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

Abstract

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). There were 182 SCD patients and 102 healthy controls. SCD patients had a significantly higher proportion of red cells containing HJB compared to controls (median 1.5% vs 0.3% respectively; P = 0.0001). The AI red cell counts were also higher among the SCD patients than the controls (median 47.5% vs 7.1% respectively; 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.

Bacteraemia Among Patients with Sickle Cell Disease in Nigeria: Association with Spleen Size and Function

“… 3 We have recently used two markers of splenic dysfunction that required simple techniques and thus can easily be performed in most resource-poor settings; the proportion of both markers, HJB- and AI-containing red cells, were higher in patients with autosplenectomy than those with visible spleens. 4 In the current study, we noted that SCD patients with bacteraemia were more likely to have higher AI and HJB red cell counts. However, the difference for both markers failed to reach statistical significance.…”

“… 5 The splenic function was assessed by manual estimation of Howell-Jolly bodies (HJB) and argyrophilic inclusion (AI) containing red cells from blood smears as previously described. 4 The splenic status was assessed using ultrasound. Data were analysed using Statistical Package for the Social Sciences (SPSS) (version 25; SPSS, Chicago, IL, USA).…”

“… 3 We recently employed the presence of two red cell-containing inclusions - Howell-Jolly bodies (HJB) and argyrophilic (silver staining) inclusion (AI) red cells - to assess splenic dysfunction among our SCD patients. 4 In the present study, we aimed to determine the prevalence and pattern of organisms causing bacteraemia among our acutely-ill SCD patients and to describe any association between bacteraemia with splenic status on ultrasound and two markers of splenic dysfunction (i.e., HJB and AI red cells)…”

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

“…Splenic function was assessed by using a manual estimation of HJBs and AI containing red cells as previously described. 17 Blood smears for HJB red cells were fixed for 1 min in methanol and stained using the classical May–Grunwald Giemsa stain procedure. Blood smears for identification of AI red cells were fixed for 3 min in formalin and stained for 20 min at 38°C in dark using the silver stain.…”

“…We recently employed the presence of two red cell containing inclusions, namely Howell–Jolly bodies (HJBs) and argyrophilic (silver staining) inclusion (AI) red cells, to assess splenic dysfunction among our patients with SCD. 17 In the present study, we aimed to investigate the variation in both markers of splenic dysfunction (i.e. HJB and AI) with age among the patients with SCD and compare results with those of healthy controls.…”