RBC Histogram as Supplementary Diagnostic Tool with Peripheral Smear Examination in Evaluating Anaemia

Background: Red blood cell(RBC) histogram provide an idea about morphological changes of red blood cells in hematological disorders. Peripheral smear examination findings are usually correlated with complete blood cell counts by automated analyzer. To known the utility and advantage of red cell histogram and correlation of microscopic examination of peripheral smear with automated histogram pattern. Methods: Blood sample was collected from 220 anemia patients in ethylene diamine tetra acetic acid (EDTA) tubes for peripheral smear examination and ran in Beckman coulter LH 780 automated hematology analyzer for obtaining histogram, complete blood count includes hemoglobin, total leucocyte count, Platelet count, red blood cell indices and red cell distribution width. This study was undertaken for a period of one month of November 2016 in department of pathology, central laboratory, narayana medical college & hospital, Nellore. Results: This study of histograms of 220 different types of anemia consisted predominantly females 154 (70%) , and males 64(30%). Maximum cases of anemia were noted in 30-40 years of age range. Microcytic hypo chromic anemia was the most common (63.63%) followed by normocytic normochromic anemia (19.4%), macrocytic hypochromic anemia (2.2%), dimorphic anemia (12.72%) and pancytopenia (1.8%). Left shifted curve and broad base mostly seen in microcytic anemia, right shift curve seen in macrocytic anemia and bimodal peak mostly seen in dimorphic anemia. Conclusion: Histogram can be used as an important screening test for hematology and can become a new parameter in the diagnosis of anemia though peripheral examination remains the definitive diagnostic test for evaluation.


Introduction
Evaluation of anemia for diagnosis and management of various red blood cell (RBC)disorders through red blood cell histogram is a vital part. Complete blood count parameters like red cell distribution width (RDW) and mean corpuscular volume (MCV) are useful along with histogram for interpretation of abnormal red blood cell morphology. [1,2] The Normal Red cell distribution curve is a bell shape and curve peak should be within the normal MCV of 80.0-100.0fl. The narrow red cell distribution curve indicates homogenous population of cells and wider redcell distribution curve indicates a heterogenous population of red cell. In megaloblastic anemia where the red blood cells are larger, the histogram curve will shift to right and the curve will move to left if the cells are smaller than normal like in microcytic anemia. After treatment of underlying cause in anemia, the curve should shift toward the normal range . In dimorphic anemia the histogram curve may show multiple peaks due to two distinct red cell populations.After treatment of the cause of an anemia curve should move toward the normal range.Even when MCV is normal, RDW is a good indicator of anisocytosis. [3] Higher RDW represents dual population of cells like small cells, some normal size cells, and immature red cells during degenerative response to anemia which are larger than normal. Histogram can be useful for monitoring the reliability of results ofanalyzer, potential causes of results and arriving at the probable diagnosis. [3]

Materials and methods
The data of 220 samples has been taken from the period of November-2016 to December-2016, central laboratory, Narayana General Hospital & Medical College, Nellore Andhrapradesh, India.
In this Retrospective study, The Red blood cell histograms of all anemic patients visiting the central diagnostic laboratory of Narayana general hospital were analyzed. The Red blood cell histogram correlated with peripheral smear interpretation when the haemoglobin is low. The spectrum of variations in Red blood cell histograms tabulated with types of anemias based on peripheral smear interpretation.
www.pacificejournals.com/apalm eISSN: 2349-6983; pISSN: 2394-6466 Complete blood count including haemoglobin , total leucocytes count (TLC), differential leucocytes count(DLC), platelet count and blood indices were obtained by Beckman coulter LH 780 automated analyser along with histogram. To maintain validity of LH 780 Automatic analysis machine quality control was done. Peripheral smear examination was also done. Normally volume of RBCs from 34fl to 150fl represents a good cell distribution ideally.
If the graph starts before 34 fl and touches the baseline before 150fl are considered to be a left shift indicates microerythrocytosis. If the graph starts after 34fl and touches the base line after 150fl are considered to be a right shift indicates macro erythrocytosis. If graph starting at 34fl and ending between 225-250 fl are considered to be a broad base. If RBCs Populations has two morphologies then the graph will have two peaks representing to their respective morphology and called as dimorphic red cells. If the RBCs population will have single lineage of cells seen, the graph will be constraint and will look like short peak.
Data was analysed using SPSS 22 and P-value was calculated using chi-square & degree of freedom. p value of 0.05 or less was considered statistically significant.
Inclusion Criteria: All cases of anemia with low haemoglobin. For men anemia is defined as haemoglobin level less than 13.5 gm/100ml and in women as haemoglobin of less than12.0 gm/100ml. Based on peripheral smear interpretation ,different types of anemias like normocytic normochromic, microcytic hypochromic, macrocytic, dimorphic and pancytopenia were included in our study.

Discussion
The RBC histogram is a graphic representation of particle size distribution in automated cell analyser. This is a standard part of complete blood picture. It provides clues in management and diagnosis of various red cell disorders and give information regarding to RBC parameters like RDW, MCH and MCV. [1,2] Normal curve is symmetrical bell shaped or Gaussian distribution. Normal curve falls within normal MCV range 80-100fl.The RBC histogram in the cell counter displays the cell ranges for RBC are between 24fl and 360fl. The instrument counts only those cells with volume sizes between 36fl to 360fl as red cells .Those cells counted in the range 24fl to 36 fl are rejected and not include in the RBC count. Normally below 36fl size space is clear, but histogram begin above base line indicates presence of small particles like microspherocytes, malaria parasite, platelet clumps, normoblast, elliptocytes, bacteria, etc. [3,4] RBC histogram follows well known coulter principle of counting and sizing red cells providing the basis for generating the histogram. This method relies on the change in conductance as each cell passes through an aperture. The change in conductance results in an electrical pulse, the amplitude of which is proportional to the cell volume. The y axis represents the number of cells per channel, with each cell being stored in the representing its size ,so that after data is further processed by the computer, and the RBC Curve is smoothed by a moving average technique and displayed on a data management system. WBCs presented in RBC channel and counted with red blood cells. RBC Histogram is affected if WBCs count is more than 50000 cells . The presence of right sided shoulder usually corresponds to reticulocytosis and a trailer of erythrocyte population on the far right of the histogram correlates to red cell agglutination .
Our study out of 220 cases showed normal curve39 (17.7%), left shift 64(29%), right shift 12 (5.45%) Broad base 83 (37.72%), short peak 6 (2.7%) and bimodal 6(7.27%). Our findings regarding to RBC histogram were correlated with other studies like sandhya [5] and Chavda J [6] (Table-6). In normocytic normochromic anemia, the red cell indices like MCV, MCH and MCHC were within the normal limits with some cases showing mild increase indices. The population of the cells would be variable in size. Like some microcytic cells with predominance of normal size cells that results in a higher deviation and hence higher RDW . In our study out of 43(19.54%) cases of normocytic normochromic anemia, 26(11.8% ) showed normal curve and 17(7.72)% showed broad base curve. These finding were correlated with study carried by Chavda J. [6] In the microcytic hypochromic anemia, MCHC may be normal but MCV and MCH are decreased. RBC population with low MCV will be shifted toward left. A broad base curve because of high RDW represents anisocytosis. RDW is also increased when there is increase number of smaller cells like in iron deficiency anemia. In present study MCV and MCH were less than normal in microcytic anemia with normal MCHC and increased RDW due to ansiopoikilocytosis as noted in peripheral smear study. Out of 140 (63.23%) cases of microcytic hypochromic anemia 7(3.18 % ) were normal, 60 (27.27% ) were left shift curve, 60 (27.27%) showed broad base curve, 7 (3.18%) showed bimodal curve histogram. and 6 ( 2.72% ) showed short peak. .These finding were correlated with study carried out by sandhya [5] and Chavda J. [6] The short peak well correlated with low haemoglobin and red cell count.
In dimorphic anemia the histogram may have 2 or more red cell populations, whereas in dual populations, the histogram has 2 distinct red cell populations. In dimorphic blood picture there may be dual population of microcytic & normocytic or normocytic & macrocytic red cells or admixture of small, normal and large cells of different sizes or admixture of patient and donor red cells. In our study in dimorphic anemia, a MCV, MCH and MCHC were normal and increased RDW due to marked ansiopoikilocytosis. The dimorphic RBC showing bimodal curve along with some cases showing left and right shifting of curve. The reason for dimorphic population may be nutrional anemia, recent blood transfusion or response therapy to nutrional anemia. [7] Out of total 28 (12.7%) cases of dimorphic anemia 9(4)% showed normal curve, 4(1.81%) showed left curve 04% showed right curve 9(4% ), broad base curve 4(1.81%) and 2 (0.9% )showed bimodal curve. These finding were correlated with study carried out by sandhya [ 5] and Chavda J. [6] In macrocytic anemia right shift with broad based curve means low Hb and macrocytic blood picture. As Causes of macrocytosis vary from benign to malignant, complete approach is essential to determine the etiology. [8,9] Macrocytosis may occur at any age, but it is more prevalent in old age. [10,11,12] In our study macrocytic anemia due to variation in size and shape of the RBC's, increased MCV, RDW, MCH were noted with normal MCHC. All 5 (2.2%) cases of macrocytic anemia showed right shift curve. This finding was correlated with Sandhya. [5] Right shift curve correlated well with increased MCV and MCH. All pancytopenia cases were broad base curve and correlated with Sandhya. [5] Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 6, November-December, 2017 Histogram plays a vital role as working tool in early stage of morphological analysis when combined with concept of normal curve and the knowledge of particular complete blood count parameters like red cell distribution width and red cell indices. Presumption of presence of fragments, microcytichypochromic, macrocytic or dimorphic red cells, and different combinations of cells is possible by observations of outlines of histograms.
In addition MCV and MCV provide an idea about distribution of red cell histogram.

Conclusion
Along with peripheral smear examination,diagnosis ofRBC disorders is supplemented by histogram and they provide guidance regarding RBCS morphology, blood indices and Hb value. While interpreting microscopic examination of peripherial smear, good idea can be obtained from reviewing histograms.The speed and reliability of analyzers allow time to evaluate abnormal blood smears and correlate with histograms with confidence and efficiency Peripherial smear interpretation was significantly correlated with histogram curve in 220 anemia cases (p value <0.001, chi square value 74.28). Histogram Changes correlated well with peripheral smear findings in majority of the cases. It is used for screening but not considered diagnostic for any pathological condition.