CE Update

The Red Cell Histogram and The Dimorphic Red Cell Population
Benie T. Constantino, SH, I; ART, MLT(CSMLS) (CML Healthcare Inc., Mississauga, Ontario, Canada)
DOI: 10.1309/LMF1UY85HEKBMIWO

Submitted 4.30.10 | Revision Received 9.27.10 | Accepted 9.27.10

Abstract

The RBC histogram is an integral part of automated hematology analysis and is now routinely available on all automated cell counters. This histogram and other associated complete blood count (CBC) parameters have been found abnormal in various hematological

conditions and may provide major clues in the diagnosis and management of significant red cell disorders. In addition, it is frequently used, along with the peripheral blood film, as an aid in monitoring and interpreting abnormal morphological changes, particularly dimorphic red cell populations. This article discusses

some morphological features of dimorphism and the ensuing characteristic changes in their RBC histograms. Keywords: histogram, dimorphic red cells, red blood cell distribution width

After reading this article, readers should be able to correlate a RBC histogram and red blood cell distribution width with microscopical findings.

Hematology exam 51102 questions and corresponding answer form are located after this CE Update on page 309.

To paraphrase an adage, 1 histogram graph is worth 1000 numbers. A large collection of data, displayed as a visual image, can convey information with far more impact than the numbers alone. In hematology, these data take on several forms, 1 of which is the RBC histogram. Visual scanning of the histogram gives a good initial sense of the range, size, shape, and other salient features of the red cell morphology. The RBC histogram, a graphic representation of particle size distribution, is now routinely available on automated cell analyzers as a standard part of automated complete blood count (CBC) analysis. This histogram in association with other CBC parameters, such as RBC distribution width (RDW) and mean corpuscular volume (MCV), has been found abnormal in various hematological conditions and may provide major clues in the diagnosis and management of significant red cell disorders.1-4 In addition, it is frequently used, along with the peripheral blood film, as an aid in monitoring and interpreting abnormal morphological changes, particularly dimorphic red cells. Table 1 lists conditions associated with dimorphic red cell changes. Before reporting CBC results, a clear distinction between dimorphic and dual populations must be clarified as they are sometimes interchangeable and confusing.

Table 1_Conditions Associated With Dimorphic Red Cells5-12
Early iron developing microcytic population Folate/vitamin B12 developing macrocytic population Post-iron treatment of iron deficiency anemia Post-iron treatment of iron deficiency with megaloblastic anemia Post-iron treatment of megaloblastic anemia Post-iron treatment of megaloblastic anemia with iron deficiency Post-iron transfusion macrocytic anemia Post-iron transfusion microcytic anemia Iron deficiency anemia with either folate or vitamin B12 deficiency Sideroblastic anemia (myelodysplasia) Hemolytic anemia (reticulocytosis, spherocytosis, fragmentation, pyropoikilocytosis) Cold/warm auto agglutination Erythropoietin-induced erythropoiesis Delayed transfusion reaction Homozygous hemoglobinopathies (admixture of many RBC forms) Myelofibrosis (admixture of extramedullary hematopoiesis) Constitutional chromosomal translocation t(11;22)(p15.5;q11.21)

Corresponding Author
Benie T. Constantino, SH, I; ART, MLT(CSMLS) btconstantino@hotmail.com

Abbreviations
CBC, complete blood count; RDW, red blood cell distribution width; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; IDA, iron deficiency anemia; RDW-CV, red blood cell distribution width coefficient of variation; thal, thalassemia; SD, standard deviation; HPP, hereditary pyropoikilocytosis

In a dimorphic picture, the histogram may have 2 or more (multiple) red cell populations, whereas in dual populations the histogram has 2 distinct red cell populations (eg, hypochromic-microcytic and normochromic-normocytic red cells). These 2 distinct populations may be comprised of either a patient’s own red cells (post-iron treatment) or a mixture of patient and donor red cells (post-iron transfusion). Although this is an arbitrary categorization, the term dimorphic is less restrictive and therefore more widely used, as it can be applied to either the dual or the multiple red cell populations. Thus, the dimorphic blood picture will look like a dual population of microcytic and normocytic (Image 1A) or normocytic and macrocytic red cells, or an admixture of small, normal, and large cells of different sizes and forms (Image 1B). Some morphological features of dimorphism and the ensuing characteristic changes in their RBC histograms are discussed in this article.
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The 256-channel pulse-height analyzer uses a number of thresholds to sort the cells into several size (volume) channels from which the histogram is formed. It is sometimes referred to as a frequency of distribution curve.3.16 Furthermore. certain conditions like the presence of fragmented red cells or red cell agglutination that could not have been identified earlier without blood film examination can now be presumably detected on the red cell histogram. membrane budding (long arrow). a sequential histogram can clearly show the progressive appearance of a new erythrocyte population well in advance of other numerical parameters. Those cells counted in the 24 fL to 36 fL range are rejected and not included in the RBC count.com data. and the RBC curve is smoothed by a moving average technique and displayed on a data management system. or as a line of frequency curve. showing a shoulder of microcytic red cells. which is elevated. Because graphics can show data in ways that are meaningful and quickly understood. and interpret empirical data that displays morphological changes graphically as points. 2) investigating the potential cause(s) of the erroneous automated results. Bimodal red cell histograms (Figure 1F. and a trailer of erythrocyte populations on the far right of the histogram correlates to red cell agglutination (Figure 1F).7 For example. It enables one to visualize. At times. and skewed data. oval macrocytes. but they may also indicate other hematological disorders as shown in Table 1. Likewise. peaks or valleys. A leftward shift of the RBC histogram signifies microcytosis (Figure 1B and Figure 1C).16 Likewise. please see reference 14. This data is further processed by the computer. with each cell being stored in the channel representing its size. the histogram is a very powerful tool in red cell morphological analysis. a histogram can provide invaluable information that may not even be apparent in the automated numerical labmedicine. Although the size ranges for RBC histograms are between 24 fL and 360 fL. subpopulations.5. The change in conductance results in an electrical pulse. elliptocytes. analyze. essential polycythemia). so that after data accumulation is completed the relative number of cells (frequency) is provided. some of the hypochromic and microcytic red cells can be identified in the histogram but may not be reflected in the numerical data. increasing from left to right. in patients with iron deficiency anemia (IDA) or megaloblastic anemia in treatment. and a rightward shift suggests macrocytosis (Figure 1D and Figure 1E). the instrument counts only those cells with volume sizes between 36 fL and 360 fL as red cells. and this may well be the first clue to a not uncommon occurrence. This method relies on the change in conductance as each cell passes through an aperture. except the RDW. and polychromasia. The RBC histogram is also abnormal. the amplitude of which is proportional to the cell volume. Each channel on the X-axis represents a specific size (volume) in femtoliter (24-360 fL). Figure 1K. iron deficiency may develop insidiously during the process of some other disease (eg. in megaloblastic anemia with developing iron deficiency. with normal cell counts and indices.3 The presence of a right-sided shoulder usually corresponds to reticulocytosis (Figure 1J). leading to quick and cost-effective decision making. They are enumerated and displayed in the histogram area between the 24 fL and 36 fL May 2011 ■ Volume 42 Number 5 ■ LABMEDICINE 301 . For a detailed explanation on the construction of histogram. as shown in Figure 1 and Figure 2. microspherocytes (regular arrow). The Y-axis represents the number of cells per channel.CE Update A     B Image 1_(A) Wright-stained peripheral blood film from post-iron treated patient showing dual population of hypochromic-microcytic (short arrow) and normochromic-normocytic red cells (long arrow). Figure 1G. Value of Histogram A histogram. (B) Peripheral blood film from post-splenectomized patient with hereditary pyropoikilocytosis showing dimorphic picture of extreme variability of the RBCs such as red cell fragments (short arrow). Figure 1L and Figure 2B to Figure 2E) are usually associated with therapeutic transfusion and/or hematinic agent response to microcytic and macrocytic anemia. a histogram can provide useful information for laboratorians in 1) monitoring the reliability of the results generated by the analyzers.15 For example. and 3) arriving at the presumptive diagnosis. is a graphic representation of a collection of data based on cell size and/or cell number depicting variations in the process.13 Figure 1A shows a typical normal RBC histogram. RBC Histogram: An Overview The well-known Coulter principle of counting and sizing red cells provides the basis for generating the histogram. It allows the users to intuitively see the visual comparison of the center and spread of data and data containing 2 or more variables: dimorphic red cells.

(B) Microcytosis. bimodal.6 54.9 79. red cell distribution wiidth-coefficient of variation. (J) Reticulocytosis.5 14. SKR. (Key hematological features of these conditions are summarized in Table 2.0 102. skewed to the right. BM.6 14.6 90. RS. (F) Cold agglutination. right-shift.CE Update RBC Histograms A       B     C D       E     F G       H     I J       K     L Figure 1_Red cell histograms in various hematological conditions. 302 LABMEDICINE ■ Volume 42 Number 5 ■ May 2011 labmedicine. MCHC (+++++) overrange. iron deficiency anemia.3 14.3 35.8 80-95 Hematological Parameters MCHC (g/L) 330 314 318 351 324 +++++ 320 285 358 298 317 330 320-360 RDW-CV (%) 14. (I) Pyropoikilocytosis. Table 2_Key Hematological Features of Various Hematological Conditions in Figure 1 Condition 1A Normal 1B Iron deficiency anemia 1C Beta thalassemia 1D Macrocytosis 1E Megaloblastic anemia 1F Cold agglutinin 1G Sideroblastic anemia 1H Beta thal major 1I Pyropoikilocytosis 1J Reticulocytosis 1K Post-iron therapy 1L Post-iron therapy Reference range HGB (g/L) 146 86 131 131 65 124 92 86 99 87 132 112 120-160 MCV (fL) 89.2 32. SKL. MCV.2 81.4 21. hemoglobin.8 127. (L) Post-iron therapy.5 31. RDW-CV. LS. (D) Macrocytosis with normal RDW.7 66. left-shift.6 60.8 110. skewed to the left. dimorphic.5 RBC Histogram Normal LS LS RS DM RS SKL DM BM DM BM LS SKR DM LS DM LS SKR DP LS SKR DP BM DP BM HGB.) Figure (A) Normal histogram. beta thal trait.2 72. megaloblastic anemia. DM.1 85. (K) Post-iron therapy. (G) Sideroblastic anemia.0 11. (C) Microcytosis. dual population.1 41.7 17. mean corpuscular hemoglobin.5-14. (H) Beta thalassemia major. (E) Macrocytosis.5 39.8 35.com . DP.

2 7 23. skewed to the left. SF. SF normal range (female)=13-145 UG/L.7 70. SL.0 <5 17.9 68.0 29.3 77. IDA.CE Update A            B RBC Histograms C            D E            F G Figure 2_Histograms of iron deficiency anemia after treatment (key hematological features are summarized in Table 3).1 61. skewed to the right.com May 2011 ■ Volume 42 Number 5 ■ LABMEDICINE 303 .9 28.5 28.8 69. BM.0 36. SKR.4 70.6 70. SKL. Table 3_Key Hematological Features in Iron Deficiency Anemia After Treatment Condition 2A Normal 2B IDA 2C IDA 2D IDA 2E IDA 2F IDA 2G IDA HGB(PT) HGB(POT) MCV(PT) Hematological Parameters MCV(POT) RDW(PT) RDW(POT) SF(PT) RBC Histogram Normal DP SKL DP BM DP BM DP BM LS DP LS SKR DM LS SKR 144 108 132 80 107 86 118 98 119 95 106 81 91 84. left shift.1 25.5 8 18. iron deficiency anemia.2 8 19. DP.6 13. post-iron treatment. labmedicine.9 22. POT.4 78.3 82.2 81. serum ferritin.9 65.8 <5 19. dual population. Figures B to G are representative examples of dimorphic (dual) red cell population response to hematinic treatment of iron deficiency anemia in 6 patients.5 25. SF (male)=27-220 UG/L.2 62. pre-treatment.9 <5 PT. bimodal.

Although their histograms are similar. These include the coincident doublet of red cells. Iron deficiency anemia is characterized by elevated RDW. or bimodal but skewed to the left or right. may be skewed to the left or right. a dotted line depicting a reference normal curve (eg. In a single population. elliptocytosis. When the distribution is not symmetric. patients showing severe anemia with hemoglobin <90 g/L and with hypochromic-microcytic red cells are most likely (80%) iron deficient. single-peaked.16 To reduce the effect of these problems. inclusion of reticulocytes with mature red cells. Simply put. aperture artifacts. or may show some combination of all of these characteristics.13.13 They develop mathematical algorithms for particle counting and produce numeric data. The normal RDW-CV is 11.7. and inclusion of leukocytes in certain diseases. Familiarity with typical RBC histograms will make the task of evaluating other notable deviations easier. in 1 way or another. allowing the lower end of the histogram to be monitored. differentiates them. A histogram may be interpreted by looking at the shape of the data distribution. the histogram may have a normal Gaussian or bell-shape curve. influence the histogram’s appearances and accordingly will have a variable effect on any measurements made from the histogram. nonlyzed RBCs. platelet clumps. the RDW is usually within range due to the homogeneity of the inherited population of erythrocytes. The curve is considered symmetric if the 2 sides of the curve coincide when folded in half or are approximately mirror images. symmetric normal curve with MCV of 96 fL and RBC distribution width coefficient of variation [RDW-CV] of 15. and “bell shaped” normal curves. parasitic organisms. 304 LABMEDICINE ■ Volume 42 Number 5 ■ May 2011 Figure 3_Calculation of RDW.0%) should be automatically superimposed on every red cell histogram so any discernible deviation from that curve can be clearly delineated for contrast.16 In IDA and beta thalassemia (thal) trait. to maximize the usefulness of the histogram. bacteria.5%.3 Nonetheless. These particles include red cell fragments. The normal RDW-SD is 39 to 47 fL. the histogram may be symmetric and/or bimodal. In a normal RBC histogram. macrothrombocytes. the information below 20% of scale on the red cell histogram are excluded (Figure 3). it is referred to as skewed wherein 1 of the 2 sides (left or right) of the curve extends much further than the other. a number of factors may affect the histogram of the aperture impedance cell distribution analysis.14 labmedicine.CE Update range. In dimorphic populations. microcytic. and the percentage of microcytosis is increased. Potentially.19. They are symmetric. which generally indicates the presence of small particles. cold agglutinin. cases involving severe anemia with homogenous microcytosis may give misleading results.13 • position of individual populations compared to normal/ typical positions • amount of separation between populations compared to normal/typical separation • relative concentration of each population compared to normal/typical concentrations • presence of unexpected or non-typical populations Figure 1A to Figure 1D are typical examples of normal.21 Analysis and Interpretations of Histograms in Figure 1 and Figure 2 The red cell histograms presented for illustration were randomly selected to represent various common hematological conditions and the different stages of post-iron IDA therapy. The centeredness and width of the histogram also define the extent of RBC variability. alteration in red cell shape. graphic data. the space below 36 fL remains clear. The tail of the distribution consists of coincident doublets and anomalous pulses. The distributional shape of the histogram can be classified as reflecting either single or dimorphic (multiple) red cell populations.16 Before taking up complex histogram graphs. as measured by the RDW. manufacturers design their instruments and reagent systems to specifically prevent and correct for interferences. In thal trait however. Normally. however. and other interfering substances such as cryoglobulin. These factors. microspherocytes.16-18 The following are important points to consider when reviewing/analyzing histograms.com . The RDW is calculated from the width of the histogram at 1 SD from the mean divided by MCV. the degree of anisocytosis. and agglutinates on the right side and platelet clumps. if the patient’s histogram strays from the reference normal range.20 In addition. it means that the histogram may be abnormal. may be widened with or without left or right shift. Although this difference is useful to some extent in distinguishing them. scatter plots. In view of this. nucleated RBCs. but in certain conditions the histogram may begin above the baseline or show a high takeoff on the far left of the curve (Figure 1G to Figure 1I). doublets and triplets. and macroglobinemia. reflecting the heterogeneity in the acquired erythrocyte populations. red cell agglutination. the beginner will find it helpful to examine the simple outline or overall pattern of a regular histogram as reflected in Figure 1A to Figure 1D. and macrocytic red cell histograms.5% to 14. the majority of each cell falls between 55 fL and 125 fL. The RDW-SD is the arithmetic width of the distribution curve measured at the 20% frequency curve. the red cell distribution curves are shifted to the left. to avoid interference in the calculations of RDW. and megathrombocytes on the left side of the histogram. These misleading data include cell coincidence. and interpretative comments that will assist or alert the users to potential incorrect results.

Likewise. by increasing the RDW) is found in 3% to 5% of adult patients. Cautionary Note When Visually Observing the higher the degrees of anisocytosis and/or poikilocytosis. It represents on the left to macrocytic cells approaching 250 fL on the the ratio of 1 SD to the mean. This is counts and the morphology may give additional information. hence the relatively high RDW-SD results.23 macrocytic red cells of <25% with MCV of may appear wider on the histogram but may give a normal 95 fL to 110 fL are morphologically graded as (1+). cytosis when the MCV is in the low normal range and anisocytosis is difficult to detect.2 The secand the MCV should be verified microscopically. However. Although both methods use right (Figure 1E). The first is referred to as RDW-CV (Figure RDW-CV and RDW-SD results for comparative review 3). it is the (2 different forms/sizes of cells in a sample). a significant denominator. this method is not recommended for for coincidental passage of cells. The more spread apart the data. It appears. the white cell and platelet histograms as well as the RDW-SD is relatively higher than the RDW-CV. it seems logical that technologists Automated counters use 2 methods to measure the reading blood films should have ready access to both the value of RDW. In most value of 127. however. that the term better and more accurate measure of anisocytosis. the folate deficiency may sometimes show a single widely distribhigher the SD and vice versa. Thus. In addition. it ± 1 SD. because the RDW-SD is a dimorphic means the occurrence of 2 types of individuals in the same species direct measure across the RBC histogram. Correlation RDW-CV. While it is by the formula restricts its capability to measure variability to convenient to associate dimorphism with a single process.8 fL counterbalances the effect of high normals. In this case. These anomalous pulses and coincident doublets are reflected ing on its average value. macrocytosis. dimorphic is broadly defined in this article to signify 2 or more (multiple) red cell especially in highly abnormal histograms. the presence of these interferences to some degree shows a better correlation as an indicator of anisocannot be avoided. those small and large abnormal cells in varying degrees of anisocytosis and/or poikilocytosis outside the ± 1 SD are included in the measurement. The analyzer automatically monitor the effect of iron therapy and to differentiate corrects results for coincidence. it exclude aberrant pulses. however.CE Update As a result. represent the genuine morphological and/or pathoWhen “dimorphic” is used. but it can affect the RDW-CV result dependAnomalous pulses: This is an interference by electrical pulses/noises but at a very low level. which is independent of MCV. hence the further reduction in the RDW-CV 50 fL-200 fL. the MCV. Note. respectively. must be emphasized that multiple interacting mechanisms are Glossary labmedicine.11 Conversely. the RDW is probably a more sensitive parameter in microcytic than in macrocytic red Macrocytosis with a MCV higher than 100 fL (and a normal cell disorders. As a result. may offset the change in the SD and reduce proportion of whom may not have anemia22 (Figure 1D). the Anisocytosis: Variation in red cell size. are above the 20% frequency variation differently. MCV (the denominator) is not a measure of dispersion. Despite the instrument’s refinement in aperture design. This reflects the ratio of 1 standard deviation (SD) to and better correlation of results. logical status of the patients. Poikilocytosis: Variation in red cell shape. the high MCV as a small tail to the right of a normal curve (Figure 1A and Figure 2A). In severe megaloblasof 172. It is also a good method to at the same time and are counted as a single cell. as it encompasses the entire spectrum of MCV values. trim line. An RDW-CV is another meauted peak of cells from very small cells and red cell fragments sure of the dispersion of data around the mean. Microcytosis will tend to elevate the RDW-CV simply by decreasing the denominaMacrocytosis tor (MCV) of the ratio. and the use of sophisticated edit circuitry to assessing highly abnormal histograms. leading to their falsely reduced blastic anemia with a RDW-CV of 35. It is a direct measurement of the width in fL at the 20% frequency level on the RBC curve Megaloblastic Anemia (Figure 3). As a result. correction value. reflected SD to measure the degree of anisocytosis. ond method of calculating the RDW is called the RDW-SD. While both results are high. as seen in deCoincident doublets: Occasionally. the word On the other hand. Put simply.an abnormal histogram of megaloof the MCV and the RDW. they measure cell as a high take-off on the left. other abnormal cells outside the ± 1 SD are excluded from the estimate hence the comparatively low RDW-CV results. and Interpreting RDW In view of the above.7% and a RDW-SD and greatly elevated values. The RDW-SD is a measure of the dispersion or scatter of Patients with macrocytic anemia due to vitamin B12/ values about the mean. the frequency distribution of red cells does not exceed the range of SD. Note a large number of small cells.8 fL. Dimorphic: According to the Webster New World College Dictionary. Acthe RDW-CV. This is because there are 2 technical meanings for the same word. IDA from thal trait. that the higher the RDWs. the context will determine the meaning. The 1 SD imposed the many faces and mechanisms of dimorphism. a population with a very small MCV between the grading level of macrocytosis (or other conditions) may appear smaller but may give a large RDW-CV. It seems to populations. In other words. changes in either the SD or MCV will influence the results. they are included in the determination Consider Figure 1E . 2 or more cells pass through an aperture veloping iron deficiency. probably because of the way dispersion is measured by the Figure 1F to Figure 1L and Figure 2B to Figure 2G portray RDW-CV formula: 1 SD divided by MCV.com May 2011 ■ Volume 42 Number 5 ■ LABMEDICINE 305 . unlike in RDW-CV. Since it is a ratio. a population with a large MCV cording to Gulati. obviously the tic anemia.

only by careful examination of the histogram. the changes in RBC counts and volumes affect all the CBC parameters.-) replace the data for RDW. using only the major population of cells. from a group of very small cells— reflected as a high take-off on the baseline of the curve. Since the doublet or triplet red cells are counted as 1 cell and large clumps of cells are excluded from the count. Pyropoikilocytosis Hereditary pyropoikilocytosis (HPP) is a congenital hemolytic disorder characterized by moderate to severe hemolytic anemia27. producing a secondary peak representing this new population of cells. to a group of normal and macrocytic cells that are equally spread on the other side.CE Update sometimes involved. a reticulocytosis (polychromasia) of this magnitude may occur. This reinforces the importance of examining the blood cell histograms and blood film in tandem in order to detect the bimodal populations. The dimorphic population message is generated when there are 2 cell size populations or there are multiple peaks in the RBC histogram pattern. The results of the red cell indices. others have suggested it is possible to detect the appearance of a new population of cells (eg. the frequency curves may vary in shapes (Figure 1F). the abnormal histogram is the result of a high titer cold agglutinin causing red cells to agglutinate and interfere with their sizing and enumeration. reticulocytes) even at just more than 5% of the total red cell population. and careful correlation with the peripheral blood morphology can a correct diagnosis be derived. and if one were to rely on indices alone.com .13. despite the abnormal histogram. nucleated RBCs. and large cells in various proportions. In other words.24 Dashes (. Since the SD is disproportionately increased. by skewing the histogram. Despite the bimodal histogram. distinct high points (Figure 1G).16. probably due to the admixture of different cell populations. This asterisk indicates that these results are unreliable and should be reviewed carefully as per laboratory protocol. Figure 1K shows a normal RDW compared to Figure 1L. These 2 histograms (Figure 1K and Figure 1L) seem to be similar in appearance: both have 2 unevenly high peak points with middle trough or valley. such 306 LABMEDICINE ■ Volume 42 Number 5 ■ May 2011 Reticulocytosis The histogram is bimodal and is skewed to the right (Figure 1J). Three to 8 days after effective iron therapy. red cell fragments. in all these examples. compounded by the very low MCV. it is clear the red cell volume histogram accurately reflects the dimorphic appearances. which has a higher RDW. A U-shape-like appearance of the curve would usually suggest the presence of 2 discrete populations. and microelliptocytes observed in this condition may cause a moderate reduction in MCV and a high take-off of the histogram on the left. The high frequency entry on the left of the histogram could be due to the effect of the high titer antibody interfering in cell separation.23 The reticulocyte count of this patient is 5. reticulocytes are slightly larger than normal mature red cells. Sideroblastic Anemia This is an example of a bimodal distribution with 2 separated small. microspherocytes. and an asterisk (*) appears on most red cell associated parameters. depending on the instrument algorithm.28 (Figure 1I). Any spurious cell population that is >1% as numerous as the red cells will influence the MCV. According to some authors. this may be the kind of picture seen in initial post-iron treatment IDA.25 However.2 When viewed microscopically. membrane budding. are within normal range.13 The minor populations and the middle trough are excluded in order to avoid interference in RDW calculation. The blood picture resembles those seen after severe burns. Hereditary pyropoikilocytosis is also considered an aggravated form of hereditary elliptocytosis. hence.4 Thus. Cold Agglutination Because this is a time-. resulting in high SD. As in Figure 1H. It is a membrane defect as suggested by the increased susceptibility to fragmentation lyses at 46°C. the 2 red cell populations must differ by less than 15% in volume. To produce a single distribution.25 It appears that the particle size distribution is very wide. However. knowledge of the possible causes of the abnormal curves.5%. The groups themselves represent different ranges of values so the entire sets of results are actually widespread. and microcytic red cells. The reason for the normal RDW is that only the major populations are included and measured for the MCV and RDW calculations. the microspherocytes. the RDW will be greatly elevated. the RDW remains increased due to increased variability of red cell volumes.16. and histogram results.20 The varying degrees of agglutination in this condition result in a variety of various histogram curves with the varying amounts of trailer of red cells seen on the far right side of the graph. in this case. it is an admixture of small cells. Although the curve appears unimodal. and agglutinin titerdependent reaction. Usually. a reticulocytosis in excess of 15% and/or a volume difference of 2 populations of less than 15% are required to affect histogram analysis. What is surprising in these curves is the difference in the RDW results. The histogram obviously shows shift to the left as it begins near the modal value on the upper left of the graph and falls away on the right side. slightly skewing the curve to the right. Some analyzers with an Interpretive Program of flags and messages may generate an RBC Agglutination flag. Generally. temperature-. 1 on either side. This may be due to the small particles seen in this disorder.24 as red cell fragments. SD is divided by MCV. the dimorphic instructive comment. however. so the MCV and RDW are that of the main population only. On the other hand. nucleated RBCs. Beta Thalassemia Major (and Other Small Particles) In this case.26 Thus. the important finding of mixed populations would be missed. RDW. other instruments still measure and calculate the RDW. producing an erroneous mean cell volume for the intact cell population. a high frequency of small cells (<50 fL) can be seen at the beginning of the histogram (Figure 1H). However. normal cells. Recall the calculation for RDW-CV. a cohort of cells even at 5% may be interpreted as another population. the instrument interpreted it as dimorphic. The labmedicine. except the hemoglobin.

Red cell histograms in the diagnosis of diseases. On the other hand. 1983.3:189-197. Patients with microcytosis due to IDA usually have a unimodal frequency curve but with appropriate therapy. NY: Technicon Instruments.3 with most studies favoring white cell histograms and their use in identifying and characterizing leukemic blast populations. such as scatter plots or histograms. The red cell histogram assists in confirming the diagnosis of IDA and in following its treatment.com May 2011 ■ Volume 42 Number 5 ■ LABMEDICINE 307 . Young M. ed. there have been very few studies on the utility of red cell histograms in identifying common hematological disorders. the pattern of red cell size changes from initial reticulocytosis to gradual normocytosis appear to be the same. electrical impedance or optical flow cytometry. There is a small subpopulation of reticulocytes that merely contribute to the right skew of the graph. From reviewing the histograms. et al. Harle JR. After 17 weeks (120 days) of effective therapy. Five to 6 weeks later. Cooper A. and spread. Bessman JD. Am J Clin Pathol. Some distributions have simple shapes. 4. both show a dual population of hypochromic-microcytic and normochromic-normocytic red cells. 1984:155-164. When combined with the concept of the normal curve and the knowledge of particular CBC parameters. full recognition of dimorphic as more than just 2 cell populations has certain practical and clinical significance. or dimorphic red cells. and the histogram is described by its shape. such as RDW and red cell indices. Figure 2A to Figure 2F are representative histograms of post-iron treatment IDA from 6 patients.4 Seven.30 Consequently. which is below the 20% frequency truncation. however.16 After 2 to 4 weeks therapy. Misleading results can occur because the frequency curve shows only the relative and not the actual number of cells in each size range. and the histogram starts to skew to the left as shown in Figure 2C and Figure 2B. attempting to do so in a skewed. a distinct population of new emerging normocytic cells with some polychromasia is evident on the graph and the blood film.13. Figure 2D shows 2 symmetric peaks representing an almost equal number of hypochromic microcytic and normochromic normocytic cells. as represented by the low peak. hemoglobin distribution width. hypochromic microcytic. 1987. Improved classification of anemias by MCV and RDW. the analysis and interpretations of histograms in this article provide good guidance in correlating histograms with microscopical findings. center.5. labmedicine. Figure 2E shows 2 unequal distinct population peaks of microcytic and normocytic cells. especially when dimorphic (multiple) populations of red cells are present. since dimorphic is usually associated with abnormal red cell populations. Hematology Beyond the Microscope. LM 1.9 At this stage. hence. especially when a dotted line of reference normal is superimposed on all automated abnormal red cell histogram results. morphological findings should be correlated with the graphical and numerical data for better interpretation of results. have been largely ignored in favor of the RDW. Fossat C. and thus may spuriously elevate the RDW. such as symmetric and skewed. et al. This pattern by itself as seen in the red cell distribution curve is meaningless unless it is compared with a reference normal curve and/or confirmed microscopically. the estimation of the number of cells from the distribution curve should be avoided. 1984. Conclusion In assessing a histogram. the relationship of the different sizes of cells can be readily delineated and contrasted. bimodal. Unfortunately. Given that dimorphic and dual populations are synonymous and may cause confusion in the reporting of results. the higher RDW value. and correlate clinical findings to histograms and other hematologic parameters with greater confidence and efficiency. one can get a good idea of what to expect when actually evaluating the peripheral blood film. 2. Arch Pathol Lab Med. Practically.CE Update middle trough. The speed and reliability of the modern analyzers allow technologists time to evaluate abnormal blood films. the MCV starts to increase and the RDW further increases. but others may be more challenging. Cell histograms: New trends in data interpretation and classification. a second population of normocytic cells appears in the majority of cases. In addition. macrocytic. in some laboratories histograms are not being used when evaluating red cell morphology.33 In fact. Gardner FH.32. Value of histograms.34 Hopefully. David M. all of which produce high returns in terms of patient health care.111:1150-1154. normocytic cells become the predominant cells.31 This is probably because graphical representation of results. and up to 15 weeks (105 days) forward. Microscopically. meaning that >15% of the total cell population are normocytic cells and the remainder is microcytic cells. and reticulocyte hemoglobin content that provide very useful information along with the red cell indices that have been traditionally used. and the minor populations. The readout on Figure 2G shows the initial result after 3 days of iron treatment. the histogram provides a concise idea not only of the different sizes of cells but also of the distribution of cells from the center (MCV) and the spread (RDW). Observation of their outlines and the relations they suggest may be so evident that the presence of fragments. New York.13 Even though the MCV can be easily located on any symmetric distribution curve. Gilmer PR Jr. In: Simpson E. as well as different combinations. a population of mostly normochromic normocytic cells and a normal MCV and RDW ensue as seen in Figure 2A. or dimorphic population is discouraged because the MCV is an average value and does not reflect the heterogeneity or the presence of different cell populations. New parameters in erythrocyte counting. can be presumed. most technologists have a limited understanding in correlating the graphic displays with the morphological findings. histograms become a practical working tool in the initial stage of morphological analysis. it appears the histograms and/ or MCV in different stages of post-iron treatment may vary. though a secondary peak is just starting to shape up as seen in Figure 2F. whatever instrument methods are used to obtain the red cell histogram. Clinically. The inclusion of minor populations will also require the inclusion of the middle trough. are excluded from the MCV and RDW calculations. consider diagnostic clues. J Med Technol.29. Williams LJ.80:322-326. 3. Barring any complications or coexistence of vitamin B12/folate deficiency. it is associated with many conditions as shown in Table 1. as these 2 CBC parameters are determined from the red cell histogram. Lawrence A. because the valley in Figure 1L has surpassed the detection level. the overall pattern is observed. Moreover. Although direct inspection of the distribution curve offers a sensitive method for detecting small populations of microcytic or macrocytic red cells. the minor and the major populations are included in the MCV and RDW calculations.

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