Since the late sixties, therapeutic or prophylactic platelet transfusion has been used to relieve hemorrhagic complications of patients with, e.g., thrombocytopenia, platelet dysfunction, and injuries, and is an essential part of the supportive care in high dose chemotherapy. Current and upcoming advances will significantly affect present standards. We focus on specific issues, including the comparison of buffy-coat (BPC) and apheresis platelet concentrates (APC); plasma additive solutions (PAS); further measures for improvement of platelet storage quality; pathogen inactivation; and cold storage of platelets. The objective of this article is to give insights from current practice to future development on platelet transfusion, focusing on these selected issues, which have a potentially major impact on forthcoming guidelines.

(1) Background: Polycythaemia is defined by an increase in haemoglobin (Hb) concentration, haematocrit (Hct) or red blood cell (RBC) count above the reference range adjusted to age, sex and living altitude. JAK2 unmutated polycythaemia is frequent but under-investigated in original publications. In this retrospective cohort study, we investigated the clinical and laboratory data, underlying causes, management and outcomes of JAK2 unmutated polycythaemia patients. (2) Methods: The hospital database was searched to identify JAK2 unmutated patients fulfilling WHO 2016 Hb/Hct criteria for PV (Hb >16.5 g/dL in men and >16 g/dL in women, or Hct > 49% in men and >48% in women, or RBC mass > 25% above mean normal predicted value) between 2008 and 2019. Clinical and laboratory data were collected and analysed. (3) Results: From 727,731 screened patients, 294 (0.04%) were included, the median follow-up time was 47 months. Epo and P50 showed no clear pattern in differentiating causes of polycythaemia. In 30%, the cause remained idiopathic, despite extensive work-up. Sleep apnoea was the primary cause, also in patients under 30. Around 20% had received treatment at any time, half of whom had ongoing treatment at the end of follow-up. During follow-up, 17.2% developed a thromboembolic event, of which 8.5% were venous and 8.8% arterial. The mortality was around 3%. (4) Conclusions: Testing for Epo and P50 did not significantly facilitate identification of underlying causes. The frequency of sleep apnoea stresses the need to investigate this condition. Idiopathic forms are common. A diagnostic flowchart based on our data is proposed here. NGS testing should be considered in young patients with persisting polycythaemia, irrespective of Epo and P50 levels.

(1) Background: Clinical and molecular data on patients with unexplained erythrocyto-sis is sparse. We aimed to analyze the clinical and molecular features of patients with congenital erythrocytosis in our tertiary reference center. (2) Methods: In 34 patients with unexplained erythrocytosis, a 13-gene Next-Generation Sequencing erythrocytosis panel developed at our center was conducted. (3) Results: In 6/34 (18%) patients, eight different heterozygous gene variants were found. These patients were, therefore, diagnosed with congenital erythrocytosis. Two patients had two different gene variants each. All variants were characterized as variants of unknown significance as they had not previously been described in the literature. The rest of the patients (28/34, 82%) had no detected gene variants. (4) Conclusions: Our experience shows that the NGS panel can be helpful in determining the reasons for persistent, unexplained erythrocytosis. In our cohort of patients with erythrocytosis, we identified some, thus far unknown, gene variants which may explain the clinical picture. However, further investigations are needed to determine the relationship between the molecular findings and the phenotype.

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Background: Real life data of underlying causes of JAK2-negative polycythemia is sparse. We aimed to analyze clinical and laboratory data of patients at our tertiary referral hospital, in order to identify the most frequent underlying causes of JAK2-negative polycythemia. Particularly, we intended to analyze the prevalence and causes of hereditary erythrocytosis. Methods: The hospital database was searched to find patients ≥15 years of age with polycythemia (inpatients and outpatients) between 1 st Oct 2008 and 31 st Jul 2019. Using a stepwise process, we focused on patients in whom a JAK2 result was available. For the diagnosis of polycythemia, including reactive and relative, the diagnostic criteria of the 2016 WHO classification for polycythemia vera were used: hemoglobin >165 g/L in men and >160 g/L in women, or hematocrit >49% in men and >48% in women. Results: Files of 727,731 patients were screened, and in 4,391 polycythemia was mentioned as a diagnosis on the electronic record. Of these, polycythemia was confirmed in 1,483 based on laboratory values. From them, 391 were tested for JAK2 mutation, and 294 were negative, thus representing our study cohort. The median age at polycythemia diagnosis was 46 years (r 15 - 89), and the majority of patients were males (N=242, 82%). The median Hb and Hct value were 172 g/L (r 157 - 224) and 51% (r 45 - 68) respectively. The patients were followed up for a median time of 4 years (r 12 days - 21 years). In 61% of patients (179/294) reactive polycythemia was diagnosed, while in 4.8% (14/294) relative polycythemia was present. In 30% of patients (89/294) the cause of polycythemia was undetermined, whereas in 70% (205/294) an underlying cause explaining polycythemia was found (Table 1). In 12 patients (4.1%), a congenital cause was identified. Among them, hemoglobinopathy or high-oxygen affinity Hb (N=8, 2.7%) were the most common (Table 1). Sleep apnea confirmed through a polysomnography (N=55, 18.7%), followed by smoking (N=51, 17.3%), increased HbCO>5% (N=14, 4.8%), respiratory diseases (N=13, 4.4%), and non-cancer kidney disease, for instance renal cysts (N=12, 4.1%), were the most common associated causes. Polysomnographies were performed on 60 patients, of whom sleep apnea was confirmed in 92% (N=55), and a significant proportion of these patients were younger than 40 years old (N=18, 33%). From all patients with undetermined causes (N=89), we focused on those with persistent polycythemia for further investigations. Accordingly, 25 patients were identified, all male and 72% (N=18) younger than 40 years of age. In 12/25 patients a congenital erythrocytosis NGS panel was performed, and in 3/12 patients 4 different heterozygous mutations were found (two of which were in one patient). All mutations were characterized as variants of unknown significance (VUS). None of these mutations were previously described in the literature, and their finding in patients with polycythemia suggests a pathogenic likelihood (Table 2). Conclusion: In one third of JAK2-negative patients with polycythemia, despite extensive workup, no underlying cause was found. Sleep apnea was the main cause of secondary forms, even in young patients, underlining the importance of performing a polysomnography in the workup of such patients. The NGS erythrocytosis panel offers easy and accessible characterization of some idiopathic forms, however there is still a majority of these patients unable to be characterized. In this cohort the investigation with NGS showed 4 mutations which have not been reported in the literature. The presence of these in patients with polycythemia suggests the probability of a relationship in its pathogenesis. Figure 1 Figure 1. Disclosures Jalowiec: Kite Pharma Gilead Sciences': Honoraria, Speakers Bureau. Rovo: AG Alexion: Research Funding; CSL Behring: Research Funding; Novartis: Research Funding; AG Alexion: Honoraria; BMS: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria; OrPhaSwiss GmbH: Honoraria; Swedish Orphan Biovitrum AG: Honoraria; Amgen: Other: Financial support for congresses and conference travel; AstraZeneca: Other; BMS: Other; Sanofi: Other; Roche: Other.

Background: Fetal red blood cells (RBC), erythroid and megakaryopoietic progenitor cells start to express the Kell (K) antigen already during the 10th gestational week (GW). Maternal anti-Kell alloimmunization (allo-anti-K-Ab) causes early (< 20th GW) and severe hemolytic disease of the fetus/newborn (HDFN) accompanied by erythroaplasia and potential thrombocytopenia. Therefore, prevention/delay of HDFN is highly relevant. Current treatment options include intra-uterine transfusions (IUT) of the fetus in case of suspected anemia, i.v. high-dose immunoglobulins (IVIG), plasma exchange and immunoadsorption (IA). Their efficacy often remains unsatisfactory and procedure related complications might occur. We present our new triple-treatment approach, combining IA + IVIG + Rituximab (Rtx) (IIR), which we successfully applied in 2 high-risk patients with allo-anti-K-Ab. Methods: The first patient was a 24 y, para 1, gravida 2, with an allo-anti-K-AB titer of 1:2000 in the 15th GW, at first presentation. Duplex-sonography showed no signs of fetal anemia. The second patient was a 40 y para 1, gravida 2. She presented with an allo-anti-K-Ab titer of 1:8000 in the 20th GW. Duplex-sonography revealed signs of severe anemia of fetus, requiring immediate IUT. Fetal K- genotyping (RT-PCR) using cell-free fetal DNA from maternal plasma confirmed a K- positive status in both fetuses. Duplex sonography monitoring was executed weekly, including the assessment of arteria cerebri media peak systolic velocity (ACM PSV). After having received informed consent of patients, we started the treatment at 14th and 21st GW in our first and second patient, respectively. IIR treatment cycles (1 cycle = 21 days) comprised 6x IA (each processing 2x the patient's plasma volume, weeks 1 + 2, every other day), followed by IVIG (1g/kg body weight) and i.v. Rtx (375 mg/m2) after the last IA of each cycle. This resulted in a total of 6 and 5 cycles for the first and second patient, respectively. We omitted Rtx for the last cycles in both patients. Results: IIR treatment resulted in a significant reduction of maternal anti K-titer to minimum of 1:32 in both patients, with a fluctuating course. In case 1 we performed a cordocentesis in the 30th GW because of an increased ACM PSV. The blood count of the fetus showed no anemia (Hb: 132 g/L) and only minimal Thrombocytopenia (145 G/L). The fetus in case 2 had already duplex- sonographic signs for a severe anemia at first presentation. Diagnostic cordocentesis confirmed a severe anemia (Hb 47 g/L) and also severe thrombocytopenia (21 G/L). Fetal alloimmune thrombocytopenia was ruled out. The first IUT of RBC has been performed right before starting IIR. Two weeks later a second IUT of RBC was necessary (fetal Hb 54 g/L), but thrombocytopenia was completely reversed (Tc: 352 G/l). The third and last IUT of RBC was performed 5 weeks later (Hb 99 g/L). Thereafter, no other signs of fetal anemia were detected. Patient one showed nausea/headache after her first IVIG-application and hypotonia/sinustachykardia during IA of the 5th cycle. IIR-treatment and IUT revealed no further adverse events. We continued treatment in both cases until the 32nd GW. Both neonates were born healthy in the 38th and 37th GW, respectively. The neonate in case 1 presented a mild anemia (Hb: 131 g/l), but normalized his Hb from 2nd day onwards. Both newborns showed a positive direct antiglobulin test (DAT), elution verified an anti-K-Ab, and their RBC were serologically positive for K. Both cases had normal Bilirubin and LDH levels, but a haptoglobin <0.1 g/l at birth. Both developed an icterus neonatorum during the first days after birth and needed phototherapy, Hb remained normal at all times. Flow cytometry of fetal lymphocytes showed a B-cell decrease (5.2%) in the first case and completely missing B-cells in the second case. Rtx level was low (2.13 µg/ml) in maternal plasma and neg. in the newborn of the first case. In the second case Rtx levels of the newborn and mother were 15,87 µg/ml and 4,5 µg/ml, respectively. In both cases, breastmilk showed no Rtx but positivity for anti-K-Ab (IgG). We observed no hemolysis of the newborns, while both were on breastfeeding. Conclusion: High-titer anti-Kell alloimmunisation causes early onset and severe HDFN and may be associated with severe thrombocytopenia. Treatment of affected pregnant women by using the Bern IIR-protocol was effective and safe in both severely affected patients. Disclosures Rovo: Novartis:Honoraria, Research Funding;CSL Behring:Research Funding;AG Alexion:Research Funding;Celgene:Honoraria, Other: financial support for congresses and conferences travel;BMS:Other: financial support for congresses and conferences travel;AstraZeneca:Other: financial support for congresses and conferences travel;Sanofi:Other: financial support for congresses and conferences travel;Amgen:Other: financial support for congresses and conferences travel;Roche:Other: financial support for congresses and conferences travel. OffLabel Disclosure: Rituximab is used for B-cell depletion in different malignant and non-malignant diseases. The off-label use concerns ist use in pregnant women with alloimmunisation.