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*Hematology Laboratory, S U M M A RY
Hospital Galdakao – Usansolo,
Galdakao, Vizcaya, Spain Introduction: Reticulocyte hemoglobin content and percentage of
†
Laboratory, Hospital hypochromic red cells are incorporated into the European best
Universitario Basurto, Bilbao,
Vizcaya, Spain practice guidelines on anemia management in chronic kidney
‡
Nephrology. HD Unit. Hospital disease. Sysmex XN analyzer (Sysmex Corporation, Kobe, Japan)
Galdakao – Usansolo, Galdakao, reports reticulocyte hemoglobin equivalent (Ret-He) and the hypo-
Vizcaya, Spain
§ chromic fraction of erythrocytes (%Hypo-He). Our aim was to
Department of Pharmacology
and Physiology, Faculty of assess the value of these parameters, in terms of the sensitivity and
Medicine, University of specificity for detecting functional iron deficiency, in hemodialysis
Zaragoza, Zaragoza, Spain (HD) patients.
Methods: Forty HD patients in the maintenance phase of erythropoi-
Correspondence:
Eloısa Urrechaga, Hematology etin therapy were included. Intravenous iron supplementation was
Laboratory, Hospital Galdakao – interrupted at least 3 weeks before recruitment. Two samples were
Usansolo, 48960 Galdakao, analyzed for each patient: the baseline after the iron-free period and
Vizcaya, Spain.
the second sample after 4 weeks of IV iron administration. Hemo-
Tel.:+ 34 94 400 7102;
Fax: + 34 94 400 7128; gram and biochemical parameters of the iron status were measured.
E-mail: eloisa.urrechagaigartua@ Patients were classified as responders or nonresponders to an iron
osakidetza.net load; responders had an increase in Hb of at least 10 g/L after iron
administration, compared to the baseline. To identify the efficiency
doi:10.1111/ijlh.12496 of the test for predicting the response to iron administration,
receiver operating characteristic analysis (ROC) was performed.
Received 16 September 2015; Results: According to the established criteria, 21 patients were
accepted for publication 8 responders and 19 nonresponders. ROC analysis results: Ret-He
March 2016 area under curve (AUC) was 0.84 (95% CI 0.64–0.93), at cutoff
30.8 pg, sensitivity 78.7%, and specificity 87.2%. % Hypo-He AUC
Keywords
was 0.78 (95% CI 0.64–0.91), at cutoff 2.4%, sensitivity 72.2%,
Iron availability, hemodialysis,
reticulocyte hemoglobin con- and specificity 88.1%.
tent, hypochromic erythrocytes, Conclusions: % Hypo-He and Ret-He are reliable parameters for the
Erythropoiesis study of erythropoiesis status in HD patients.
360 © 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38, 360–365
E. URRECHAGA ET AL. | PERCENTAGE OF HYPOCHROMIC ERYTHROCYTES AND RETICULOCYTE HEMOGLOBIN 361
© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38, 360–365
362 E. URRECHAGA ET AL. | PERCENTAGE OF HYPOCHROMIC ERYTHROCYTES AND RETICULOCYTE HEMOGLOBIN
the NKF-K/DOQI (National Kidney Foundation, Kid- Serum iron, transferrin, ferritin, and sTfR were
ney Disease Outcomes Quality Initiative) guidelines. assayed in a chemical analyzer Cobas c 711 (Roche
All of them were treated with intermittent in center Diagnostics, Mannheim, Germany).
hemodialysis (standard 4-h bicarbonate dialysis, three
times per week), receiving erythropoietin (rHuEPO) for
Statistical analysis
at least 3 months, and were in the maintenance phase
of their treatment, with stable doses (2000 U/week) for Statistical software package SPSS (SPSS; Chicago, IL,
at least 4 weeks. Patients were receiving folate (5 mg USA) version 19.0 for windows was applied for statis-
orally twice a week) and vitamin B12 (1000 lg three tical analysis of the results.
times a week) supplement and iron therapy (intra- The differences between responders and nonre-
venous iron sucrose), to maintain the iron availability sponders were evaluated using Student’s t-test; P val-
and TSAT > 20%. IV iron administration was inter- ues less than 0.05 were considered to be statistically
rupted at least 3 weeks before recruitment. The study significant.
began at the end of the washout iron-free period: At To identify the efficiency of the test and the opti-
this point, hematological and iron monitoring parame- mal cutoff for predicting the response to iron adminis-
ters were measured (baseline). tration, receiver operating characteristic analysis
The second sample for each patient was analyzed (ROC) was performed.
after 4 weeks of the IV iron administration, 100 mg
iron sucrose at each dialysis session for 4 weeks.
R E S U LT S
Patients were classified as responders or nonrespon-
ders to iron load according to their Hb increment. According to the established criteria, 21 patients were
Those who had an increase in Hb of at least 10 g/L at responders and 19 nonresponders. Their clinical and
the end of iron loading compared to the baseline were pharmacological conditions are listed in Table 1 and
considered responders, while nonresponders presented mean values together with standard deviation for ana-
a difference in Hb of <10 g/L. lytical data in Table 2.
Hb, RBC, Ret-He, and % Hypo-He were statistically
different in responders and nonresponders,
Laboratory methods
P < 0.0001, while ferritin and TSAT had no differ-
Samples were obtained in the course of routine analy- ences (P = 0.888 and 0.522 respectively).
sis, collected in EDTA anticoagulant tubes (Vacu- Changes after administration of IV iron were
tainerTM Becton-Dickinson, Rutherford, NJ, USA), recorded. In the responders group, Hb mean incre-
and were run in the Sysmex XN analyzer Sysmex ment was 15 g/L. The change in parameters related to
Corporation, Kobe, Japan) within 6 h of collection. Hb content is summarized in Table 3.
Table 1. Baseline characteristic of patients. Values are reported as mean (standard deviation)
ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor blocker; rHuEPO, recombinant human
erythropoietin.
© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38, 360–365
E. URRECHAGA ET AL. | PERCENTAGE OF HYPOCHROMIC ERYTHROCYTES AND RETICULOCYTE HEMOGLOBIN 363
© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38, 360–365
364 E. URRECHAGA ET AL. | PERCENTAGE OF HYPOCHROMIC ERYTHROCYTES AND RETICULOCYTE HEMOGLOBIN
The cutoffs obtained correlate with those published needed to detect significant descent after iron admin-
for the RBC extended parameters reported by the Sys- istration [33].
mex XE series analyzers. TSAT is currently used to monitor IV iron therapy,
The cutoff obtained for Ret-He, 30.8 pg, is around as a marker of iron functionally available for erythro-
the value currently accepted for the detection of func- poiesis [13, 14]; our results show that, based on TSAT
tional iron deficiency, not only for Sysmex XE series values, the clinician cannot recognize a responder
analyzers, but also for Siemens and Abbott counters patient.
[17, 18, 21, 30]. The combined use of TSAT <20% and sTfR
Kim et al. evaluated the same Siemens parameter >1.5 mg/L has been proposed to improve functional
as a marker of iron deficiency and predictor of iron deficiency detection in dialysis patients suspected
response to intravenous iron in hemodialysis patients of having inflammatory conditions [34], but this is an
[31]; Miwa et al., the Ret-He responses postiron sup- expensive test; for this reason, the new parameters of
plementation [32]; and both studies found more the hemogram reported with no additional cost seem
marked differences than traditional measurements promising.
after iron supplements; our results show similar XN analyzer has been recently launched, so we
trends. tried to verify that the clinical decision limits of its
The cutoff 2.7% has been proposed for %Hypo-He markers of hypochromia are in concordance with
in HD patients, in correlation with %Hypo reported those of XE 5000.
by Siemens analyzers [21], and 3.6% when the com- A limitation of the study is that we have focused on
parison was made with soluble transferrin receptor parameters of iron status, but factors other than low
[22]. iron stores have been recognized to hamper the efficacy
The efficiency of %Hypo-He was better than that of ESA therapy, that is high turnover bone disease and
obtained for MCH and MCHC. These indices are the hyperparathyroidism [35], so prospective studies are
mean values of the global red cell population; the needed, including more variables, to verify these results
contribution to the hypochromic subset is diluted by in dialyzed patients in different clinical situations.
the majority of normochromic population. The % The conclusion is that Ret-He and %Hypo-He
Hypo-He presents the advantage to directly quantify reported by Sysmex XN analyzer seems to be an
hypochromic erythrocytes, so the changes in this preferable alternative in the routine practice. Both
important subset can be detected and evaluated could be useful in assessing functional iron deficiency
accordingly. and therefore improve anemia management in
Nevertheless, %Hypo-He in the group of responder patients receiving HD and could help to guide clini-
patients presented no significant differences between cians in their iron management decisions. In conjunc-
the baseline and the second sample. The explanation tion with standard parameters, it could enable the
could be that the gap between both samples was only diagnosis of the patients who will benefit from ther-
4 weeks, but due to red cell lifespan, 6 to 8 weeks are apy rapidly and accurately.
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Hemoglobin Equivalent: an indicator of Efficacy and safety of intravenous iron
© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38, 360–365