Evaluation of Erythrocyte Disorders with Mean Corpuscular Volume (MCV) and Red Cell Distribution Width (RDW)

Azad K.L.¹, Dwivedi S.K.¹, Lokwani D.P.², Bansal A.K.¹, Shrivastav P.K¹., Chandrakar S.K.² and Sachdeva N.²

¹Govt. Medical College, Jagdalpur (India) 494001.

²NSCB Medical College, Jabalpur.

ABSTRACT:

The Red Cell Distribution width (RDW) which provides a Quantitative measure of heterogeneity of red cells in the peripheral Blood and the mean Corpuscular Volume (MCV) are part of the routine red cell indices reported by automated blood analysis. The study evaluated 250 cases with a wide range of erythrocyte disorders and determined the diagnostic utility of the Red Cell Distribution width (RDW) in relation to the Mean Corpuscular Volume (MCV). Six different groups of erythrocyte disorders by Mean Corpuscular Volume (MCV) and Red Cell Distribution Width (RDW) values are described: Low Mean Corpuscular Volume (MCV) /normal red cell distribution width (RDW), Low Mean Corpuscular Volume (MCV) /High Red Cell distribution width (RDW), normal Mean Corpuscular Volume (MCV) / Normal Red Cell Distribution width (RDW), Normal Mean Corpuscular Volume (MCV) /high Red Cell Distribution width (RDW). High Mean Corpuscular Volume / normal Red Cell Distribution width, High Mean Corpuscular Volume / High Red Cell Distribution width.

This combination established a use full differential diagnosis of erythrocyte disorders. The data provides a base line against which future studies of infants and children can be compared, though each Laboratory has to verify its own norms, it should be cautioned that different electronic counters yield different. Red Cell Distribution width (RDW) values, so there have to be when reporting reference values. The Red Cell Distribution width (RDW) may find its best use as a guide in the differential diagnosis of anemia rather than as a definitive test per se. In 1957 a study group of World Health Organization ( W.H.O.) has expressed the view that in order to get a comprehensive picture of disease or health problem more and more studies have to be carried out, Garg Narendra K.(1). This prompted the authors to undertake this study.

KEYWORDS: Mean Corpuscular Volume, Red Cell Distribution Width, Iron Deficiency Anemia.

INTRODUCTION:

Erythrocyte disorders are traditionally into two groups Anaemia and Polycythemia and are characterized respectively by a decreased (erythrocytopenia) or increased (erythrocytosis) size of the red cell mass.

Automated analysis of the blood has made the erythrocyte indices more accurate reproducible and readily available even without experienced morphologists. In the past poor intra-observer and inter-observer reproducibility and lack of skill in blood smear interpretation limited their use.

An additional parameter obtained by analysis of blood sample in fully automated cell counters is red cell distribution width (RDW). RDW is an estimate of erythrocyte anisocytosis or heterogeneity. RDW is the coefficient of variation (CV) of the distribution of individual red cell volume.

RDW-CV = Histogram width at ISD/MCV. The RDW-CV is less sensitive to early macrocytosis. In contrast, the RDW-SD is very sensitive to the small proportion microcytes of macrocytes. Red Cell indices generated by automated cell counter is superior and accurate then peripheral smear morphology studied by haematologist.

Uses of automatic procedure for determination of cell count and indices have enabled us to erythrocytes size and its distribution. In Iron Deficiency Anaemia (IDA), there is marked dispersion in the red cell volume (size), so that the RDW increase where as it generally remains within normal limit in Anaemia of Chronic Disorder (ACD).

The initial classification of anaemia can improved substantially by including RDW and histograms of RCV as there variables become part to the routine blood counts. Without going to invasive and expensive method of investigation we can screen the various red cell disorders like Iron Deficiency Anemia, Sickle Cell Anemia, Thalasemia, B₁₂ deficiency anemia etc. In recent past results of such studies were published and in 1957 a study group of World Health Organization (WHO) has expressed the view that in order to get a comprehensive picture of disease (health problem), more and more such studies have to be carried out, Garg Narendra K. (1). This prompted the authors to undertake this study to evaluate of erythrocyte disorders with mean corpuscular volume (MCV) and red cell distribution width (RDW), which is less expensive, less time consuming, less invasive procedure and easy to carry out to early screening the population for the diseases like Iron and B-₁₂ Defficiency Anaemia, Haemolytic Anaemia like Sickle cell Anaemia and other haemoglobinopathies.

MATERIALS AND METHODS:

A prospective study was carried out in Central Lab Deptt. of Pathology, Netaji Subhash Chandra Bose, Medical College, Jabalpur (M.P.) India.

Initially all patients coming to Central lab for CBC by automated cell counter was screened. Those with Hb < 12.5 were selected. A possible path physiologic entity was assigned. Now the peripheral smear was screened and a specific etiology (Category or Cases) was assigned. These were done as follows: -

Based on these, appropriate diagnostic tests mentioned above were advised. Those patients’s for which these test results are available were assigned specific diagnosis. Thus the data obtained from cell counter were studied for specific etiologic diagnosis.

A control study for RDW was done using data from 125 non anaemic (Hb > 12.5) cases.The data thus collected were compiled and analyzed using standard statistical methods and inferences were drawn.

Determination of HbF and Hb A2 Level

The respective bands are identified by comparing with the position control bands on the Hb electrophoresis strip. These bands are then cut and eluted in distilled water. The eluted hemoglobin is allowed to stand at room temperature for 15-20 minutes and is red by colorimeter at 413 nm filter Distilled water is taken as blank. Then quantitative levels of HbA2 and HbF are calculated.

Serum Iron (Randox Daytona):

They were analyzed in a closed system of fully automatic Randox Daytona biochemistry analyzer. The reagent kits used were Iron buffer and Chromogen.

Cellulose acetate electrophoresis:

“Electrophoresis is the movement of charged particles in an electrical field. At ph 8.9 hemoglobin which is a negatively charged protein migrates towards the anode in an electrical field. During electrophoresis various hemoglobins separate due to charge difference causes by structural variation thereby allowing their identification.”

Serum B₁₂ Assay:

This test was unavailable locally. For the purpose of this study we advised 75 cases for S. B₁₂ assay.

Sample: The samples used were 3ml serum. These were frozen 0 to -5⁰ C in ordinary refrigerator. All were shipped frozen to reference lab at Bombay for assay.

FINDINGS:

In this study, only two parameters (RDW and MCV) screen the various red cell disorders with sensitivity and specificity inspite of using others red cell indices/Parameter and also comparison with the findings of other study who used the above parameters.

In this study 250 cases were studied with 128 female patients and 122 male patients.

40 cases (16%) were of pediatric age group. Maximum 20.8% cases were seen in 15-24 yrs. Age group with females being twice the number of males in this group. Above 45 yrs. Males were 50% more commonly affected than females. All age groups in both sexes were represented in this study. (Table - I)

Table – I: Age and Sex wise distribution of the studied cases

Age group (yrs.)	Sex		Total
	M	F
0-14	27	13	40
15-24	17	35	52
25-34	13	25	38
35-44	21	27	48
45-54	22	7	29
55-65	9	13	22
>65	13	8	21
Total	122	128	250

Table – II: Correlation of S. Iron with Degree of Anemia in Nutritional anemia

		S. Iron (µmol/l)			No. of Cases
		< 10.6	10.6 – 28.3	> 28.3
Hb (gm %)	< 6	15 (6+2.5 %)	9 (37.5)	-	24
	6 – 9	32 (66.6)	15 (31.3)	1 (2.1)	48
	9 – 12	31 (67.4)	9 (19.0)	6 (12.6)	46
	>12	-	4 (100)	-	4
		78	37	7	122

Table – III: Correlation of S. Iron with MCV, MCH and RDW in Iron Deficiency Anemia

	MCV (fl)			MCH (pg)			RDW (%)		Total
S. Iron (µmol/l) (< 10.6)	< 76	76 - 96	> 96	< 76	27 - 32	> 32	13 - 16	> 16
	59 (75.6)	19 (24.4)	-	65 (83.4)	13 (16.6)	-	19 (24.4)	59 (75.6)	78

On analysis of the collected data it has been revealed that approximately 2/3^rd i.e. 78 (64.0%) of cases of 122 anaemia regardless of degree showed low serum iron level. Significantly, 30.3 % cases with anemia also had normal serum iron level while all cases without anemia had normal S. iron (Table - II).

(Table - III) revealed that 75.6 % iron deficiency anemia (IDA) (Peripheral Smear Picture-I) had microcytosis majority 83.4 % of IDA had hypochromia 75.6 % if IDA cases had increased RDW, 24.4 % cases of IDA diagnosed by low S. iron level didn’t show microcytosis. Table further revealed that 16.6 % of cases of IDA diagnosed by low S. iron level did not show hypochromasia as determined by MCH.

Peripheral Smear Picture-I

Table – IV: Correlation of variation of MCV and degree of anemia in case of IDA

(S. Iron < 10.6)

		MCV (fl)			Total
		< 76	76 - 96	> 96
Hb (gm %)	< 6	8 (53.3)	7 (46.7)	-	15
	6 - 9	26 (81.3)	6 (18.8)	-	32
	9 - 12	25 (83.3)	5 (16)	-	30
	> 12	0	1	-	1
	Total	59	19	Nil	78

(Table - IV) showed the comparision of variation of MCV for different degree of anemia in cases of IDA. The following were evident – 75.6 % of cases across of all degree showed microcytosis while 24.4 % showed normocytosis, No case shows macrocytosis (MCV > 96 f1).

That more than 80 % of case of mild and moderate anemia showed microcytosis but this came down to 53 % in cases suffers from severe anemia.

(Table – V) revealed that in mild cases of IDA only 60 % of cases show raised RDW. In all 100 % cases of suffering from moderate to severe anemia there was increased in RDW. An increased in RDW was noticed in 97.5 % cases of (Table - VI) all cases of IDA

Table – V: Comparison of variation in values of RDW (cut off at 16 %) in different degrees of anemia in cases of IDA

		RDW		Total
		13 – 16 (%)	> 16 (%)
Hb (gm %)	< 6	0	15 (100)	15
	6 - 9	0	32 (100)	32
	9 - 12	18 (60)	12 (40)	30
	> 12	1 (100)	0	1
	Total	19 (24.4)	59 (76.6)	78

Table – VI: Comparison of variation in values of RDW (cut off at 14 %) in different degrees of anemia in cases of IDA

		RDW		Total
	Hb (gm %)	13 – 14 (%)	> 14 (%)
Hb (gm %)	< 6	-	15 (100)	15
	6 - 9	-	32 (100)	32
	9 - 12	2 (6.7)	28 (93.3)	30
	> 12	0	1	1
	Total	2 (2.5 %)	76 (97.5 %)	78

(Table – VII) revealed that 1/3 showed low serum B₁₂ level macrocytosis (PS Picture – II and Bone Marrow Picture - III) was observed in 60 % cases of vit. B₁₂ deficiency, 40 % had normocytic RBCs. Increased MCH was seen in all these cases and increased RDW is present in significant majority (80 %) of cases.

PS Picture – II and Bone Marrow Picture – III

(Figure - I) showed the correlation of Hb electrophoresis with MCV, MCH and RDW in haemolytic anemia. Out of 53 cases of haemolytic anemia, 8 (15.09 %), 26 (49.05 %), 19 (35.82 %) cases were suffering from sickle cell Trait (PS Picture - IV), sickle cell disease andThalasemia trait (PS Picture - V) respectively.

PS Picture – IV

PS Picture - V

It was further notices that there was low MCV and high RDW feature in 71.6 % / 38 out 53 / cases. A normal to high MCH was seeming less than in order 22 % with 78 % cases being by hypochromic.

Table VII (57) and (58) All 250 cases studied, were categorized on the basis of MCV and RDW as follows MCV – Below 76 (f₁) Microcytosis 76 – 96 (f₁) – Microcytosis and > 96 (f₁) Microcytosis RDW was considered normal 16 % and increased ≥ 16 % (58) on account of the above results all 250 studied cases were categorized on follows: -

	Cases %
1. Microcytic heterogenous (↓ MCV ↑ RDW) 2. Microcytic non heterogenous (↓ MCV (N) RDW) 3. Normocytic heterogenous ((N) MCV ↑ RDW) 4. Normocytic non heterogenous ((N) MCV (N) RDW) 5. Macrocytic heterogenous (↑ MCV ↑ RDW) 6. Macrocytic non heterogenous (↑ MCV (N) RDW)	90 - 36.0 43 - 17.2 57 - 22.8 44 - 17.6 15 - 6.0 01 - 0.4 ---------------- 250 - 100.0

57 Abnormal MCV (both microcytosis and normocytosis) was a feature of 22.8 % cases, RDW was abnormal (>16 %) in 162/25 64.8 % of cases. When cases of normocytosis were subclassified on basis of RDW further 57 cases were found as abnormal increasing the field of abnormal cases 46 %.

INTERPRETATIONS:

As present study revealed that serum iron fails to categorize up to 1/3 case of nutritional anemic, a single test in case of IDA. Kim S.K. et al (4) has also demonstrated that s. iron as a single assay had 60 % sensitivity in diagnostic care of IDA. Uchida (2) observed that there is increased RDW as a screening test for IDA over serum iron as ferentia.

Table – VII: Correlation of serum Vit. B₁₂ with MCV, MCH and RDW

	MCV (fl)			MCH (pg)			RDW (%)		Total
S. B12 (pg/ml) (<157)	< 76	76 - 96	> 96	< 76	27 - 32	> 32	13 - 16	> 16
	-	10 (40 %)	15 (60 %)	-	-	25 (100 %)	5 (20 %)	20 (80 %)	25

Table – VIII: Major Classes of anemia based on MCV and RDW

MCV (Low) (n = 133)		MCV (N) (n = 101)		MCH (H) (n = 16)
RDW (N)	RDW (H)	RDW (N)	RDW (H)	RDW (N)	RDW (H)
43 (17.2)	90 (36)	44 (17.6)	57 (22.8)	1 (0.4)	15 (6)
Microcytic non-heterogenous	Microcytic non-heterogenous	Normocytic non-heterogenous	Normocytic heterogenous	Microcytic non-heterogenous	Microcytic heterogenous
RDW (N) = 88 RDW (H) = 162

Seward et al (5) found that 83 % with low serum had low MCV as offered 75 % in the present study. Artaza et. al. (3) found that RDW is more sensitive than MCV which is contrary to the findings of the present study were both these parameters equally affected. Kim S.K. et al (4) had observed that MCV had a sensitivity of 90 % for detecting IDA if cut off was < 70 % and increase RDW is 83 % cases in comparision to the cut off < 76 and sensitivity. Fialon et al (6), Burk and Arenz et. al. (7) noted increased RDW in 72 % and 90 % cases respectively which is more or less similar to the findings of the present study (75 %) Uchinda (2) observed that higher the cut off value for RDW, the lower is the sensitivity which is in accordance of the findings of the present study. Bessman et al (8) found low MCV and high RDW as predictive of IDA. Monzon C.M. et al (9) noted that increased MCV and RDW was a feature of B₁₂ deficiency. Gupta et al (10) found that B₁₂ deficiency causes increase in both RDW and MCV which is more or less is accordance of the findings of the present study.

Seward et al (5) observed that MCV along was poor in identifying patient’s with B₁₂/folate deficiency, 12 % patient’s with low B₁₂ level had low MCV also, But in present study normal MCV is 40 % cases. This high figure may reflect underlying concomitant IDA. Chen L. et al (11) has found the high sensitivity and specificity of these changes i.e. decreased MCV and increased RDW is Hemolytic anemia, which is similar to the present study where more than 2/3 of cases had low MCV and high RDW. Hypochromia was also observed in 71 % of cases. Robert et al (12) also observed that highest and earliest changes in RDW were seen in hemolytic anemia. Bansal A.K. et. al. (13) also observed similar findings in his study.

Bansal A.K. et. al. (14) in their study of the epidemiological pattern of sickle cell disease in Bastar noted that it has broken the myths that sickle has not been observed in the Muslims population in India. Schwieger et al (15) found that 82 % cases had increased RDW and MCV as a discriminatory factor was not significant in sickle cell anemia as compared to 94 % in the present study and microcytosis was seen 60 % cases where as 40 % were normocytic. Thus red cell heterogeneity is a predominant feature in case of sickle cell anemia. Sayed et al (16) RDW increase in sickle cell anemia similarly Monzon et al (9) observed that sickle cell disease was characterized by normal MCV but raised RDW, which is similar in the findings of the present study. Webster et al (19) Monzon et al (9) demonstrate that increases RDW was a feature of SCD only while in the present study this was seen in predominant number of case of both SCD and SCT. This is however because of function of existing reticulocytosis isn the patient’s. Flynn et al (18) noted that increased red cell heterogeneity was seen in nearly ½ of their cases of β-thaalasemia traits Baquar et al (19) compared to 32 % in the present study. This may be done to concomitant IDA. Laso et al (20) had also noted that RDW was below 16 % in pure cases of BTT and icreased RDW was found when IDA co-exists. Shrivastava P.K. and Bansal A.K. (21) in their study of Sickle Cell disorder among Tribal and Non tribal community of Bastar India noted that 3.04% of the sickle cell suffers were Muslims and Chistians.

Artaza and Carbia (3) also observed in their study that presence of increased RDW is more sensitive than changes of MCV to dieffidiotiate the begging of IDA. Although RDW is considered to be a great help in differencing two very important causes of microcytic Hypochromic anemia i.e. IDA and β-thaalasemia trait. Amongst 1/3^rd of cases in present study show increased RDW in contrast to expected low or no heterogeneity, which was seen in 68 % of cases.

Thus the findings of the different studies conducted by different authors from time to time throughout the globe were more or less similar to the findings of the present study.

CONCLUSION:

From above observation and discussions, the authors reached to the conclusion that fairly sensitive and specific diagnostic approach can eliminate unnecessaryand expensive and invasive investigation for classification; evaluation and monitoring of various types of anemia in large number of cases and only in few cases expensive Conservatory test are required.

ACKNOWLEDGEMENT:

The authors express their cordial thanks to Mr. Anand Singh Kanwar, Lab-Technician, Deptt. of Community Medicine, Govt. Medical College, Jagdalpur (C.G.) for his neat and excellent typing, Special thanks to my wife Mrs. Preeti Azad.

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Received on 21.04.2011

Modified on 20.05.2011

Accepted on 10.06.2011

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Research J. Pharmacology and Pharmacodynamics. 4(1):Jan. - Feb., 2012, 25-30