Howell-Jolly Research Articles

Autoimmune manifestation in splenic atrophy presented with toxic shock syndrome: a case report

Splenic atrophy is an uncommon diagnosis, associated with autoimmune gastrointestinal disorders and other well-characterized connective tissue diseases. We would like to contribute a case report to support the association evidence of the real-world data. To our best knowledge, there is no similar case of splenic atrophy with the presentation of streptococcal toxic shock syndrome reported. Our patient was initially detected with atrophic spleen via CT scan and subsequently diagnosed with systemic sclerosis. Hyposplenism should be suspected in patients with adult-onset infections caused by encapsulated bacteria, especially if autoantibodies are present. Our patient received her pneumococcal vaccination before discharge and was followed in the clinic for further vaccination education and health check-up. Learning points: 1) Acquired splenic atrophy is a rare condition that may be suspected from persistent isolated thrombocytosis after the resolution of sepsis and Howell-Jolly bodies from peripheral blood film; 2) The pathophysiological mechanism of splenic atrophy in the context of autoimmune disorders remains unknown; 3) A high index of suspicion towards the evaluation of splenic function is required if a patient presented with community-acquired encapsulated organism bacteraemia; 4) Vaccination against encapsulated bacterial agents should be performed in patients with hyposplenism.

Focal spleen lesions in loiasis: A pilot study in Gabon.

Infection with the filarial nematode Loa loa, endemic in Central and Western Africa, has been associated with increased morbidity and mortality. A number of reports described the presence of spleen nodules, originating from degenerating microfilariae, in humans and animals infected with L. loa. The long-term consequences of this process on individuals chronically exposed to infection in terms of spleen function and possible link with excess mortality are unknown. The aim of this study was to evaluate the prevalence of focal spleen lesions, their evolution over time, and markers of spleen function, in individuals with L. loa infection living in highly endemic areas of Gabon. This was a cross-sectional study followed by a longitudinal study of the subset of individuals with spleen nodules. Two hundred sixteen participants from Ngounié and Moyen-Ogooué provinces of Gabon, reporting a history of eyeworm migration and/or Calabar swelling, were included. Participants were categorized into infected microfilaraemic with low (N = 74) and high (N = 10) microfilaraemia, and symptomatic amicrofilaraemic (N = 132), based on blood microscopy. Howell-Jolly bodies in erythrocytes, as indirect marker of spleen functional impairment, were within normal ranges. On ultrasound, no evident signs of spleen fibrosis or hypotrophy were observed. Multiple spleen hypoechoic centimetric macronodules were observed in 3/216 participants (1.4%), all with microfilaraemic L. loa infection (3.4% of microfilaraemics); macrondules disappeared at the 6-months follow-up examination in 2/3 individuals. Spleen hypoechoic micronodules, persisting at the 6-months follow-up, were detected in 3/216 participants (1.4%), who were all amicrofilaraemic. Transitory spleen macronodules are present in a small but consistent proportion of individuals with microfilaraemic loiasis, appearing a rather benign phenomenon in terms of impact on spleen morphology and function. Their occurrence should be taken into consideration to avoid misdiagnosis and mistreatment. Prevalence and significance of spleen micronodular ultrasound patterns in the general population would be also worth evaluating.

Impaired IgM Memory B Cell Function Is Common in Coeliac Disease but Conjugate Pneumococcal Vaccination Induces Robust Protective Immunity.

Coeliac disease (CD) is associated with hyposplenism, an acquired impairment of spleen function associated with reduced IgM memory B cells and increased susceptibility to serious pneumococcal infection. Little is known about the immune implications of hyposplenism in CD or the optimal pneumococcal vaccination strategy. In this study, the immune effects of hyposplenism in CD, and the accuracy of screening approaches and protective responses induced by two different pneumococcal vaccines were examined. Active and treated CD cohorts, and healthy and surgically splenectomised controls underwent testing for the presence of Howell-Jolly bodies and pitted red cells, spleen ultrasound, and immune assessment of IgM memory B cell frequency and IgM memory B cell responses to T cell-dependent (TD) or T cell-independent (TI) stimulation. Responses following conjugate (TD) and polysaccharide (TI) pneumococcal vaccination were compared using ELISA and opsonophagocytic assays. Although hyposplenism is rare in treated CD (5.1%), functional B cell defects are common (28-61%) and are not detected by current clinical tests. Conjugate pneumococcal vaccination induced superior and sustained protection against clinically relevant serotypes. Clinical practice guidelines in CD should recommend routine pneumococcal vaccination, ideally with a conjugate vaccine, of all patients in lieu of hyposplenism screening.

Malaria Infection in Patients with Sickle Cell Disease in Nigeria: Association with Markers of Hyposplenism.

Malaria is considered an important cause of morbidity and mortality among people living with sickle cell disease (SCD). This has partly been attributed to the loss of splenic function that occurs early in the disease process. We conducted a cross-sectional study and determined the frequency of malaria infection among SCD patients and explored the association with spleen's presence on ultrasonography and spleen function assessed using the frequency of Howell-Jolly bodies (HJBs). A total of 395 participants consisting of 119 acutely-ill SCD patients, 168 steady-state SCD controls, and 108 healthy non-SCD controls were studied. The prevalence of Plasmodium falciparum parasitemia was 51.3% in acutely-ill SCD patients, 31.7% in steady-state SCD controls, and 11.0% in the healthy non-SCD controls; however, the mean parasite density was significantly higher in the non-SCD controls compared to both SCD groups (p = 0.0001). Among the acutely-ill SCD patients, the prevalence of clinical malaria and severe malaria anemia were highest in children <5 years of age. The prevalence of parasitemia (p = 0.540) and parasite density (p = 0.975) showed no association with spleen presence or absence on ultrasonography. Similarly, the frequency of HJB red cells was not associated with the presence of parasitemia (p = 0.183). Our study highlights the frequency and role of malaria infection in acutely-ill SCD patients, especially in those younger than five years. Although we have found no evidence of an increased risk of malaria parasitemia or parasite density with markers of hyposplenism, the role played by an underlying immunity to malaria among SCD patients in malaria-endemic region is not clear and needs further studies.

Spleen function is reduced in individuals with NR5A1 variants with or without a difference of sex development: a cross-sectional study.

NR5A1 is a key regulator of sex differentiation and has been implicated in spleen development through transcription activation of TLX1. Concerns exist about hypo- or asplenism in individuals who have a difference of sex development (DSD) due to an NR5A1 disease-causing variant. We aimed to assess spleen anatomy and function in a clinical cohort of such individuals and in their asymptomatic family member carriers. Cross-sectional assessment in 22 patients with a DSD or primary ovarian insufficiency and 5 asymptomatic carriers from 18 families, harboring 14 different NR5A1 variants. Spleen anatomy was assessed by ultrasound, spleen function by peripheral blood cell count, white blood cell differentiation, percentage of nonswitched memory B cells, specific pneumococcal antibody response, % pitted red blood cells, and Howell-Jolly bodies. Patients and asymptomatic heterozygous individuals had significantly decreased nonswitched memory B cells compared to healthy controls, but higher than asplenic patients. Thrombocytosis and spleen hypoplasia were present in 50% of heterozygous individuals. Four out of 5 individuals homozygous for the previously described p.(Arg103Gln) variant had asplenia. Individuals harboring a heterozygous NR5A1 variant that may cause DSD have a considerable risk for functional hyposplenism, irrespective of their gonadal phenotype. Splenic function should be assessed in these individuals, and if affected or unknown, prophylaxis is recommended to prevent invasive encapsulated bacterial infections. The splenic phenotype associated with NR5A1 variants is more severe in homozygous individuals and is, at least for the p.(Arg103Gln) variant, associated with asplenism.

Intra-erythrocytic vacuoles in asplenic patients: elusive genesis and original clearance of unique organelles.

The spleen plays a dual role of immune response and the filtration of red blood cells (RBC), the latter function being performed within the unique microcirculatory architecture of the red pulp. The red pulp filters and eliminates senescent and pathological RBC and can expell intra-erythrocytic rigid bodies through the so-called pitting mechanism. The loss of splenic function increases the risk of infections, thromboembolism, and hematological malignancies. However, current diagnostic tests such as quantification of Howell-Jolly Bodies and splenic scintigraphy lack sensitivity or are logistically demanding. Although not widely available in medical practice, the quantification of RBC containing vacuoles, i.e., pocked RBC, is a highly sensitive and specific marker for hyposplenism. The peripheral blood of hypo/asplenic individuals contains up to 80% RBC with vacuoles, whereas these pocked RBC account for less than 4% of RBC in healthy subjects. Despite their value as a spleen function test, intraerythrocytic vacuoles have received relatively limited attention so far, and little is known about their origin, content, and clearance. We provide an overview of the current knowledge regarding possible origins and mechanisms of elimination, as well as the potential function of these unique and original organelles observed in otherwise "empty" mature RBC. We highlight the need for further research on pocked RBC, particularly regarding their potential function and specific markers for easy counting and sorting, which are prerequisites for functional studies and wider application in medical practice.

The Value of IgM Memory B-Cells in the Assessment of Splenic Function in Childhood Cancer Survivors at Risk for Splenic Dysfunction: A DCCSS-LATER Study.

Childhood cancer survivors (CCS) who received radiotherapy involving the spleen or total body irradiation (TBI) might be at risk for splenic dysfunction. A comprehensive screening test for examining splenic dysfunction is lacking. We investigated whether IgM memory B-cells could be used to assess splenic dysfunction in CCS who received a splenectomy, radiotherapy involving the spleen, or TBI. All CCS were enrolled from the DCCSS-LATER cohort. We analyzed differences in IgM memory B-cells and Howell-Jolly bodies (HJB) in CCS who had a splenectomy (n = 9), received radiotherapy involving the spleen (n = 36), or TBI (n = 15). IgM memory B-cells < 9 cells/µL was defined as abnormal. We observed a higher median number of IgM memory B-cells in CCS who received radiotherapy involving the spleen (31 cells/µL, p=0.06) or TBI (55 cells/µL, p = 0.03) compared to CCS who received splenectomy (20 cells/µL). However, only two CCS had IgM memory B-cells below the lower limit of normal. No difference in IgM memory B-cells was observed between CCS with HJB present and absent (35 cells/µL vs. 44 cells/µL). Although the number of IgM memory B-cells differed between splenectomized CCS and CCS who received radiotherapy involving the spleen or TBI, only two CCS showed abnormal values. Therefore, this assessment cannot be used to screen for splenic dysfunction.

Clinical and laboratory factors associated with splenic dysfunction among patients with sickle cell disease in a malaria endemic region.

Although loss of splenic function is the expected natural course for individuals with sickle cell disease (SCD), factors such as high HbF and coexistence of alpha thalassemia may ameliorate this process. We evaluated factors associated with two surrogate markers of spleen dysfunction, namely Howell-Jolly bodies (HJBs) and argyrophilic inclusion (AI) red cell counts, among patients with SCD. Cross-sectional data of 182 patients with SCD (median age 11 y; 1-45 y) and 102 normal controls (median age 12 y; 1-32 y) were evaluated. Blood tests including full blood count, serum chemistry and high-performance liquid chromatography were performed. The HJB and AI red cell counts were performed on peripheral blood smears. The percentages of HJB and AI red cells rose significantly with increasing age in the SCD group. On regression analysis, the frequency of HJB red cells associated positively with mean corpuscular hemoglobin (MCH) (β=0.289; p=0.001) and negatively with HbF (β=-0.259; p=0.002). The AI red cell counts also associated positively with MCH (β=0.321; P=0.001) and negatively with HbF (β=-0.242; p=0.020). Data from this study indicate that the negative association of HbF with both markers of splenic dysfunction among our patients with SCD residing in a malaria endemic region is similar to findings elsewhere of its ameliorating effect on splenic dysfunction.

Are accessory spleen screening and resection in refractory immune cytopenia an effective strategy or a waste of resources?

Splenectomy remains an effective treatment for refractory immune cytopenia (RIC), which encompasses immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). Accessory spleens (AS) have been described without identifying specific risk factors. We retrospectively analyzed patients with RIC after splenectomy who underwent splenic scintigraphy (SS) at our institution. Seventy-one patients were included. Sixty-two patients had ITP, five had AIHA, and four had Evans syndrome. Seventy-five percent (n = 53) were women. Eleven patients (15.5%) had an AS detected by SS. A complete response (CR) to first-line steroids (odds ratio (OR) 5.75, 95% confidence interval (CI) 1.37-24.14, p = 0.017) and the absence of Howell-Jolly bodies (HJB) in peripheral blood smear (PBS) (OR 11.37, 95% CI 2.70-47.85, p = 0.001) were found to be risk factors. Patients with both elements had a higher rate of AS (83.3%) when compared to those with one or no factors (p < 0.001). Eight patients (73%) underwent an accessory splenectomy: seven (87.5%) achieved a CR, and none had perioperative complications. The presence of HJB in PBS changed from 25 to 87.5% after accessory splenectomy. We recommend the search for an AS via SS in patients with RIC due to ITP, who had a CR to corticosteroids and the absence of HJB in PBS. Accessory splenectomy is a safe and effective procedure.

Evaluation of two red cell inclusion staining methods for assessing spleen function among sickle cell disease patients in North-East Nigeria.

The loss of splenic function is associated with an increased risk of infection in sickle cell disease (SCD); however, spleen function is rarely documented among SCD patients in Africa, due partly to the non-availability of sophisticated techniques such as scintigraphy. Methods of assessing splenic function which may be achievable in resource-poor settings include counting red blood cells (RBC) containing Howell Jolly Bodies (HJB) and RBC containing silver-staining (argyrophilic) inclusions (AI) using a light microscope. We evaluated the presence of HJB-and AI-containing RBC as markers of splenic dysfunction among SCD patients in Nigeria. We prospectively enrolled children and adults with SCD in steady state attending outpatient clinics at a tertiary hospital in North-East Nigeria. The percentages of HJB-and AI-containing red cells were estimated from peripheral blood smears and compared to normal controls. There were 182 SCD patients and 102 healthy controls. Both AI- and HJB-containing red cells could be easily identified in the participants blood smears. SCD patients had a significantly higher proportion of red cells containing HJB (1.5%; IQR 0.7%-3.1%) compared to controls (0.3%; IQR 0.1%-0.5%) (P <0.0001). The AI red cell counts were also higher among the SCD patients (47.4%; IQR 34.5%-66.0%) than the control group (7.1%; IQR 5.1%-8.7%) (P < 0.0001). The intra-observer reliability for assessment of HJB- (r = 0.92; r2 = 0.86) and AI- containing red cells (r = 0.90; r2 = 0.82) was high. The estimated intra-observer agreement was better with the HJB count method (95% limits of agreement, -4.5% to 4.3%; P = 0.579).We have demonstrated the utility of light microscopy in the assessment of red cells containing-HJB and AI inclusions as indices of splenic dysfunction in Nigerian SCD patients. These methods can be easily applied in the routine evaluation and care of patients with SCD to identify those at high risk of infection and initiate appropriate preventive measures.

Howell-Jolly Body-Like Inclusions in Coronavirus Disease 2019 (COVID-19): Possible Novel Findings.

During COVID-19 pandemic, it is difficult to distinguish febrile patient infected by SARS-CoV-2 or bacterial causes. Howell-Jolly bodies are a well-known entity found in red blood cells. They are nuclear fragments, composed of deoxyribonucleic acid, commonly observed in the peripheral blood smears of hyposplenic or asplenic patients. Recently, similar inclusions often referred to as Howell-Jolly body-like inclusions (HJBLIs) have been reported in the neutrophils of patients with acquired immune deficiency syndrome (AIDS) and COVID-19 patient. To explore whether HJBLIs in peripheral blood smear could differentiate between patients with confirmed SARS-CoV-2 and bacterial pneumonia. We performed cross-sectional study using secondary data from COVID-19 database and re-evaluated peripheral blood smears to identify HJBLIs. We included confirmed COVID-19 adults age >18 years who were hospitalized in Dr. Hasan Sadikin General Hospital, Bandung, Indonesia from March 1st 2020-May 31st 2020. We also examined peripheral blood smears in patients with confirmed bacterial pneumonia as a control group. Clinical characteristics including disease severity, CURB-65 score, comorbidity, and the present of HJBLIs in peripheral blood smears were evaluated. Overall, 33 patients were included: 22 were confirmed COVID-19 and 11 were confirmed bacterial pneumonia. The median (interquartile range) age in COVID-19 and patients with bacterial pneumonia were 53 years (40-64) vs 57 years (53-71), respectively. Compared with patients with bacterial pneumonia, HJBLIs were significantly higher in COVID-19 patients [21/22 (80.8%) vs 5/11 (45.5%), p 0.001]. Howell-Jolly body-like inclusions could be a potential feature to help differentiate between COVID-19 and bacterial pneumonia.

High Incidence of Intra-Cardiac Supra-Centimetric Thrombosis and Embolic Complications in Sickle Cell Disease Patients with Dual Lumen Ports

In Sickle Cell Disease (SCD) patients with cerebral vasculopathy and/or other severe complications, a transfusion program is indicated. A sustainable venous access is vital for patients engaged in these programs (5-10% of patients with SCD). Dual lumen ports (Vortextechnology) have emerged has a way to bypass the frequent local complications associated with arteriovenous fistulae. Between July 2015 and December 2020, a dual lumen port was implanted in 27 SCD patients in transfusion program at Necker Hospital. Over a 30 months period, we observed 20 venous thromboembolic events (VTE) in 12 patients (44%): 18 were voluminous intracardiac thrombosis (Fig 1), asymptomatic (n=15) or complicated of pulmonary embolism (n=3), one jugular thrombosis and one superior cellar syndrome, leading to long-term curative anticoagulation, except in cases of contraindication such as Moya-Moya. VTE were diagnosed on average 18 months after implantation (range 1 to 29 months). This observation has considerably limited the use of the device, even though no complications were observed in 56% of patients, suggesting that a subgroup of patients may be at lower risk of thrombosis and could still benefit from a Vortex. We conducted a clinical study aiming to identify potential biological markers related to Vortex thrombosis in SCD patients. Among the biological markers, alterations of thrombin generation and fibrino-formation, as well as potentially procoagulants items altered in SCD, such as microvesicules (MV), red blood cell (RBC) deformability and the intensity of splenic dysfunction were explored. Between January and June 2021, 11 healthy donors and 58 patients with SCD treated by erythrapheresis in the biotherapy department of Necker Hospital were included, 23 patients with a Vortex with (n=12) or without (n=11) intra-cardiac and/or other VTE and 36 patients without a Vortex (including 8 with a history of VTE). Thrombin generation Assay (TGA) in plasma and standard coagulation assays were performed on pre-transfusion sample, including Prothrombin Time (PT), activated partial thromboplastin time (APTT) and fibrinogen, dosage of FII, FV, FVII, FX and FXII, Protein C (PC), Protein C (PS), Antithrombin (AT) dosage. All samples were pretreated by activated carbon for removal of Direct Oral Anticoagulants from plasma. Quantification and characterization of plasma MV, determination of erythrocyte deformability and point of sickling (ektacytometry with oxygenscan) and assessment of splenic function (quantification of pocked RBC and Howell Jolly bodies) were also performed. We found a significant decrease in the mean levels of PC (69.7 vs 109.1%, p=0.0002) and PS (58.4 vs 99.4, p<0.0001) in SCD patients when compared to healthy donors while AT levels were only slightly decreased (91 vs 98,6%, p=0,04). We also observed a significant decrease in vitamin-K dependent coagulation factors: FII (77.5 vs 100.6%, p<0,0001), FVII (61.9 vs 84.5% p=0.005) and FX (71.8 vs 96.8%, p=0.0001) while FV (80.1 vs 93.8% p=0.02) was only slightly decreased and fibrinogen levels were comparable (2.62 vs 2.57g/L p=0.86). No significant differences were noted between SCD patient groups (with or without thrombosis). Thrombin generation Assay (TGA) in plasma showed an increase in endogenous thrombin potential (ETP; 714 vs 451nM.min, p=0.04) and Peak (161 vs 95 nM.min, p=0.03) in SCD patients compared to healthy donors but no differences were found between patient groups. Assessment of splenic function, point of sickling in oxygenscan and deformability of RBC measured by ektacytometry showed no differences between patient groups. Patients with SCD had a significant increase of circulating CD235+ MV (93268 vs 15 207/µL, p<0.0001), Annexin 5 MV (473553 vs 248404 /µL) and CD235+ erythrocyte ghosts (1757 vs 61/µL) compared to healthy donors. Again, no differences were seen between patient study groups. In conclusion, no biological predictive factors for intra cardiac Vortex thrombosis could be demonstrated by our study despite an extensive study of biological factors known to date to favor thrombosis. Given the high incidence of intracardiac thrombosis and the impossibility to date of predicting the risk of occurrence, we recommend that Vortex technology be reserved for patients who are already receiving effective anticoagulant therapy or for whom no other solution can be found, while maintaining a close surveillance of echocardiography.

The protective effect of the spleen in sickle cell patients. A comparative study between patients with asplenia/hyposplenism and hypersplenism.

Sickle cell disease (SCD) is caused by a point mutation in the beta-globin gene. SCD is characterized by chronic hemolytic anemia, vaso-occlusive events leading to tissue ischemia, and progressive organ failure. Chronic inflammatory state is part of the pathophysiology of SCD. Patients with SCD have extremely variable phenotypes, from mild disease to severe complications including early age death. The spleen is commonly injured in SCD. Early splenic dysfunction and progressive spleen atrophy are common. Splenomegaly and hypersplenism can also occur with the loss of the crucial splenic function. Acute, life-threatening spleen-related complications in SCD are well studied. The association of laboratory parameters with the spleen status including hyposplenism, asplenia, and splenomegaly/hypersplenism, and their implication in vaso-occlusive crisis and long-term complications in SCD remain to be determined. We evaluated the association between the spleen status with clinical and laboratory parameters in 31 SCD patients: Group a) Patients with asplenia/hyposplenism (N = 22) (including auto-splenectomy and splenectomized patients) vs. Group b) patients with splenomegaly and or hypersplenism (N = 9). Laboratory studies included: Complete Blood Count, reticulocyte count, iron metabolism parameters, C Reactive Protein (CRP), Hb variant distribution, and D-dimer. Metabolic and morphological red blood cell (RBC) studies included: density gradient (by Percoll), glucose consumption, lactate release, and K+ leakage, fetal RBC (F-Cells) and F-Reticulocytes, annexinV+, CD71+, oxidative stress measured by GSH presence in RBC and finally Howell Jolly Bodies count were all analyzed by Flow Cytometry. Scanning electron microscopy analysis of RBC was also performed. Patients with asplenia/hyposplenism showed significantly higher WBC, platelet, Hematocrit, hemoglobin S, CRP, D-dimer, Gamma Glutamyl Transferase (GGT), cholesterol, transferrin, annexin V+ RBCs, CD71+ RBCs, together with a markedly lower F Reticulocyte levels in comparison with splenomegaly/hypersplenism patients. In summary, important differences were also found between the groups in the studied RBCs parameters. Further studies are required to elucidate the effect of the spleen including hyper and hypo-splenia on laboratory parameters and in clinical manifestations, vascular pathology, and long-term complications of SCD. The benefits and risks of splenectomy compared to chronic transfusion need to be evaluated in clinical trials and the standard approach managing hypersplenism in SCD patients should be re-evaluated.

P-025: ABNORMAL MITOCHONDRIA RETENTION IN MATURE RED BLOOD CELLS OCCURS NOT ONLY IN SS BUT ALSO SC PATIENTS AND IS ASSOCIATED WITH HEMOLYTIC MARKERS

Purpose: Sickle cell disease (SCD), the most frequent genetic disease worldwide, results from a unique point mutation leading to the synthesis of the abnormal hemoglobin S (HbS). Under hypoxic condition, HbS polymerization causes a mechanical distortion of red blood cells (RBCs) called sickling, which is accompanied by a decrease of their deformability and their life span. SCD is characterized by a wide variability of clinical expression and a complex pathophysiology including vaso-occlusive processes, intravascular hemolysis as well as pro-inflammation, pro-coagulant and vaso-constrictive states. Recently, the abnormal mitochondria retention in mature RBCs has been documented in small series of SS patients but the biological consequences of this abnormality have not been fully documented and not been documented in other sickle cell syndromes. In the present study, we analyzed mature RBC mitochondria retention in both SS and SC patients, the second most frequent sickle cell syndrome, taking into account the biological/clinical features associated with these abnormal RBCs. Materials and methods: Eighty-nine adult SCD patients were included: 35 SS patients treated with hydroxyurea (HU), 15 SS patients not treated with HU and 39 SC patients. All of them were at steady-state and regularly followed by the sickle center of Guadeloupe. Twenty-one Guadeloupean adult controls (AA) were also included. Mature RBCs containing mitochondria were identified by flow cytometry using MitoTracker® probe as well as thiazole orange and anti-CD71 antibody to discriminate total and stress reticulocytes respectively. Correlations between the percentage of mature mitochondria+-RBCs and the following parameters were then analyzed: RBC deformability (Ektacytometry), reticulocytes (both total and CD71+) counts and levels of hemoglobin, total bilirubin, aminotransferase aspartate and lactate dehydrogenase levels using routine procedures. The impact of splenic function was evaluated using Howell-Jolly bodies, assessed by classic May-Grünwald Giemsa smears, as a marker of splenic dysfunction. We also analyzed the relationships between mitochondria+-RBCs and HU treatment as well as the rates of hyper vaso-occlusive (painful vaso-occlusion, acute chest syndrome, osteonecrosis) and hyper hemolytic (micro/macro-albuminuria, leg ulcer) complications. Results: Abnormal mitochondria+ RBCs were detected not only in SS but also in SC patients, although at a less extend. In both sickle cell syndromes, positive correlations with mitochondria+ RBCs were detected with hemolysis markers (percentage of total and stress reticulocytes). In addition, a negative relationship with hemoglobin level was detected in SS patients. A negative correlation was also detected between these abnormal mitochondria+ RBCs and RBC deformability. In contrast, no association between the presence of this RBC sub-population and clinical manifestations, hydroxyurea treatment or Howell-Jolly bodies was detected. Conclusion: In the present report, we demonstrated that abnormal retention of mitochondria in mature RBCs occurred not only in SS but also in SC patients. We also presented evidence that this phenotype could be related to hemolysis rate. Further studies are warranted to reveal the molecular mechanisms involved. E. BERNIT declares a conflict of interest: Consultancy, Expert: for NOVARTIS PHARMA SAS

Hematologic and biochemical reference intervals of brown-throated sloths (Bradypus variegatus).

The brown-throated sloth, Bradypus variegatus, is a common species endemic to South and Central America. Nonetheless, maintaining these animals in captivity can be challenging, and very few institutions manage to do so. The São Paulo Zoological Park Foundation is in a remnant of the Atlantic rainforest in the middle of São Paulo, the largest city in Brazil. This forest fragment has a population of B. variegatus that is geographically isolated and yet to be studied. Assessing the health status of individuals remains difficult due to the lack of reference intervals (RIs) for hematologic and biochemical variables for this species. We aimed to establish hematologic and biochemical RIs in a population of wild B. variegatus living in the largest remnant of Atlantic rainforest in São Paulo city, Brazil. Blood samples from 25 individuals of wild B. variegatus were collected and analyzed for 20 hematologic and 21 biochemical variables, using standard laboratory techniques. Each variable was statistically analyzed according to the American Society for Veterinary Clinical Pathology guidelines. The results obtained for each variable were statically analyzed, making it possible to obtain descriptive statistics for all hematologic and biochemical variables. RIs were determined for 16 hematologic variables. During the microscopic analysis, we observed anisocytosis, polychromatophils, Howell-Jolly bodies, macroplatelets, and reactive lymphocytes. The RIs and descriptive statistics described here establish important baseline numbers that could be essential for the management and treatment of both captive and wild B. variegatus sloths.

Phenotypic analysis of erythrocytes in sickle cell disease using imaging flow cytometry.

The morphology and other phenotypic characteristics of erythrocytes in sickle cell disease (SCD) have been analyzed for decades in patient evaluation. This involves a variety of techniques, including microscopic analysis of stained blood films, flow cytometry, and cell counting. Here, we analyzed SCD blood using imaging flow cytometry (IFC), a technology that combines flow cytometry and microscopy to enable simultaneous rapid-throughput analysis of cellular morphology and cell-surface markers. With IFC, we were able to automate quantification of poikilocytes from SCD blood. An important subpopulation of poikilocytes represented dense cells, although these could not be distinguished from other poikilocytes without first centrifuging the blood through density gradients. In addition, CD71-positive RBCs from SCD patients had two subpopulations: one with high CD71 expression and a puckered morphology and another with lower CD71 expression and biconcave morphology and presumably representing a later stage of differentiation. Some RBCs with puckered morphologies that were strongly positive for DAPI and CD49d were in fact nucleated RBCs. IFC identified more phosphatidylserine-expressing red cells in SCD than did conventional flow cytometry and these could also be divided into two subpopulations. One population had diffuse PS expression and appeared to be composed primarily of RBC ghosts; the other had lower overall PS expression present in intense, punctate dots overlying Howell-Jolly bodies. This study demonstrates that IFC can rapidly reveal and quantify RBC features in SCD that require numerous tedious methods to identify conventionally. Thus, IFC is likely to be a useful technique for evaluating and monitoring SCD.

S112: COMPARATIVE SCORE OF HOWELL-JOLLY BODIES IN SICKLE CELL DISEASE PATIENTS ACCORDING TO GENOTYPE, AGE, SPLENECTOMY OR STEM CELL TRANSPLANTATION HISTORY

Background: Presence of Howell-Jolly bodies is associated with splenic dysfunction which progressively appears during aging or after splenectomy in patients with sickle cell disease. Improvement of splenic function has been found in several transplanted patients for SCD (Ferster 1993, Bernaudin 2007) encouraging our team to propose partial splenectomy to patients with hypersplenism and available matched-sibling donor. Aims: The goal of the present study was to evaluate the outcome of the splenic function from birth to adult age and the impact of splenectomy (partial or total) in SCD patients transplanted or not. Patients and Methods: In the pediatric Créteil SCD-cohort, including 907 SCD patients (111 SC, 47 Sb+, 725 SS, 21 Sb0 and 3 SDPunjab), Howell-Jolly bodies score (from 0 to 4) was prospectively assessed and recorded at each annual check-up. This study included 5013 annual check-ups (637 in SC/Sb+ and 4376 in SS/Sb0 patients) from Jan 1991 to July 2019. Results: Howell-Jolly bodies mean score increased during aging in SCD patients but was significantly lower in SC/Sb+ than in SS/Sb0 patients at each age of assessment (Figure 1). During the 2nd year of life, mean (SD Howell-Jolly bodies score was 0.34 (0.61) in SC/Sb+ vs 0.69 (0.82) in SS/Sb0 (P=0.01) while during the 18th year it was 0.61 (0.61) in SC/Sb+ vs 1.33 (1.1) in SS/Sb0 children (P<0.001). Among 749 SCA children, 86 were splenectomized at mean (SD) age of 6.4 years (3.9). As expected, mean (SD) Howell-Jolly bodies score was significantly higher at check-up in splenectomized than in non-splenectomized SCA-patients: 1.73 (1.2) vs 1.1 (1.0), p<0.001. However, the difference between both populations decreased during aging because of the progressive functional splenic dysfunction in non-splenectomized patients (Figure 2) Among SCA-children, 138 were transplanted with matched sibling donor at mean (SD) age of 9.0 years (4.9y). At each age of assessment, Howell-Jolly bodies score was strongly significantly (p<0.001) lower in transplanted than in non-transplanted children (Figure 3). At check-up performed during the 18th year of life, mean (SD) Howell-Jolly bodies score was 1.57 (1.0) in 241 non transplanted vs 0.54 (0.9) in 70 transplanted children (p<0.001) and the score in transplanted children was significantly positively correlated with the age at transplant (r=0.335, p=0.002). Seven children with splenic sequestration history or hypersplenism and available MSD-donor had partial splenectomy before transplant in order to try to preserve splenic function. Mean (SD) Howell-Jolly bodies score was 1.83 (1.2) before and was nul after transplant in all seven children.Conclusion: This cohort study clearly shows that splenic dysfunction increases during aging in SCA children and that stem cell transplantation allows to preserve splenic function especially since the transplant is performed early. In children with indication of splenectomy for recurrent splenic sequestrations or hypersplenism who have an available matched sibling donor, the present study encourages to propose partial vs total splenectomy just before stem cell transplantation in order to preserve splenic function.

Splenic auto-transplantation after splenectomy for trauma: evaluation of a new technique.

Splenic autotransplantation is a promising method to recover splenic functions after traumatic splenectomy. However, it is associated with several postoperative complications, such as subphrenic abscess, intestinal obstruction and torsion of the omentum with necrosis of the implanted splenic tissues. Therefore, the aim of this study is to evaluate a new splenic autotransplantation technique that could overcome those complications. A single segment of the spleen was implanted inside a pedunculated omental pouch and fixed in the native site of the spleen in 15 patients who underwent splenectomy for abdominal trauma. This group of patients was compared with the next 17 patients who underwent splenectomy alone. Additionally, splenic functions of the patient and control groups were evaluated 3months using peripheral blood smear [the presence of Howell-Jolly (HJ) bodies] and abdominal contrast-enhanced computed tomography (CECT). All patients who underwent splenic autotransplantation showed evidence of a well-vascularised splenic tissue on CECT and normal peripheral blood smear without HJ bodies 3months postoperatively. No postoperative complications related to splenic autotransplantation were observed, and platelet count after 3months was significantly higher in patients who underwent splenectomy only (p=0.04). Splenic autotransplantation using the aforementioned technique could restore splenic functions with minimum postoperative complications related to the procedure.

Germline 3p22.1 microdeletion encompassing RPSA gene is an ultra-rare cause of isolated asplenia

BackgroundIsolated Congenital Asplenia (ICA, OMIM #271400) is a rare, life-threatening abnormality causing immunodeficiency, which is characterized by the absence of a spleen. Diagnosis should be completed in early childhood and antibiotic prophylaxis applied with additional vaccinations.Case presentationWe report the case of a six-month old girl with hematologic abnormalities and asplenia documented in imaging, with Howell-Jolly bodies in peripheral blood smear. Targeted Next Generation Sequencing screening did not reveal any pathogenic variant in genes associated with congenital asplenia. Since absence of the spleen was found by imaging, high-resolution copy number variations detection was also performed using genomic Single Nucleotide Polymorphism microarray: a heterozygous 337.2 kb deletion encompassing the RPSA gene was observed, together with SLC25A38, SNORA6, SNORA62 and MOBP genes. Despite haploinsufficiency of SLC25A38, SNORA6, SNORA62 and MOBP, no change in the clinical picture was observed. A search of available CNV databases found that a deletion of the RPSA locus seems to be unique and only duplications were found in this region with the frequency of less than 0.02%.ConclusionsCopy number variations in RPSA gene locus are ultrarare cause of isolated asplenia. Furthermore, since the patient does not present any concomitant clinical features, it would appear that haploinsufficiency of SLC25A38, SNORA6, SNORA62 and MOBP genes does not affect the phenotype of patients. However, to confirm this thesis a longer follow-up of the patient’s development is needed.

Revisiting Spleen Function and Pneumococcal Risk in Children with Hemoglobin SC Disease

Spleen dysfunction and susceptibility to pneumococcal infection is a well known feature in homozygous sickle cell disease (HbSS), whilst to date splenic function in hemoglobin SC disease (HbSC) has been poorly investigated. The aim of this study was to analyze spleen function in children with HbSC disease using a high-throughput validated method (1) and to examine if the current recommendations regarding pneumococcal risk are appropriate in this population.Spleen function was evaluated using a flow cytometry quantification of red blood cells (RBCs) with Howell-Jolly bodies (HJBs), in a cross-sectional study of patients at steady state during an outpatient visit in an expert center. Quantification of HJB-RBCs was performed in children with HbSC disease aged < 10 years and compared to children with HbSS disease or healthy children of the same age groups, or splenectomized children. Additional exploratory analysis was performed according to age (under or above the age of 5 years old) and treatment group (hydroxyurea).The median (Q1-Q3) HJB-RBCs count was 16 (11-28.25) /100.000 RBCs in 40 HbSC children (Figure 1). This result was not statistically different from the control group of 22 healthy children (p=0.96) nor in subgroups < or ≥ 5 years old, indicating that children with HbSC under 10 years have a preserved splenic function. Expectedly, the HJB-RBCs counts differed significantly from splenectomized children (419 (296-489)/100.000 RBCs, n=15, p<0.0001).By contrast, among the 53 HbSS children, the median HJB-RBCs count was 134 (29-216) /100.000 RBCs, differing significantly from HbSC children (p<0.0001). In HbSS children, HJB-RBCs counts increased significantly with age (r=0.30, p=0.03), showing important variability among subjects but did not reach the level found in splenectomized patients suggesting that complete loss of spleen function occurs presumably later in a majority of children in this population. Treatment with hydroxyurea did not significantly impact HJB-RBCs counts in a subgroup analysis in HbSS children.The result of this study suggests that spleen function in children under 10 years old with HbSC is not altered. The routine administration of prophylactic penicillin to young children with SC disease may therefore be questioned. Similarly, fever in children with HbSC under 3 years old may not require parenteral antibiotics as it is generally currently recommended by analogy to children with HbSS. Functional or anatomical asplenia in children with HbSC is delayed compared to those with HbSS at least after the first decade of life. Future large cohort studies using similar methodology will allow better evaluation of the pneumococcal risk in adolescents and adults with Hb SC disease.Bibliography(1) El Hoss S, Dussiot M, Renaud O, Brousse V, El Nemer W. A novel non-invasive method to measure splenic filtration function in humans. Haematologica. oct 2018;103(10):e436-9. [Display omitted] DisclosuresEl Nemer: Hemanext: Consultancy.