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ORIGINAL ARTICLE  
Year : 2015  |  Volume : 58  |  Issue : 3  |  Page : 307-309
Mean reticulocyte volume enhances the utility of red cell mean sphered cell volume in differentiating peripheral blood spherocytes of hereditary spherocytosis from other causes


1 Department of Transfusion Medicine and Immunohaematology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Laboratory Haematology and Molecular Genetics, TATA Medical Center, Kolkata, West Bengal, India
3 Department of Laboratory Services, Artemis Health Institute, Gurgaon, India

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Date of Web Publication14-Aug-2015
 

   Abstract 

Context: Mean sphered cell volume (MSCV) and mean reticulocyte volume (MRV) are additional reticulocyte parameters generated while processing the blood samples on Beckman coulter LH 755 in the reticulocyte mode using the volume, conductivity and scatter technology. It has been observed that the difference between mean corpuscular volume (MCV) and MSCV is higher in the cases of hereditary spherocytosis (HS) and this difference is increasingly being utilized as a screening tool for spherocytes. In addition now there have been new observations that reticulocyte volume in cases of HS is less as compared to normal reticulocyte. Aims: Our aim was to test the usefulness of reticulocyte parameters like MSCV and MRV in distinguishing cases of HS and autoimmune hemolytic anemia (AIHA). Materials and Methods: This is a retrospective and partly prospective study where peripheral blood ethylenediaminetetraacetic acid samples from cases of HS (n = 57) and AIHA (n = 29) were processed on LH 755 in both the differential and the reticulocyte mode. The data generated were analyzed and compared with data from normal healthy donors (n = 46). Results: Using an algorithm of MCV - MSCV >10 and MRV - MSCV <25, a sensitivity of 84.2% and specificity of 94.7% was observed in cases of HS. Conclusions: With the reticulocyte analysis, we may now have a simple and cheap additional tool for screening of HS.

Keywords: Autoimmune hemolytic anemia, hereditary spherocytosis, mean corpuscular volume, mean reticulocyte volume, mean sphered cell volume

How to cite this article:
Nair SC, Arora N, Jain S, Inbakumar D, Mammen J, Sitaram U. Mean reticulocyte volume enhances the utility of red cell mean sphered cell volume in differentiating peripheral blood spherocytes of hereditary spherocytosis from other causes. Indian J Pathol Microbiol 2015;58:307-9

How to cite this URL:
Nair SC, Arora N, Jain S, Inbakumar D, Mammen J, Sitaram U. Mean reticulocyte volume enhances the utility of red cell mean sphered cell volume in differentiating peripheral blood spherocytes of hereditary spherocytosis from other causes. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Jun 5];58:307-9. Available from: http://www.ijpmonline.org/text.asp?2015/58/3/307/162836



   Introduction Top


Spherocytes the red blood cells with decreased membrane surface area have number of physiological abnormalities. These include alterations in cell shape and cellular deformability. [1] These physiological changes and the splenic sequestration apparently contribute to the shortened survival of these spherocytes in cases of hemolytic anemia. [2] Spherocytic hemolytic anemia can have varied etiology but the principal and the common causes are hereditary spherocytosis (HS) and autoimmune hemolytic anemia (AIHA). The mechanism of formation of these spherocytes described is different in both these disorders. In HS the mutations in genes encoding red cell membrane proteins such as spectrin, ankyrin, band 3, protein 4.2 etc., cause an intrinsically unstable membrane vesiculations, which result in loss of cell membrane whereas in AIHA the interaction between the red cell membrane bound immunoglobulin with Fc receptors of the phagocytic cells leads to partial phagocytosis of the red cell surface area and this leads to the formation of spherocytes. [3]

It has been observed in some of the recent studies that the reticulocyte volume in HS is less as compared to reticulocytes in normal/AIHA individuals. As the origin of spherocyte in HS is believed to be due to decreased surface area of the HS cell membrane it was suggested that this cell surface loss actually begins at the reticulocyte stage in HS. Hence if reticulocytes in HS are indeed abnormal and small, documentation of the presence of small dehydrated reticulocytes in the context of spherocytosis could be potentially useful in distinguishing the spherocytes of HS from those of AIHA in the peripheral blood of patients suspected to have HS. [2],[4],[5] This hypothesis actually forms the basis of this study.

This study was undertaken to study the utility of various reticulocyte research parameters like mean corpuscular volume (MCV), mean sphered cell volume (MSCV) and mean reticulocyte volume (MRV) obtained on LH 755 in the above context. Our aim was to analyze whether any of these reticulocyte features can be helpful in distinguishing HS from AIHA on cell counters. This protocol was based on some of the findings of Chiron et al. (MCV - MSCV > 10 as an indicator of spherocytosis) and those of Da Costa et al. [2],[6]


   Materials and Methods Top


Blood samples drawn from 86 patients and 46 normal healthy donors were analyzed in this study. None of these patients were transfused during the preceding 6 months. The 86 patients included cases of nonsplenectomised HS (n = 57) (age range 0-50 years, 32 males and 24 females) and pretreated AIHA (n = 29) cases with positive direct coomb's test (age range 0-75 years, 13 males and 16 females). Most of the HS cases were above 5 years of age (n = 49). Diagnosis of HS was based on clinical history, physical examination and the results of the laboratory (complete blood count, blood smears showing the presence of spherocytes, increased reticulocyte count, increased bilirubin, increased red cell osmotic fragility, family history of hemolysis and negative direct coomb's test). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was not available in any of our patients. This is partly retrospective partly prospective, single center observational study. The data generated was analyzed, sensitivity and specificity was calculated.

Peripheral blood samples were collected by venipuncture in K2EDTA. All these samples were analyzed within 8 h after collection. Red cell morphology on blood smears was evaluated using Wright-Geimsa stain, using optical light microscopy. Red blood cell counts, reticulocyte counts and indices were determined using an automated blood analyzer (LH 755, Beckman Coulter). Reticulocyte analysis on Beckman coulter uses new methylene blue stain, a non flurochrome dye, to precipitate the residual RNA. The function of this stain is to identify and delineate the reticulocyte from mature red cells. An acidic hypo osmotic ghosting solution is then introduced. This ghosting solution spheres the cells, clears the hemoglobin and stains the RNA of the reticulocyte. The resulting sphered cells are then classified as either mature red cells or reticulocyte using the volume, conductivity and scatter technology. This analysis on the LH 755 generates research parameters like MSCV and MRV in addition to other known parameters.

The average volume of all events classified as reticulocyte is referred to as MRV. The average volume of all red cell events - both mature and reticulocyte is referred to as MSCV.

Normal red cells are able to undergo an osmotic expansion whereas spherocytes after reaching a critical osmotic volume fragment resulting in very low MSCV. Difference between MCV and MSCV has been suggested to be useful screening tool for HS and other conditions having spherocytes in the peripheral blood.


   Results Top


The following parameters in all the three groups (normal healthy donors, HS and AIHA) were studied.

  1. Difference between MCV and MSCV (MCV - MSCV) [Table 1]
    Table 1: Difference between MCV and MSCV (MCV — MSCV)


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  2. Difference between MRV and MSCV (MRV - MSCV) [Table 2]
    Table 2: Difference between MRV and MSCV (MRV — MSCV)


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  3. Difference between MCV and MRV (MCV < MRV) [Table 3].
    Table 3: Difference between MRV and MCV (MRV < MCV)


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It was observed that majority of the cases of HS (50/57; 87.8%) and those of AIHA (23/29; 79.3%) had an MCV-MSCV difference of > 10 whereas none of the normal healthy donors (0/46) had a value of MCV - MSCV of > 10 [Table 1]. The other significant observation was regarding MRV - MSCV [Table 2], that the majority of the cases of (54/57; 94.7%) HS and healthy donors (42/46; 91.3%) had an MRV - MSCV difference of < 25, whereas (25/29; 86.2%) AIHA cases had a MRV - MSCV difference of >25.

By combining these 2 parameters and following an algorithm of MCV - MSCV >10 and MRV - MSCV < 25 [Table 4] it was observed that (48/57) cases of HS had these 2 combined findings and none of the normal cases had both these findings. Only 4/29 cases of AIHA had these 2 combined findings. Using an algorithm of MCV - MSCV > 10 and MRV - MSCV < 25 a sensitivity of 84.2% and specificity of 94.7% was observed in cases of HS.
Table 4: MCV — MSCV >10 and MRV — MSCV < 25


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Now while looking for any significant difference in the three groups in terms of MCV - MRV a very interesting and significant observation came to the picture. Almost 56% (n = 32) of the HS cases had an MRV, which was less than MCV [Table 3] whereas the normal cases had MRV greater than MCV.


   Discussion Top


Hemolytic anemia characterized by spherocytic red cells with reduced membrane surface area is a feature of HS and some forms of AIHA. In the present study, the difference between MCV and MSCV and difference between MRV and MSCV allowed us to analyze the particular behavior of spherocytes and the reticulocytes respectively. HS and AIHA cases with spherocytes in the peripheral blood showed a decrease in MSCV and in majority of cases MCV - MSCV > 10 allowed us to suspect and detect spherocytes in these cases. This parameter has been shown to be quite a reliable parameter to detect spherocytes in the peripheral blood of cases of HS with a sensitivity of 71% and 100% in the studies by Chiron et al., 1999 and Broséus et al. 2010 respectively. [6],[7] Adding to this information are the parameters like MRV and the difference between MRV and MSCV, which further allow us to suspect the etiology of some of these spherocytic anemia. Hence using an algorithm of MCV - MSCV >10 and MRV - MSCV < 25 a sensitivity of 84.2% and specificity of 94.7% was observed in cases of HS.

In HS the spherocytes formation has been attributed to increase propensity of red cells to shed membrane during their journey in circulation due to improper assembly of the membrane proteins where as in AIHA it is due to the partial phagocytosis of the circulating red cell membrane bound complexes by macrophages. [3],[8] It was proposed by Da Costa et al. that surface loss in HS but not in AIHA is already present at the circulating reticulocyte stage and the HS reticulocyte from the beginning has decreased cell volume. [2] This is a very significant observation and along with other reticulocute parameters on the cell counter LH 755 can be utilized to detect and differentiate cases of HS and AIHA. So in a setting of peripheral blood having spherocytes if the reticulocyte volume is low, then the chances of this being a case of HS are very high.

Da Costa et al. suggested that reticulocytes of HS and AIHA are different in terms of volume and the HS reticulocytes are smaller. These findings have been similar to our observations whereby we observed that (32/57; 56%) of the cases of HS had an MRV, which was less than MCV which does suggest that perhaps reticulocyte of HS is indeed small. This parameter along with other reticulocyte parameters on the cell counter LH 755 can thus be utilized to suspect and differentiate cases of HS from AIHA.

To the best of our knowledge, this is the first study looking at reticulocyte research parameters and trying to propose an algorithm [Figure 1] for the differentiation of HS and AIHA on cell counters. This data also supports the hypothesis that the reticulocytes in HS are defective and can imply that surface area loss in HS, is already present at the circulating reticulocyte stage which is contrary to the existing concepts that the surface loss occurs during the journey of the red blood cells in the circulation. This is a single center observational study and these are the initial observations where some of the parameters look very promising but to come into clinical practice they would need to be tested by different groups, at different places and need to be validated in a larger cohort.
Figure 1: Proposed cell counter approach to a suspected case of hereditary spherocytosis


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   References Top

1.
Chasis JA, Agre P, Mohandas N. Decreased membrane mechanical stability and in vivo loss of surface area reflect spectrin deficiencies in hereditary spherocytosis. J Clin Invest 1988;82:617-23.  Back to cited text no. 1
    
2.
Da Costa L, Mohandas N, Sorette M, Grange MJ, Tchernia G, Cynober T. Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia. Blood 2001;98:2894-9.  Back to cited text no. 2
    
3.
Ferreira JA, Feliu E, Rozman C, Berga L, Bombi JA, Marti M, et al. Morphologic and morphometric light and electron microscopic studies of the spleen in patients with hereditary spherocytosis and autoimmune haemolytic anaemia. Br J Haematol 1989;72:246-53.  Back to cited text no. 3
    
4.
Salomao M, Chen K, Villalobos J, Mohandas N, An X, Chasis JA. Hereditary spherocytosis and hereditary elliptocytosis : a0 berrant protein sorting during erythroblast enucleation. Blood 2010;116:267-9.  Back to cited text no. 4
    
5.
Manwani D. Throwing out the baby. Blood 2010;116:154-5.  Back to cited text no. 5
    
6.
Chiron M, Cynober T, Mielot F, Tchernia G, Croisille L. The GEN.S : a0 fortuitous finding of a routine screening test for hereditary spherocytosis. Hematol Cell Ther 1999;41:113-6.  Back to cited text no. 6
    
7.
Broséus J, Visomblain B, Guy J, Maynadié M, Girodon F. Evaluation of mean sphered corpuscular volume for predicting hereditary spherocytosis. Int J Lab Hematol 2010;32:519-23.  Back to cited text no. 7
    
8.
Groom AC. The Microcirculatory Society Eugene M. Landis award lecture. Microcirculation of the spleen : n0 ew concepts, new challenges. Microvasc Res 1987;34:269-89.  Back to cited text no. 8
    

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Correspondence Address:
Dr. Neeraj Arora
Department of Laboratory Haematology and Molecular Genetics, TATA Medical Center, Kolkata - 700 156, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.162836

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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]

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