Heparin-induced thrombocytopenia: effect of heat inactivation of patient's serum on functional flow cytometric assay results.

BACKGROUND: Heat inactivation of a patient's sample is not systematically performed in the diagnostics of heparin-induced thrombocytopenia (HIT). Some authors recommend that the patient's sample is heat-inactivated to avoid the effect of thrombin on platelet activation in a functional assay. Others do not find this additional step essential or even advise against it. MATERIAL AND METHODS: An enzyme-linked immunosorbent assay (ELISA) and flow cytometry-based functional assay with CD62P as a marker of platelet activation were performed. Forty-seven patients with suspected HIT and three healthy controls were included in the study. Each serum sample was divided into two aliquots: one was heat-inactivated and the other was not. Both aliquots were tested in parallel using the same donor platelets from four randomly selected individuals. We designed an index of platelet activation for both protocols to assess platelet activation in the assay and to compare the results. RESULTS: We observed a higher percentage of platelet activation in heat-inactivated compared to non-heat-inactivated sera. This phenomenon was seen in low and high heparin steps, although it did not occur for all samples. There were discrepant results in seven samples, which tested negative in the non-heat-inactivated protocol and positive in the heat-inactivated protocol. There was no case in which the result of a non-heat-inactivated aliquot was positive and the corresponding heat-inactivated aliquot was negative. DISCUSSION: Due to the higher percentages of donor platelet activation, all seven non-compliant cases met our current criteria for a positive result. However, those results were probably false-positive based on ELISA optical density value and 4T score. Therefore, in the current settings, heat inactivation of serum is not suitable for our flow cytometric functional assay since it can cause an elevated risk of creating false-positive results.

Severe intracranial haemorrhage in neonatal alloimmune thrombocytopenia due to antibodies against human platelet antigen 1b: Case report and literature review.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a rare life-threatening disorder, leading to severe thrombocytopenia and potentially bleeding, with intracranial haemorrhage (ICH) being the most serious complication. We report on a FNAIT case with fourth-degree ICH that arose due to antibodies against human platelet antigen (HPA)-1b. The male infant, born to an otherwise healthy mother, presented with severe signs of ICH soon after delivery. Since only moderate thrombocytopenia was noted and there were no active signs of bleeding, the infant did not receive intravenous immunoglobulins (IVIg) or platelet transfusion. Spontaneous recovery of platelets was noted on the eighth day of life, but permanent neurological impairment remained as a consequence of ICH. We report the results of HPA and human leukocyte antigen (HLA) antibodies in the mother's and the infant's sera, the family's HPA genotype and the mother's HLA genotype, and summarise previously described cases of FNAIT due to anti-HPA-1b antibodies in the literature. FNAIT with severe ICH due to anti-HPA-1b antibodies is rarely diagnosed. An association between HLA genes and sensitization to HPA-1b antibodies was not demonstrated. The severity of FNAIT and the occurrence of ICH is often difficult to predict. In this case, the infant presented with moderate thrombocytopenia and ICH, with subsequent permanent consequences.

K antigens on neonatal red blood cells blocked by anti-K with titer of 32.

CONCLUSIONS: The blocking of red blood cell (RBC) antigens occurs when potent maternal antibodies bind to antigens on fetal or neonatal RBCs, causing them to be negative when typed with human IgM antisera. This phenomenon is rare; when it does occur, the antibody is usually of a high titer. This reported finding is typically due to anti-D, with rare reports describing false-negative K phenotyping due to blocking by maternal anti-K. We report a case of a potent anti-K with a titer of 32 that blocked K antigens on neonatal RBCs, causing them to phenotype as K-. The neonate also had clinically significant anemia (i.e., hemolytic disease of the newborn) due to the anti-K.

Haemolysis, pure red cell aplasia and red cell antibody formation associated with major and bidirectional ABO incompatible haematopoietic stem cell transplantation.

BACKGROUND: Acute and delayed haemolysis, alloimmunisation and pure red cell aplasia (PRCA) are potential complications after ABO incompatible haematopoietic stem cell transplantation (HSCT). The aims of this study were to investigate acute and delayed red blood cell (RBC) antibody-associated complications, including haemolysis, PRCA and alloimmunisation in major and bidirectional ABO incompatible HSCT. MATERIALS AND METHODS: We retrospectively examined the transplant courses of 36 recipients of bone marrow or peripheral blood stem cells from ABO incompatible donors and evaluated the current practice of performing plasmapheresis in patients with higher isoagglutinin titres. We investigated the role of ABO incompatibility in haematopoietic recovery, transfusion requirements, alloimmunisation and PRCA. RESULTS: Laboratory signs of acute haemolysis were noted in five (14%) patients, one (3%) of whom had clinically overt haemolysis. Patients with haemolysis had IgM titres ≥1:8 and received >16 mL of RBC in the HSCT. In patients with higher titres, plasmapheresis performed prior to the transplant prevented acute haemolysis. Delayed haemolysis was not recorded in the follow up. Haematopoietic recovery and transfusion requirements did not differ notably between patients with and without haemolysis. De novo RBC antibodies were detected in two (5.5%) patients after HSCT, and PRCA was noted in one (3%) patient. DISCUSSION: Carried out with adequate graft processing, plasmapheresis and blood component support, haemolysis is not a common complication after HSCT. Our results confirm that the occurrence of haemolysis depends on larger RBC volumes and higher isoagglutinin titres. Despite the reduction of patients' isoagglutinin titres by plasmapheresis, we still noted a critical combination for the development of laboratory signs of haemolysis (IgM titre ≥1:8 and RBC volume >16 mL). De novo immunisation to RBC antigens and PRCA are rare events following ABO incompatible HSCT.

[ISKRA guidelines on sore throat: diagnostic and therapeutic approach--Croatian national guidelines]. / Smjernice iskra za grlobolju: dijagnosticki i terapijski pristup--Hrvatske nacionalne smjernice.

Sore throat is most commonly caused by viruses, but when caused by bacteria, the most important is group A streptococcus (GAS). The aim of these guidelines is to determine optimal treatment for streptococcal sore throat and reasonable indications for tonsillectomy, as well as recommend how to differentiate streptococcal infection for which antibiotics are justified, from numerous other sore throats where antibiotics wont have a significant effect on disease course, but might contribute to bacterial resistance to antibiotics. The development of the guidelines was initiated by the Interdisciplinary Section for Antibiotic Resistance Control (ISKRA) of the Croatian Ministry of Health and Social Welfare in accordance with the principles of AGREE (Appraisal of Guidelines for Research and Evaluation) methodology which means that the guidelines are the result of consensus between all interested professional societies and institutions. For streptococcal sore throat diagnostics, the Working Group recommends evaluation of clinical presentation according to Centor criteria and for patients with Centor score 0-1, antibiotic therapy is not recommended nor bacteriological testing, while for patients with Centor score 2-4 bacteriological testing is recommended (rapid test or culture) as well as antibiotic therapy in case of positive result. The drug of choice for the treatment of streptococcal tonsillopharyngitis is oral penicillin taken for ten days (penicillin V) or in case of poor patient compliance benzathine penicillin G can be administered parenterally in a single dose. Other antibiotics (macrolides, clindamycin, cephalosporins, co-amoxiclav) are administered only in case of hypersensitivity to penicillin or in recurrent infections. Tonsillectomy is a widely accepted surgical procedure that decreases the number of sore throats in children and should be performed only if indications for this procedure are established. Absolute indications include five or more streptococcal infections per year, tonsillitis complications, permanent respiratory tract obstruction, obstructive sleep apnea syndrome and suspected tonsillar malignancy. Relative indications include chronic tonsillitis and occlusion disturbances.

[ISKRA guidelines on antimicrobial treatment and prophylaxis of urinary tract infections--Croatian national guidelines]. / ISKRA smjernice antimikrobnog lijecenja i profilakse infekcija mokracnog sustava--hrvatske nacionalne smjernice.

These guidelines refer to diagnosis, antimicrobial treatment and prophylaxis of urinary tract infections in adults and children older than 12 years of age and cover lower urinary tract in females, uncomplicated pyelonephritis, complicated UTI with or without pyelonephritis, asymptomatic bacteriuria and recurrent UTI. These guidelines do not cover sexually transmitted diseases. The guidelines are primarily intended for use by general practitioners and specialists working in primary health care and hospitals. The members of the Working Group for the development of guidelines on antimicrobial treatment and prophylaxis of urinary tract infections were appointed by the Croatian Ministry of Health and Social Welfare. The project was financially supported by the Dutch government and professional assistance was provided by international consultants. The evidence for this guidelines is based on a systematic review of the literature, local antibiotic resistance data, the existing clinical protocols on the treatment and prophylaxis of UTIs, as well as suggestions and comments made by colleagues physicians during more than 50 continuous medical education courses held in the last three years on antimicrobial treatment and prophylaxis of UTIs. Draft version of the guidelines was available for comments on the web site http://iskra.bfm.hr and during the two-month piloting period the guidelines were widely presented to general practitioners, specialists working in primary care and hospitals--urologists, gynecologists, infectious disease specialists, nephrologists. The final version of the guidelines was approved by the Intersectoral Coordination Mechanism for the Control of Antimicrobial Resistance (ISKRA) Board.