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

Introduction 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. Methods 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. Results 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). Conclusion 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. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NA ### Funding Statement none to declare ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: University of Maiduguri teaching Hospital REC Liverpool School of Tropical medicine REC I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The data underlying this article are available in the manuscript, figures, and tables. Further request can be addressed to the corresponding author