Study of RBC Histograms and Its Correlation with Etiopathogenesis and Other Parameters in Various Anemias

The pluripotent stem cells which are present in the bone marrow renew by its own and differentiate into mature cells. These stem cells undergo division by the presence of erythropoietin, where the nucleus is extruded out from the cell during the end of differentiation, thereby retaining cytoplasmic RNA to form a reticulocyte. The reticulocyte i s a precursor to red blood cell and on losing the RNA it matures into a Red Blood Cell. The present study aimed to analyze the correlation between the automated histogram patterns along with morphological features of RBC‘s prepared from peripheral smear examination in different types of anemia. viz., MCV, MCH, MCHC & RWDCV.


INTRODUCTION
Anemia is a condition in which thephysiological function have neo been supplied with adequate oxygen due to the reduced number of blood red blood cells (RBC) or any hindrances in their oxygen carrying capacity. It may vary with a number of factors including age, sex, altitude,

Original Research Article
smoking, and pregnancy status. Iron deficiency is the predominant cause of anemia occurring globally besides other important conditions particularly deficiencies in folate, vitamin A and B12 synthesis.Apart from these, chronic inflammation, parasitic infections, and inherited disorders can also cause severe anemia in people. Complete blood count (CBC) using automated analyser and microscopic peripheral smear examination are the main diagnosis methods. Complete blood count (CBC) i s a routine test conducted now days to evaluate the concentration & count of various cellular components of blood such as RBC count, WBC count, platelet count, Hb, haematocrit, red cell indices, differential count( WBC), Mean platelet volume, histograms of RBC, WBC, platelets & red cell distribution width. In recent days, many of the laboratories have replaced the traditional methods in haematology by automated analysers & automated data [1]. As there is advancement in the technology of automated analyzers with increase in i t s precision, the rate of manual peripheral smear review have been declining [2]. RBC histogram i s a symmetrical bell -shaped curve, a diagrammatic representation for better understanding & interpretation of various anemias. But it is still limited in day to day use as the technologists are unaware & only a few have understanding in correlating & interpreting [1]. Width of red blood cells, haemoglobin distribution and its width and reticulocyte cell count are the parameters that have gained popularity as they provide useful information & a less importance i s given to scatter plots & histograms [3,4,5].

MATERIALS AND METHODS
This is a prospective study conducted in the department of pathology, Sree Balaji Medical College and Hospital after getting Institutional Ethical Committee approval. Patients were sourced from the clinical departments. A total of 500 patients with anemia were studied for 24 months since October 2015 to September 2017.

Inclusion Criteria
Patients presenting with anemia with hemoglobin levels, l less than 11.5 gm%, were included for the study irrespective of patients age group.

Exclusion Criteria
Patients with a recent history of blood transfusion were excluded from the study. Samples with inadequate e quantity (< 3 ml) for analysis and study were excluded.

Staining of Thin Blood Smear
Leishmans stain was used in this study for the staining of blood smear. Leishmans stain is added drop by drop to the s l ide and it is left to wait over a period of two minutes, which helps in fixation of the blood smear. Double the quantity of buffered / distilled water is added to the s l ide and mixed gently for 8 minutes. I t i s washed in a slow stream of running tap water and left to dry. After drying, the s l ide is studied under oil emersion lens of the microscope [6-9].

Statistical Data Analysis
The data collected in the study were assorted using Microsoft excel and analysis of the data was done using IBMSPSS statistics software, Pearson' s Chi square test was used wherever appropriate.

RESULTS
This present prospective study was carried out in the department of Pathology, Sree Balaji Medical College & Hospital for 24 months duration started from October 2015 to September 2017. A total of 500 cases of anemia that belonged to the inclusion criteria were studied.

Age of the Study Group
The study comprised of a population with the age group ranging from 1 to 93 years. Major cases of patients were between 41 to 50 years of age.

Sex Distribution
The majority of the study population were females which contributed 61% of the total.

Classification of Anaemia Based on Haemoglobin
Based on the levels of haemoglobin, anaemia is classified as Mild, Moderate & Severe.

Anemia based on RBC histogram pattern
Histogram patterns of all 500 cases were s tudied and among this

Correlation of RBC histogram patterns with peripheral smear study
The histogram patterns obtained for each case is correlated with the peripheral smear and tabulated for statistical analysis.

Microcytic hypochromic anaemia
Among the cases which were reported as Microcytic Hypochromic Anemia 87.8% cases showed Left Shift in the histogram patt ern & 6.6% showed normal curve .

Macrocytic anaemia
Among the cases which were reported as Macrocytic Anaemia 86.9% cases showed Broad Base curve in the histogram pattern & 13.1% showed Right shift curve.

Dimorphic anaemia
Among the cases which were rep orted as Dimorphic Anaemia 37.2% cases showed Broad Base curve in the histogram pattern & 24.7% showed Left shift curve.

Normocytic normochromic anaemia
Among the cases which were reported as Normocytic Normochromic Anaemia 84.3% cases showed Normal curve in the histogram pattern & 15 .7% showed Left shift curve.

Prevalence of anemia based on Mean Corpuscular Volume (MCV)
In  In dimorphic anemia the peripheral smear usually shows a dual population of microcytes and normocytes; macrocytes and normocytes; sometimes a mixture microcytes, normocytes and macrocytes. The histogram pattern varies showing a change in the centre of the curve and the width of the curve. The red cell indices may or may not be normal. Hence diagnosis purely based on automated parameters will be sometimes misleading in thiscondition. Thusperipheralsmear study of the morphology of red cells in correlation will help in interpreting the appropriate diagnosis. Earlier a similar finding was done was they have stated that dual population of RBCs can be identified by histograms in most cases.

Comparative analysis of various anemia's represented by peripheral smear with red cell indices
In all the 500 cases of our study, red cell indices (MCV, MCH, MCHC) was recorded from the automated analyser BC-5380 Mind ray which also provided the histogram. The red cell parameters were compared a nd correlated with the peripheral smear f indings of each sample. The values of MCV were categorized as microcytic, normocytic/ dimorphic, macrocytic. Cases with the MCV value < 80 f l were labeled as microcytic. Cases that were in the normal range of MCV (8 to 100 f l ) were labeled as normocytic or dimorphic. Other cases which had an MCV above 100 f l were labeled as macrocytic. A similar method of labeling was applied for the MCH values of all the cases. Microcytic -< 26 pg; normocytic/ dimorphic -26 to 34 pg; macrocytic -> 34 pg.
On applying s tatistical analysis and correlation with two variables one was anemia based on peripheral smear and the other was anemia based on red cell indices. We obtained P values in the correlations in which some showed significant correlation and a few showed a significant difference. Made a s tudy in which they s tated that the MCV l evel was normal in 61% of anaemic patients among their s tudy population. In our s tudy 36.6% of the total 500 anemia cases had a normal MCV. This shows that MCV alone cannot be used as an independent sensitive parameterto classify anemia. This was s tudied and they proposed few probable reasons stating that MCV is a mean value and i t does not represent the various red cell population present in the blood sample. MCV is insensitive when the microcytes and macrocytes are very few in number. MCH and MCHC also gives a very l i t t le information.
This concludes that all cases with decreased haemoglobin levels require a peripheral s mear examination for diagnosis. In 2005 Barbara J. Bain [42] is tated that in the current age of automation and even during the age of molecular analysis peripheral smear examination will remain as the most important diagnostic method and along with the latest modern investigative methods peripheral smear examination will also be in light. In comparison to peripheral blood smear examination visual examination of RBC histograms i s usually more sensitive and objective in identifying the presence of group of cells that are few in population and morphologically varied in size

Red cell distribution width -a parameter for interpretation in anemia
RDW i s an additional parameter provided by the automated haematology analys ersnowadays .Inour study majority of the cases were microcytic hypochromic anemias by peripheral smear study. Among these cases majority had a high RDW. Anisocytosis usually gives a high RDW value. Thus RDW helps in identifying anisocytosis in conditions where the MCV is not in ab normal range, like Early I ron Deficiency Anaemia where diagnosis is difficult. In this study, previously we have discussed that cases interpreted as dimorphic anemia had a majority of broad base histograms which explained the presence multiple population of RBC and this i s described as a high rate of anisocytosis which will automatically reflect as an increase in RDW. showed that the RDW was increased in case of microcytosis; stated that when there is a higher degree of anisopoikilocytosis there is an increase in RDW. These studies are in concordance with our study.

CONCLUSION
The RBC histogram obtained from the automated haematology analyser provides valuable information in view to the diagnosis of various anemia's. Only a few s tudies have been done on RBC histograms to reveal i t s importance whereas, much more importance was given to WBC histograms to bring it to light for the diagnosis of leukaemia and blast cell population. Ourstudy was doneinapurposetoidentify the significance of correlation between RBC histograms and peripheral smear studies in various anemias such as microcytic hypochromic type, macrocytic type, dimorphic type and normocytic normochromic anemia type. The analysis and results showed that there is a significant correlation. However, there is a complex histogram pattern distribution in dimorphic anemia that makes the histogram pattern analysis a non -independent parameter; peripheral s mear must be done in such cases as they are in good correlation with histograms.This concludes that peripheral smear examination i s an important diagnostic tool even in the period of molecular and automated analysis. As a supplementary to peripheral smear examination RBC histogram and other criteria such as MCV, MCH, MCHC and RDW can be used in diagnosis of various anemias.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
The study was approved by the Institutional Ethics Committee of Sree Balaji Medical College and Hospital.