INTRODUCTION:

Anemia is classified as microcytic, normocytic, or macrocytic based on red cell morphology and parameters such as mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC). Another classification delves into the cause, such as production failure, red blood cell destruction, blood loss, and so on, which necessitates extensive research and expense¹. A more recent and kinetic method is based on the bone marrow response to anemia based on the reticulocyte parameters².

Beckman Coulter DXH800 measures reticulocytes by staining with a non-fluorescent dye new methylene blue which precipitates the RNA. A hypo-osmotic agent is added to clear the hemoglobin, highlight the RNA, and cause sphering of the cells. The cells are then analyzed and characterized according to optical light scatter, impedance, and radiofrequency to differentiate red blood cells from reticulocytes. Amongst the parameters, reticulocyte count (%) and absolute reticulocyte count are the most widely studied parameters with their role proven in the determination of marrow response to anemia, erythropoietic status following therapeutic interventions as well as in the classification of anemia³. Immature reticulocyte fraction has gained more recognition in recent times due to its utility in monitoring bone marrow engraftment⁴, marrow recovery⁵, erythropoiesis in kidney disease, retroviral disease, myelodysplasia^6,7,8,9, monitoring the effect of treatment in nutritional anemias,¹⁰ diagnosis of hemolytic anemia¹¹, aplastic anemia^12,13,14 and screening for hereditary spherocytosis¹⁵.

The other parameters are studied in a few conditions like higher mean reticulocyte volume (MRV) in thalassemia compared to iron deficiency anemia and increase in MRV following hematinic in iron deficiency anemia^16,17. Decrease in MRV on the treatment of megaloblastic anemia¹⁸, a lower mean sphered cell volume in hereditary spherocytosis¹⁹, and red cell size factor as the measure of iron availability in erythropoiesis and its early decrease in iron deficiency²⁰. The present study aims to study the distribution of these automated reticulocyte parameters derived by Beckman coulter DXH800 in nutritional deficiency anemias.

MATERIALS AND METHODS:

It is a retrospective, single-center, record-based study done in the pathology department of a tertiary care center. An archival search for all cases with reticulocyte analysis was done from the period of 1st Oct 2017 to 30th Sept 2018.

Inclusion criteria: All cases (Hb <13g/dL in males, <11g/dL in females) with a biochemically proven diagnosis of Iron deficiency anemia (IDA) [serum iron <33 micro gm/dl, total iron binding capacity> 400microgm/dl and serum ferritin <13nanogm/ml], Megaloblastic anemia (MA) [serum vitamin B12 <197picogm/ml, serum folate < 4.5nanogm/ml], and Mixed deficiency anemia (deficiency of both iron and vitamin B12), as case groups. All cases without anemia registered for health checkups (without comorbidities) as a comparison non-anemic group.

Exclusion criteria: All cases <18years, pregnant women, cases with comorbidities, multifactorial anemia, incomplete workup.

The demographic and treatment data were retrieved from medical records. The various reticulocyte parameters derived from Beckman Coulter DXH800 include: Reticulocyte percentage, RET% = All Reticulocyte Events/All Red Cell Events, Absolute reticulocyte count, ARC = (Reticulocyte% x RBC count)/100, Immature reticulocyte fraction, IRF = Reticulocyte Events (channels 3-10)/All Reticulocyte Events, Mean reticulocyte volume, MRV = Average Volume of All Retic Events, High light scatter reticulocytes, HLR% = % Retic Events (channels 3-10)/ All Red Cell Events, High light scatter reticulocyte count, HLR# = (HLR% x RBC count)/100, Mean sphered cell volume, MSCV = (HLR% x RBC count)/ 100, Reticulocyte distribution width, RDWr = (RDWr SD/MRV) x 100, RDWr-SD = SD of Reticulocyte Population, Red cell size factor, RSF = Square Root of MCV x MRV.

The statistical analysis in this study was carried out using SPSS software (IBM, Chicago, version 20) and Microsoft Excel Office 2016. Descriptive analysis was done for all the data. An Independent sample T-test to detect statistically significant deviation between the groups and sub-groups was performed. P-value <0.05 was considered statistically significant. Comparison of the means of reticulocyte parameters between all the groups and subgroups was done by F test of Analysis of variance (ANOVA) and post hoc test using Bonferroni adjustments. A two-tailed Pearson method was used for calculating correlation coefficients.

Ethics:

All procedures performed in the current study were approved by IRB (IEC 620/2017, 19.9.2017) in accordance with the 1964 Helsinki declaration and its later amendments.

RESULT:

The included cases were categorized as: Non-anemic group (n=68), IDA (n=146), MA (n=119), and mixed deficiency anemia (n=32).

Iron deficiency anemia:

The mean age was 46.84years with female predisposition [females (65.8%) and males (34.2%)]. There was a statistically significant increase in RET%, ARC, IRF, HLR%, HLR#, and RDWr, a significant reduction was seen in MRV, MSCV, and RSF. The descriptive statistical analysis is summarized in Table 1. On calculating Pearson correlation coefficient (2-tailed), there was a significant (p<0.001) positive correlation between Hb and MRV (r=0.461), MSCV (r=0.421) and RSF (r=0.701). There was a significant inverse correlation between Hb and RET% (r=-0.526), IRF (r=-0.5), HLR% (r=-0.557) and RDWr (r=-0.424). No significant correlation was seen between Hb and ARC (r=-0.147), HLR# (r=-0.113) and RDWr-SD (r=-0.094).

Of the 146 cases, 41 were receiving treatment. IDA on treatment had significantly higher RET%, ARC, IRF, HLR%, and HLR# (Table 2) in comparison with the without treatment group.

Megaloblastic anemia:

Among the 119 patients with MA, 63 cases had vitamin B12 deficiency, 7 had folate deficiency, and 49 cases had a deficiency of B12 and folate. All reticulocyte parameters were significantly increased, except IRF (Table 1). There was a significant positive correlation between Hb and ARC (r=0.33). An inverse correlation was seen between Hb and RET% (r=-0.336), MRV (r=-0.526), IRF (r=-0.584), HLR% (r=-0.405), RDWr (r=-0.535) and RDWr-SD (r=-0.688). No significant correlation was seen between Hb and HLR# (r=-0.048) and MSCV (r=-0.233).

Among the 119 patients of MA, 53 were on treatment. MA without treatment had increased MRV, MSCV, RSF, RDWr, and RDWr-SD. MA on treatment had significantly higher values for RET%, ARC, IRF, HLR%, and HLR# and significantly lower values of MRV, RDWr, and RDWr-SD. (Table 2).

Mixed deficiency anemia:

There was a statistically significant increase in RET%, ARC, IRF, HLR%, HLR#, and RDWr and a reduction in MRV, MSCV, and RSF in comparison with the non-anemic group (Table 1). Among the 32 patients with mixed anemia, 13 were on treatment. Mixed anemia on treatment had significantly higher values for RET%, ARC, and HLR# while the remaining parameters had no significant difference (Table 3).

When comparing the reticulocyte parameters between the groups of IDA, MA, and mixed deficiency anemia, the statistically significant parameters were RET%, MRV, IRF, MSCV, HLR%, RSF, RDWr, and RDWr-SD. All these parameters were significantly higher in MA than in the other two groups.

Table 1: Statistical analysis of reticulocyte parameters in non-anemia, iron deficiency anemia, megaloblastic anemia, and mixed deficiency anemia.

Parameters	Non-anemic group (n=68)		Iron deficiency anemia (n=146)			Megaloblastic anemia (n=119)			Mixed deficiency anemia (n=32)
	Mean	SD	Mean	SD	p	Mean	SD	p	Mean	SD	p
Hb (g/dL)	13.77	1.25	7.6	2.2		7.58	2.55		7.13	2.42
Hct (%)	40.03	3.19	24.69	6.14		22.34	8.36		23.22	7.01
RET (%)	1.31	1.28	1.87	0.88	0.0002*	3.96	4.7	0.0001**	2.28	1.49	0.0011***
ARC(x10⁶/mL)	0.0543	0.0194	0.069	0.029	0.0002*	0.0735	0.0769	0.0448**	0.076	0.044	0.0009***
MRV (fL)	107.03	7.18	96.21	9.35	0.0001*	146.99	26.01	0.0001**	101.5	18.26	0.0321***
IRF (ratio)	0.366	0.084	0.46	0.09	0.0001*	0.5363	0.1591	0.92	0.467	0.103	0.0001***
MSCV (fL)	81.84	4.86	66	9.21	0.0001*	99.42	13.3	0.0001**	69.85	12.45	0.0001***
HLR% (%)	0.4372	0.2156	0.895	0.554	0.0001*	2.4	3.4	0.0001**	1.15	1.04	0.0001***
HLR# (x10⁶/mL)	0.0204	0.0097	0.036	0.029	0.0001*	0.0434	0.0526	0.0005**	0.0373	0.0309	0.0001***
RSF (fL)	95.9	4.28	78.21	8.11	0.0001*	123.96	18.02	0.0001**	82.45	15.4	0.0001***
RDWr (%)	25.64	1.87	27.75	3.14	0.0001*	29.64	3.35	0.0001**	27.96	3.02	0.0001***
RDWr-SD(fL)	27.44	2.63	26.61	3.02	0.0528	43.65	10.95	0.0001**	28.06	3.89	0.35

*Significant at p<0.05- IDA vs non-anemic group, ** significant at p<0.05- MA vs non-anemic group, *** significant at p<0.05- mixed deficiency anemia vs non-anemic group

Table 2: Comparison of anemia groups (iron deficiency anemia, megaloblastic anemia) based on treatment history

	Iron deficiency anemia					Megaloblastic anemia
	Without treatment (n=105)		With treatment (n=41)			Without treatment (n=66)		With treatment (n=53)
Parameter	Mean	SD	Mean	SD	p	Mean	SD	Mean	SD	p
RET (%)	1.56	0.63	2.65	0.03	<0.001*	1.46	0.82	7.07	5.62	<0.001*
ARC(x10⁶/mL)	0.0574	0.0157	0.0983	0.0328	<0.001*	0.0293	0.0163	0.1286	0.0867	<0.001*
MRV (fL)	95.35	8.7	98.41	10.64	0.106	152.02	27.41	140.72	22.87	0.016**
IRF (ratio)	0.434	0.088	0.511	0.0753	<0.001*	0.505	0.143	0.575	0.171	0.018**
MSCV (fL)	65.65	9.95	66.89	6.99	0.317	99.73	13.81	99.03	12.75	0.777
HLR% (%)	0.7021	0.3808	1.3888	0.6239	<0.001*	0.792	0.608	4.392	4.295	<0.001*
HLR# (x10⁶/mL)	0.0295	0.0301	0.0511	0.0213	<0.001*	0.016	0.014	0.078	0.062	<0.001*
RSF (fL)	77.74	8.02	79.44	8.29	0.265	126.8	19.7	120.43	15.14	0.055
RDWr (%)	27.77	3.3	27.71	2.74	0.919	30.63	5.24	28.41	2.79	0.004**
RDWr-SD (fL)	26.41	3.18	27.12	2.51	0.159	46.58	12.25	40	7.76	<0.001*

*Significant at p<0.001, ** significant at p<0.05

Table 3: Comparison of mixed deficiency anemia groups based on treatment history.

	Mixed deficiency anemia
	Without treatment (n=19)		With treatment (n=13)
Parameter	Mean	SD	Mean	SD	p
RET (%)	1.74	0.63	3.07	2.01	0.037**
ARC(x10⁶/mL)	0.058	0.019	0.101	0.057	0.019**
MRV (fL)	98.75	17.83	105.52	18.84	0.317
IRF (ratio)	0.449	0.093	0.492	0.116	0.273
MSCV (fL)	67.92	12.66	72.67	12.07	0.294
HLR% (%)	0.807	0.438	1.65	1.437	0.06
HLR# (x10⁶/mL)	0.027	0.013	0.053	0.042	0.049**
RSF (fL)	80.52	15.12	85.28	15.98	0.406
RDWr (%)	28.34	3.06	27.42	3.01	0.406
RDWr-SD (fL)	27.72	4.17	28.56	3.53	0.544

*Significant at p<0.001, **significant at p<0.05

DISCUSSION:

The role of reticulocytes in the evaluation of anemia is an under-unexplored territory. The majority of the studies done have focused on RET%, ARC, and IRF following chemotherapy induction, and marrow engraftment to look for these parameters reflecting recovery. Their role in nutritional anemia has not been widely documented. The first response to anemia is the increase in RET%, ARC, and immature reticulocytes (IR), which also signifies a responsive marrow to anemia and therapeutic interventions. In the present study, the correlation between Hb and the various reticulocyte parameters in IDA shows a positive correlation between Hb and MRV, MSCV, and RSF indicating a falling Hb to be associated with smaller reticulocytes and lower RSF. There was an inverse correlation between Hb and RET%, IRF, HLR%, and RDWr reflecting the bone marrow response to increased erythropoietin stimulation by falling Hb. Elevated RET% and IR in iron deficiency can be attributed to the increase in cytoplasmic levels of transferrin receptor mRNA thereby leading to an increase in the IR, compensatory erythroid expansion and increased IR production from the bone marrow^19,20. RSF is an indicator of restricted erythropoiesis and is reduced in IDA due to deficiency in heme synthesis caused by the restriction of iron supply. The present study shows that RSF has the strongest correlation making it the most useful marker for detecting iron-restricted erythropoiesis. This has also been suggested by Urrechaga E et al^21,22, and Osta V et al²³.

While comparing the cases of treatment and non-treatment groups of IDA, there were significantly higher values for RET%, ARC, IRF, HLR%, and HLR# in cases on treatment. MRV on treatment became normal but was not statistically significant. MSCV and RSF also improved slightly. Zini G et al²⁰and Davis BH et al²⁴have also stated response to treatment by increased RET% and IR. Many of these cases were on oral iron tablets, which might be the reason for the slow improvement of volumetric parameters, and RSF compared to the other studies with intravenous iron treatment²⁰. Brugnara et al¹⁴ have documented oral iron to be ineffective in immediate improvement of iron-deficient erythropoiesis (detected by reduced MRV and RSF), and better results by intravenous iron in patients with markedly reduced serum ferritin.

In MA, there was an inverse correlation between Hb and RET%, MRV, IRF, HLR%, RSF, RDWr, and RDWr-SD. RDWr-SD had the highest correlation, followed by IRF and MRV. In vitamin B12 or folate deficiency, the unbalanced cell growth and impaired cell division causes macroreticulocytosis due to defects in DNA synthesis. This results in larger and more IR production in the marrow, indicated by the raised MRV and IRF. This was also observed in the present study, with higher MRV and IRF levels, although the IRF rise was not statistically significant. Studies by Torres GA et al²⁵, Balci YI et al²⁶, and Ceylan C et al²⁷ also reported an increased MRV (with varying normal cut-offs). Similar to the present study (high normal IRF), Torres GA et al²⁵ also reported increased IRF compared to controls. The cases on treatment exhibited significantly increased RET%, ARC, IRF, HLR%, and HLR#, indicating an expected bone marrow erythropoietic response to treatment. Reduced MRV post-treatment was similar to report by d'Onofrio G et al¹⁶. They documented an inversion of the MRV/MCV ratio, but this was not seen in the present study.

Mixed anemia presents with decreased Hb with a decreased/ normal MCV, directing towards IDA/ thalassemia in case of microcytosis and anemia of chronic disease in case of normocytic anemia²⁶. The present study explored the utility of reticulocyte parameters in conjunction with the RBC parameters to aid the diagnosis. The cases of mixed anemia showed a decrease in MCV, MSCV, and RSF, making it similar to IDA. Although RSF, has not been studied previously in mixed anemia, the study by Balci et al²⁶has reported decreased reticulocyte hemoglobin content in mixed anemia, which can explain the decreased RSF (a marker for iron-restricted erythropoiesis) in the present study. This group had a normal MRV as opposed to patients with pure IDA. One possibility for this could be that concomitant MA, which causes macroreticulocytosis, neutralized the MRV value. On the treatment of mixed anemia, there was an increase in all reticulocyte parameters, though only RET%, ARC, and HLR# were statistically significant. HLR% almost doubled in value but was not statistically significant. The mean RET% was concordant with the documentation by Balci et al²⁶ but does not agree with the mean MRV value. The mean ARC in the present study was comparable to the low normal ARC reported by Poorana PP et al²⁸. As much literature is not available for reticulocyte parameters in mixed deficiency anemia, these results could not be completely validated, and therefore we urge caution before interpretation.

CONCLUSION:

The present study provides descriptive analyses of less studied reticulocyte parameters in nutritional anemias. The study strengthens the utility of RSF as a marker of iron-restricted erythropoiesis, inverse relation of MRV with Hb in MA, and a low RSF and higher MRV in differentiating IDA from mixed deficiency anemia. Irrespective of the etiology, RET%, ARC, HLR%, and HLR# can be utilized for monitoring therapy in nutritional anemias.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 06.08.2022 Modified on 24.02.2023

Research J. Pharm. and Tech 2023; 16(12):5896-5900.

DOI: 10.52711/0974-360X.2023.00955