0.001). Those patients with elevated platelet PAIgG also had elevated platelet albumin, and this relationship was irrespective of the total platelet protein content or mean platelet volume. It is possible that the increased platelet-bound IgG in ITP reflects an increase in platelet surface area or contaminating platelet fragments that are not manifested as an increase in platelet volume or total platelet protein. Alternatively, a platelet membrane abnormality may occur in ITP that results in the uptake of significant amounts of plasma proteins. Either possibility implies that not all of the IgG on platelets from patients with ITP is pathologic IgG.     

Relationship between platelet volume and anti-platelet autoantibodies in idiopathic thrombocytopenic purpura

We used flow cytometry to explore the relationship between platelet volume and anti-platelet autoantibodies in 71 patients with idiopathic thrombocytopenic purpura (ITP). An increase in platelet volume was found more frequently in patients with a platelet count of less than 20,000/microliters. Platelet volume was larger in patients without anti-GPIIb/IIIa autoantibodies than in patients with these autoantibodies. Furthermore, the platelet count was significantly lower in patients without anti-GIIb/IIIa autoantibodies than in the patients with these autoantibodies. There was a positive correlation between a large platelet volume in patients with a platelet count of less than 30,000/microliters and high platelet-associated IgM levels. These results suggest that the platelet volume is related to the severity of thrombocytopenia in ITP.     

The relation of thrombokinetics to bone marrow megakaryocytes in idiopathic thrombocytopenic purpura (ITP).

In 23 patients with untreated idiopathic thrombocytopenic purpura (TP), the relation of thrombokinetics to quantitative determinations of megakaryocytes in bone marrow sections was studied. The megakaryocytes were classified into maturation stages, and platelet sizes were determined. Megarkaryocyte number and volume per microliter of bone marrow were significantly higher in ITP as compared to controls. The megakaryocyte number and volume were inversely related to the peripheral platelet count. Platelet production rate was significantly increased in ITP and related to the megakaryocyte number and volume. The megakaryocytes were shifted towards more immature forms in ITP, suggesting an increased turnover rate of the expanded recognizable metakaryocyte compartment. Platelet size was significantly increased in ITP, and the mean platelet diameter was 1.6 times normal. There was a significant relationship between platelet size and platelet production rate, as well as an inverse relationship between platelet size and platelet mean life-span (MLS). There was also a significant correlation between platelet size and the proportion of young megakaryocytes.     

Analysis of anti-platelet antibody and platelet volume in idiopathic thrombocytopenic purpura

We investigated platelets and plasma from patients with idiopathic thrombocytopenic purpura (ITP) to elucidate the antigenic determinants at which their autoantibodies are directed, and studied the relationship between anti-platelet antibody and platelet volume. We used flow cytometry to detect platelet-associated IgG (PAIgG), C3 (PAC3), IgM (PAIgM) and platelet volume, and also to determine the binding rate of monoclonal anti-platelet antibodies in patients with ITP. The following results were obtained. 1. Both anti-GPIIb/IIIa autoantibodies (21 of 71 patients) and anti-GPIb autoantibodies (3 of 71 patients) were found in ITP. 2. The decrease in platelet count in patients without anti-GPIIb/IIIa autoantibodies was significant. 3. The increase in platelet volume was found more frequently in patients with a platelet count less than 50,000 and in untreated patients. 4. There was a positive correlation between the platelet volume and PAIgM in patients with a platelet count less than 30,000 and high levels of PAIgM.     

Pathophysiological significance of simultaneous measurement of reticulated platelets, large platelets and serum thrombopoietin in non-neoplastic thrombocytopenic disorders

An automated reticulocyte counter using flowcytometric analysis, the R-3000 (Sysmex Inc. Kobe, Japan), has recently been modified to determine reticulated platelets (RPs) and large platelets (LPs). We measured frequencies of RPs, LPs in total platelet count and serum thrombopoietin concentration comprehensively in non-neoplastic thrombocytopenic patients with immune thrombocytopenic purpura (ITP, n = 23), aplastic anemia (AA, n = 21), liver cirrhosis (LC, n= 17), and hematologically normal subjects (control, n = 151). ITP was characterized as high frequencies of both RP and LP, AA as high RP frequency and elevated thrombopoietin concentration, and LC as no difference compared with control. Interestingly, the frequency of RP appeared to depend on total platelet count rather than the cause of thrombocytopenia, while the frequency of LP appeared to depend much less on total platelet count. Furthermore, significant positive correlations were observed between frequencies of RP and LP in control, ITP and LC subjects, in whom bone marrow stem cells are intrinsically normal. However, there was no such correlation in AA patients with stem cell deficiency, suggesting that this correlation might be a useful new parameter for detecting qualitatively abnormal platelets. Measurement of RP and LP is thus useful for elucidating the pathophysiology of thrombocytopenic disorders.     

Platelet volume and intraplatelet adenine nucleotides in various hematologic disorders

By recent advanced techniques, blood platelets have proved to be varied in size and metabolism in various hematologic disorders. We examined platelet volume and intraplatelet adenine nucleotides in 36 patients with various hematologic disorders in order to clarify the quantitative platelet abnormalities. Platelet volumes were smaller in patients with acute leukemia and aplastic anemia, and larger in patients with immune thrombocytopenic purpura (ITP). The amount of intraplatelet ADP was decreased and ATP/ADP ratio was increased in acute leukemia, aplastic anemia and myeloproliferative disorders (MPD), which strongly suggested the presence of storage pool deficiency in these patients. Intraplatelet ADP per volume was decreased in acute leukemia, aplastic anemia and MPD, and ATP per volume was decreased in aplastic anemia. ATP content was increased in ITP in proportion to the increased platelet volume. These parameters were examined in 36 patients with the following hematologic disorders: 7 acute leukemia treated with cytotoxic chemotherapy, 5 aplastic anemia, 3 paroxysmal nocturnal hemoglobinuria (PNH), 9 immune thrombocytopenic purpura (ITP), 5 hypersplenism and 7 myeloproliferative disorders (MPD).     

Platelets in idiopathic thrombocytopenic purpura are increased in size but are of normal density

To determine whether platelet size and volume are related to one another or to platelet age, subpopulations of platelets from patients with idiopathic thrombocytopenic purpura (ITP) have been produced on the basis of density using Percoll gradients. The density distribution of platelets from patients with ITP and from patients with other forms of thrombocytopenia (thought to be nonimmune in nature) was the same as in normal controls. However, the platelets in each density subpopulation from ITP patients were increased in size. beta-thromboglobulin (beta TG) content of platelets from each patient group and the normals increased with density and tended to be higher in ITP than in normal controls. beta TG concentration per unit platelet volume and its level in plasma were similar in ITP patients and in normal controls. This suggests that the apparently normal density of ITP platelets was not a result of degranulation of large, dense platelets. Thus platelet size and density are independently determined and the increased size of platelets in immune thrombocytopenia may be the result of abnormalities in their production.     

A rapid quantitation of platelet-associated IgG by nephelometry

Platelet-associated IgG (PAIgG) was measured by a simple rapid nephelometric technique using washed solubilized platelets and commercially available, prestandardized reagents. Normal subjects with normal platelet counts had PAIgG levels of 2.1-6.7 fg/platelet. Subjects with idiopathic immune thrombocytopenic purpura (ITP) had levels of 7.2-43.3 fg/platelet. Ninety percent of ITP patients had values exceeding 2 SD units of the mean of normal subjects. Elevated values were also found in 17% of patients with recovered ITP, patients with SLE with and without thrombocytopenia, patients with thrombocytopenia occurring during septicemia, and patients with IGg myeloma. Results can be obtained within several hours of receipt of blood specimen, and are similar to the reports that used more complex techniques.     

Flow cytometric analysis of platelet activation under calcium ion‐chelating conditions

Platelet activation and aggregation results in factitious counting and sizing in routine haematology testing. In this study, the possibility of platelet activation in anticoagulated solutions was examined. Whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. Blood treated with ethylenediaminetetraacetic acid (EDTA) exhibited platelet activation both pre‐ and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre‐ and postagitation. Heparin‐treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA‐dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, platelet activation involving platelet size change can occur in the absence of calcium ions in blood treated with EDTA.     

Platelet size for distinguishing between inherited thrombocytopenias and immune thrombocytopenia: a multicentric,real life study

The most frequent forms of inherited thrombocytopenia (IT) are characterized by platelet size abnormalities and it has been suggested that this parameter is useful for their differentiation from immune thrombocytopenia (ITP). Recently, a monocentric study identified cut‐off values for mean platelet volume (MPV) and mean platelet diameter (MPD) with good diagnostic accuracy in this respect. To validate these cut‐off values in a different and larger case series of patients, we enrolled 130 subjects with ITP and 113 with IT in six different centres. The platelet count and MPV was each measured by the instrument routinely used in each institution. In some centres, platelet count was also measured by optical microscopy. MPD was evaluated centrally by image analysis of peripheral blood films. The previously identified cut‐off value for MPV had 91% specificity in distinguishing ITP from inherited macrothrombocytopenias (mono and biallelic Bernard‐Soulier, MYH9‐related disease), while its sensitivity was greatly variable depending on the instrument used. With an appropriate instrument, specificity was 83%. The diagnostic accuracy of MPD was lower than that obtained with MPV. We concluded that MPV is a useful parameter for differentiating ITP from IT provided that it is measured by appropriate cell counters.     

Platelet associated IgG, platelet mean life span and treatment with intravenous immunoglobulin in idiopathic thrombocytopenic purpura

The clinical significance of platelet associated IgG in ITP detected by direct platelet suspension immunofluorescence test (PSIFT) was studied. The platelet mean life span (MLS) was measured with 111In-labelled platelets in 17 adult patients. All the patients had shortened platelet MLS. The direct PSIFT was positive in 14 patients. Patients were initially treated with prednisone; 12 patients with poor response to the drug were splenectomised. 8 of these 12 patients were treated with intravenous immunoglobulin (IvIg) before splenectomy. The response to IvIg was as good or better in the 3 patients with negative PSIFT, than in the 5 patients with positive PSIFT.     

Abnormal platelet function and arachidonate metabolism in chronic idiopathic thrombocytopenic purpura

We observed several patients with chronic idiopathic thrombocytopenic purpura (ITP) whose bleeding times were more prolonged than would have been expected from their platelet counts. To investigate this further, we performed in vivo and in vitro platelet function studies, assessed arachidonate metabolism, and measured platelet-associated IgG (PAIGG) in seven patients with chronic ITP. The bleeding times of three of the patients were prolonged for greater than 7 min, and all of these patients had impaired platelet aggregation and abnormal platelet arachidonic acid metabolism as reflected by increased production of the lipoxygenase product HETE and a concomitant decrease in cyclooxygenase products, TXB2 and HHT (p less than 0.001). The abnormalities noted were not due to concomitant drug ingestion, since they were present on repeated evaluation. There was no relationship between the platelet count and the bleeding time; however, there was a significant inverse correlation between the bleeding time and TXB2 production in all patients evaluated (r = 0.81; p less than 0.05). There was no relationship between the level of platelet-associated IgG and any parameter of platelet aggregation or arachidonate metabolism. The abnormalities noted should be looked for in the individual patient with chronic ITP, since the bleeding tendency is exacerbated by the superimposed impairment of platelet function even at platelet counts of greater than 50,000/cu mm, levels generally regarded as "safe."     

Relationship between size and thiazole orange fluorescence of platelets in patients undergoing high-dose chemotherapy.

The reticulated platelet count relies upon the assumption that newly formed platelets contain a residual amount of RNA which selectively binds the dye thiazole orange (TO) and greatly enhances its fluorescence signal. It has, however, recently been shown that almost half of the platelet TO-signal is derived from the labelling of dense-granule nucleotides. It is therefore possible that the higher TO fluorescence of young platelets partially derives from the higher granule content due to their larger volume. To investigate the relationship between platelet size and TO fluorescence we studied 13 patients with high-risk breast cancer undergoing high-dose chemotherapy. Mean platelet volume, platelet distribution width, platelet-large cell ratio, membrane content of glycoprotein Ib and IIb-IIIa and platelet aggregation were significantly greater during resolution than during development of thrombocytopenia, suggesting a prevalence of young and old platelets respectively. Mean TO fluorescence per cell was higher in the platelet population enriched in young cells than in that enriched in old cells, but this difference was no longer observed when the ratio TO signal/platelet size was examined. Moreover, RNase treatment and platelet degranulation reduced TO fluorescence to a similar extent in platelet populations enriched in young or old cells. Therefore our data suggest that the higher TO signal of young platelets is derived, to a significant extent, from their larger volume and granule content.