Presumptive Glucocorticoid-Induced Refractory Hypocalcemia in a Dog with Idiopathic Immune-Mediated Hypoparathyroidism, Thrombocytopenia, and Hemolytic Anemia.

Clinically relevant hypocalcemia is a well-documented complication of glucocorticoid administration in people with hypoparathyroidism. The current report describes the phenomenon in a dog. A 7 yr old neutered male Pomeranian was diagnosed with immune-mediated thrombocytopenia, immune-mediated hemolytic anemia, and primary hypoparathyroidism. This dog required long-term parenteral calcium gluconate to prevent clinical hypocalcemia despite appropriate doses of oral calcitriol and calcium carbonate. This is the first description of clinically significant presumptive glucocorticoid induced hypocalcemia in a dog with primary hypoparathyroidism.

Cord-derived mesenchymal stem cells therapy for liver cirrhosis in children with refractory Henoch-Schonlein purpura: A case report.

RATIONALE: To explore the curative effect of human umbilical cord-derived mesenchymal stem cell (ucMSC) therapy for patients with liver cirrhosis complicated with immune thrombocytopenia and refractory Henoch-Schonlein purpura (HSP). PATIENT CONCERNS: A 12-year-old boy presented to our hospital with an 11-month history of purpura on the skin of both lower limbs accompanied by thrombocytopenia. The patient had a history of repeated swelling and painful dorsum pedis, followed by skin redness. DIAGNOSIS: Bone marrow slides showed megakaryocyte maturation disorder. Based on the pathology and drug abuse history, he was diagnosed with nodular cirrhosis, secondary allergic purpura, and thrombocytopenia, etiologies related to his drugs and an immune dysfunction. INTERVENTIONS: ucMSC transplantation was performed, the liver damaging drugs were discontinued, and the appropriate liver immunosuppressive drugs were administered. ucMSCs were injected 8 times/wk in 2 months, with a median cell count of 5.65 × 10/L, ranging from 5.48 to 5.98 × 10/L. OUTCOMES: As the patient's skin rash resolved, his platelets gradually increased to >150 × 10/L and liver transaminase levels gradually decreased to a normal level. Ultrasonography of the abdomen indicated that the round nodules in the liver decreased in size and that the spleen thickness also decreased. LESSONS: This is a unique case of significant HSP with associated thrombocytopenia in a patient with liver cirrhosis. Long-term oral administration of excessive herbal medicine may cause liver damage. We believe that ucMSCs provide a novel approach for the treatment of liver cirrhosis.

Icaritin Improves Antibody-Induced Thrombocytopenia in a Mouse Model by Regulating T-cell Polarization.

Previous studies have shown that icaritin (ICT) has significant protective effects on immune thrombocytopenia (ITP), and the present study aimed to discuss the mechanism of this protective effect from the aspect of regulating T-cell polarization by an antibody-induced ITP mice model. Mice were given rat anti-mouse CD41 antibody (MWReg30) by intraperitoneal injection for 7 d to produce ITP model. At the same time, ICT was administrated at 10 mg/kg/d orally for 9 d. Peripheral blood platelets were counted by hematology analyzer. Spleen index was also tested. Spleen T-helper cell (Th), cytotoxic T-cell (CTL), Th1, Th2, Th17, regulatory T-cell (Treg), and follicular helper T-cell (Tfh) were quantified by flow cytometry. Serum Th1/Th2/Th17 cytokines were tested by mouse Th1/Th2/Th17 cytometric bead array (CBA) kit and transforming growth factor beta (TGF-ß) were analyzed by enzyme-linked immunosorbent assay (ELISA) kit. The results indicated that ICT (10 mg/kg) protected against MWReg30-induced ITP, as evidenced by increased blood platelets and decreased spleen index. In addition, the imbalance of Th/CTL in ITP mice spleen was regulated by ICT. Meanwhile, ICT inhibited Th1, Th17, and Tfh and improved Th2 and Treg in ITP mice spleen. Furthermore, the results of CBA and ELISA suggested that ICT decreased serum Th1- and Th17-related cytokines and increased Th2 cytokines, as well as promoted the release of TGF-ß. These results demonstrated that the protective effect of ICT on ITP was mediated by regulating T-cell polarization.

Low-level light treatment ameliorates immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an immune-mediated acquired bleeding disorder characterized by abnormally low platelet counts. We reported here the ability of low-level light treatment (LLLT) to alleviate ITP in mice. The treatment is based on noninvasive whole body illumination 30 min a day for a few consecutive days by near infrared light (830 nm) transmitted by an array of light-emitting diodes (LEDs). LLLT significantly lifted the nadir of platelet counts and restored tail bleeding time when applied to two passive ITP models induced by anti-CD41 antibody. The anti-platelet antibody hindered megakaryocyte differentiation from the progenitors, impaired proplatelet and platelet formation, and induced apoptosis of platelets. These adverse effects of anti-CD41 antibody were all mitigated by LLLT to varying degrees, owing to its ability to enhance mitochondrial biogenesis and activity in megakaryocytes and preserve mitochondrial functions in platelets in the presence of the antibody. The observations argue not only for contribution of mitochondrial stress to the pathology of ITP, but also clinical potentials of LLLT as a safe, simple, and cost-effective modality of ITP.

Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies.

BACKGROUND: Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows ("BNP dams"). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and ß-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. RESULTS: Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced. CONCLUSION: This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.

Novel method to dissociate platelet clumps in EDTA-dependent pseudothrombocytopenia based on the pathophysiological mechanism.

BACKGROUND: EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is an in vitro phenomenon of platelet clumping that leads to spuriously low platelet counts by automatic hematology analyzers. The mechanism is not clearly defined, but is known as an immunologically mediated phenomenon due to the presence of EDTA-dependent antiplatelet auto-antibodies that induce platelet clumping. The purpose of this study was to identify antiplatelet antibodies in EDTA-PTCP samples and to design a method to dissociate platelet clumps based on the pathophysiological mechanism. METHODS: The antiplatelet antibody was investigated using direct and indirect immunofluorescent flow cytometric methods in 23 EDTA-anticoagulated whole blood (WB) samples and 12 serum samples of EDTA-PTCP patients, respectively. A novel mixture containing 9 mmol/L CaCl(2) and 0.1 unit/L sodium heparin, that provides calcium replacement while curbing coagulation, was designed to dissociate platelet clumps. The effect on dissociation was demonstrated in 26 samples of EDTA-PTCP and compared with the established method of kanamycin supplementation. RESULTS: The direct test was positive for IgM and IgG antiplatelet antibody in 60.9% and 4.4% of patients, respectively [mean median fluorescence intensity (MFI) of 223.9 and 128.4, respectively]. The indirect test was positive for IgM antiplatelet antibody in 58.3% of patients (mean MFI of 123.4). The novel method dissociated the platelet clumps with a mean increased platelet count of 242.3% and was equivalent in efficiency to kanamycin supplementation. CONCLUSIONS: The novel method is an easily applicable and efficient measure that allows dissociation of platelet clumps, based on the pathophysiological mechanism of EDTA-PTCP.

Rivaroxaban--an oral, direct Factor Xa inhibitor--has potential for the management of patients with heparin-induced thrombocytopenia.

Rivaroxaban is an oral, direct activated Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. Currently available anticoagulants include unfractionated heparin (UFH) and low molecular weight heparins (LMWHs); however, their use can be restricted by heparin-induced thrombocytopenia (HIT). HIT is usually caused by the production of antibodies to a complex of heparin and platelet factor-4 (PF4). This study was performed to evaluate, in vitro, the potential of rivaroxaban as an anticoagulant for the management of patients with HIT. UFH, the LMWH enoxaparin, fondaparinux and the direct thrombin inhibitor argatroban were tested to enable comparative analyses. Rivaroxaban did not cause platelet activation or aggregation in the presence of HIT antibodies, unlike UFH and enoxaparin, suggesting that rivaroxaban does not cross-react with HIT antibodies. Furthermore, rivaroxaban did not cause the release of PF4 from platelets and did not interact with PF4, unlike UFH and enoxaparin. These findings suggest that rivaroxaban may be a suitable anticoagulant for the management of patients with HIT.

Relative efficacy of intravenous immunoglobulin G in ameliorating thrombocytopenia induced by antiplatelet GPIIbIIIa versus GPIbalpha antibodies.

Intravenous immunoglobulin G (IVIG) is used to treat idiopathic thrombocytopenic purpura (ITP). Although many patients benefit from IVIG, some are refractory to this therapy. ITP is characterized by platelet clearance mediated primarily by antiplatelet antibodies against GPIIbIIIa and/or the GPIbalpha complex. These 2 groups of antibodies may induce ITP through different mechanisms. We tested the hypothesis that IVIG may not be equally effective in preventing ITP caused by anti-GPIIbIIIa versus anti-GPIbalpha antibodies in mice. Thrombocytopenia was induced in BALB/c mice using monoclonal antibodies against either mouse GPIIbIIIa (JON1, JON2, and JON3) or GPIbalpha (p0p3, p0p4, p0p5, p0p9, and p0p11). Pretreatment with IVIG significantly ameliorated ITP in all anti-GPIIbIIIa-injected animals. Conversely, IVIG failed to prevent ITP in all anti-GPIbalpha-treated mice, except for p0p4. These results were repeated in C57BL/6 mice, and with different IVIG preparations. These data in mice suggest that patients with ITP mediated by anti-GPIbalpha antibodies may be less responsive to IVIG treatment.

Bivalirudin anticoagulation for a patient with hypercoagulable immune syndromes undergoing mitral valve surgery.

Unfractionated heparin has been a near universal anticoagulant for cardiac surgery; however it is contraindicated in heparin-induced thrombocytopenia type II. Alternative anticoagulants such as bivalirudin (a direct thrombin inhibitor) are being utilized. Bivalirudin was successfully used in an immunologically complex patient (diagnoses of heparin-induced thrombocytopenia type II, systemic lupus erythematosus, antiphospholipid syndrome, and dialysis-dependent renal failure) requiring cardiopulmonary bypass. Thrombotic events are common in antiphospholipid syndrome patients undergoing cardiac surgery utilizing high-dose heparin. This may represent unrecognized heparin-induced thrombocytopenia type II. Our patient did not experience perioperative thrombotic or bleeding complications. The possible cross-reactivity between heparin induced thrombocytopenia type II and antiphospholipid syndrome has not been investigated.

Argatroban therapy does not generate antibodies that alter its anticoagulant activity in patients with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated syndrome that can lead to limb- and life-threatening thrombosis. Argatroban, a small synthetic molecule (Argatroban; GlaxoSmithKline, Philadelphia, PA), and lepirudin, a protein of non-human origin (Refludan; Aventis, Bridgewater, NJ), are direct thrombin inhibitors that have been used successfully for anticoagulant therapy in HIT patients. It has been reported that between 44-74% of lepirudin-treated HIT patients develop drug-specific antibodies that either enhance or suppress the anticoagulant activity of lepirudin. By contrast, there have been no reported patient experiences suggestive of unexpected loss or enhancement of argatroban's anticoagulant effect in clinical trials, including those in HIT patients, or in postmarketing safety surveillance of over 4,800 patients treated in Japan. To confirm the lack of antibodies in argatroban-treated patients with HIT, we examined plasma for anticoagulant-altering activity and reviewed dosing patterns of re-exposed patients. Paired, pre-therapy and post-therapy (> or =7 days) plasma pools exhibited comparable in vitro anticoagulant responses (aPTT and antithrombin activity) to argatroban supplementation. Argatroban at 5 microg/mL similarly prolonged aPTTs of normal plasma pretreated with IgG isolated from pre-therapy versus post-therapy plasma (P>0.6). In trials, mean argatroban doses during initial therapy versus re-exposure were not different among individuals anticoagulated for the treatment or prophylaxis of thrombosis (P=0.60) or during percutaneous coronary interventions (P=0.79), with no discernable pattern of suppression or enhancement of argatroban anticoagulation. Consistent with the lack of reported patient experiences suggestive of unexpected loss or enhancement of argatroban's anticoagulant effect across clinical trials and post-marketing safety surveillance, these data support the lack of anti-argatroban antibodies that affect drug activity in argatroban-treated HIT patients.

A functional platelet fibrinogen receptor with a deletion in the cysteine-rich repeat region of the beta(3) integrin: the Oe(a) alloantigen in neonatal alloimmune thrombocytopenia.

This report describes a new low-frequency alloantigen, Oe(a), responsible for a case of neonatal alloimmune thrombocytopenia (NAIT). In a population study none of 600 unrelated blood donors was an Oe(a) carrier. By immunochemical studies the Oe(a) antigen could be assigned to platelet glycoprotein (GP) IIIa. Sequencing of GPIIIa complementary DNA from an Oe(a) (+) individual showed deletion of a lysine residue at position 611 (DeltaLys(611)). Analysis of 20 Oe(a) (-) and 3 Oe(a) (+) individuals showed that the DeltaLys(611) form of GPIIIa was related to the phenotype. Anti-Oe(a) reacted with the DeltaLys(611), but not with the wild-type isoforms on stable transfectants expressing GPIIIa, indicating that DeltaLys(611) directly induces the expression of Oe(a) epitopes. Under nonreducing conditions the Pro(33)DeltaLys(611) variant migrated with a slightly decreased molecular weight compared to the Pro(33)Lys(611) isoform suggesting that DeltaLys(611) has an influence on the disulfide bonds of GPIIIa. The Pro(33)DeltaLys(611) GPIIIa could undergo conformational changes and bind to fibrinogen in a similar manner as the Pro(33)Lys(611) isoform. No difference was found in the tyrosine phosphorylation of pp125(FAK), suggesting that DeltaLys(611) has no effect on integrin function. In contrast to all other low-frequency antigens, the DeltaLys(611) isoform was associated with the HPA-1b, but not with the high frequency HPA-1a allele. Comparison with GPIIIa DNA from nonhuman primates indicated that the HPA-1a allele represents the ancestral form of GPIIIa. It can be assumed that the Oe(a) form did arise as a result of a mutational event from an already mutated GPIIIa allele.

In vitro efficacy of platelet glycoprotein IIb/IIIa antagonist in blocking platelet function in plasma of patients with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an important complication following administration of heparin. Platelet activation and aggregation induced by heparin/platelet factor 4/immunoglobulin complexes are thought to be the underlying mechanism for this condition, so it was hypothesized that abciximab (a humanized murine monoclonal antibody directed against the glycoprotein IIb/IIIa receptor) would prevent heparin-induced platelet aggregation and activation in plasma from patients with HIT. Platelet aggregation was tested in vitro with platelet-poor plasma (obtained from 23 patients with HIT), platelet-rich plasma (from normal donors with known reactivity), heparin (0.5 U/ml), and ascending doses of abciximab (0.07-0.56 microg/ml). The ability of abciximab to prevent platelet activation was also evaluated using flow cytometry (P selectin expression, mepacrine release, microparticle formation) and platelet factor 4 immunoassay. In vitro, abciximab inhibited heparin-induced platelet aggregation in a dose-dependent fashion (IC50 0.103 microg/ml) and inhibited microparticle formation, the expression of P-selectin, release of mepacrine and platelet factor 4. These findings suggest that abciximab may be useful in treatment of patients with HIT and warrants further clinical evaluation.

[Heparin-induced thrombopenia: significance and difficulties of precise identification of the immunologic mechanism]. / Thrombopénies induites par l'héparine: intérêt et difficultés de l'identification précise d'un mécanisme immunologique.

Heparin-induced thrombocytopenia (HIT) is a drug induced immunohematologic adverse reaction which is a rare but potentially very severe accident. Its diagnosis is important for epidemiologic and drug surveillance studies and in order to decide the most appropriate treatment. Its importance is enhanced since there is no gold standard diagnostic criteria. In clinical practice the diagnosis is based on a group of criteria related to clinical events and laboratory tests. We have established a score based on anamnestic criteria which allowed us to evaluate and compare two different laboratory tests: a platelet aggregation test (PAT) and a test for the detection of heparin dependent antibodies (Heparin Platelet Induced Antibodies or HPIA). The functional test PAT which is commonly used in expert laboratories detects antibodies inducing platelet aggregation in the presence of heparin. The HPIA test more recently developed is an ELISA test which detects antibodies directed at heparin-platelet factor 4 complexes. The relative value of theses two methods for the diagnosis of HIT is not well documented. We have analysed the results of these two tests in 273 consecutive patients with a suspicion of HIT. The results were concordant in 70% of patients. In selecting the patients with the lowest and the highest probability of HIT according to the score, PAT was found a more sensitive and HPIA a more specific test than the other. At low probability PAT is more often positive than HPIA 18% and 9% respectively. No test is 100% reliable, the specificity being limited for both tests since in about 20% of cases one or both tests are negative contrasting with a highly probable HIT. In this last group of patients, PAT was more frequently positive (86%) than HPIA (72%). Both tests are negative in 6% of patients suggesting the existence of presently unknown antigenic targets. Considering a group of 19 patients with a high probability of HIT, we have found antibodies against IL-8 or NAP-2 in only 7 patients. The discrepancy between a HPIA positive and a PAT negative encountered in 8% of patients may be explained by the existence of IgA or IgM immunoglobulins since in contrast to IgG they are unable to promote platelet aggregation via the CD32 platelet membrane receptor. This work suggests than neither test is 100% reliable and that they play a complementary role in the diagnosis of HIT. The potential advantage of using both tests should be confirmed in complementary studies

Diltiazem-associated thrombocytopenia.

Nine days after starting diltiazem therapy for new-onset atrial fibrillation, a patient's platelet count had diminished to 61 x 10(3)/mm3, and 2 weeks later the nadir was reached at 23 x 10(3)/mm3. No hypersensitivity reaction otherwise was noted, and other hematologic values were unaffected. The patient's platelet counts were not affected by platelet transfusions. Bone marrow examination showed normal to increased numbers of megakaryocytes, suggesting peripheral destruction. After discontinuing diltiazem and administering high-dose immunoglobulin infusions, the platelet count returned to normal. This case suggests an immune-mediated cause for thrombocytopenia after exposure to diltiazem.