High mannose N-glycans (HMGs) are the base structures in the biosynthesis of glycoproteins and are typically found on the endoplasmic reticulum and Golgi bodies 1. In 2021, Cao et al. reported that patches of HMGs (Man5-9GlcNAc2) are externalized on the surface of the red cell membrane of diseased or oxidized cells and bind to the mannose receptor (CD206) on phagocytes 2. The receptor binding is considered a key stage in the process of erythrocyte removal, which predominately takes place in the spleen. Disease states, such as sickle cell disease (SCD) or severe malarial infection, upregulate mannose presentation on the cell surface, with the HMGs associated with high molecular weight fragments containing spectrin. Increased HMG expression leads to phagocytosis of infected red cells and the clearance of older or disease-associated red cells. Cao et al. in 2022 also noted that red cells lose plasma membrane as they mature and HMGs may be involved in this process in the spleen 3. HMG levels in erythrocytes from healthy controls were significantly lower compared to patients who had undergone splenectomy, and the HMG levels correlated well with manual pit counts (r = 0.75–0.85), which are known to be associated with spleen dysfunction. Increased levels of HMGs were detected in many patients with functional hyposplenism and it was concluded that HMGs are a useful marker for spleen function, which can be routinely measured using flow cytometry. We developed a clinical diagnostic assay to assess spleen function using flow cytometry to measure HMGs on red cells from the peripheral blood, using Galanthus nivalis lectin as the targeting probe 4. In healthy patients, the level of HMG expression in erythrocytes was low compared to adult SCD patients, who lose spleen function before adulthood, and individuals that had undergone splenectomy. We also found a strong correlation between HMG expression and other markers of hyposplenism, such as pit counts 5. A threshold of > 36% difference between the mean fluorescence of the healthy control to the sample under investigation provided a test with 93% sensitivity and 100% specificity for detection of splenic dysfunction. All results were concordant with the findings by Cao et al., and all results suggested that HMG expression was a marker of red cell age, with older red cells externalizing more HMG and therefore marking themselves for removal by specialist cells in the spleen. We developed a semi-automated magnetic bead-based separation method to purify reticulocytes from 1 mL of EDTA blood using a CD71 OKT-9 antibody (eBioscience #13-0719-82) and the KingFisher Flex (ThermoFisher) platform. Flow cytometry analysis of the CD71 positive population showed that the purity was 94.9% ± 3.4% (n = 4; CD235+/CD45−), indicating they were of predominantly a pure erythrocytic lineage. We wanted to understand if there is differential HMG expression between reticulocytes and erythrocytes. Reticulocytes were isolated from healthy individuals and SCD patients, and HMG externalization was measured using flow cytometry as previously reported 4. The mean fluorescence level of the reticulocyte populations was high for both the healthy controls and the SCD groups compared to erythrocytes (Figure 1A), with the SCD group having higher fluorescence. Healthy reticulocytes had ×2.3 more HMG fluorescence than healthy erythrocytes, compared to ×2.1 more fluorescence in the SCD group. Reticulocytes from SCD samples had significantly higher levels of HMGs compared to healthy controls (p = 0.0053), consistent with spleen dysfunction in the SCD population (Figure 1B). Comparing mean cell fluorescence, HMG levels are higher in reticulocytes than erythrocytes in both healthy individuals and SCD patients (Figure 1A,B). The results suggest that as reticulocytes mature into erythrocytes they reduce the level of HMGs externalized, and this is more pronounced in the healthy population because of the functioning spleen. In the SCD population, spleen function is limited or lost and therefore HMG expression remains high. Reticulocyte maturation is thought to take 1–2 days and occurs primarily in the spleen's red pulp 6, 7. To test this, we re-measured HMG levels on the same blood samples from healthy controls at days 0 and 1, and did not observe any changes in HMG expression on CD71 positive cells (data not shown). This suggests that maturation does not take place ex vivo and that supports the idea that a period of time is required to be spent in the spleen to complete maturation into erythrocytes. As we want to use HMG expression to assess spleen function, we wanted to check that high levels of reticulocytes would not affect the assay. The best disease to assess this in is hereditary spherocytosis (HS) as there is chronic hemolytic anemia with reticulocytosis but with normal spleen function. In two patients tested, spleen function was found to be normal, however, the mean fluorescence intensity of HMG expression positively correlated with the level of reticulocytes in peripheral blood (Table 1). Suggesting that, in patients with normal spleen function, HMG expression is influenced by the presence of elevated levels of reticulocytes in peripheral blood but not sufficient to affect the assessment of spleen function. It has been speculated that HMG externalization is a stress marker linked to available energy inside the red cell, and the same process arises in the bone marrow and peripheral blood 3. As stem cells express high levels of HMGs 8, it is worth considering that HMG expression may be a biological requirement in the bone marrow, supporting the formation of erythroblastic islands which form around a central macrophage 7. As reticulocytes are released from the bone marrow into circulation, we do not know if the HMGs are removed by macrophages or if the interaction causes “flipping” of the red cell membrane to internalize them. In the latter case, HMGs may only be lost as the erythrocyte matures and loses cell membrane (including CD71). With an energy deficit, the membranes' lowest energy state is with externalized HMGs and therefore, as erythrocytes age, the membrane flips and externalizes HMGs, eventually marking them for phagocytosis. In conclusion, we have identified HMGs on the surface of early CD71 expressing red cells and we believe them to be a subpopulation that has left the bone marrow and are mid-journey to the spleen for maturation. Their presence does not affect the use of HMG expression to assess spleen function but higher levels of reticulocytosis will reduce the fold-change measured to determine spleen dysfunction. The reason why early reticulocytes expressed high levels of HMGs is still up for debate. However, red cells do externalize more HMGs as they age and this marks them for removal by the spleen. Therefore, presentation of HMGs on red cells is a marker for both immature red cells starting their journey in the blood, as well as aged cells at the end of their life. A.A.K. and B.C. planned the experimental work, performed data analyses, and wrote the manuscript. B.C. and D.C.R. supervised the study. All authors contributed equally to the reviewing and editing of the manuscript. This work was supported by Synnovis LLP Innovation Accelerator Fund Progressive Award P050 and Agile Award A018. The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Khan et al. (Tue,) studied this question.