Flow Cytometric Assessment of AKT Signaling in Platelet Activation: An Alternative Diagnostic Tool for Small Volumes of Blood.

INTRODUCTION: The diagnosis of platelet function disorder in children is challenging. Light transmission aggregometry is the gold standard for platelet function disorders. However, large blood volumes are required. Currently, there are no existing tools for the diagnosis of platelet function disorders that use small blood volumes. AKT signaling plays a central role in platelet activation during hemostasis and might be visualized by flow cytometry. METHODS: Platelet-rich plasma obtained by centrifugation of citrated blood from healthy volunteers was activated with arachidonic acid, thrombin receptor activating peptide-6 (TRAP-6), collagen, adenosine diphosphate ADP, collagen-related peptide (CRP), and epinephrine. After platelet activation, the phosphorylation of AKT was assessed by flow cytometer using a Navios cytometer. RESULTS: Healthy volunteers showed a reproducible phosphorylation of AKT upon activation. In comparison to nonactivated platelets, we documented an increase in pAKT expression with all agonists. Especially TRAP-6 and CRP caused considerable increase in percentage of pAKT expression throughout all the tested healthy volunteers. CONCLUSION: An activation of the AKT-signal pathway by different agonists can clearly be detected on the flow cytometer, indicating that the visualization of signaling in platelets by flow cytometry might be an efficient alternative for light transmission aggregometry to test platelet function in children.

Miller-Fisher syndrome after COVID-19: neurochemical markers as an early sign of nervous system involvement.

Miller-Fisher syndrome (MFS) is classified as a variant of Guillain-Barré syndrome (GBS), accounting for 5%-25% of all GBS cases. Since the coronavirus disease-2019 (COVID-19) outbreak, increasing evidence has been reported of the neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, affecting both the central and peripheral nervous system. Here we report the clinical course, detailed cerebrospinal fluid (CSF) profile including CSF/blood antibody status, and neurochemical characteristics of a patient with a typical clinical presentation of MFS after a positive SARS-CoV-2 infection test.

MR-imaging pattern is not a predictor of occult atrial fibrillation in patients with cryptogenic stroke.

BACKGROUND: To date, insertable cardiac monitors (ICMs) are the most effective method for the detection of occult atrial fibrillation (AF) in cryptogenic stroke. The overall detection rate after 12 months, however, is low and ranges between 12.4 and 33.3%, even if clinical predictors are considered. Ischemic stroke patients due to cardiogenic embolism present with particular lesion patterns. In patients with cryptogenic stroke, MR-imaging pattern may be a valuable predictor for AF. METHODS: This is an MRI-based, retrospective, observational, comparative, single-center study of 104 patients who underwent ICM implantation after cryptogenic stroke. The findings were compared to a reference group with related stroke etiology, i.e., 166 patients with embolic stroke due to AF detected for the first time by long-term ECG. Lesion patterns were evaluated with regard to affected territories, distribution (cortical, lacunar, scattered), lesion volume, and lesion size (diameter of the lesion size > 20 mm). RESULTS: The MR-imaging analysis of acute ischemic lesions yielded no association between AF and lesion size or volume, arterial vessel distribution, or the number of affected territories. There was no significant difference between the cohorts regarding ischemic patterns (cortical lesions, scattered lesions, and lacunar infarcts). An important clinical inference of our findings is that 10% (2 of 20) of cases in the ICM group in whom AF was detected had a lacunar infarct pattern. Similar results were shown in cases of ischemic stroke patients with AF detected for the first time by long-term ECG, with 10.9% (16 of 147) of them showing lacunar infarcts. The analysis of chronic MRI lesions revealed no differences between the groups in the rate of chronic lesions, arterial vessel distribution, or the number of affected territories. Left atrial size (LA size) and the presence of atrial runs in long-term ECG were independently associated with AF. CONCLUSIONS: In this MRI-based analysis of patients with cryptogenic stroke who had received ICM implantation, the detection rate of AF in patients with ICM was not related to the imaging pattern. In addition, the lacunar infarct pattern should not be an exclusion criterion for ICM insertion in patients with cryptogenic stroke. ICM insertion in patients with cryptogenic stroke should not be evaluated solely on the basis of reference to infarct patterns.

Diagnosis of inherited platelet disorders on a blood smear: a tool to facilitate worldwide diagnosis of platelet disorders.

Essentials There are many hereditary platelet disorders (HPD) but diagnosing these is challenging. We provide a method to diagnose several HPDs using standard blood smears requiring < 100 µL blood. By this approach, the underlying cause of HPD was characterized in ~25-30% of referred individuals. The method facilitates diagnosis of HPD for patients of all ages around the world. SUMMARY: Background Many hereditary thrombocytopenias and/or platelet function disorders have been identified, but diagnosis of these conditions remains challenging. Diagnostic laboratory techniques are available only in a few specialized centers and, using fresh blood, often require the patient to travel long distances. For the same reasons, patients living in developing countries usually have limited access to diagnosis. Further, the required amount of blood is often prohibitive for pediatric patients. Objectives By a collaborative international approach of four centers, we aimed to overcome these limitations by developing a method using blood smears prepared from less than 100 µL blood, for a systematic diagnostic approach to characterize the platelet phenotype. Methods We applied immunofluorescence labelling (performed centrally) to standard air-dried peripheral blood smears (prepared locally, shipped by regular mail), using antibodies specific for proteins known to be affected in specific hereditary platelet disorders. Results By immunofluorescence labelling of blood smears we characterized the underlying cause in 877/3217 (27%) patients with suspected hereditary platelet disorders (HPD). Currently about 50 genetic causes for HPD are identified. Among those, the blood smear method was especially helpful to identify MYH9 disorders/MYH9-related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia and gray platelet syndrome. Diagnosis could be established for GATA1 macrothrombocytopenia, GFI1B macrothrombocytopenia, ß1-tubulin macrothrombocytopenia, filamin A-related thrombocytopenia and Wiskott-Aldrich syndrome. Conclusion Combining basic and widely available preanalytical methods with the immunomorphological techniques presented here, allows detailed characterization of the platelet phenotype. This supports genetic testing and facilitates diagnosis of hereditary platelet disorders for patients of all ages around the world.

Anti-platelet factor 4/heparin antibodies in patients with impaired graft function after liver transplantation.

BACKGROUND: Heparin, the standard perioperative anticoagulant for the prevention of graft vessel thrombosis in patients undergoing liver transplantation (LT), binds to the chemokine platelet factor 4 (PF4). Antibodies that are formed against the resulting PF4/heparin complexes can induce heparin-induced thrombocytopenia. LT is a clinical situation that allows the study of T-cell dependency of immune responses because T-cell function is largely suppressed pharmacologically in these patients to prevent graft rejection. OBJECTIVES: To investigate the immune response against PF4/heparin complexes in patients undergoing LT. PATIENTS AND METHODS: In this prospective cohort study, 38 consecutive patients undergoing LT were systematically screened for anti-PF4/heparin antibodies (enzyme immunoassay and heparin-induced platelet aggregation assay), platelet count, liver function, and engraftment. RESULTS: At baseline, 5 (13%) of 38 patients tested positive for anti-PF4/heparin IgG (non-platelet-activating) antibodies. By day 20, an additional 5 (15%) of 33 patients seroconverted for immunoglobulin G (two platelet-activating) antibodies. No patient developed clinical heparin-induced thrombocytopenia. Two of six patients with graft function failure had anti-PF4/heparin IgG antibodies at the time of graft function failure. Graft liver biopsy samples from these patients showed thrombotic occlusions of the microcirculation. CONCLUSIONS: Anti-PF4/heparin IgG antibodies are generated despite strong pharmacologic suppression of T cells, indicating that T cells likely have a limited role in the immune response to PF4/heparin complexes in humans.

MYH9 related platelet disorders - often unknown and misdiagnosed.

MYH9 related platelet disorders are a relatively rare cause of thrombocytopenia. Located on chromosome 22, the MYH9 gene encodes the motorprotein non-muscular myosin heavy chain IIA (NMMHCIIA). Heterozygous defects in this gene lead to 4 different autosomal dominant syndromes namely May-Hegglin anomaly, Epstein syndrome, Fechtner syndrome and Sebastian platelet syndrome. All 4 syndromes are characterized by macrothrombocytopenia and a mild bleeding tendency. Depending on the position of the causative mutation within the gene, the risk increases for syndromic manifestations such as renal failure, hearing loss and pre-senile cataract. Mutations in the neck region of the NMMHCIIA protein are more likely associated with these comorbidities than mutations in the N- or C-terminal part of the gene. MYH9 related platelet disorders should be excluded in patients with chronic thrombocytopenia and large platelets. Most sensitive for diagnosis/exclusion are immunofluorescence studies using a blood smear. The biggest risk for these patients is ineffective but potentially harmful treatment based on the misdiagnosis of immune thrombocytopenia. This review provides a workflow for diagnosis and treatment of MYH9 related thrombocytopenia.

[Heparin-induced thrombocytopenia]. / Heparininduzierte Thrombozytopenie.

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that carries an increased risk of thromboembolic complications. HIT is caused by platelet-activating antibodies directed against a complex of platelet factor 4 (PF4) and heparin. HIT typically manifests in the second week after initiation of heparin therapy with a platelet count reduction of more than 50% of the highest level after the start of heparin administration as well as thromboembolic events. The clinical probability can be calculated by the 4 T's score. The laboratory diagnosis of HIT is based on confirmation of PF4/heparin antibodies or on functional tests that provide evidence of heparin-dependent platelet-activating antibodies. A low 4 T's score and negative HIT test virtually rule out the presence of HIT. Patients with acute HIT require anticoagulation with a compatible anticoagulant in a therapeutic dose. The drugs currently available for this include the direct thrombin inhibitors argatroban, lepirudin, bivalirudin, and desirudin and the indirect factor Xa inhibitors danaparoid and fondaparinux.

Heparin-induced thrombocytopenia: towards standardization of platelet factor 4/heparin antigen tests.

BACKGROUND: Laboratory confirmation of heparin-induced thrombocytopenia (HIT) is based on detection of heparin-dependent platelet-activating antibodies. Platelet factor 4 (PF4)/heparin enzyme-immunoassays (EIA) are a widely available surrogate for platelet-activating antibodies. OBJECTIVE: Defining the optical density (OD) reactivity profiles of a PF4/heparin EIA in reference subject and patient populations and the correlation of the EIA results (expressed in OD units) with the prevalence of platelet-activating antibodies. PATIENTS/METHODS: Using quantile regression we determined the 97.5th percentile of PF4/heparin-immunoglobulin G (IgG) EIA reactivities in non-heparin-treated individuals [blood donors (n = 935)] and patients before heparin therapy (n = 1207). In patients with suspected HIT, we compared the correlation of EIA-IgG reactivities (Greifswald laboratory; n = 2821) and the heparin-induced platelet activation assay (HIPA) with the correlation of reactivities of another EIA-IgG (McMaster laboratory; n = 1956) with the serotonin-release assay (SRA). RESULTS: PF4/heparin-IgG EIA OD reactivities had a lower OD 97.5th percentile in blood donors compared with patient groups before heparin treatment (P < 0.001). The percentage of sera testing positive in the functional assays strongly correlated with PF4/heparin-IgG EIA OD reactivities in both laboratories with very similar results (correlation coefficient > 0.9) when normalized OD ranges (maximum OD divided by 10) were used instead of absolute OD values. CONCLUSIONS: Results of PF4/heparin-IgG EIA should not be reported as only positive or negative as there is no single acceptable cut-off value. Instead, reporting PF4/heparin-IgG EIA OD results in ranges allows for risk-stratified prediction for presence of platelet-activating antibodies. Use of normalized OD ranges permits a standardized approach for inter-laboratory comparisons.

Heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT), typically occurring in the second week of heparin therapy, is an antibody-mediated adverse drug reaction associated with increased thrombotic risk. The most important antigens are located on platelet factor 4 (PF4)/heparin complexes. HIT is always caused by platelet-activating antibodies, but not all PF4/heparin-reactive antibodies cause HIT. Thus, tests have a high negative, but only a moderate, positive predictive value. Clinical suspicion of HIT requires cessation of heparin and substitution with an alternative anticoagulant. As these drugs have an increased bleeding risk, they should be used in therapeutic doses only if HIT is considered very likely. Avoiding/postponing coumarin is crucial in minimizing microthrombotic complications. Recent studies of HIT immunobiology suggest that HIT mimics immunity against repetitive antigens, as are relevant in microbial defense. Thus, understanding HIT may help unravel why host defenses can trigger autoimmunity.

False-positive tests for heparin-induced thrombocytopenia in patients with antiphospholipid syndrome and systemic lupus erythematosus.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy that can be associated with arterial or venous thrombosis and is caused by antibodies against platelet factor 4 (PF4)-heparin complex. Patients with antiphospholipid syndrome (APS) have been reported with positive tests for PF4-heparin complex antibodies by antigen assay. Whether such patients can be treated with heparin is a dilemma. OBJECTIVES: To determine the incidence and nature of the HIT immune reaction in patients with APS and/or systemic lupus erythematosus (SLE). METHODS: Antibodies against PF4-heparin complex were assayed by particle gel immunoassay (PaGIA), or enzyme immunoassay (EIA) with or without an excess of heparin. EIA for PF4 alone was also performed. Functional assays for HIT, that is, heparin-induced platelet activation (HIPA) and heparin-induced platelet aggregation, were also performed. RESULTS: In 32 of 42 patients (76.2%) with APS, APS and SLE, SLE, or SLE with antiphospholipid antibodies, EIA IgG or PaGIA for PF4-heparin complex antibodies were positive. Of these 32 samples, 26 (81.3%) tested positive for anti-PF4 antibodies. All 24 samples that were positive for PF4-heparin complex by EIA IgG were also positive for EIA IgG in the presence of heparin excess, and all were negative by the HIPA and heparin-induced platelet aggregation tests. CONCLUSION: A large proportion of patients with APS and/or SLE give false-positive HIT antigen test results that are presumably related to autoantibodies against PF4, which can be distinguished from true HIT antibodies by EIA for PF4-heparin complexes tested with heparin excess, and by functional assays.

[Clinical aspects of hypophyseal diseases from the viewpoint of the ophthalmologist]. / Klinik von Hypophysenerkrankungen aus augenärztlicher Sicht.

The anatomy of the chiasmal region and the visual symptoms due to pituitary tumors will be presented from the viewpoint of an ophthalmologist. The importance of perimetry in every patient with otherwise unexplained loss of vision is stressed. Other tests (swinging flash light test, colour vision, screening ophthalmoscopy) are mentioned as well. An ophthalmologist should participate in the diagnosis as well as in the follow-up after treatment of any case with pituitary tumors.

[How accurate is video refraction measurement in infants in comparison with skiascopy? How much do the values change within a half a year?]. / Wie genau ist die Videorefraktionsmessung beim Kleinkind im Vergleich zur Skiaskopie? Wie ändern sich die Werte innerhalb eines halben Jahres?

BACKGROUND: Isotropic photorefraction (Atkinson et al., 1981) is a method used to screen the refraction in infants. The aim of this study is to analyze its limitations as a measuring instrument. MATERIALS AND METHODS: 215 children aged between 5 and 12 months were examined in cycloplegia. The results were subsequently compared (1) to the results of streak retinoscopy also performed at the same time and (2) to photorefraction performed a second time, 6 months later (with 145 children). RESULTS: Spherical refraction: The mean spherical values differed by 0.70 dpt through photorefraction methods, compared to retinoscopy. The standard deviation (SD) was 0.67 dpt. The values of the photorefraction differed therefore in 95% of the children within a range of 2.7 dpt (or +/- 2 SD). Compared to the second photorefractive measurement 6 months later, the average refraction showed a small myopic shift (-0.45 dpt). Astigmatism: The errors of measurement were more pronounced here. Extreme differences of up to 9 dpt were found in single cases. The mean difference, compared to retinoscopy, was 0.73 dpt. The standard deviation (SD) was 0.90 dpt. In 95% of the children, the values of photo-refraction differed therefore by a range of 3.6 dpt (or +/- 2 SD). Compared to the second photorefractive measurement 6 months later, the average refraction showed a mean change of 0.73 dpt. CONCLUSIONS: Our photorefractive measurements showed errors in the range of 1-2 dpt for the spherical refraction, and in the range of 2-3 dpt for astigmatism. In 5% of the children examined, even greater aberrations were found. As long as the aim of photorefraction is restricted to disclosing high refractive errors, this method is judged suitable for the refractive screening of infants. For more precise refraction, and for the prescription of spectacles, we believe that is should be supplemented by a second examination technique, such as retinoscopy or automated refractometry.

[Video refraction measurement in the first year of life]. / Videorefraktionsmessung im ersten Lebensjahr.

BACKGROUND: 426 children were examined, using the isotropic photorefraction method (Atkinson et al. 1981). The aim of this study was to analyze whether the frequency of convergent strabismus and/or amblyopia would increase, particularly in cases of high ametropia, and whether the early prescription of spectacles would be beneficial. The preliminary results of this study are presented here. MATERIALS AND METHODS: 426 children aged between 5 and 12 months were examined. Family history, particularly involving strabismus, ametropia and amblyopia, was ascertained and taken into consideration. RESULTS: Spherical refraction: 92% of the children were emmetropic or slightly hyperopic (< or = +2.5 D). 3.4% were hyperopic (> +2.5 D spherical equivalent) and 4.6% were myopic (0.9 > or = -2.0 D). Astigmatism: 85% had no or mild (< or = 1.5 D) astigmatism. Values greater than 3.5 D were rarely seen. Anisometropia: 67% of the children had no side-difference and only 2.4% had anisometropia with values greater than 1.5 D. Family history/orthoptic findings: 2.6% of the examined population had strabismus. In 12.2% of all the cases one or more first degree relatives had strabismus. Hyperopia and strabismus were found more frequently in this latter group, namely hyperopia (> +2.5 D spherical equivalent) in 13.6% and strabismus in 11.5%. CONCLUSIONS: Refractive errors greater than 2.5 D were seldom seen in this study, and yet were more frequently detected in families with a history of strabismus. In our opinion, isotropic photorefraction is a method most suitable to screening these especially high-risk groups.