Hematopathology / RETICULOCYTE PARAMETER (RET-Y) OF SYSMEX XE 2100 IN SIDEROPENIC ANEMIA

The New Reticulocyte Parameter (RET-Y) of the Sysmex XE 2100 Its Use in the Diagnosis and Monitoring of Posttreatment Sideropenic Anemia
Mauro Buttarello, MD,1 Valeria Temporin, MD,1 Renato Ceravolo, MD,2 Giorgio Farina, MD,1 and Pietro Bulian, MD3
Key Words: Reticulocyte parameters; Mean reticulocyte hemoglobin content; Mean reticulocyte volume; Hematology analyzer; Iron deficiency anemia
DOI: 10.1309/W65295DTUWK7U1HH

Abstract
To verify their clinical usefulness in diagnosis and early response to therapy of sideropenic anemia, we compared the behavior of the reticulocyte parameter (RET-Y), a raw measure dependent on size and content of the cell, generated by the Sysmex XE 2100, with the mean reticulocyte volume (MCVr) and mean reticulocyte hemoglobin content (CHr) from the Bayer ADVIA 120 in healthy subjects and patients with iron deficiency anemia. Correlations were high (r = 0.88 and r = 0.94, respectively). All parameters varied significantly as early as 48 hours after the start of intravenous iron therapy (mean differences of 17.4% [RET-Y], 4.5% [MCVr], and 9.5% [CHr]). Sudden decreases in those parameters at interruption of therapy indicate the reappearance of sideropenic erythropoiesis. The receiver operating characteristic curve demonstrated a high degree of efficiency in differentiating moderate or severe iron deficiency anemia from the healthy state. The best association between sensitivity and specificity was at a cutoff of channel 1,624 for RET-Y and 104.5 fL for MCVr (negative and positive predictive values, respectively, of 99.6% and 96.5% for RET-Y and 98.7% and 93.3% for MCVr). RET-Y is correlated closely with CHr and is useful for diagnosis and early monitoring after the administration of intravenous iron.

Reticulocyte counts have once again acquired great interest and importance following the introduction of instruments that use dyes specific for RNA. This has resulted in precise and accurate counts even at low reticulocyte concentrations. The latest generation of automated analyzers provides additional information on reticulocytes, such as the immature reticulocyte fraction (IRF) and other reticulocyte indices, eg, mean reticulocyte volume (MCVr) and mean reticulocyte hemoglobin content (CHr) Table 1. After leaving the bone marrow, reticulocytes, under normal conditions, require approximately 24 hours to become mature erythrocytes. The blood concentration of reticulocytes represents a quantitative measure of erythropoiesis, while the reticulocyte parameters provide real-time information about reticulocyte quality. To date, the most widely studied of the reticulocyte indices is the CHr. The hemoglobin content is considered to be constant throughout the lifetime of erythrocytes and circulating reticulocytes1 unless structural changes take place that compromise the amount of cytoplasm or cause cellular fragmentation. During intramedullary development, reticulocytes actively synthesize hemoglobin.2,3 The CHr, which directly reflects the recent hemoglobin synthesis in bone marrow precursors, is a measure of adequate iron stores. This is more useful than stainable iron, which is a rough estimate of deposits in the reticuloendothelial system.4 On the one hand, the importance of this parameter is linked to its ability to enable the diagnosis of sideropenic erythropoiesis, even when biochemical markers such as ferritin or transferrin saturation are inadequate (ie, in inflammation and anemia of chronic disease), and on the other hand, it is linked to its usefulness in monitoring early
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Table 1 Reticulocyte Parameters

Analyzer Abbott Cell Dyn 4000, Abbott, Santa Clara, CA ABX Pentra 120 Retic, ABX, Montpellier, France Bayer ADVIA 120, Bayer Diagnostics, Tarrytown, NY Beckman Coulter LH 750, Miami, FL Sysmex XE 2100, Sysmex, Kobe, Japan Reticulocyte Immaturity Fractions IRF 3-Population/IRF 3-Population/IRF IRF 3-Population/IRF Reticulocyte Cellular Indices MCVr MCVr, CHr, CHCr, dispersion indices MSRV RET-Y

CHCr, mean reticulocyte hemoglobin concentration; CHr, mean reticulocyte hemoglobin content; IRF, immature reticulocyte fraction; MCVr, mean reticulocyte volume; MSRV, mean sphered reticulocyte volume; RET-Y, mean channel value of the forward scatter histogram within the reticulocyte population.

response to intravenous therapy and avoiding the risk of iron overload. Low CHr values indicate sideropenic erythropoiesis in patients undergoing dialysis. This parameter is more suited than biochemical markers for the monitoring of iron administration in these patients.5,6 The functional iron deficiency that appears in healthy subjects treated with erythropoietin manifests with early reduction of the CHr compared with baseline values. In this case, intravenous iron administration prevents the formation of hypochromic reticulocytes, increasing the value of the CHr.7 This parameter has been proven useful in diagnosing sideropenic anemia in patients affected by chronic inflammatory disease in whom traditional markers of iron metabolism are ineffective.8 The CHr is the most useful index of iron deficiency and sideropenic anemia in pediatric patients.9 Its usefulness also has been shown in monitoring the response to iron supplements administered orally or intravenously.10 Fewer studies are available concerning the clinical usefulness of the MCVr. In subjects with depleted iron stores, this parameter increases rapidly when iron supplements are administered and decreases quickly during iron-deficient erythropoiesis.10 The MCVr decreases quickly, and reticulocytes are smaller than circulating RBCs in macrocytosis treated with vitamin B12 and folic acid.11-13 It also has been noted during the follow-up of transplant recipients that inversion of the ratio of MCVr and the mean volume of mature erythrocytes (normally MCVr/MCV > 1) occurs during conditioning chemotherapy (due to a decrease in MCVr). The success of the marrow transplant is indicated early by normalization of

this ratio.12 The MCVr multiplied by the number of reticulocytes gives the value of hematocrit-reticulocytes used to evaluate possible abuse of erythropoietin in sports.14 Thus, it is possible to see how the MCVr might have clinical usefulness that overlaps that of the CHr, although they are not completely equivalent. The main limitation to general use of the CHr is its availability only on Bayer ADVIA 120 counters (Bayer Diagnostics, Tarrytown, NY). The MCVr or similar size parameters, on the other hand, even though measured by different methods and with evident problems of standardization,15 can be obtained with 4 of the 5 most modern automated counters on the market Table 2. We hope that the manufacturers of these instruments can solve standardization and calibration problems, thus making the results from the various counters comparable. The aims of the present study were to assess concordance between the parameter RET-Y, which is a forward light scatter measure correlated with the size and content of the reticulocyte as calculated by the Sysmex XE 2100 (Sysmex, Kobe, Japan), and the MCVr and CHr generated by the Bayer ADVIA 120 and compare their clinical usefulness in diagnosis and monitoring in a group of subjects with iron deficiency anemia.

Materials and Methods

Analyzers The Sysmex XE 2100 (software version 00-17) is a fully automated fluorescence flow cytometer using a polymethine

Table 2 Methods for Reticulocyte Size Determination*

Analyzer ABX Pentra 120 Retic Bayer ADVIA 120 Beckman Coulter LH 700 Sysmex XE 2100
*

Technology Direct current volume Forward light scatter Direct current volume Forward light scatter

Cell Condition Native Sphered Sphered Native

Units fL fL fL Channel No.

For proprietary information, see Table 1. This measure is related only partially to the size of the reticulocyte.

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dye that can bind to cytoplasmic RNA to permit reticulocyte counting. By using forward light scatter, the instrument can measure a signal proportional to the size of RBCs and reticulocytes and provide their respective mean values (RBC-Y and RET-Y) in arbitrary units (channel numbers). The ADVIA 120 uses Oxazine 750 to stain reticulocyte RNA and measures absorbance. This cytometer, like the XE 2100, uses forward light scatter; however, because the cells have been sphered previously, the Mie theory16 can be used to decouple the relation between size and index of refraction and produce a volume signal (reticulocyte volume in femtoliters). Because this counter can measure the hemoglobin concentration of individual reticulocytes, it is possible to obtain hemoglobin content as the product of hemoglobin concentration of individual reticulocytes reticulocyte volume and, therefore, calculate an average value (CHr) expressed in picograms. This instrument also furnishes the percentage of reticulocytes with hemoglobin content below a certain threshold, set at 27 pg by the manufacturer. Both analyzers can provide the immature reticulocyte fraction. The counters were calibrated initially and checked daily according to the manufacturers instructions and with materials provided by the respective manufacturers. Reference Ranges in Health We analyzed 80 specimens from healthy subjects, selected according to National Committee for Clinical Laboratory Standards criteria,17 in parallel and in duplicate to calculate the corresponding reference intervals, during a period of 4 weeks at the laboratory of Clinical Pathology of the Geriatric Hospital of Padua, Italy. Specimens During a period of 6 months, specimens obtained from 29 patients (29-78 years, 23 women and 6 men) with iron deficiency anemia and attending an outpatient clinic for intravenous iron therapy were analyzed. Therapy consisted of saccharated iron oxide (VENOFER, VIFOR International, St Gallen, Switzerland) equivalent to 100 mg of iron, administered slowly, intravenously each morning, Monday through Friday, for 2 consecutive weeks. Blood samples were obtained before therapy was started (day 0) and on days 1, 2, 3, 4, 7, 8, 9, 10, and 11. To study the imprecision of the parameters evaluated (RET-Y, MCVr, CHr) 15 specimens were selected to cover a wide range of values. Each of these specimens was analyzed repeatedly 7 times (at 20-minute intervals between counts). All specimens were obtained in the morning under fasting conditions, into vacuum tubes containing K2 EDTA (Vacutainer, Becton Dickinson, Plymouth, England), were kept at room temperature and analyzed within 4 hours. The gold standard used to define iron deficiency anemia was the
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response to iron therapy (hemoglobin concentration increase of at least 0.75 g/dL [7.5 g/L] by the 11th day). Statistical Analysis The reference intervals were calculated by using a nonparametric method.18 Imprecision was evaluated by using imprecision profiles. The diagnostic efficiency of the MCVr and RET-Y was verified by several methods: (1) noting the first day to show a significant increase in measured parameters from day 0; results were compared initially by using the Friedman test and subsequently with the Wilcoxon 2-tailed test; (2) constructing a receiver operating characteristic curve (ROC) and calculating the area under the curve19; and (3) calculating the sensitivity, specificity, and negative and positive predictive values with confidence intervals.

Results
The reference intervals in health for the MCVr and CHr in the adult population, reported in Table 3, are in accordance with previously published values.8 For RET-Y, the only data presently available are those of Briggs et al20 on 40 subjects, which indicate an upper limit greater than ours (channel 1,963 vs channel 1,820); this also is true for the CHr (36.3 vs 33.4 pg). These differences probably are due to the different populations studied. The results of the imprecision showed that the coefficients of variation for the RET-Y and CHr are approximately 1% for all concentrations, while for MCVr they approach 2% for values less than 100 fL. Imprecision is, therefore, negligible. The results for days 0 and 11 are summarized in Table 4. The correlation between the RET-Y and CHr for all subjects (n = 109) was better (r = 0.94) than that between the size parameters RET-Y and MCVr (r = 0.88). The average behavior of size parameters studied Figure 1 indicated a variation (+4.5% for MCVr and +17.4% for RET-Y) with respect to time 0 that became statistically significant at the second day (P < .05 for MCVr and P < .01 for RET-Y), even if the values still did not enter the normal range. This behavior overlaps that of the CHr (+9.5% at day 2; P < .01). The average values at day 2 were influenced by the presence of preexisting reticulocytes (in conditions of anemia, their mean circulating life span is greater than 24 hours, which is typical of a normal hemoglobin concentration) and probably by the continued release of some hypochromic reticulocytes. This can be confirmed by the persistence of a large percentage of reticulocytes with CHr values less than 27 pg (and, therefore, with low hemoglobin content) at day 2 (average, 65.6%; range, 43.0%-86.6%), with respect to the average of 81.6% (range, 66.3%-91.9%) at time 0. The
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Table 3 Reticulocyte Parameters in 80 Healthy Subjects

Range Parameter Mean reticulocyte hemoglobin content (pg) Mean reticulocyte volume (fL) Reticulocytes with mean hemoglobin content < 27 pg (%) RET-Y (channel No.) Median 31.1 110.8 8.9 1,740 Mean 31.0 111.3 10.5 1,741 2.5 Percentile 28.2 104.5 2.10 1,661 97.5 Percentile 33.4 119.6 26.7 1,820

RET-Y, mean channel value of the forward scatter histogram within the reticulocyte population.

Table 4 Significant CBC Count Parameters and Reticulocyte Cellular Indices in Iron Deficiency Anemia on Day 0 and 11 Days From Start of Therapy
Mean Parameter Hemoglobin, g/dL (g/L) Mean cell volume, m3 (fL) Mean reticulocyte hemoglobin content (pg) Mean reticulocyte volume (fL) RET-Y (channel No.) Immature reticulocyte fraction* Day 0 9.1 (91) 76 (76) 22.2 95.9 1,347 0.20 Day 11 9.9 (99) 78.8 (78.8) 29.2 109.9 1,746 0.22 Day 0 6.5-10.9 (65-109) 63.8-85.0 (63.8-85.0) 16.0-25.0 84.0-104.8 1,044-1,626 0.10-0.28 Range Day 11 7 .5-11.7 (75-117) 66.4-88.5 (66.4-88.5) 22.5-33.6 98.7-119.4 1,502-1,896 0.12-0.35

RET-Y, mean channel value of the forward scatter histogram within the reticulocyte population. * According to the Bayer ADVIA 120, Bayer Diagnostics, Tarrytown, NY.

A
2,000

B
140

1,800 RET-Y (Channel No.)

130

120 MCVr (fL) 0 5 Days 10 15 1,600

110

1,400

100 1,200

90

1,000

80 0 5 Days 10 15

Figure 1 Mean channel value of the forward scatter histogram within the reticulocyte population (A; RET-Y) and mean reticulocyte volume (B; MCVr) response during intravenous iron therapy. A, Arrow, +17 .4% (P < .01). B, Arrow, +4.5% (P < .05). Squares indicate the mean; error bars, the range.

percentage difference at day 2 on average was greater for RET-Y than for MCVr. Figure 2 compares variations (from day 0 to day 11) in MCVr, RET-Y, CHr, and percentage of reticulocytes with
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low hemoglobin content found in 3 clinical situations: Figure 2A shows data for a patient in whom therapy was interrupted over a weekend; Figure 2B, for a patient in whom therapy was uninterrupted; and Figure 2C, for a
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130

2,000

35

100

120

1,800 RET-Y (Channel No.) % of Reticulocytes % of Reticulocytes % of Reticulocytes 30 CHr (pg) 75

MCVr (fL)

110

1,600

50 25 25 20

100 1,400 90 1,200 80 1,000 0 5 Days 10 15

0 0 5 Days 10 15

B
130 2,000 35 100

120 1,750 MCVr (fL) 110 1,500 100 RET-Y (Channel No.) 30 CHr (pg) 75

50 25 25 20

90

1,250

80 1,000 0 5 Days 10 15

0 0 5 Days 10 15

C
130 1,300 35 100

120

1,200 30 75 RET-Y (Channel No.)

MCVr (fL)

110

CHr (pg)

1,100

50 25 25 20

100 1,000 90 900 80 800 0 5 Days 10 15

0 0 5 Days 10 15

Figure 2 Reticulocyte response during intravenous iron therapy for patients with the indicated conditions. A, Suspension of therapy over a weekend. B, Uninterrupted therapy. C, Iron deficit associated with -thalassemia trait. Diamonds, mean channel value of the forward scatter histogram within the reticulocyte population (RET-Y); squares, mean reticulocyte volume (MCVr); circles, mean reticulocyte hemoglobin content (CHr); asterisks, percentage of reticulocytes with CHr <27 pg.
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patient with iron deficiency associated with the thalassemia trait. These graphs demonstrate at least 3 phenomena: (1) The MCVr, RET-Y, and CHr generally move in parallel, even when there is sudden inversion in the direction of the curve, while the percentage of reticulocytes with low hemoglobin moves in the opposite direction, ie, the proportion of reticulocytes with low hemoglobin falls as the MCVr, RET-Y, and CHr increase (except for the patient with combined iron deficiency and thalassemia trait (Figure 2C). (2) In most patients, a peak is reached for the 3 parameters at approximately day 7 (often with RET-Y preceding the peak of MCVr). (3) By day 11, the reticulocyte parameters have entered the reference range in health in 24 of the 29 patients. Of the 5 exceptions, the values for 3 patients reached normal levels by day 7 and regressed to increase slowly once again without reaching normal on day 11. In the remaining 2 patients, values did not reach normal levels. Further study revealed that they had the -thalassemia trait associated with iron deficiency (Figure 2C). The IRF had a different behavior: It increased at day 1, decreased on day 2, and later continued to increase until reaching the maximum value at day 5. Afterward, the IRF decreased steadily until day 11. The final value reentered the reference interval (<0.25)21 for 21 subjects. The ROC curve indicated an extremely high diagnostic efficiency for the MCVr and RET-Y (areas under the curve, 0.997 and 0.999, respectively) for iron deficiency anemia. From the analysis of the ROC curve, it is possible to calculate the cutoff that gives the optimum association between sensitivity and specificity. This is channel 1,624 for RET-Y (sensitivity, 99%; specificity, 98.7%; negative predictive value, 99.6% [confidence interval, 93.5-100]; and positive predictive value, 96.5% [CI, 76.6-99.9]) on the Sysmex XE 2100 and 104.5 fL for the MCVr (sensitivity, 96.6%; specificity, 97.5%; negative predictive value, 98.7% [CI, 91.699.9], and positive predictive value, 93.3% [CI, 71.8-99.0]) on the Bayer ADVIA 120.

Discussion
Sideropenic anemia is the most common form of anemia, with a prevalence that varies in different parts of the world from 12% to 43%.22 The diagnosis and monitoring of response to therapy presumes the availability of highly specific and sensitive tests that also are useful to prevent the negative side effects of iron overload. The traditional biochemical indices (ferritin, transferrin saturation) are influenced by other clinical conditions such as inflammation, hepatic diseases, malignant neoplasms, chronic disease, and oral contraceptive use.
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Laboratory tests such as reticulocyte indices, which might provide direct information concerning hemoglobin synthesis in bone marrow precursors, are of great interest. Many studies1,5,6,9,10,12,13 have demonstrated the clinical usefulness of the CHr for identifying sideropenic erythropoiesis, iron deficiency anemia, and functional iron deficiency, to the point that this test has become referred to as the gold standard for these clinical conditions.23 However, this parameter is strictly instrument-dependent. The search for reticulocyte parameters that can have clinical usefulness that equals or mimics the CHr and that can be generated by most automated cytometers is perceived as a general need. The forward scatter signal measure by the XE 2100 (RET-Y) is a step in this direction. The first important observation is the high correlation of the RET-Y with the CHr (r = 0.94). This is in complete accord with previously published data.20 The correlation of RET-Y with MCVr was marginally less good (r = 0.88). It seems likely that the forward scatter on which the RET-Y depends is related to cell size and to cell content (mainly). In this patient series, it is important to note the significant increase on the second day after beginning iron therapy. Such a rapid increase in size (and in IRF) can be explained at least partially by the release of less mature stress macroreticulocytes. The analogous increase in the CHr and the comparable decrease in the percentage of reticulocytes with low hemoglobin content (CHr, <27 pg), however, might be due to an increased production of hemoglobin by the bone marrow reticulocytes or by the involvement of latestage erythroblasts. Because certain patients have these variations after only 24 hours from the start of intravenous therapy, the time is too short to hypothesize that these variations are due to the differentiation of new reticulocytes with normal hemoglobin content from normalized precursors. The presence of a sudden decrease in the parameters at days 8 and 9 could be caused by the reappearance of irondeficient erythropoiesis due to the suspension of therapy over the weekend. Because the latency period of this phenomenon is 2 to 3 days at most, there can be more than 1 explanation: (1) the release of hypochromic reticulocytes that represent the final stages of development of precursors with low hemoglobin content already present before beginning therapy or (2) a sudden reduction in hemoglobin synthesis in the final stage of maturation owing to an insufficient supply of iron with respect to iron demand. In an attempt to determine the reason, 4 additional patients were enrolled, 2 of whom received a double dose of iron on days 3 and 4 with interruption of therapy on days 5 and 6 and 2 of whom received therapy without interruption (therefore, with the same total dosage). The results showed persistence of the decline in the RET-Y, MCVr, and CHr on days 8 and 9 in the first 2 patients and lack of this decrease in patients receiving
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uninterrupted therapy (Figure 2B). The second hypothesis is, therefore, more likely to explain the production of smaller reticulocytes with lower hemoglobin content, which depends on the reduced availability of iron rather than on the production of hypochromic reticulocytes derived from hypochromic precursors. The reticulocyte response would seem to be more efficient with uninterrupted therapy rather than in cases of equivalent dosage administered with interruption. Analysis of the ROC curve demonstrated high diagnostic efficiency for the RET-Y (analogous to the MCVr) in cases of moderate or severe iron deficiency anemia. Further studies are necessary to evaluate the ability to recognize cases of sideropenic erythropoiesis in the absence of or with a low level of anemia. We can confirm that in all subjects studied there was a high correlation between the RET-Y and MCVr and between the RET-Y and CHr, even if the latter is not a size parameter but reflects hemoglobin content. Probably the RET-Y parameter depends on size and content of the cell. Still more interesting is the fact that at diagnosis and during monitoring of patients treated with intravenous iron, these parameters can provide equivalent information for the clinician. This also is true for the study of pathophysiologic erythropoiesis in these patients.
From the 1Clinical Pathology Laboratory, Geriatric Hospital, Department of Laboratory Medicine, Padua, Italy; 2Outpatient Clinic, Geriatric Hospital, Padua; and 3Department of Laboratory Medicine, S. Maria degli Angeli Hospital, Pordenone, Italy. Address reprint requests to Dr Buttarello: Dipartimento di Medicina di Laboratorio, Servizio di Medicina di Laboratorio, Azienda Ospedaliera, via Giustiniani 2, 35100 Padova, Italia; or mbuttarello@yahoo.it. Acknowledgments: We are grateful to Claudio Franceschini and Marco Rimoldi, DASIT SpA, Milan, Italy, for providing the instrument and reagents; Carmen Fasolato, RN, and Attilio Rambaldi, RN, nurses of the outpatient clinic; and Alfredo Businaro, laboratory technician.

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