The Red Cell Histogram and the Dimorphic Red Cell Population

CE Update

Submitted 4.30.10 | Revision Received 9.27.10 | Accepted 9.27.10 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

Abstract conditions and may provide major clues in some morphological features of dimorphism The RBC histogram is an integral part of the diagnosis and management of significant and the ensuing characteristic changes in their automated hematology analysis and is now red cell disorders. In addition, it is frequently RBC histograms. routinely available on all automated cell used, along with the peripheral blood film, as counters. This histogram and other associated an aid in monitoring and interpreting abnormal Keywords: histogram, dimorphic red cells, red complete blood count (CBC) parameters have morphological changes, particularly dimorphic blood cell distribution width been found abnormal in various hematological red cell populations. This article discusses

After reading this article, readers should be able to correlate a RBC Hematology exam 51102 questions and corresponding answer form histogram and red blood cell distribution width with microscopical are located after this CE Update on page 309. findings.

Table 1_Conditions Associated With Dimorphic To paraphrase an adage, 1 histogram graph is worth 5-12 1000 numbers. A large collection of data, displayed as a visual Red Cells image, can convey information with far more impact than the numbers alone. In hematology, these data take on several Early iron developing microcytic population forms, 1 of which is the RBC histogram. Visual scanning Folate/vitamin B12 developing macrocytic population Post-iron treatment of iron deficiency anemia of the histogram gives a good initial sense of the range, size, Post-iron treatment of iron deficiency with megaloblastic anemia shape, and other salient features of the red cell morphology. Post-iron treatment of megaloblastic anemia The RBC histogram, a graphic representation of particle Post-iron treatment of megaloblastic anemia with iron deficiency size distribution, is now routinely available on automated cell Post-iron transfusion macrocytic anemia analyzers as a standard part of automated complete blood Post-iron transfusion microcytic anemia count (CBC) analysis. This histogram in association with Iron deficiency anemia with either folate or vitamin B12 deficiency other CBC parameters, such as RBC distribution width Sideroblastic anemia (myelodysplasia) (RDW) and mean corpuscular volume (MCV), has been Hemolytic anemia (reticulocytosis, spherocytosis, fragmentation, pyropoikilocytosis) found abnormal in various hematological conditions and may Cold/warm auto agglutination provide major clues in the diagnosis and management of sig- Erythropoietin-induced erythropoiesis nificant red cell disorders.1-4 In addition, it is frequently used, Delayed transfusion reaction along with the peripheral blood film, as an aid in monitoring Homozygous hemoglobinopathies (admixture of many RBC forms) and interpreting abnormal morphological changes, particu- Myelofibrosis (admixture of extramedullary hematopoiesis) larly dimorphic red cells. Constitutional chromosomal translocation t(11;22)(p15.5;q11.21) Table 1 lists conditions associated with dimorphic red cell changes. Before reporting CBC results, a clear distinc- tion between dimorphic and dual populations must be clari- fied as they are sometimes interchangeable and confusing. 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). Corresponding Author These 2 distinct populations may be comprised of either a Benie T. Constantino, SH, I; ART, MLT(CSMLS) patient’s own red cells (post-iron treatment) or a mixture of [email protected] 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 Abbreviations to either the dual or the multiple red cell populations. Thus, CBC, complete blood count; RDW, red blood cell distribution width; the dimorphic blood picture will look like a dual popula- Image 1A MCV, mean corpuscular volume; MCHC, mean corpuscular hemo- tion of microcytic and normocytic ( ) or normocytic globin concentration; IDA, iron deficiency anemia; RDW-CV, red and macrocytic red cells, or an admixture of small, normal, Image 1B blood cell distribution width coefficient of variation; thal, thalas- and large cells of different sizes and forms ( ). Some semia; SD, standard deviation; HPP, hereditary pyropoikilocytosis morphological features of dimorphism and the ensuing characteristic changes in their RBC histograms are discussed in this article.

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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). (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), microspherocytes (regular arrow), membrane budding (long arrow), elliptocytes, oval macrocytes, and polychromasia.

RBC Histogram: An Overview data.5,15 For example, in megaloblastic anemia with developing The well-known Coulter principle of counting and sizing iron deficiency, some of the hypochromic and microcytic red red cells provides the basis for generating the histogram. This cells can be identified in the histogram but may not be reflected method relies on the change in conductance as each cell passes in the numerical data, and this may well be the first clue to a through an aperture. The change in conductance results in not uncommon occurrence. Likewise, iron deficiency may de- an electrical pulse, the amplitude of which is proportional to velop insidiously during the process of some other disease (eg, the cell volume. The 256-channel pulse-height analyzer uses a essential polycythemia), with normal cell counts and indices, number of thresholds to sort the cells into several size (volume) except the RDW, which is elevated. The RBC histogram is also channels from which the histogram is formed. Each channel abnormal, showing a shoulder of microcytic red cells.16 on the X-axis represents a specific size (volume) in femtoliter Furthermore, a histogram can provide useful information (24-360 fL), increasing from left to right. The Y-axis represents for laboratorians in 1) monitoring the reliability of the re- the number of cells per channel, with each cell being stored in sults generated by the analyzers; 2) investigating the potential the channel representing its size, so that after data accumula- cause(s) of the erroneous automated results; and 3) arriving at tion is completed the relative number of cells (frequency) is the presumptive diagnosis.7 For example, certain conditions provided. This data is further processed by the computer, and like the presence of fragmented red cells or red cell agglutina- the RBC curve is smoothed by a moving average technique and tion that could not have been identified earlier without blood displayed on a data management system.13 Figure 1A shows a film examination can now be presumably detected on the red typical normal RBC histogram. For a detailed explanation on cell histogram.3,16 Likewise, in patients with iron deficiency the construction of histogram, please see reference 14. anemia (IDA) or megaloblastic anemia in treatment, a sequen- tial histogram can clearly show the progressive appearance of a new erythrocyte population well in advance of other numerical parameters.3 Value of Histogram The presence of a right-sided shoulder usually corresponds A histogram, as shown in Figure 1 and Figure 2, is a to reticulocytosis (Figure 1J), and a trailer of erythrocyte popu- graphic representation of a collection of data based on cell lations on the far right of the histogram correlates to red cell size and/or cell number depicting variations in the process. It agglutination (Figure 1F). A leftward shift of the RBC histo- is sometimes referred to as a frequency of distribution curve. gram signifies microcytosis (Figure 1B and Figure 1C), and a Because graphics can show data in ways that are meaningful rightward shift suggests macrocytosis (Figure 1D and Figure and quickly understood, the histogram is a very powerful tool 1E). Bimodal red cell histograms (Figure 1F, Figure 1G, Figure in red cell morphological analysis. It enables one to visualize, 1K, Figure 1L and Figure 2B to Figure 2E) are usually associated analyze, and interpret empirical data that displays morpholog- with therapeutic transfusion and/or hematinic agent response to ical changes graphically as points, peaks or valleys, or as a line microcytic and macrocytic anemia, but they may also indicate of frequency curve. It allows the users to intuitively see the other hematological disorders as shown in Table 1. visual comparison of the center and spread of data and data Although the size ranges for RBC histograms are between containing 2 or more variables: dimorphic red cells, subpopu- 24 fL and 360 fL, the instrument counts only those cells with lations, and skewed data, leading to quick and cost-effective volume sizes between 36 fL and 360 fL as red cells. Those decision making. cells counted in the 24 fL to 36 fL range are rejected and At times, a histogram can provide invaluable information not included in the RBC count. They are enumerated and that may not even be apparent in the automated numerical displayed in the histogram area between the 24 fL and 36 fL

labmedicine.com May 2011 ■ Volume 42 Number 5 ■ LABMEDICINE 301 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. (Key hematological features of these conditions are summarized in Table 2.) Figure (A) Normal histogram, (B) Microcytosis, iron deficiency anemia, (C) Microcytosis, beta thal trait, (D) Macrocytosis with normal RDW, (E) Macrocytosis, megaloblastic anemia, (F) Cold agglutination, (G) Sideroblastic anemia, (H) Beta thalassemia major, (I) Pyropoikilocytosis, (J) Reticulocytosis, (K) Post-iron therapy, (L) Post-iron therapy.

Table 2_Key Hematological Features of Various Hematological Conditions in Figure 1

Hematological Parameters Condition HGB (g/L) MCV (fL) MCHC (g/L) RDW-CV (%) RBC Histogram 1A Normal 146 89.6 330 14.4 Normal 1B Iron deficiency anemia 86 54.7 314 21.6 LS 1C Beta thalassemia 131 66.0 318 14.5 LS 1D Macrocytosis 131 102.8 351 14.3 RS 1E Megaloblastic anemia 65 127.8 324 35.7 DM RS SKL 1F Cold agglutinin 124 110.6 +++++ 17.5 DM BM 1G Sideroblastic anemia 92 90.9 320 39.2 DM BM LS SKR 1H Beta thal major 86 79.6 285 32.1 DM LS 1I Pyropoikilocytosis 99 60.2 358 41.5 DM LS SKR 1J Reticulocytosis 87 72.2 298 31.3 DP LS SKR 1K Post-iron therapy 132 81.1 317 14.8 DP BM 1L Post-iron therapy 112 85.8 330 35.0 DP BM

Reference range 120-160 80-95 320-360 11.5-14.5

HGB, hemoglobin; MCV, mean corpuscular hemoglobin; RDW-CV, red cell distribution wiidth-coefficient of variation; DM, dimorphic; BM, bimodal; DP, dual population; LS, left-shift; RS, right-shift; SKR, skewed to the right; SKL, skewed to the left; MCHC (+++++) overrange.

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RBC Histograms A B

C D

E F

Figure 2_Histograms of iron deficiency anemia after treatment (key hematological features are summarized in Table 3). Figures B to G are representative examples of dimorphic (dual) red cell population response to hematinic treatment of iron deficiency anemia in 6 patients.

Table 3_Key Hematological Features in Iron Deficiency Anemia After Treatment

Hematological Parameters

Condition HGB(PT) HGB(POT) MCV(PT) MCV(POT) RDW(PT) RDW(POT) SF(PT) RBC Histogram

2A Normal 144 84.7 13.9 Normal 2B IDA 108 132 70.3 82.8 22.1 25.5 8 DP SKL 2C IDA 80 107 69.2 81.1 18.0 29.2 7 DP BM 2D IDA 86 118 61.3 77.4 23.0 36.0 <5 DP BM 2E IDA 98 119 70.4 78.9 17.9 28.2 8 DP BM LS 2F IDA 95 106 65.6 70.2 19.5 28.8 <5 DP LS SKR 2G IDA 81 91 62.9 68.6 19.5 25.9 <5 DM LS SKR

PT, pre-treatment; POT, post-iron treatment; IDA, iron deficiency anemia; DP, dual population; BM, bimodal; SKL, skewed to the left; SKR, skewed to the right; SL, left shift; SF, serum ferritin. SF normal range (female)=13-145 UG/L; SF (male)=27-220 UG/L.

labmedicine.com May 2011 ■ Volume 42 Number 5 ■ LABMEDICINE 303 CE Update range, however, allowing the lower end of the histogram to The following are important points to consider when be monitored. Normally, the space below 36 fL remains clear, reviewing/analyzing histograms.13 but in certain conditions the histogram may begin above the • position of individual populations compared to normal/ baseline or show a high takeoff on the far left of the curve typical positions (Figure 1G to Figure 1I), which generally indicates the presence • amount of separation between populations compared to of small particles. These particles include red cell fragments, normal/typical separation microspherocytes, nucleated RBCs, nonlyzed RBCs, elliptocy- • relative concentration of each population compared to tosis, macrothrombocytes, platelet clumps, bacteria, parasitic normal/typical concentrations organisms, and other interfering substances such as cryoglobu- • presence of unexpected or non-typical populations lin, cold agglutinin, and macroglobinemia.7,16-18 Figure 1A to Figure 1D are typical examples of normal, microcytic, and macrocytic red cell histograms. They are symmetric, single-peaked, and “bell shaped” normal curves. Analysis and Interpretations of Histograms The curve is considered symmetric if the 2 sides of the curve in Figure 1 and Figure 2 coincide when folded in half or are approximately mirror im- The red cell histograms presented for illustration were ages. When the distribution is not symmetric, it is referred to randomly selected to represent various common hematological as skewed wherein 1 of the 2 sides (left or right) of the curve conditions and the different stages of post-iron IDA therapy. extends much further than the other. In a normal RBC histo- A histogram may be interpreted by looking at the shape of the gram, the majority of each cell falls between 55 fL and 125 fL. data distribution. The distributional shape of the histogram The tail of the distribution consists of coincident doublets can be classified as reflecting either single or dimorphic (mul- and anomalous pulses.16 tiple) red cell populations. In a single population, the histo- In IDA and beta thalassemia (thal) trait, the red cell gram may have a normal Gaussian or bell-shape curve, may distribution curves are shifted to the left, and the percentage be widened with or without left or right shift, may be skewed of microcytosis is increased. Although their histograms are to the left or right, or may show some combination of all of similar, the degree of anisocytosis, as measured by the RDW, these characteristics. In dimorphic populations, the histogram differentiates them. Iron deficiency anemia is characterized by may be symmetric and/or bimodal, or bimodal but skewed to elevated RDW, reflecting the heterogeneity in the acquired the left or right. The centeredness and width of the histogram erythrocyte populations. In thal trait however, the RDW is also define the extent of RBC variability. usually within range due to the homogeneity of the inherited Potentially, a number of factors may affect the histogram population of erythrocytes. Although this difference is useful of the aperture impedance cell distribution analysis. These to some extent in distinguishing them, cases involving severe include the coincident doublet of red cells, red cell agglutina- anemia with homogenous microcytosis may give misleading tion, inclusion of reticulocytes with mature red cells, altera- results.3 Nonetheless, patients showing severe anemia with tion in red cell shape, and inclusion of leukocytes in certain hemoglobin <90 g/L and with hypochromic-microcytic red diseases. These factors, in 1 way or another, influence the cells are most likely (80%) iron deficient.21 histogram’s appearances and accordingly will have a variable effect on any measurements made from the histogram.16 To reduce the effect of these problems, manufacturers design their instruments and reagent systems to specifically prevent and correct for interferences.13 They develop mathematical algorithms for particle counting and produce numeric data, graphic data, scatter plots, and interpretative comments that will assist or alert the users to potential incorrect results.19,20 In addition, to avoid interference in the calculations of RDW, the information below 20% of scale on the red cell histogram are excluded (Figure 3). These misleading data include cell coincidence, aperture artifacts, doublets and triplets, and agglutinates on the right side and platelet clumps, and mega- thrombocytes on the left side of the histogram.13,16 Before taking up complex histogram graphs, 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. Familiarity with typical RBC histograms will make the task of evaluating other notable deviations easier. In view of this, to maximize the usefulness of the histogram, a dotted line depicting a reference normal curve (eg, symmetric normal curve with MCV of 96 fL and RBC distribution width coefficient of variation [RDW-CV] of 15.0%) should be automatically superimposed on every red cell histogram so any Figure 3_Calculation of RDW. The RDW is calculated from the width discernible deviation from that curve can be clearly delineated of the histogram at 1 SD from the mean divided by MCV. The normal for contrast. Simply put, if the patient’s histogram strays from RDW-CV is 11.5% to 14.5%. The RDW-SD is the arithmetic width of the reference normal range, it means that the histogram may the distribution curve measured at the 20% frequency curve. The be abnormal. normal RDW-SD is 39 to 47 fL.14

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As a result, other abnormal cells outside the Glossary ± 1 SD are excluded from the estimate hence the Anisocytosis: Variation in red cell size. comparatively low RDW-CV results. In addition, the MCV (the denominator) is not a measure of disper- Anomalous pulses: This is an interference by electrical pulses/noises but at sion, but it can affect the RDW-CV result depend- a very low level. These anomalous pulses and coincident doublets are reflected ing on its average value. In this case, the high MCV as a small tail to the right of a normal curve (Figure 1A and Figure 2A). In most value of 127.8 fL counterbalances the effect of high normals, the frequency distribution of red cells does not exceed the range of SD, hence the further reduction in the RDW-CV 50 fL-200 fL. Despite the instrument’s refinement in aperture design, correction value. Thus, this method is not recommended for for coincidental passage of cells, and the use of sophisticated edit circuitry to exclude aberrant pulses, the presence of these interferences to some degree assessing highly abnormal histograms. However, it cannot be avoided. shows a better correlation as an indicator of anisocytosis when the MCV is in the low normal range Coincident doublets: Occasionally, 2 or more cells pass through an aperture and anisocytosis is difficult to detect, as seen in de- at the same time and are counted as a single cell. The analyzer automatically veloping iron deficiency. It is also a good method to corrects results for coincidence. monitor the effect of iron therapy and to differentiate IDA from thal trait. Dimorphic: According to the Webster New World College Dictionary, the word On the other hand, 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, it is the (2 different forms/sizes of cells in a sample). Note, however, that the term better and more accurate measure of anisocytosis, dimorphic is broadly defined in this article to signify 2 or more (multiple) red cell populations. This is because there are 2 technical meanings for the same word. especially in highly abnormal histograms. It seems to When “dimorphic” is used, the context will determine the meaning. represent the genuine morphological and/or patho- logical status of the patients, as it encompasses the Poikilocytosis: Variation in red cell shape. entire spectrum of MCV values. In other words, unlike in RDW-CV, those small and large abnormal cells in varying degrees of anisocytosis and/or poikilocytosis outside the ± 1 SD are included in the measurement, hence the relatively high RDW-SD re- Cautionary Note When Visually Observing sults. It appears, however, that the higher the RDWs, and Interpreting RDW the higher the degrees of anisocytosis and/or poikilocytosis. In view of the above, 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. The first is referred to as RDW-CV (Figure RDW-CV and RDW-SD results for comparative review 3). This reflects the ratio of 1 standard deviation (SD) to and better correlation of results. the MCV. Since it is a ratio, changes in either the SD or MCV will influence the results. Microcytosis will tend to elevate the RDW-CV simply by decreasing the denominator (MCV) of the ratio. As a result, the RDW is probably a Macrocytosis more sensitive parameter in microcytic than in macrocytic red Macrocytosis with a MCV higher than 100 fL (and a normal cell disorders.11 Conversely, macrocytosis, by increasing the RDW) is found in 3% to 5% of adult patients, a significant denominator, may offset the change in the SD and reduce proportion of whom may not have anemia22 (Figure 1D). Ac- the RDW-CV. Put simply, a population with a large MCV cording to Gulati,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+). Correlation RDW-CV. Likewise, 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.2 The sec- and the MCV should be verified microscopically. ond method of calculating the RDW is called the RDW-SD, which is independent of MCV. It is a direct measurement of the width in fL at the 20% frequency level on the RBC curve (Figure 3). Megaloblastic Anemia 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 more spread apart the data, the folate deficiency may sometimes show a single widely distrib- higher the SD and vice versa. An RDW-CV is another mea- uted peak of cells from very small cells and red cell fragments sure of the dispersion of data around the mean. It represents on the left to macrocytic cells approaching 250 fL on the the ratio of 1 SD to the mean. Although both methods use right (Figure 1E). Note a large number of small cells, reflected SD to measure the degree of anisocytosis, they measure cell as a high take-off on the left, are above the 20% frequency variation differently. trim line. As a result, they are included in the determination Consider Figure 1E - an abnormal histogram of megalo- of the MCV and the RDW, leading to their falsely reduced blastic anemia with a RDW-CV of 35.7% and a RDW-SD and greatly elevated values, respectively. In severe megaloblas- of 172.8 fL. While both results are high, obviously the tic anemia, the white cell and platelet histograms as well as the RDW-SD is relatively higher than the RDW-CV. This is counts and the morphology may give additional information. 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. The 1 SD imposed the many faces and mechanisms of dimorphism. While it is by the formula restricts its capability to measure variability to convenient to associate dimorphism with a single process, it ± 1 SD. must be emphasized that multiple interacting mechanisms are

labmedicine.com May 2011 ■ Volume 42 Number 5 ■ LABMEDICINE 305 CE Update sometimes involved. The dimorphic population message is as red cell fragments, nucleated RBCs, microspherocytes, and generated when there are 2 cell size populations or there microcytic red cells, producing an erroneous mean cell vol- are multiple peaks in the RBC histogram pattern.13,24 ume for the intact cell population. Although the curve appears unimodal, the instrument interpreted it as dimorphic, probably due to the admixture of different cell populations. Any spurious cell population that is >1% as numerous as the red Cold Agglutination cells will influence the MCV, RDW, and histogram results.26 Because this is a time-, temperature-, and agglutinin titer- Thus, in all these examples, only by careful examination of dependent reaction, the frequency curves may vary in shapes the histogram, knowledge of the possible causes of the abnor- (Figure 1F). A U-shape-like appearance of the curve would mal curves, and careful correlation with the peripheral blood usually suggest the presence of 2 discrete populations, 1 on morphology can a correct diagnosis be derived. either side. However, in this case, the abnormal histogram is the result of a high titer cold agglutinin causing red cells to agglutinate and interfere with their sizing and enumeration. Some analyzers with an Interpretive Program of flags and Pyropoikilocytosis messages may generate an RBC Agglutination flag.24 Dashes Hereditary pyropoikilocytosis (HPP) is a congenital (- -) replace the data for RDW, and an asterisk (*) appears hemolytic disorder characterized by moderate to severe hemo- on most red cell associated parameters. This asterisk indicates lytic anemia27,28 (Figure 1I). Hereditary pyropoikilocytosis is that these results are unreliable and should be reviewed care- also considered an aggravated form of hereditary elliptocy- fully as per laboratory protocol. tosis. It is a membrane defect as suggested by the increased On the other hand, despite the abnormal histogram, other susceptibility to fragmentation lyses at 46°C. The blood instruments still measure and calculate the RDW, using only picture resembles those seen after severe burns. As in Figure the major population of cells.13 The minor populations and the 1H, the microspherocytes, nucleated RBCs, red cell frag- middle trough are excluded in order to avoid interference in ments, membrane budding, and microelliptocytes observed RDW calculation. The high frequency entry on the left of the in this condition may cause a moderate reduction in MCV histogram could be due to the effect of the high titer antibody and a high take-off of the histogram on the left. However, the interfering in cell separation. Since the doublet or triplet red RDW remains increased due to increased variability of red cells are counted as 1 cell and large clumps of cells are excluded cell volumes, resulting in high SD. Recall the calculation for from the count, the changes in RBC counts and volumes affect RDW-CV; SD is divided by MCV. Since the SD is dispro- all the CBC parameters, except the hemoglobin.20 The varying portionately increased, compounded by the very low MCV, degrees of agglutination in this condition result in a variety of the RDW will be greatly elevated. The histogram obviously various histogram curves with the varying amounts of trailer of shows shift to the left as it begins near the modal value on red cells seen on the far right side of the graph. the upper left of the graph and falls away on the right side, slightly skewing the curve to the right.

Sideroblastic Anemia This is an example of a bimodal distribution with 2 sepa- Reticulocytosis rated small, distinct high points (Figure 1G). To produce a The histogram is bimodal and is skewed to the right (Fig- single distribution, the 2 red cell populations must differ by ure 1J). Usually, this may be the kind of picture seen in initial less than 15% in volume.16,25 It appears that the particle size post-iron treatment IDA. Three to 8 days after effective iron distribution is very wide, from a group of very small cells— therapy, a reticulocytosis (polychromasia) of this magnitude reflected as a high take-off on the baseline of the curve, to a may occur, producing a secondary peak representing this new group of normal and macrocytic cells that are equally spread population of cells. Generally, reticulocytes are slightly larger on the other side. The groups themselves represent different than normal mature red cells.23 The reticulocyte count of this ranges of values so the entire sets of results are actually wide- patient is 5.5%. According to some authors, a reticulocytosis spread. In other words, it is an admixture of small cells, nor- in excess of 15% and/or a volume difference of 2 populations mal cells, and large cells in various proportions. The results of less than 15% are required to affect histogram analysis.16,25 of the red cell indices, however, are within normal range, and However, others have suggested it is possible to detect the ap- if one were to rely on indices alone, the important finding pearance of a new population of cells (eg, reticulocytes) even at of mixed populations would be missed. This reinforces the just more than 5% of the total red cell population, by skewing importance of examining the blood cell histograms and blood the histogram.4 Thus, depending on the instrument algorithm, film in tandem in order to detect the bimodal populations.2 a cohort of cells even at 5% may be interpreted as another When viewed microscopically, it is clear the red cell volume population, hence, the dimorphic instructive comment. histogram accurately reflects the dimorphic appearances. 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. What is surprising in these curves is the difference in the RDW results. Despite the bi- Beta Thalassemia Major modal histogram, Figure 1K shows a normal RDW compared (and Other Small Particles) to Figure 1L, which has a higher RDW. The reason for the In this case, a high frequency of small cells (<50 fL) can normal RDW is that only the major populations are included be seen at the beginning of the histogram (Figure 1H). This and measured for the MCV and RDW calculations, so the may be due to the small particles seen in this disorder, such MCV and RDW are that of the main population only. The

306 LABMEDICINE ■ Volume 42 Number 5 ■ May 2011 labmedicine.com CE Update middle trough, which is below the 20% frequency truncation, Although direct inspection of the distribution curve and the minor populations, as represented by the low peak, offers a sensitive method for detecting small populations of are excluded from the MCV and RDW calculations. The microcytic or macrocytic red cells, the estimation of the num- inclusion of minor populations will also require the inclu- ber of cells from the distribution curve should be avoided. sion of the middle trough, and thus may spuriously elevate Misleading results can occur because the frequency curve the RDW. On the other hand, because the valley in Figure shows only the relative and not the actual number of cells in 1L has surpassed the detection level, the minor and the major each size range.13 Even though the MCV can be easily located populations are included in the MCV and RDW calculations; on any symmetric distribution curve, attempting to do so in hence, the higher RDW value. Microscopically, both show a a skewed, bimodal, or dimorphic population is discouraged dual population of hypochromic-microcytic and normochro- because the MCV is an average value and does not reflect the mic-normocytic red cells. heterogeneity or the presence of different cell populations. Figure 2A to Figure 2F are representative histograms of When combined with the concept of the normal curve and post-iron treatment IDA from 6 patients. The red cell histo- the knowledge of particular CBC parameters, such as RDW gram assists in confirming the diagnosis of IDA and in fol- and red cell indices, histograms become a practical working lowing its treatment. Patients with microcytosis due to IDA tool in the initial stage of morphological analysis. Observation usually have a unimodal frequency curve but with appropriate of their outlines and the relations they suggest may be so evi- therapy, a second population of normocytic cells appears in dent that the presence of fragments, hypochromic microcytic, the majority of cases. The readout on Figure 2G shows the macrocytic, or dimorphic red cells, as well as different combina- initial result after 3 days of iron treatment. There is a small tions, can be presumed. In addition, the histogram provides a subpopulation of reticulocytes that merely contribute to the concise idea not only of the different sizes of cells but also of right skew of the graph.9 At this stage, the MCV starts to the distribution of cells from the center (MCV) and the spread increase and the RDW further increases.16 (RDW), as these 2 CBC parameters are determined from the After 2 to 4 weeks therapy, a distinct population of new red cell histogram.13,29,30 Consequently, whatever instrument emerging normocytic cells with some polychromasia is evi- methods are used to obtain the red cell histogram, electrical dent on the graph and the blood film, though a secondary impedance or optical flow cytometry, the relationship of the peak is just starting to shape up as seen in Figure 2F. Figure different sizes of cells can be readily delineated and contrasted, 2E shows 2 unequal distinct population peaks of microcytic especially when a dotted line of reference normal is superim- and normocytic cells, meaning that >15% of the total cell posed on all automated abnormal red cell histogram results. population are normocytic cells and the remainder is micro- From reviewing the histograms, one can get a good idea cytic cells. Five to 6 weeks later, Figure 2D shows 2 symmetric of what to expect when actually evaluating the peripheral blood peaks representing an almost equal number of hypochromic film. Unfortunately, most technologists have a limited under- microcytic and normochromic normocytic cells.4 Seven, and standing in correlating the graphic displays with the morpholog- up to 15 weeks (105 days) forward, normocytic cells become ical findings.31 This is probably because graphical representation the predominant cells, and the histogram starts to skew to the of results, such as scatter plots or histograms, have been largely left as shown in Figure 2C and Figure 2B. After 17 weeks (120 ignored in favor of the RDW, hemoglobin distribution width, days) of effective therapy, a population of mostly normochro- and reticulocyte hemoglobin content that provide very useful mic normocytic cells and a normal MCV and RDW ensue as information along with the red cell indices that have been tradi- seen in Figure 2A. Barring any complications or coexistence of tionally used.5,32,33 In fact, in some laboratories histograms are vitamin B12/folate deficiency, it appears the histograms and/ not being used when evaluating red cell morphology. or MCV in different stages of post-iron treatment may vary; Moreover, there have been very few studies on the utility however, the pattern of red cell size changes from initial re- of red cell histograms in identifying common hematological ticulocytosis to gradual normocytosis appear to be the same. disorders,3 with most studies favoring white cell histograms and their use in identifying and characterizing leukemic blast populations.34 Hopefully, the analysis and interpretations of histograms in this article provide good guidance in correlating Conclusion histograms with microscopical findings. In assessing a histogram, the overall pattern is observed, The speed and reliability of the modern analyzers allow and the histogram is described by its shape, center, and technologists time to evaluate abnormal blood films, consider spread. This pattern by itself as seen in the red cell distribu- diagnostic clues, and correlate clinical findings to histograms tion curve is meaningless unless it is compared with a refer- and other hematologic parameters with greater confidence ence normal curve and/or confirmed microscopically. Some and efficiency, all of which produce high returns in terms of distributions have simple shapes, such as symmetric and patient health care. LM skewed, but others may be more challenging, especially when dimorphic (multiple) populations of red cells are present. Given that dimorphic and dual populations are synony- 1. Bessman JD, Gilmer PR Jr, Gardner FH. Improved classification of anemias by mous and may cause confusion in the reporting of results, full MCV and RDW. Am J Clin Pathol. 1983;80:322-326. recognition of dimorphic as more than just 2 cell populations 2. Williams LJ. Cell histograms: New trends in data interpretation and has certain practical and clinical significance. Clinically, it is classification. J Med Technol. 1984;3:189-197. associated with many conditions as shown in Table 1. Practi- 3. Fossat C, David M, Harle JR, et al. New parameters in erythrocyte counting. cally, since dimorphic is usually associated with abnormal red Value of histograms. Arch Pathol Lab Med. 1987;111:1150-1154. cell populations, morphological findings should be correlated 4. Lawrence A, Young M, Cooper A, et al. Red cell histograms in the diagnosis of with the graphical and numerical data for better interpretation diseases. In: Simpson E, ed. Hematology Beyond the Microscope. New York, NY: of results. Technicon Instruments; 1984:155-164.

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5. Kakkar N, Makkar M. Red cell cytograms generated by an ADVIA 120 20. Constantino BT, Cogionis B. High mean corpuscular hemoglobin automated hematology analyzer: Characteristic patterns in common concentration: Its causes and effects on automated CBC results. Can J Med Lab hematological conditions. LabMed. 2009;40:549-555. Sci. 2007;69:113-126. 6. Novak R. Q & A What is the significance of two distinct RBC populations aside 21. Constantino BT. The evaluation and differentiation of hypochromic microcytic from the obvious transfusion? In: Savage RA, ed. CAP Today. September 2005. red blood cells of thalassemia trait and iron deficiency anemia. Can J Med Lab 7. Gulati GL, Hyun BH. The automated CBC. A current perspective. Hematol Sci. 1999;61:112-121. Oncol Clin North Am. 1994;8:593-603. 22. Brigden ML. A systematic approach to macrocytosis: Sorting out the causes. 8. Rees MI, Worwood M, Thompson PW, et al. Red cell dimorphism in a young Postgrad Med. 1995;97:171-184. man with a constitutional chromosomal translocation t(11;22)(p15.5;q11.21). 23. Gulati G. Blood Cell Morphology Grading Guide. Chicago, IL: ASCP Press; Br J Haematol. 1994;87:386-395. 2009:10-11. 9. Bessman D. Erythropoiesis during recovery from iron deficiency: Normocytes 24. Troubleshooting Guide Sysmex XE-Series Automated Hematology Systems and macrocytes. Blood. 1977;50:987-993. Document number: MKT-40-1010. Sysmex America Inc.; 2004. 10. Bessman JD. Erythropoiesis during recovery from macrocytic anemia: 25. Bessman JD. Heterogeneity of red cell volume: Quantitation, clinical macrocytes, normocytes, and microcytes. Blood. 1977;50:995-1000. correlations, and possible mechanisms. Johns Hopkins Med J. 1980;146:226-230. 11. Fishleder AJ, Hoffman GC. Automated hematology: Counts and indices. Lab 26. Park KI, Kim KY. Clinical evaluation of red cell volume distribution width Management. 1984;22:21-36. (RDW). Yonsei Med J. 1987;28:282-290. 12. Bessman JD, Banks D. Spurious macrocytosis, a common clue to erythrocyte cold 27. Ramos MC, Schafernak KT, Peterson LC. Hereditary pyropoikilocytosis: A rare agglutinins. Am J Clin Pathol. 1980;74:797-800. but potentially severe form of congenital hemolytic anemia. J Pediatr Hematol 13. Beckman Coulter LH 780 on line IB072841. Beckman Coulter Education Oncol. 2007;29:128-129. Center, Miami Lakes, FL; 2007. 28. Cochran DL, Burnside LK. Detecting and identifying hereditary 14. Munro BH. Statistical Methods for Health Care Research. 5th ed. New York, NY: pyropoikilocytosis. LabMed. 1999;30:26-29. Lippincott Williams and Wilkins; 2005, 29. ADVIA 120 Hematology System “Technology and cytogram interpretation.” 15. Bate I, Bains BJ. Basic hematological techniques. In: Bates I, Bain BJ, Lewis Bayer Diagnostics, Tarrytown, NY; 2005. SM, eds. Dacie and Lewis Practical Hematology. 9th ed. London: Churchill 30. CELL DYN Sapphire TM System Operator Manual. Abbott Park, IL: Abbott Livingstone; 2001:19-46. Laboratories; 2005. 16. Rowan RM. Blood Cell Volume Analysis—A New Screening Technology for the 31. Sullivan E. Hematology analyzer: From workhorse to thoroughbred. LabMed. Hematologist. London: Albert Clark and Company; 1983:43-55. 2006;37:273-278. 17. Chaplin H, Glazer H, Hockett R, et al. Clinicopathologic conference: 32. Radtke H, Meyer T, Kalus U, et al. Rapid identification of iron deficiency Abdominal pain, total intravascular hemolysis, and death in a 53-year-old in blood donors with red cell indexes provided by Advia 120. Transfusion. woman. Am J Med. 1990;88:667-674. 2005;45:5-10. 18. Bessman JD. Red blood cell fragmentation. Improved detection and 33. Marković M, Majkić-Singh N, Subota V, et al. Reticulocyte hemoglobin content identification of causes. Am J Clin Pathol. 1988;90:268-273. in the diagnosis of iron deficiency anemia. Clin Lab. 2004;50:431-436. 19. Steele BW, Wu NC, Whitcomb C. White blood cell and platelet counting 34. Krause JR, Costello RT, Krause J, et al. Use of the Technicon H-1 in the performance by hematology analyzers: A critical evaluation. Lab Hematol. characterization of leukemias. Arch Pathol Lab Med. 1988;112:889-894. 2001;7:255-266.

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