Flow cytometry analysis of platelet antibodies

We have devised assays to detect both circulating alloantibodies to platelets (indirect assay) and platelet-association IgG and IgM (direct assay) using a flow cytometric technique. A variety of patients with immune thrombocytopenia were studied. Employment of a confocal lens in the flow cytometer increased the discrimination power of the instrument. Patients with autoimmune thrombocytopenia (idiopathic thrombocytic purpura [ITP], systemic lupus erythematosus (SLE), lymphoma, leukemia, and drug-induced thrombocytopenia showed a significant increase in platelet-associated antibody. Circulating antibodies to platelets (alloantibodies) were demonstrated in cases of platelet refractoriness and neonatal isoimmune purpura. Day-today precision of the assays ranged from 3% to 6% (coefficient of variation). No interference was shown in the presence of hemoglobin (5 g/L), triglycerides (10 g/L), or polyclonal and monoclonal immunoglobulinemia (50 g/L: IgG, IgA, IgM). The sensitivity of the direct assay was 500 attograms of IgG or IgM platelet.     

Recurrent venous thrombosis following free flap surgery: The role of heparin-induced thrombocytopenia

Complications following free tissue transfer have been well established in the literature. Common and rare causes of free flap failure must be addressed by the treating surgeon when microvascular patency is threatened. With the evolution and prevalence of microsurgery, ‘rare’ causes of free flap failure will become increasingly frequent. A high index of suspicion must be established in patients with multiple failed operative interventions. A case of recurrent free flap failure secondary to heparin-induced thrombocytopenia is presented in a patient with a history of squamous cell carcinoma of the floor of the mouth, and a long-standing history of alcohol and tobacco consumption.     

Hematopoietic turnover index in reactive and neoplastic bone marrow lesions: quantification by apoptosis and PCNA labeling

 In order to determine the dynamics of hematopoietic cell turnover, proliferative activity and incidence of apoptosis (programmed cell death) were evaluated in bone marrow trephine biopsies. Selection of patients (20 in each group) included in addition to a control group, idiopathic thrombocytopenia (ITP), reactive thrombocytosis (TH), secondary polycythemia-smokers' polyglobuly (PG), primary (essential-hemorrhagic) thrombocythemia (PTH), polycythemia vera (PV), and finally acute myeloid leukemia (AML). Apoptosis was demonstrated by the in situ end-labeling technique (ISEL) and proliferative activity by applying the monoclonal antibody PC10 raised against proliferating cell nuclear antigen (PCNA). To assess dynamic features of hematopoiesis, an index was calculated consisting of the ratio between PCNA-positive nuclei and the apoptotic cell fraction. This factor was termed the hematopoietic turnover index (HTI). Morphometric analysis revealed that the HTI was significantly increased in AML and PV. According to cell culture studies both disorders are characterized by either a prevalent proliferation of the myeloid or erythroid cell mass. On the other hand, PG, PTH, and TH showed no relevant enhancement of this index in comparison to the control specimen. In vitro experiment results are in keeping with the finding that PG and PTH are not associated with a significant expansion of the erythroid lineage (CFU-E). Similar to ITP and TH, in PTH megakaryocyte proliferation (CFU-MEG) is the predominant feature of cell turnover. Differences between PTH and TH are in line with the reduced in vitro formation of CFU-MEG in the latter disorder. In conclusion, our in situ study on turnover rates of the bone marrow in various neoplastic and reactive lesions extends previous experimental data on hematopoietic cell kinetics. Received: 10 March 1997 / Accepted: 18 May 1997     

Splenic irradiation in HIV-related thrombocytopenia

Splenic irradiation has been used in patients with HIV-related thrombocytopenia. This retrospective review deals with four patients treated with low dose splenic irradiation. All patients had an increase in platelet count and tolerated the treatment without side effects. However, the treatment response lasted for several months only.     

Atypical hemolytic uremic syndrome in human immunodeficiency virus-1-infected children

We describe the clinical and pathological findings of the hemolytic uremic syndrome (HUS) in two children with human immunodeficiency virus (HIV) infection. Both patients presented with microangiopathic hemolytic anemia, thrombocytopenia, and subsequently developed renal failure. The diagnosis of HUS was confirmed by renal histopathology in both patients. None of these children presented with bloody diarrhea, evidence of circulating antibody response to Escherichia coli O157 lipopolysaccharide, or other known risk factors for HUS, except for the presence of HIV infection. Each patient was treated with intravenous plasma infusion and renal replacement therapy. Their clinical course was characterized by non-oliguria and lack of significant hypertension throughout the acute phase of the disease. Despite these favorable clinical parameters, both patients developed end-stage renal failure. The etiology of this atypical HUS characterized by poor renal survival remains unknown and the role of HIV infection in its pathogenesis, although possible, is unclear. Received March 5, 1996; received in revised form and accepted October 15, 1996     

Chronic isolated macrothrombocytopenia with autosomal dominant transmission: a morphological and qualitative platelet disorder

Abstract: We studied 47 subjects belonging to 13 unrelated families with a history of mild haemorrhagic diathesis and chronic thrombocytopenia. 36 patients presented some degree of thrombocytopenia: 7/36 (19%) had slight thrombocytopenia (100–150×10⁹/L); 26/36 (72%) had mild thrombocytopenia (50–100×10⁹/L) and 3/36 (8%) had severe thrombocytopenia (<50×10⁹/L). No correlation was observed between platelet count and the degree of haemorrhagic diathesis, which was mild in the majority of patients. Transmission was autosomal dominant. Platelet anisocytosis, increased percentage of large platelets and absence of leukocyte inclusions were observed in 26/30 (87%) of the examined blood smears. The ultrastructural appearance of platelets was normal. Megakaryocytes appeared normal in number in 10/10 patients, but showed asynchronous nuclear-cytoplasm maturation and mainly nonlobulated nuclei. Platelet aggregation was studied in 26 patients and either increased or decreased curves were variably observed in response to different aggregating agents. Platelet-associated IgG (PAIgG) was increased in 18/31 (58%) patients, while serum autoantibodies against platelet glycoproteins Ib/IX or IIb/IIIa were demonstrable in only 1 case. An increased expression of platelet surface glycoproteins Ib and IIb/IIIa, as studied by murine monoclonal antibodies binding in 17 cases, was observed. Platelet survival performed by 111In-oxine-labelled autologous platelets was normal in the 3 studied patients. Congenital macrothrombocytopenia confirms to be a distinct clinical disorder for which the name of “chronic isolated hereditary macrothrombocytopenia” is proposed.     

病毒性肝炎患者血小板减少与脾脏肿大和血小板生成素的关系

目的研究病毒性肝炎患者血小板减少、脾脏肿大及血清血小板生成素(TPO)水平之间的关系,以探讨病毒性肝炎患者血小板减少的发病机制。方法应用腹部彩色B超测量58例病毒性肝炎并血小板减少症患者(A组)以及48例病毒性肝炎血小板正常患者(B组)和20例健康志愿者(C组)的脾脏大小,并采用酶联免疫吸附法测定其血清TPO水平。结果A组脾脏厚度(50.49±13.58mm)明显大于B组(38.45±8.14mm,P<0.01)和C组(32.25±3.73mm,P<0.01)。血小板数与脾脏大小呈负相关(r=-0.553,P<0.01)。血清TPO水平A组(88.05±17.09pg/mL)明显低于B组(100.20±17.63pg/mL,P<0.01)和C组(108.96±25.90pg/mL,P<0.01)。血小板数与血清TPO水平呈正相关(r=0.407,P<0.01)。结论病毒性肝炎血小板减少与脾脏肿大、血清TPO水平下降有关。     

Early onset of autoimmunity in MRL/++ mice following immunization with beta 2 glycoprotein I.

Antiphospholipid antibodies (aPL) are associated with thrombosis, thrombocytopenia and recurrent fetal loss in humans and in some animal models. Immunization with beta 2 glycoprotein I (beta 2GPI) induced aPL production in normal rabbits and mice. However, the association of these antibodies with disease manifestations remains controversial. To determine whether induction of aPL by beta 2GPI immunization in an autoimmune strain of mice (MRL/++) would result in acceleration of clinical and serological autoimmune disease manifestations, three groups of 8-week-old female mice were studied. One group was immunized with beta 2GPI, and one with ovalbumin (OVA); the third was not immunized. After two booster injections, sera were analysed for the presence of anticardiolipin (aCL) and anti-DNA by ELISA and anti-nuclear antibody (ANA) by immunofluorescence. Mice were studied for thrombocytopenia, proteinuria, fecundity rates, litter sizes and the development of central nervous system dysfunction. Elevated levels of aCL, anti-DNA and ANA were detected in all beta 2GPI-immunized, in three OVA-immunized, and in none of the unimmunized mice. The anti-DNA antibodies were inhibited by CL micelles, suggesting cross-reactivity between aCL and anti-DNA. Platelet counts, fecundity rates and litter size were reduced in beta 2GPI-immunized but not in OVA-immunized or unimmunized mice. None of the mice developed neurological dysfunction or significant proteinuria over a 10-week period post-immunization. These findings suggest that beta 2GPI immunization induces aPL in MRL/++ mice associated with accelerated autoimmune manifestations resembling the antiphospholipid syndrome.     

Diagnostic performance of aPS/PT antibodies in neuropsychiatric lupus and cardiovascular complications of systemic lupus erythematosus

Abstract

Background: Systemic lupus erythematosus (SLE) is associated with a constellation of complications affecting multiple organs, including neuropsychiatric manifestations (NPSLE) and ischaemic events, leading to increased long-term morbidity. Antiphospholipid antibodies (aPL) are a major determinant of vascular inflammation and thromboembolic risk. The diagnostic role of anti-phosphatidylserine/prothrombin (aPS/PT) antibodies in this setting is incompletely defined.

Aim: To verify whether aPS/PT add to diagnostics and disease stratification in patients with SLE with or without other aPL.

Methods: 131 consecutive patients were studied, including 20 patients with SLE and secondary antiphospholipid syndrome (APS). aPS/PT IgG and IgM were assessed through ELISA and patients were stratified based on the presence of other aPL, on their clinical and laboratory features at time of blood sampling and on their clinical history. Synthetic indices of disease activity, chronic damage and cardiovascular risk were calculated at time of venipuncture.

Results: Fifty-one (38.9%) patients with SLE had aPS/PT and 15 (11.5%) patients had aPS/PT as the only aPL (aPS/PT-only). aPS/PT-only patients had a significantly higher prevalence of NPSLE than quadruple aPL-negative patients (p?=?.007). Patients with aPS/PT were more likely to have a history of ischaemia, thrombocytopenia and Libman–Sacks’ endocarditis. The presence of aPS/PT also associated with previous accrual of at least one damage item (p?=?.043), but had limited predictive values for damage progression in the short term.

Conclusion: aPS/PT antibodies provide non-redundant information that could contribute to risk assessment and stratification of patients with SLE.     

Effect of Antiphospholipid Antibodies on β2Glycoprotein I-Phospholipid Interaction

PROBLEM: β₂ glycoprotein I (β₂GPI) physiologically binds to negatively charged phospholipids (PLs) and is a natural regulator of the coagulation cascade. Thrombotic clinical complications and recurrent fetal loss associated with autoimmune antiphospholipid (aPL) antibodies are thought to be related to their binding to β₂GPI-PL complex and interference with the physiological function of β₂GPI. METHOD OF STUDY: To investigate the effect of aPL on β₂GPI-PL interaction, we studied the binding of biotinylated β₂GPI to cardiolipin (CL) by enzyme-linked immunosorbent assay (ELISA) in the presence and absence of purified aPL immunoglobulin G (IgG) antibodies. RESULTS: Adding five different aPL IgG antibodies with different levels of aPL activity isolated from the sera of five patients with aPL-associated recurrent fetal death greatly increased the binding of biotinylated β₂GPI to CL-coated plates. The optical densities (ODs) were 0.635, 0.890, and 1.265 in the presence of three aPL IgG antibodies, compared to 0.425 in the absence of aPL IgG. In contrast, normal human IgG had no enhancing effect. The OD was 0.480 and 0.425, respectively. The enhancement of β₂GPI binding to CL by aPL IgG correlated with the titers of aPL antibodies. The use of phosphate-buffered saline with increasing salt concentrations as a washing buffer for the ELISA resulted in more stable binding of β₂GPI to PL in the presence of aPL IgG. CONCLUSIONS: These findings suggest that the binding of autoimmune aPL antibodies to β₂GPI-PL complex results in abnormally tighter interaction between β₂GPI and PLs, which may lead to physiological dysfunction of β₂GPI as a regulator of coagulation.