Extreme Reticulocytosis After Splenectomy in a Patient With Hemoglobin Mizuho.

BACKGROUND: Patients with Hb Mizuho may be splenectomized at a young age to decrease their need for blood transfusions. OBSERVATIONS: Transfusion-dependency decreased dramatically in a 4-year-old white boy with Hb Mizuho after splenectomy. Surprisingly, he developed reticulocytosis (>1000×10 9 /L) with a peak reticulocyte percentage of 49%, and erythrocyte abnormalities, including Heinz bodies, Howell-Jolly bodies, and basophilic stippling. Manual reticulocyte counting and flow cytometric measurement with anti-CD71 antibodies supported a truly elevated reticulocyte count. CONCLUSIONS: We propose possible explanations for the extreme reticulocytosis that arose postsplenectomy and compare the reticulocyte count in the present case with previously published cases.

Principales modificaciones asociadas a la esplenectomía / Main modifications associated with splenectomy

Introducción. El bazo es un órgano linfoide implicado en el reconocimiento antigénico, la depuración de patógenos y la remoción de eritrocitos envejecidos o con inclusiones citoplasmáticas. La esplenectomía es una técnica utilizada tanto para el diagnóstico (linfomas), el tratamiento (trombocitopenia inmune, anemia hemolítica adquirida) y la curación (microesferocitosis hereditaria) de diversas enfermedades. Métodos. Describir los principales cambios hematológicos y complicaciones asociadas al procedimiento de esplenectomía. Discusión. Los cambios posteriores a la esplenectomía pueden ser inmediatos, como la aparición de cuerpos de Howell-Jolly, la trombocitosis y la presencia de leucocitosis durante las primeras dos semanas. Otras complicaciones tempranas incluyen la presencia de trombosis, en especial en pacientes con factores de riesgo secundarios (edad, sedentarismo, manejo hospitalario, obesidad) o un estado hipercoagulable (diabetes, cáncer, trombofilia primaria), siendo tanto el flujo de la vena porta como el volumen esplénico los principales factores de riesgo para su aparición. Las complicaciones tardías incluyen la alteración en la respuesta inmune, aumentando el riesgo de infecciones por bacterias encapsuladas, en conjunto con una reducción en los niveles de IgM secundario a la ausencia de linfocitos B a nivel de bazo. Debido al riesgo de infecciones, principalmente por Streptococcus pneumoniae, la esplenectomía parcial se ha considerado una opción. Conclusión. Una adecuada valoración de la indicación de esplenectomía y la identificación precoz de complicaciones posoperatorias son fundamentales para reducir la mortalidad asociada a la esplenectomía

Introduction. The spleen is a lymphoid organ involved in antigen recognition, pathogen clearance, and removal of aged erythrocytes or those with cytoplasmic inclusions. Splenectomy is a technique used for diagnosis (lymphomas), treatment (immune thrombocytopenia, acquired hemolytic anemia), and cure (hereditary microspherocytosis) of various diseases. Methods. To describe the main hematological changes and complications associated with the splenectomy procedure. Discussion. Changes after splenectomy can be considered immediate: the appearance of Howell-Jolly bodies, thrombocytosis, and leukocytosis during the first two weeks. Other complications include the presence of thrombosis, especially in patients with risk factors (age, sedentary lifestyle, long hospital stay, obesity) or a hypercoagulable state (diabetes, cancer, primary thrombophilia), with both portal vein flow and splenic volume being the main risk factors for its appearance. Late complications include altered immune response, increased risk of infections by encapsulated bacteria, and a reduction in IgM levels secondary to the absence of B lymphocytes in the spleen; due to the risk of diseases mainly by Streptococcus pneumoniae, partial splenectomy has been considered an option. Conclusion. An adequate assessment of the indication for splenectomy and the early identification of complications are essential to reduce the mortality associated with splenectomy

[Hyposplenism, an underrecognised immune diffeciency: Is sonography helpful?] / "Hyposplenie", eine weitgehend unerkannte Immunschwäche: Ist die Sonografie hilfreich?

The immunodeficiency syndrome of functional hyposplenia/asplenia is frequently unrecognized. The gold standard procedure for the diagnosis of this disease is technetium-99m scintigraphy of heat-denatured erythrocytes and the detection of pitted erythrocytes or Howell-Jolly bodies. Initial work suggests that this clinical presentation is associated with a small spleen. The aim of this review is to describe the value of B-mode and contrast-enhanced ultrasound in the identification of immunodeficiency caused by functional hyposplenism.

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.

Potential role of Howell-Jolly bodies in identifying functional hyposplenism: a prospective single-institute study.

Although patients with cancer and immunosuppression are at a risk of functional hyposplenism, how to detect it promptly remains unclear. Since hyposplenism allows erythrocytes with nuclear remnants (Howell-Jolly bodies [HJBs]) to appear in the peripheral blood, HJB detection by a routine microscopic examination may help identify patients with functional hyposplenism. This prospective study was thus performed to determine the underlying diseases in patients who presented with HJBs. Of 100 consecutive patients presenting with HJBs, 73 had a history of splenectomy. The remaining 27 had hematologic cancer (n = 6, 22%), non-hematologic cancer (n = 8, 30%), hepatic disorders (n = 4, 15%), premature neonates (n = 3, 11%), hemolytic anemia (n = 2, 7%), autoimmune disorders (n = 2, 7%) and miscellaneous diseases (n = 2, 7%), and their prior treatments included chemotherapy (n = 8, 30%), steroids (n = 7, 26%) and molecular-targeted therapy (n = 3, 11%). Among the 27 patients, 22 had computed tomography scans available: 3 (14%) had underlying diseases in the spleen, and the remaining 19 (86%) were all found to have a decreased splenic volume, including 11 (50%) with more than 50% of the ideal value. The present findings suggest that HJB detection identifies patients with potentially functional hyposplenism who should receive appropriate interventional treatment, such as vaccination and prophylactic antibiotics.

[Emergence of Howell-Jolly bodies in a patient with splenic hypoplasia complicated by fulminant pneumococcal infection].

We report the case of a patient with fulminant pneumococcal infection along with the presence of Howell-Jolly bodies (HJBs) and splenic hypoplasia at the onset. A 71-year-old man developed fever during outpatient chemotherapy for IgG-κ multiple myeloma and was diagnosed with septic shock due to invasive pneumococcal infection. HJBs were observed on peripheral blood smears at this visit. Computed tomography revealed marked hypoplasia of spleen, suggesting the presence of hyposplenic function. Antibacterial therapy was initiated and the pneumococcal infection was cured; however, there was no notable change in his splenic hypoplasia. Splenic hypoplasia can be associated with fatal infections; hence, care should be taken when it is found in the elderly and in patients with cancer and those receiving immunosuppressive treatment. Even today, when automated hematology analyzers have become common, not all patients with hematological diseases have peripheral blood smears checked with a normal optical microscope. This study suggests that HJBs may be useful for simple and rapid screening of splenic hypofunction. The importance of detecting HJBs in peripheral blood smears with a normal optical microscope should be re-recognized.

[Benefit of Howell-Jolly bodies detection: finding of an acquired hyposplenism in a patient with Goodpasture syndrome]. / Intérêt de la détection des corps de Howell-Jolly : hyposplénisme acquis dans le cadre d'un syndrome de Goodpasture.

Howell-Jolly bodies are intraerythrocytic inclusions corresponding to a small portion of chromatin. Red blood cells that contain these nuclear remnants are removed from the circulation by the spleen. In most cases, presence of Howell-Jolly bodies on a blood smear is the result of functional asplenia and splenectomy. Observation. We report incidental finding of numerous Howell-Jolly bodies in a patient followed by the nephrology department. This microscopic observation of blood smear led to a diagnostic imaging and to the evidence of a reduced spleen, possibly favoured by a history of Goodpasture syndrome in this renal transplant patient without splenectomy. Vaccination and antibioprophylaxy were proposed to prevent infectious risk linked to this splenic hypoplasia. Conclusion. Seeking of Howell-Jolly bodies could be made in every condition associated with a risk of splenic hypoplasia to prevent infectious risk.

Newly recognized Anaplasma sp. in erythrocytes from Gopher Tortoises (Gopherus polyphemus).

BACKGROUND: In 2015, a previously unrecognized intracytoplasmic erythrocytic inclusion was discovered in anemic wild-caught adult gopher tortoises (Gopherus polyphemus). Subsequently, molecular diagnostics revealed this inclusion to be a novel Anaplasma sp. OBJECTIVES: The goal of this study was to morphologically characterize these erythrocytic inclusions by light and transmission electron microscopy (TEM). METHODS: Blood samples were taken from two car-injured wild-caught gopher tortoises for the preparation of Wright-Giemsa stained smears and TEM specimens. CBC data were serially performed and morphologically examined during treatment periods. RESULTS: Studies revealed a moderate to severe anemia with moderate regeneration as indicated by polychromasia and the presence of immature erythroid precursors. In addition, on light microscopy, one to two variably-sized round basophilic stippled paracentral erythrocytic inclusions were present per cell in both animals and involved 10%-25% of erythrocytes. TEM identified the intraerythrocytic inclusions as discrete membrane-bound cytoplasmic vacuoles (morulae) containing membrane-bound bacterial subunits that were of variable size, shape, and electron density. Serial hematologic data indicated complete remission of the infection in response to a single long-term course of doxycycline. CONCLUSIONS: The presence of a regenerative anemia in gopher tortoises from Florida revealed a newly recognized bacterial species that has morphologic characteristics similar to members of the genus Anaplasma.

Spleen Organoid Units Generate Functional Human and Mouse Tissue-Engineered Spleen in a Murine Model.

Introduction: Splenectomy is common after trauma or hematologic disease, and alters immune protection against pathogens, which may lead to fulminant infection with high mortality. Yet the spleen has demonstrable regenerative capacity and cells might be recovered and reimplanted at the time of injury or excision to avoid these risks. Methods: Tissue-engineered spleen (TESp) was generated from ActinGFP mice (mTESp) or human donor spleen (hTESp) through implantation of spleen organoid units (spleen OU), in NOD/SCID mice with concurrent splenectomy, on a biodegradable scaffold. Explants were evaluated and blood smears were obtained to investigate the presence of target cells or Howell-Jolly bodies, which are erythrocyte sequelae of asplenia. Results: TESp was generated from mouse (mTESp) and human (hTESp) donor cells with essential splenic components: red and white pulp with trabeculae. mTESp and hTESp demonstrated green fluorescent protein- or lamin-positive costaining with proliferating cell nuclear antigen, CD4, and CD11c, identifying proliferative donor cells and key immune components of the spleen of donor origin. Animals with hTESp and mTESP combined with splenectomy had significantly fewer Howell-Jolly bodies on blood smears than controls. Conclusion: TESp from mouse and human donor cells can be generated by 4 weeks and contains donor immune cells identified by CD4 and CD11c. TESp reduces postsplenectomy erythrocyte inclusions, indicating possible function. Impact Statement Overwhelming postsplenectomy infection is rare but highly mortal. Tissue-engineered spleen (TESp) was generated from murine (mTESp) and human (hTESp) donors and appeared histologically similar to native spleen. Both mTESp and hTESp demonstrated proliferative cells of donor spleen origin. Importantly, functional cells were demonstrated on imaging with a corresponding reduction in the number of erythrocyte inclusions in blood smears that are typically identified in patients with asplenia and indicate a lack of clearance by functional spleen tissue. Taken together, these findings indicate that this approach might be clinically relevant as a future human therapy.

Insights into determinants of spleen injury in sickle cell anemia.

Spleen dysfunction is central to morbidity and mortality in children with sickle cell anemia (SCA). The initiation and determinants of spleen injury, including acute splenic sequestration (ASS) have not been established. We investigated splenic function longitudinally in a cohort of 57 infants with SCA enrolled at 3 to 6 months of age and followed up to 24 months of age and explored the respective contribution of decreased red blood cell (RBC) deformability and increased RBC adhesion on splenic injury, including ASS. Spleen function was evaluated by sequential 99mTc heated RBC spleen scintigraphy and high-throughput quantification of RBCs with Howell-Jolly bodies (HJBs). At 6 and 18 months of age, spleen filtration function was decreased in 32% and 50% of infants, respectively, whereas the median %HJB-RBCs rose significantly (from 0.3% to 0.74%). An excellent correlation was established between %HJB-RBCs and spleen scintigraphy results. RBC adhesion to laminin and endothelial cells increased with time. Adhesion to endothelial cells negatively correlated with splenic function. Irreversibly sickled cells (ISCs), used as a surrogate marker of impaired deformability, were detected at enrollment and increased significantly at 18 months. %ISCs correlated positively with %HJB-RBCs and negatively with splenic uptake, indicating a relationship between their presence in the circulation and spleen dysfunction. In the subgroup of 8 infants who subsequently experienced ASS, %ISCs at enrollment were significantly higher compared with the asymptomatic group, suggesting a major role of impaired deformability in ASS. Higher levels of %HJB-RBCs were observed after the occurrence of ASS, demonstrating its negative impact on splenic function.

Evaluation of Thalassaemia Screening Tests in the Antenatal and Non-Antenatal Populations in Singapore.

INTRODUCTION: Haemoglobinopathy testing is performed for carrier screening and evaluation of microcytic anaemia. We evaluated the effectiveness of thalassaemia screening tests at our institution and suggest ways of improving the testing algorithm. MATERIALS AND METHODS: A total of 10,084 non-antenatal and 11,364 antenatal samples with alkaline gel electrophoresis (AGE), capillary electrophoresis (CE), haemoglobin H (HbH) inclusion test, mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) were retrospectively reviewed. A subgroup of 187 samples with genetic testing was correlated with HbH inclusions and MCH/ MCV. The effect of iron deficiency on percentage hemoglobin A2 (HbA2) was studied. RESULTS: HbH inclusion test showed low sensitivity of 21.43% for α-thalassaemia mutations but higher sensitivity of 78.95% for --SEA deletion. By receiver operating characteristic (ROC) analysis, MCH ≤28 pg or MCV ≤80 fl for non-antenatal samples and MCH ≤27 pg or MCV ≤81 fl for antenatal samples had >98% sensitivity for HbH inclusions. Above these thresholds, the probability that HbH inclusions would be absent was <99% (negative predictive value [NPV] >99%). MCH ≥28 pg had 100% sensitivity (95% CI 95.63%-100%) for α-thalassaemia mutations and 97.68% calculated NPV in the antenatal population. Detection of haemoglobin variants by CE correlated highly with AGE (99.89% sensitivity, 100% specificity). Severe iron deficiency reduced HbA2 in hemoglobin (P <0.001) and α-thalassaemia (P = 0.0035), but not in ß-thalassaemia. CONCLUSION: MCH/MCV thresholds have adequate sensitivity for α-thalassaemia in the antenatal population, and genotyping plays an important role as HbH inclusion test shows low sensitivity. CE without AGE, may be used as initial screening for haemoglobin variants. Our study provides contemporary data to guide thalassaemia screening algorithms in Singapore.

Howell-Jolly Body-Like Inclusions in Neutrophils of Patients With Myelodysplastic Syndrome: A Novel Correlation.

CONTEXT.­: Howell-Jolly body-like inclusions have been previously associated with patients who are human immunodeficiency virus (HIV) infected, taking antiviral medications, and immunosuppressed. These inclusions appear in neutrophils and resemble Howell-Jolly bodies of normoblasts in abnormal erythropoiesis. In granulocytes, they are thought to represent detached nuclear fragments produced from dysplastic granulopoiesis. To the best of our knowledge, no association of Howell-Jolly body-like inclusions and myelodysplastic syndrome (MDS) has been reported previously. OBJECTIVE.­: To establish an unprecedented correlation of Howell-Jolly body-like inclusions in patients with MDS. DESIGN.­: Eleven bone marrow cases from patients diagnosed with MDS and 20 bone marrow cases with no significant pathologic alterations were retrospectively reviewed. Detailed medical record review was performed to ensure none of the patients had a history of HIV, or was taking immunosuppressants and/or antiviral medications. RESULTS.­: Eight of 11 cases (72%) from the study group show detached intracytoplasmic inclusions in a minority (<5%) of mature neutrophils consistent with Howell-Jolly body-like inclusions. No Howell-Jolly body-like inclusions were identified in the control group. Notably, none of the selected patients had a history of HIV or was taking immunosuppressants and antiviral medications. CONCLUSIONS.­: In review of the literature, Howell-Jolly body-like inclusions seem to correlate with immunosuppression and antiviral therapies with nucleoside analogs. We propose that the formation of Howell-Jolly body-like inclusions is the consequence of dysplasia, and hence its correlation not only with the abovementioned conditions, but also with MDS. The inclusions are, however, seen in only a minority of white cells (<5%), which is probably why they were not brought to practicing pathologists' awareness in the past. This study aims to raise awareness and correlate the presence of Howell-Jolly body-like inclusions in MDS.

Evaluation of Thalassaemia Screening Tests in the Antenatal and Non-Antenatal Populations in Singapore

INTRODUCTION@#Haemoglobinopathy testing is performed for carrier screening and evaluation of microcytic anaemia. We evaluated the effectiveness of thalassaemia screening tests at our institution and suggest ways of improving the testing algorithm.@*MATERIALS AND METHODS@#A total of 10,084 non-antenatal and 11,364 antenatal samples with alkaline gel electrophoresis (AGE), capillary electrophoresis (CE), haemoglobin H (HbH) inclusion test, mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) were retrospectively reviewed. A subgroup of 187 samples with genetic testing was correlated with HbH inclusions and MCH/ MCV. The effect of iron deficiency on percentage hemoglobin A2 (HbA2) was studied.@*RESULTS@#HbH inclusion test showed low sensitivity of 21.43% for α-thalassaemia mutations but higher sensitivity of 78.95% for deletion. By receiver operating characteristic (ROC) analysis, MCH ≤28 pg or MCV ≤80 fl for non-antenatal samples and MCH ≤27 pg or MCV ≤81 fl for antenatal samples had >98% sensitivity for HbH inclusions. Above these thresholds, the probability that HbH inclusions would be absent was 99%). MCH ≥28 pg had 100% sensitivity (95% CI 95.63%-100%) for α-thalassaemia mutations and 97.68% calculated NPV in the antenatal population. Detection of haemoglobin variants by CE correlated highly with AGE (99.89% sensitivity, 100% specificity). Severe iron deficiency reduced HbA2 in hemoglobin ( <0.001) and α-thalassaemia ( = 0.0035), but not in β-thalassaemia.@*CONCLUSION@#MCH/MCV thresholds have adequate sensitivity for α-thalassaemia in the antenatal population, and genotyping plays an important role as HbH inclusion test shows low sensitivity. CE without AGE, may be used as initial screening for haemoglobin variants. Our study provides contemporary data to guide thalassaemia screening algorithms in Singapore.

Crystals and Fatty Acid Abnormalities Are Not Present in Circulating Cells From Choroideremia Patients.

Purpose: To confirm whether choroideremia (CHM) is a systemic disease characterized by blood lipid abnormalities and crystals found in, or associated with, circulating peripheral blood cells of patients. Methods: Peripheral blood samples obtained from three subjects with confirmed mutations in the CHM gene and three age-matched normal controls were processed for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Fatty acids from plasma of nine male CHM subjects were analyzed and compared to reference values for a sample from a Canadian population. Results: Intracellular crystals were not observed in the cells from choroideremia-affected males. No crystals were found adherent to the external plasma membrane of red blood cells. Fatty acid profiles of patients were similar to reference values, with the exception of lower levels of nervonic acid. Conclusions: This investigation failed to observe crystals previously reported in peripheral circulating blood cells derived from CHM subjects, and showed no significant fatty acid abnormalities, not supporting the view of CHM as a systemic disease.

A novel candidate species of Anaplasma that infects avian erythrocytes.

BACKGROUND: Anaplasma spp. are Gram-negative obligate intracellular bacteria transmitted by ticks. Even though numerous studies have detected DNA from Anaplasma spp. in the blood of birds, thus far mammals were the only vertebrates demonstrated to serve as competent hosts to these organisms. We report a novel candidate species of Anasplasma that was associated with cytoplasmic inclusions in the erythrocytes of an African penguin (Spheniscus demersus) in South Africa. METHODS: Cytoplasmic inclusions were morphologically characterized from freshly-produced blood smears, and phylogenetic analysis of 16S rRNA and groEL genes were used to evaluate the evolutionary relationships of the organism to other Anaplasmataceae. RESULTS: Dark-purple round or oval inclusions consistent with Anaplasmataceae morulae were observed in the cytoplasm of erythrocytes. Phylogenetic trees produced using different methods agreed that the organism detected in this study belongs to the genus Anaplasma, and suggested that it is most closely related to the cluster comprising A. centrale, A. capra, A. marginale and A. ovis. We propose provisionally naming the strain detected in this study as "Candidatus Anaplasma sphenisci". CONCLUSIONS: This is the first species of Anaplasma shown to produce cytoplasmic inclusions in avian cells, opening the possibility that cytoplasmic inclusions in avian erythrocytes that had previously been attributed to Aegyptianella sp. might in fact correspond to Anaplasma. Further studies on the molecular biology of avian-infecting Anaplasmataceae will be valuable to provide insight into the evolution and epidemiology of these organisms.