Reticulocyte Counting in Thalasemia

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ABSTRACT

The reticulocyte count represents an important test in thestudy of marrow erythropoietic activity. Automated techniqueshave led to a significant improvement in the accuracy and preci-sion of reticulocyte enumeration compared with manual counting.This study compared automated reticulocyte counting by 2 tech-nologies (Coulter Gen S and Sysmex R-2000) in samples from45 healthy subjects and 66 thalassemic patients (21 hemoglobin[Hb] H, 8 Hb H/Constant Spring [CS], 20 -thalassemia[-thal]/Hb E nonsplenectomy, and 17 -thal/Hb E splenecto-my). There was no statistically significant difference between the2 methods, although the Sysmex R-2000 showed a slightly higher

mean reticulocyte count (RC). The overall correlation between the2 instruments was r= 0.877. RCs in all thalassemia groups weresignificantly higher than in control subjects (P< .0001). There

was no significant difference among-thalassemia syndromes,although absolute RCs of Hb H/CS patients had the highest val-ues, indicating better compensatory erythropoiesis. Compared

with nonsplenectomized-thal/Hb E patients, splenectomized-thal/Hb E patients had a higher percentage of reticulocytes, ahigher absolute RC, a higher percentage of high-light-scatter retic-ulocytes, and greater mean reticulocyte volume, mean sphered cell

volume, and high-fluorescence-intensity rat io. Patients with-thal/Hb E showed a more severe degree of ineffective erythro-poiesis than patients with -thalassemic syndromes. In conclu-sion, we have shown good agreement between the 2 automatedreticulocyte counting technologies. Lab Hematol. 2000;6:73-78.

KEY WORDS: Reticulocyte count Thalassemia Coulter Gen S Sysmex R-2000

INTRODUCTION

The reticulocyte count (RC) is the fundamental routine meas-urement used to evaluate the erythropoietic activity of bone mar-row. Although the manual counting method is inexpensive andrelatively simple to perform, it has certain major limitations. Thetechnique is time-consuming, is subject to statistical error becauseof the small number of cells counted, and has significant inter-observer variation [1-4]. Development of automated counting byflow cytometry has tremendously increased the accuracy and preci-sion of counting reticulocytes and determining their maturity [5-8].Different maturity indexes, such as the reticulocyte maturationindex (RMI) or the different maturational fractions obtained by the

Sysmex R series automated counter (Toa Medical Electronics,Kobe, Japan), have been designed based on the relative amount ofcellular RNA. The Sysmex counter provides information aboutreticulocyte maturation based on the different reflecting intensitiesrelative to RNA content, divided into low-fluorescence-intensityratio (LFR), middle-fluorescence-intensity ratio (MFR), and high-fluorescence-intensity ratio (HFR). The HFR contains the mostimmature reticulocytes, MFR contains immature reticulocytes, andLFR contains mature reticulocytes. In the Sysmex R-3500, theimmature reticulocyte fraction (IRF), which is equal to MFR plusHFR, is also reported. Recently, Coulter (Hialeah, FL) developedan automated method using the new methylene blue (NMB) pro-cedure for use with the Gen S hematology analyzer. In addition toproviding normal parameters, the Coulter Gen S also provides

parameters such as mean reticulocyte volume (MRV), IRF, high-light-scatter reticulocytes (HLR) (percentage and absolute value),and mean sphered cell volume (MSCV), which is the average of thetotal red blood cells (RBCs) in the reticulocyte analysis. These newparameters have generated clinical interest, and some studies haveevaluated their possible role in the study of anemia [9] and in mon-itoring post-therapeutic hematologic recovery [10].

The thalassemias are a heterogeneous group of genetic hemoglo-bin disorders resulting from reduced synthesis of- and -globinchains [11]. Clinically, they are divided into 3 groups: thalassemiaminor, which is the symptomless thalassemia heterozygote; thalassemia

Reticulocyte Counting in Thalassemia Using Different

Automated Technologies

PORNVAREE LAMCHIAGDHASE,1 KOVIT PATTANAPANYASAT,2WANNA MUANGSUP3

1Department of Clinical Microscopy, Faculty of Medical Technology;2Center of Excellence for Flow Cytometry, Office for Researchand Development, Faculty of Medicine, Siriraj Hospital;3Department of Medicine, Faculty of Medicine, Siriraj Hospital,Mahidol University, Bangkok, Thailand

Laboratory Hematology 6:73-78

2000 Carden Jennings Publishing Co., Ltd.

73

Correspondence and reprint requests: Pornvaree Lamchiagdhase, Depart-

ment of Clinical Microscopy, Faculty of Medical Technology, Mahidol Uni-

versity, Siriraj Hospital Campus, Bangkoknoi, Bangkok 10700, Thailand

(e-mail: [email protected])

(Received November 19, 1999; accepted April 4, 2000)

ISLH

Official Publication


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intermedia, characterized by moderate anemia; and transfusion-dependent thalassemia major. Although the molecular biology of thegenetic defect has been studied extensively, knowledge of RBCpathology is still insufficient for understanding the prematureremoval of abnormal RBCs in the marrow and peripheral circula-tion. Patients suffer from anemia because of untimely RBC destruc-tion in the bone marrow and spleen. Compensatory erythropoiesis

is still exacerbated by premature RBC destructionso-called inef-fective erythropoiesis in the marrow. Evaluation of erythropoiesis isa very interesting approach to investigating pathogenesis of anemia.

In Thailand, -thalassemia, -thalassemia, abnormal hemoglo-bin (Hb) E, and Hb Constant Spring (CS) are common [11-14].In the present study, we compared manual reticulocyte counts andreticulocyte counts performed on the Coulter Gen S and the Sys-mex R-2000 in various genotypes of thalassemia.

MATERIALS AND METHODS

Samples and SubjectsForty-five healthy adults (21 men, 24 women) aged 26 8 years

who were not on medication and had normal hematologic data

by automated complete blood count (CBC) were used as the con-trol group in this study. We also examined 66 thalassemic patientsaged 34 12 years, all with clinical symptoms of thalassemiaintermedia. Criteria for the diagnosis was based on previousreports [12,13]. The thalassemic group included 21 Hb Hpatients (-thalassemia [-thal] 1/-thal 2; 4 men, 17 women),8 Hb H/CS patients (-thal 1/Hb CS; 4 men, 4 women), and20 -thal/Hb E patients (6 men, 14 women), all of whom werenonsplenectomized and had no blood transfusions for at least3 months before this study. Seventeen splenectomized -thal/Hb Epatients (7 men, 10 women) were also included.

Blood samples were collected and placed in K2EDTA anticoagu-lant and analyzed for CBC, blood smear, and RC within 4 hours.

Manual Reticulocyte CountingSupravital staining of unfixed RBCs with NMB was performed

by mixing 100 L whole blood with 100 L of 1% NMB solutionin a 12 75-mm tube [15]. After incubation at room temperaturefor 15 minutes, the dilution was remixed, and a wedge smear wasperformed for each sample. The number of reticulocytes per1000 RBCs was determined microscopically. A reticulocyte wasdefined as a RBC containing at least 2 granules of reticulum. Theabsolute RC was calculated from the RBC count using CoulterGen S, and the reticulocyte production index (RPI) was calculatedmanually. The RPI (also known as shift correction) is a generalindicator of the rate of RBC production above normal in anemia.

Automated Reticulocyte Counting by Coulter Gen SThe performance characteristics are described with the Coulter

Gen S reticulocyte measurement in RETIC mode. Briefly, 34 Lwhole blood is automatically aspirated and incubated with 166 LNMB to precipitate the basophilic RNA in reticulocytes [16].Hemoglobin and unbound stain are removed by adding a clearingreagent, which leaves clear, spherical, mature RBCs and darklystained reticulocytes. Stained reticulocytes are differentiated frommature RBCs and other cell populations by direct currentmeasurements for volume, conductivity, and light scatter (VCS).Cells (3.2 104) are counted to measure the following parameters:

RET%: The number of reticulocytes per 100 RBCs, directly meas-ured and reported as a percentage of RBCs.

RET#: The absolute number of reticulocytes, calculated fromRET% and RBC number and expressed as 106 cells/L or109 cells/L.

IRF: Immature reticulocyte fraction, calculated from the ratioof HLR and total reticulocytes.

HLR%: High-light-scatter reticulocytes, directly measured andreported as a percentage of RBCs.

HLR#: The absolute number of HLR, calculated from HLR% andRBC number and expressed as 106 cells/L or 109 cells/L.

MRV: Mean reticulocyte volume, the average volume of individ-ual reticulocytes, expressed in fL.

MSCV: Mean sphered cell volume, the average of total RBCs inthe reticulocyte analysis, expressed in fL.

Automated Reticulocyte Counting by Sysmex R-2000The Sysmex R-2000 is a fully automated reticulocyte counter.

The measuring principle of the system is based on flow cytometrycombined with hydrodynamic focusing. EDTA blood (100 L) isaspirated, and intracellular RNA is stained by auramine O, which is

fluorescent under argon laser light [17]. The reticulocyte popula-tion is further subdivided into LFR, MFR, and HFR. The percent-age of reticulocytes is given as the sum of LFR, MFR, and HFR.

Approximately 3.2 104 RBCs are usually analyzed for each sam-ple. The IRF is manually calculated by MFR plus HFR.

Statistical AnalysisFor statistical analysis, SPSS/PC+ version 4.0 (Microsoft, Red-

mond, WA) was used to calculate descriptive statistics (mean, SD,coefficient of variation, and range). Comparisons of statistical dif-ference between reticulocyte counting methods were performed

with Friedman 2-way analysis of variance and Wilcoxon signedrank test. The simple linear regression and correlation coefficient(r) were also determined. Among the groups of patients, Kruskal-

Wallis 1-way analysis of variance and the Mann-WhitneyU testwere used. The threshold for statistical significance for all compar-isons was chosen as P= .05.

RESULTS

The RCs of healthy subjects and thalassemic patients are sum-marized in Table 1. The percentage reticulocytes and absolute RCsof the patients were significantly higher than those of normal sub-

jects. In healthy subjects, the percentage reticulocytes did not differbetween men and women, but the absolute RC in men was higher(P< .0001). The comparison of the manual RC and the 2 automat-ed RCs showed no difference in the thalassemic group. Among thethalassemic patients, however, percentage reticulocytes and absoluteRCs of Hb H/CS were high by the manual method, and splenec-tomized -thal/Hb E patients showed the highest values whenusing the Sysmex R-2000. In addition, the correlation between themanual RC and the 2 automated RCs was good (P= .001), withr= 0.874 and r= 0.816 for the manual RC versus the 2 automatedmethods and r= 0.877 between the 2 automated methods (Figure).The percentage reticulocytes and absolute RCs of Hb H/CS weresignificantly higher than those of the other group of nonsplenec-tomized patients. However, when results were compared betweenthe 2 types of Hb H diseases, there was no statistically significant

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difference in any parameter or method used. Splenectomized-thal/Hb E reticulocyte percentages were significantly higher(P= .001) than the 2 types of Hb H on the Sysmex R-2000.

Table 2 shows the mean values and SDs of IRF, the percentageand absolute HLR obtained by the Coulter Gen S, and the percent-age of LFR, MFR, and HFR obtained by the Sysmex R-2000 inhealthy subjects and thalassemic patients. In 2 types of Hb Hdiseases, none of the parameters obtained by the 2 automated meth-ods showed a significant difference. However, nonsplenectomized-thal/Hb E showed significantly lower IRF, HLR, MRV, MSCV,MFR, and HFR compared with -thalassemia and splenectomized-thal/Hb E; only the LFR percentage was comparable.

For the immature fraction using the Sysmex R-2000 (Table 2),only LFR (P= .01) and MFR (P= .001) of Hb H patients weresignificantly different from those of nonsplenectomized andsplenectomized -thal/Hb E patients. However, when comparingnonsplenectomized and splenectomized -thal/Hb E, the LFR andMFR did not show any significant difference (P> .05).

The RPI, MRV, HLR#, MFR, HFR, and IRF of Hb H/CSpatients were significantly higher than those of Hb H and non-splenectomized -thal/Hb E patients. However, when results

were compared between the 2 types of Hb H diseases, there wasno statistically significant difference in any parameter or methodused. The mean percentage reticulocytes, absolute RC, HLR#,and MRV were slightly higher than those of Hb H patients, butin contrast, all reticulocyte values except IRF, LFR, and MFR ofnonsplenectomized -thal/Hb E patients were significantly lowerthan those of other patients.

Table 3 demonstrates the average of RBC size and reticulocytes.MRV was consistently higher than mean corpuscular volume(MCV), with a mean difference in healthy subjects of 27.9 7.5 fL.In splenectomized -thal/Hb E patients, the highest MRV was

found, with a mean difference of 60.7 14.2 fL. The ratio ofMCV to MRV in every group was >1. MRV of splenectomized-thal/Hb E patients was significantly higher than that of otherpatients, with the mean volume ratio (MRV:MCV) of those cellpopulations being approximately 1.8 0.2.

Correlation between percentage reticulocytes and absolute RCwith IRF, HLR, MRV, and MSCV is shown in Table 4. Poor corre-lation was found with IRF when using the 2 instruments, but

Automated Reticulocyte Count in Thalassemia 75

TABLE 1. Reticulocyte Count of Healthy Subjects Using the Manual Method, the Coulter Gen S, and the Sysmex R-2000*

n Manual Coulter Gen S Sysmex R-2000

Reticulocyte count, %

Healthy subjects

Men 21 1.0 0.3 (0.5-1.6) 1.1 0.3 (0.6-1.7) 1.2 0.3 (0.7-2.3)

Women 24 1.0 0.3 (0.4-1.7) 1.2 0.4 (0.7-1.7) 1.1 0.4 (0.7-2.0)Total 45 1.0 0.3 (0.4 -1.7) 1.1 0.4 (0.6-1.7) 1.2 0.4 (0.7-2.3)

Hb H 21 7.5 2.6 (3.4-13.4) 7.1 2.2 (3.6-11.2) 7.0 2.7 (2.7-12.9)

Hb H/CS 8 9.5 2.3 (6.3-12.3) 7.4 1.4 (5.0-10.0) 8.6 3.5 (5.8-16.2)

-Thal/Hb E (NS) 20 3.3 1.1 (1.4-5.9) 3.9 1.0 (2.3-6.7) 2.5 0.9 (1.3-4.8)

-Thal/Hb E (S) 17 9.2 4.6 (4.4-19.5) 8.5 4.6 (3.4-21.1) 11.4 4.5 (4.0-21.5)

Total 111 4.5 4.1 (0.4-19.5) 4.3 3.6 (0.5-21.1) 4.5 4.4 (0.5-21.6)

Absolute reticulocyte count,109/L

Healthy subjects

Men 21 53 16 (25-87 ) 58 15 (35-92) 65 18 (44-78)

Women 24 44 17 (26-83) 53 16 (24-91) 49 20 (23-99)

Total 45 48 17 (25-87) 55 16 (24-92) 57 20 (23-99)

Hb H 21 297 94 (116-552) 269 91 (37-407) 306 119 (105-651)

Hb H/CS 8 407 156 (263-649) 310 67 (218-388) 389 219 (208-853)

-Thal/Hb E (NS) 20 144 43 (38-194) 144 43 (67-221) 105 36 (49-163)-Thal/Hb E (S) 17 278 135 (126-529) 221 99 (64-404) 342 123 (163-633)

Total 111 167 146 (18-649) 154 111 (24-407) 170 156 (21-853)

*Data are means SD (range). Hb indicates hemoglobin; CS, Constant Spring;-thal,-thalassemia;NS, nonsplenectomy; S, splenectomy.

Correlation of reticulocyte count (RC) as determined by theCoulter Gen S and Sysmex R-2000.


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percentage and absolute HLR on the Coulter Gen S were signifi-cantly correlated with MFR and IRF on the Sysmex R-2000.

DISCUSSION

The peripheral blood RC is commonly used as an indicator ofthe erythropoietic activity of bone marrow. Manual counting withthe microscope usually gives results with a high degree of inaccura-cy and imprecision and a large inter- and intraobserver variability.

Automated counting has overcome those limitations, and the avail-ability of reticulocyte maturation indexes, based on the measure-ment of RNA content, extends the clinical utility of reticulocytecounting. Using 2 different principles of automated reticulocytecounting, we compared automated reticulocyte counting with themanual method in thalassemic and healthy subjects.

Correlation analysis of manual RC versus the Coulter Gen Sand Sysmex R-2000 produced r values of 0.874 and 0.816,respectively. Correlation analysis between the 2 automated instru-ments gave an rvalue of 0.877. Kessler et al [18] reported goodcorrelation between Coulter Gen S and manual RC (r= 0.800),Coulter MAXM (r= 0.936), and R-1000 (r= 0.890). Kojima etal [19] and Tatsumi et al [20] also found good correlations

between manual RCs and Sysmex R-1000, with rvalues of 0.945and 0.893, respectively.

The percentage reticulocytes and absolute RCs of thalassemicpatients were significantly higher than those of normal subjects.High RCs in Hb H and Hb H/CS patients have been reportedusing the manual microscope procedure [13,21]. In this study, nomethods showed statistically significant differences between Hb Hand Hb H/CS patients. However, RPI of Hb H/CS patients (3.6)

was significantly higher than that of Hb H patients (2.0). This find-ing is similar to findings in our previous report (Hb H = 1.1; HbH/CS = 2.3) [22] and to the findings of Khuhapinant et al (Hb H =1.97; Hb H/CS = 3.62) [23]. RPI is an indicator of the rate of RBCproduction and increases in response to anemia. An RPI value >3generally indicates an adequate bone marrow response to anemia,

whereas an RPI value


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difference when we compared these measurements in the non-splenectomized -thal/Hb E patients. However, when we comparednonsplenectomized and splenectomized -thal/Hb E patients, onlythe HLR% and HFR showed statistically significant differences.This result suggests that the spleen may play an important role inpooling reticulocytes before releasing them into the circulation [25].

A marked increase in reticulocytes was found in splenectomized-thal/Hb E patients, especially on the Sysmex R-2000. However,there was no statistically significant difference between the manualRC (9.2 4.6) and that of the Coulter Gen S (8.5 4.6), and RCs

with both methods were significantly lower than those with the Sys-mex R-2000 (11.4 4.5) (P= .0012 and .0037, respectively). Oneexplanation is that the supravital stain used for the Coulter Gen S issimilar to that used for manual RC but different from that for theSysmex R-2000, which uses a different fluorochrome. Villamor et al[26] presented evidence that when using flow cytometrybasedmethods, white blood cells could erroneously be identified as reticu-locytes, which may affect the reticulocyte differential and RMI. Thisresult occurs because the fluorochrome can stain both RNA andDNA. The presence of Howell-Jolly bodies and nucleated red bloodcells, as seen in splenectomized patients, can also interfere with auto-mated RC [27], resulting in high numbers. Our data show no statis-tically significant difference among methods in Hb H and HbH/CS patients, but there was a significant difference in nonsplenec-tomized and splenectomized -thal/Hb E patient values.

For MRV, we found no difference in the 2 types of Hb H dis-eases. However, in splenectomized -thal/Hb E patients, the MRVand MRV:MCV ratio were significantly higher. This finding sug-gests that the spleen plays an important role in modifying the sizeof immature RBCs and supports previous findings that maturationin abnormal erythropoiesis of thalassemia patients is similar to thatof normal subjects, except after splenectomy [28,29].

In summary, the present investigation demonstrated a linearrelationship between manual RC and 2 automated instruments

with no statistically significant difference between these methods.RCs by automated methods are useful not only for providing pre-cise counts, but also for determining RBC maturation. This is espe-cially true of the Coulter Gen S, which uses the same principle instaining reticulocytes as the manual method and has less interfer-ence from inclusion-containing RBCs. By using these 2 automatedtechnologies to evaluate erythropoietic activity in thalassemia, wefound that both thalassemia intermedia and nonsplenectomized-thal/Hb E had severe ineffective erythropoiesis when compared

with 2 types of Hb H diseases. -Thal/Hb E splenectomizedpatients showed a marked increase in reticulocyte number withincreasing MRV and immature reticulocytes.

ACKNOWLEDGMENTS

The authors wish to thank PCL Holding Co. Ltd., Thailand,for support with the reagents and all the cost of publication. Wealso thank Janice M. Darden for reading the manuscript and

Amphun Chaiphan and Sirintorn Mahem for technical assistance.

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Automated Reticulocyte Count in Thalassemia 77

TABLE 4. Correlation Between Reticulocyte Count and Other Parameters of 111 Samples Using the Coulter Gen S and the Sysmex R-2000*

Coulter Gen S Sysmex R-2000

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P= .01.

Note. Italicized numbers represent nonsignificant figures.


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