Recovery from Neutropenia Can Be Predicted by the Immature Reticulocyte Fraction Several Days Before Neutrophil Recovery in Autologous Stem Cell Transplant Recipients

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ORIGINAL ARTICLE Recovery from neutropenia can be predicted by the immature reticulocyte fraction several days before neutrophil recovery in autologous stem cell transplant recipients

ML Grazziutti1, L Dong1, MH Miceli1, M Cottler-Fox2, SG Krishna1, A Fassas1, F van Rhee1, BM Barlogie1 and EJ Anaissie1

1Myeloma Institute of Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR, USA and 2Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

The duration of neutropenia (absolute neutrophil count Introduction (ANC) p100/ll)identifies cancer patients at risk for infection. A test that precedes ANCX100/ll would be of Infection is a serious complication of antineoplastic therapy clinical value. The immature reticulocyte fraction (IRF) particularly in the setting of profound and prolonged reflects erythroid engraftment and hence a recovering myelosuppression.1 The severity of myelosuppression has marrow. We evaluated the IRF as predictor of marrow been evaluated as absolute neutrophil count (ANC)p100/ recovery among 90 myeloma patients undergoing their ml, absolute monocyte count (AMoC)p100/ml,2,3 expected first and second (75 patients)melphalan-based autologous resolution of neutropenia in o10 days and ‘early evidence stem cell transplantation (Mel-ASCT). The time to IRF of marrow recovery.4 Unfortunately, it is difficult to predict doubling (IRF-D)preceded ANC X100/ll in 99% of time to resolution of neutropenia, and ‘early evidence of patients after the first Mel-ASCT by (mean7s.d.) marrow recovery’4 is not a clearly defined end point. A test 4.2371.96 days and in 97% of the patients after the that can predict marrow recovery in an individual patient second Mel-ASCT by 4.1171.95 days. We validated earlier than ANCX100/ml would be of clinical value. these findings in a group of 117 myeloma patients and 99 An increase in oral mucosal neutrophils precedes patients with various disorders undergoing ASCT with neutrophil recovery by 3 days5 but is time consuming. different conditioning regimens. We also compared the Other predictive tests of marrow recovery include hypophos- time to hypophosphatemia and to absolute monocyte phatemia6 and hypouricemia,7 both of which develop in countX100/ll to the time to ANCX100/ll. These approximately 60% of patients upon marrow recovery6,7 but markers were reached prior to this ANC end point in 55 precede ANCX500/ml by only 2–3 days and can be altered and 25% of patients but were almost always preceded by by phosphate replacement, renal failure and/or allopurinol IRF-D. We conclude that the IRF-D is a simple, therapy. inexpensive and widely available test that can predict Increasing numbers of circulating immature reticulocytes marrow recovery several days before ANCX100/ll. has been proposed as a marker of marrow recovery.8,9 Bone Marrow Transplantation (2006) 37, 403–409. During erythropoiesis, reticulocytes are released into the doi:10.1038/sj.bmt.1705251; published online 9 January circulation where they gradually lose their RNA, mature 2006 and evolve into erythrocytes. A higher proportion of Keywords: neutropenia; autologous stem cell transplant; circulating immature reticulocytes (high RNA content) immature reticulocyte fraction; bone marrow engraftment indicates recovering marrow activity and is quantitated by automated hematology cell analyzers.8,9 The fluorescence intensity of the entire reticulocyte population was initially reported as the reticulocyte maturation index or the mean fluorescent index.10,11 Reticulocytes have now been grouped into low, middle (MFR) or high fluorescent region (HFR) corresponding to the lower, middle and higher RNA content, respectively. The immature reticulocyte fraction Correspondence: Dr EJ Anaissie, Myeloma Institute of Research and Therapy, University of Arkansas for Medical Sciences, 4301 West (IRF) measures the MFR and HFR populations and is Markham Slot 776, Little Rock, AR 72205, USA. more reproducible than the HFR.12 E-mail: [email protected] We compared the time to doubling of IRF (IRF-D) to Preliminary results of this manuscript have been presented in abstract time to ANCX100/ml in 90 consecutive myeloma patients form at the 43rd Annual meeting of the Infectious Disease Society of America. San Francisco, October 6–9, 2005 undergoing melphalan-based tandem autologous stem Received 16 September 2005; revised and accepted 11 November 2005; cell transplantation (Mel-ASCT). The IRF-D was found published online 9 January 2006 to be a reliable predictor of ANC recovery and preceded Immature reticulocytes predict neutrophil recovery ML Grazziutti et al 404 ANCX100/ml by several days in the overwhelming majority Table 1 Kinetics of marrow recovery (immature reticulocyte of patients. We also validated the predictive role of the fraction) in relation to neutrophil engraftment in 99 patients under- IRF-D in two cohorts, one of 99 patients with various going autologous stem cell transplantation for various disorders underlying disorders undergoing ASCT with different according to underlying disease and conditioning regimen conditioning regimens and another of 117 myeloma Underlying disease N Days IRF-D P-value patients who were treated with Mel-ASCT. In this latter preceded group of myeloma patients, we compared the time to IRF- ANC4100/ml X D with the time to AMoC 100/ml and time to a 20% drop Non-Hodgkin’s lymphoma 40 5.3373.36 0.23 in serum phosphate over 24 h to the time to ANCX100/ml. Hodgkin’s disease 21 3.973.3 The effect of red blood cell transfusion and hemoglobin Waldenstrom’s macroglobulinemia 14 4.2172.52 (Hgb) level on IRF was also analyzed for the entire study AL-amyloidosis 10 472.91 7 population. Solid tumors 7 4.71 3.25 Others 8 6.7572.66

Conditioning regimen Patients and methods BEAM 53 4.8173.48 0.48 High-dose melphalan 25 4.2872.59 Others 22 5.4173.03 This study was conducted at the Myeloma Institute for Research and Therapy, the University of Arkansas for Medical Sciences, Little Rock, Arkansas. Institutional BEAM ¼ BCNU, etoposide, cytarabine and melphalan.14 Review Board approval was obtained for this study. Others: BEAM-based ¼ 11 patients (with addition of rituximab, velcade or fludarabine); melphalan-based ¼ five patients (with addition of fludarabine, gemcitabine, others); etoposide plus carboplatin (five patients), cytoxan/ Patient population busulfan (one patient). Derivation set: multiple myeloma, prospective. We conducted a prospective evaluation of 90 consecutive patients with newly diagnosed myeloma enrolled in our Total Therapy III protocol (February 2004–April 2005) and who were undergoing Mel-ASCT. The induction phase of Blood cell determinations Total Therapy III consists of two cycles of VDT- For measuring blood cells including the IRF, the Abbott PACE (bortezomib, dexamethasone, thalidomide, cisplatin, Cell-Dyn 4000 cell analyzer, (Abbott Diagnostic, Santa doxorubicin, cyclophosphamide and etoposide) with stem Clara, CA, USA) was used between 1998 and December cell collection followed by tandem Mel-ASCT. All 90 2004, and was subsequently replaced by the Sysmex XE- patients received their first Mel-ASCT and 75 received their 2100 Automated Hematology Analyzer Line (Sysmex second Mel-ASCT at the time of this report. All patients America Inc., Mundelein, IL, USA) (December 2004– had daily ANC and IRF values from day 0 ASCT to current). The ranges of normal IRF values for these two ANCX500/ml. units were 0.13–0.31 and 0.04–0.12, respectively.

Validation set – Group 1: multiple myeloma, retrospective. We conducted a retrospective review of the medical records of Definitions 382 consecutive patients (October 1998–December 2002) IRF: Two IRF end points taken after IRF nadir were used: with newly diagnosed myeloma enrolled in our Total (a) IRF doubling time (IRF-D) defined as the first of 2 Therapy II protocol13 which relies on four cycles of consecutive days in which the IRF value doubled and (b) induction chemotherapy followed by tandem Mel-ASCT. IRF normal value (IRF-N) which referred to normalization The 117 patients who had daily ANC, AMoC, serum of the IRF by two consecutive days. phosphate and IRF values from day 0 ASCT to Neutrophil recovery: ANCX100/ml. ANCX500/ml during their first Mel-ASCT were analyzed. Monocyte recovery: AMoCX100/ml. Hypophosphatemia: A 20% drop over 24 h in serum Validation set – Group 2: diseases other than myeloma, phosphate levels (PO4-20% drop). retrospective. We also reviewed the medical records of 188 patients (January 1998–July 2005) who underwent ASCT for diseases other than myeloma and received various Statistical analysis conditioning regimens. Of these patients, 99 had daily IRF All analyses were performed with SAS 9.1 (SAS Institute and ANC values from day 0 ASCT to ANCX500/ml. Inc., Cary, NC, USA). Significant level was chosen as 0.05. The underlying disease for this group of 99 patients We used the Student’s t-test to measure the difference included non-Hodgkin’s lymphoma (40%), Hodgkin’s between time to IRF-D and time to ANCX100/ml and disease (21%), Waldenstrom’s macroglobulinemia (14%), paired Student’s t-test to compare the IRF values before amyloidosis (10%), leukemia, solid tumor and others and after red blood cells transfusion. We used ANOVA to (15%). Conditioning regimens consisted of BEAM test for differences in IRF-D among patients with various (BCNU, etoposide, cytarabine and melphalan)14 (53%), underlying diseases and conditioning regimens (validation melphalan (70–200 mg/m2) (21%) and others (22%) set), and for differences in IRF-D according to Hgb levels (Table 1). divided in tertiles.

Bone Marrow Transplantation Immature reticulocytes predict neutrophil recovery ML Grazziutti et al 405 Results l m 2.6 1.1 2.78 Kinetics of ANC and reticulocytes after ASCT 1.4 7 7 7 7 1 to+6) 2 to+8) 100/ 2 to +10) Derivation set: 90 myeloma patients, 165 transplants, 3 to +17) À À 4 À À

prospective (Table 2). The IRF-D preceded ANC4100/ml Days IRF-N in 99% of the 90 patients undergoing their ﬁrst Mel-ASCT preceded ANC and in 97% of these 75 patients undergoing their second Mel-ASCT. The IRF-D preceded ANCX100/mlbyamean l melphalan-based autologous

7 7 m of 4.23 1.96 days and 4.11 1.95 days after the ﬁrst and cell transplants for various 4.84 3.6 1.97 2.2 2.41 2.9 3.46 4.08 ¼ 7 7 7

second Mel-ASCT, respectively. IRF-N preceded 7 500/ 2 to +9) (2; À ANCX100/ml in 99% of the patients after the ﬁrst and 4 Days IRF-D second Mel-ASCT by a mean time of 2.971.4 and 2.271.1 preceded ANC days, respectively.

Validation set – Group 1: 117 myeloma patients, 117 l m 2.793.25 7.6 7.83 1.95 4.88 transplants, retrospective. The IRF-D preceded ANCX 1.96 5.18 7 7 7 7 100/ 3 to +8) (5; 1 to +15) (7; 0–31) (3; 7 1 to +17) (7; 0–21) (4;

100/ml in 98% of patients by a mean of 5.49 2.79 days À 4 À À

X Days IRF-D

while the IRF-N preceded ANC 100/ml in 93% of preceded ANC patients, by a mean of 4.0872.78 days (Table 2). normalization of the IRF; Mel-ASCT

Validation set – Group 2: 99 non-myeloma patients, 99 ¼ l m 2.2 5.49 2.06 5.41 0.74 4.11 transplants, retrospective. The kinetics of marrow recov- 0.91 4.23 7 7 7 7 ery for the 99 patients in this group were comparable to 100/ those observed in the derivation set. The IRF-D preceded 4 ANCX100/ml in 95% of patients by a mean of 5.4173.21 days (Table 2). These results were observed among patients with different underlying diseases and conditioning regimens (Table 1). 2.4 12.73 2.72 11.31 1.82 10.56 1.66 11.37

X 7 7 7 The IRF-N preceded ANC 100/ml in 97% of patients 7 by a mean of 3.672.6 days.

Analysis of the results obtained with the two cell Days to IRF-D Days to ANC analyzers (patients transplanted before and after December 2004) yielded comparable results (data not shown). l m 2.3 7.24 2.14 5.92 1.13 6.45 Effect of red blood cell transfusion (RBCTx) on 1.05 7.13 7 7 7 7 100/ doubling of immature reticulocyte fraction; IRF-N (6; 3–9) (6; 3–14) (10; 9–12) (4; 7.3 IRFvalues o (7; 4–17) (6; 1–17) (11; 7–21) (5; (7; 4–19) (7; 4–18) (13; 9–24) (5; (6; 3–10) (7; 3–11) (11; 10–16) (4; 0–10) (5; 1–17) (3; The effect of RBCTx on IRF was evaluated by comparing ¼ the IRF values obtained immediately before RBCTx (same day) and the IRF values on the subsequent day after each

RBCTx administered. The period of evaluation included 99 7.56 ¼ n the day of ASCT infusion (day 0) till 1 day before doubling 75 5.68 90 5.53 ¼ ¼

of the IRF. A total of 38 RBCTx were given during this n period for the entire patient population. The IRF values n (mean7s.d.) before and after the 38 RBCTx were 0.1270.1 and 0.1370.09, respectively (P ¼ 0.63).

Effect of Hgb level on time to IRF-D We also examined the relationship between Hgb level and immature reticulocyte fraction; IRF-D ¼ 117 time to IRF-D by dividing patients into Hgb tertiles within s.d. (median; range). ¼ each group of patients and overall (Table 3). Higher Hgb 7 n Group 2, non-myeloma ASCT, Myeloma, 2nd Mel-ASCT, levels were associated with signiﬁcantly earlier time to IRF- D overall and within each group of patients except for the derivation set of the 75 patients undergoing their second Mel-ASCT. This latter group had signiﬁcantly higher Hgb levels than the other groups. As a result of the delayed time

to IRF-D among patients with lower Hgb levels, the period Kinetics of marrow recovery (immature reticulocyte fraction) in relation to neutrophil engraftment in 306 patients undergoing 381 autologous stem between time to IRF-D and to ANCX100/ml was shorter for these patients. For the overall population, the IRF-D absolute neutrophil count; IRF ¼ preceded the ANCX100/ml in each Hgb tertile (ﬁrst tertile Validation set, retrospective Group 1, myeloma 1st Mel-ASCT, stem cell transplantation. Table 2 Patient populationDerivation set, prospective Myeloma, 1st Mel-ASCT, The results are expressedANC by mean Days with ANC Hgbo9.7 mg/dl; second tertile Hgb 9.7–11.1 mg/dl and underlying disorders

Bone Marrow Transplantation Immature reticulocytes predict neutrophil recovery ML Grazziutti et al 406 Table 3 Effect of hemoglobin level on time of doubling of the immature reticulocyte fraction (IRF-D)

Patient population Tertiles of N Days to IRF- ANOVA hemoglobin D mean7s.d. level (mg/dl) (median; range) P-value Comparison of 1st, 2nd and 3rd tertilesa by t-test

Derivation set, Myeloma, ﬁrst Mel-ASCT, n ¼ 90 o9.6 32 7.8771.2 0.0026 1st vs others 0.0014 prospective (8; 5–10) 9.6–11.1 31 7.0371.84 1st vs 2nd 0.037 (7; 3–11) 411.1 27 6.4171.6 2nd vs 3rd 0.13 (6; 3–10) Myeloma, second Mel-ASCT, n ¼ 75 o10.5 28 6.7571.62 0.26 1st vs others 0.19 (7; 4–9) 10.5–12 25 6.4872.12 1st vs 2nd 0.57 (6; 4–12) 412 22 5.9571.21 2nd vs 3rd 0.3 (6; 3–8)

Validation set, Group 1, myeloma ﬁrst Mel-ASCT, n ¼ 117 o9.6 39 7.8271.82 0.0041 1st vs others 0.001 retrospective (8; 4–12) 9.6–11 43 6.7272.39 1st vs 2nd 0.014 (6; 4–18) 411 35 6.3171.6 2nd vs 3rd 0.37 (6; 4–12) Group 2, non-Myeloma ASCT, n ¼ 99 o9.4 35 6.5772.19 0.0015 1st vs others 0.06 (7; 1–11) 9.4–10.5 32 6.5973.06 1st vs 2nd 0.9 (6; 1–17) 410.5 33 4.5572.4 2nd vs 3rd 0.0017 (4; 1–9)

Overall o9.7 127 7.2872 o0.0001 1st vs others o0.0001 (7; 1–12) 9.7–11.1 121 6.5572.46 1st vs 2nd 0.006 (1–18) 411.1 129 5.9571.79 2nd vs 3rd 0.021 (1–10)

a1st tertile ¼ lowest hemoglobin level; 2nd tertile ¼ intermediate hemoglobin level; 3rd tertile ¼ highest hemoglobin level. Bold characters indicate statistically signiﬁcant values.

third tertile Hgb411.1 mg/dl) by 4.3972.63; 5.1172.79 Discussion and 5.672.32 days, respectively (P ¼ 0.001). Our results indicate that the doubling of the IRF from nadir can predict myeloid engraftment several days before Other predictors of myeloid recovery ANCX100/ml among patients undergoing ASCT for a The time to IRF-D and IRF-N was also compared to the variety of underlying diseases and with different condition- time to AMoCX100/ml and to PO4-20% drop among the ing regimens. 117 myeloma patients of the ﬁrst validation set. The mean Earlier studies have suggested that circulating immature time to AMoCX100/ml was 13.3973.5 days. Only 25% of reticulocytes can predict neutrophil recovery following patients reached AMoCX100/ml before ANCX100/mlbya chemotherapy. These studies however, utilized different mean of 1.4870.09 days while 32% of patients reached methods for measuring immature reticulocytes and/or both targets simultaneously. The remaining 43% of compared the IRF to ANCX500/ml and not to the more patients had an earlier recovery of ANCX100/ml than clinically relevant ANCX100/ml (Table 4).10,15–22 Since AMoCX100/ml. The time to AMoCX100/ml never pre- there are different types and manufacturers of blood count ceded IRF-D or IRF-N. analyzers with different reference values and calibration The PO4-20%-drop was reached before ANCX100/ml parameters,9,23 end points such as doubling (IRF-D) or in 65 patients (55 %) by a mean of 2.7271.66 days. The normalization (IRF-N) of the IRF can be used regardless mean time to PO4-20%-drop for these 65 patients was of equipment and would simplify and standardize result 9.672.18 days. The PO4-20%-drop was the ﬁrst single interpretation among investigators. variable predicting myeloid recovery in seven patients Other predictors of marrow recovery, AMoCX100/ml only (6%). and hypophosphatemia6 were not as predictive of marrow

Bone Marrow Transplantation Table 4 Immature reticulocytes as markers of engraftment after chemotherapy and hematopoietic transplantation

Parameter Immature reticulocyte end point Patients reaching reticulocyte end point/total Mean days the immature reticulocyte end point preceded Reference patients neutrophil or platelet engraftment

Allo-BMT Auto-BMT Auto-SCT ANCa PLTb

Mean RNA ﬂuorescence of reticulocytesc Reticulocyte maturation index (RMI) 20% increase from nadir — 12/20 — 3 — 8 42 22/37 — 2 10 15 Mean ﬂuorescence index (MFI) Doubling from nadir 22/22 17/20 — Allo-BMT ¼ 6.5 Allo-BMT ¼ 5.5 16 Auto-BMT ¼ 3 Auto-BMT ¼ 6 MFIX10 30/30 — 30/30 3 4 21

Fraction of immature reticulocytes Highly ﬂuorescent fraction (HFR)d X5% — — 6/13 CD34+ CD34+ selected ¼ 1 non- — 22 selected; 14/15 elected ¼ 4 non-selected X500/ml 27/27 7/21 — Allo-BMT ¼ 5—17 Auto-BMT ¼ 1 3 successive increases from nadir 8/14 10/22 — 5 — 18 X3% 30 — 30 1 2 21

Immature reticulocyte fraction (IRF)e 10% 30/30 — 30/30 3 4 21 X5% 26/29 54/58 — Allo-BMT ¼ 5—19 Auto-BMT ¼ 6 f Doubling from nadir (2 consecutive days) — — 371/381 Prospective: — Current Grazziutti recovery ML neutrophil predict reticulocytes Immature study Myeloma, 1st ASCT ¼ 4.2 Myeloma, 2nd ASCT ¼ 4.1

Retrospective: al et Myeloma, 1st ASCT ¼ 5.5 Non-myeloma ¼ 5.4

IRFg Doubling from nadir 10/10 9.5 8.5 20

Volume of reticulocytesh Mean reticular volume (MRV)/mean Abrupt increase 22/22 17/20 — Allo-BMT ¼ 8.5 Allo-BMT ¼ 7.5 16 corpuscular volume (MCV) Auto-BMT ¼ 4 Auto BMT ¼ 7 MRV 4110 ﬂ 30 — 30 3 4 21

ANC ¼ absolute neutrophil count; PLT ¼ platelets; BMT ¼ bone marrow transplantation; Auto ¼ autologous; Allo ¼ allogeneic; SCT ¼ stem cell transplantation. aANC engraftment endpoint 500 cells/ml except for the study of Davis8 whose ANC endpoint was 50 cells/ml and the present study: ANC endpoint of 100 cells/ml. bPLT engraftment endpoint 20,000 cells/ml. cHigher proportion of immature reticulocytes reflected by an increased mean fluorescence. oeMro Transplantation Marrow Bone d(Higher RNA content), increased fraction of HFR. eIncreased IRF (HFR+MFR). fTotal of 306 patients and 381 ASCT: 90 myeloma patients after first Mel-ASCT transplant, 75 also received 2nd Mel-ASCT, and 99 non-myeloma patients receiving one ASCT. gIncreased reticulocyte volume. hNon-fluorescent method, used new methylene blue as a dye. 407 Immature reticulocytes predict neutrophil recovery ML Grazziutti et al 408 recovery as IRF-D or IRF-N. We therefore recommend the profound neutropenia following high-dose myelosuppressive use of IRF-D or IRF-N for an earlier prediction of marrow chemotherapy. Am J Hematol 2003; 72: 13–19. recovery and have adopted the use of the IRF in our daily 6 Raanani P, Levi I, Holzman F, Grotto I, Brok-Simoni F, clinical practice. Avigdor A et al. Engraftment-associated hypophosphatemia – Red blood cell transfusion did not appear to affect the the role of cytokine release and steep leukocyte rise post Bone Marrow Transplant IRF values. However, the time to IRF-D was one day stem cell transplantation. 2001; 27: 311–317. longer for patients with lower Hgb levels probably 7 Uckan D, Cetin M, Dida A, Batu A, Tuncer M, Tezcan I. reflecting a poor bone marrow reserve. Even among these Hypophosphatemia and hypouricemia in pediatric allogeneic patients, the IRF-D still preceded ANC recovery. bone marrow transplant recipients. Pediatr Transplant 2003; 7: Routine measurement of the IRF following chemother- 98–101. apy is likely to have a significant impact on patient 8 Davis BH, Bigelow NC. Flow cytometric reticulocyte quanti- management. A neutropenic patient with fever and fication using thiazole orange provides clinically useful increasing IRF values is expected to reach ANCX100/ml reticulocyte maturity index. Arch Pathol Lab Med 1989; 113: within an average of 4 days and could be managed more 684–689. conservatively, and without additional testing or intensive 9 Lacombe F, Lacoste L, Vial JP, Briais A, Reiffers J, Boisseau MR et al. Automated reticulocyte counting and immature empirical therapies,4 unless otherwise clinically warranted. reticulocyte fraction measurement. Comparison of ABX By contrast, a febrile neutropenic patient with persistently PENTRA 120 Retic, Sysmex R-2000, flow cytometry, and low IRF values is more likely to have prolonged manual counts. Am J Clin Pathol 1999; 112: 677–686. neutropenia and can therefore be targeted for intensive 10 Davis BH, Bigelow N, Ball ED, Mills L, Cornwell III GG. diagnostic, prophylactic and therapeutic measures, if Utility of flow cytometric reticulocyte quantification as a clinically indicated. The IRF-N can be particularly useful predictor of engraftment in autologous bone marrow trans- in patients lacking the daily reticulocyte values needed to plantation. Am J Hematol 1989; 32: 81–87. calculate the IRF-D. 11 Davis BH. Immature reticulocyte fraction (IRF): by any name, Our findings also have implications for clinical research a useful clinical parameter of erythropoietic activity. Lab including the identification of high-risk patients for Hematol 1996; 2: 2–8. enrollment into clinical trials of antimicrobial agents. 12 Chang CC, Kass L. Clinical significance of immature reticulocyte fraction determined by automated reticulocyte Concerns about including patients with impending marrow counting. Am J Clin Pathol 1997; 108: 69–73. recovery into trials evaluating the efficacy of antimicrobial 13 Shaughnessy Jr J, Tian E, Sawyer J, McCoy J, Tricot G, agents have hampered our ability to assess the true efficacy Jacobson J et al. Prognostic impact of cytogenetic and of the agent used vs the beneficial effect of resolving interphase fluorescence in situ hybridization-defined chromo- neutropenia. The IRF tests will likely enhance our ability to some 13 deletion in multiple myeloma: early results of total determine the role of these two factors and thus potentially therapy II. Br J Haematol 2003; 120: 44–52. reduce the sample size required for such studies.24,25 14 Martin A, Perez-Simon JA, Caballero MD, Lopez-Holgado N, In conclusion, our data suggest that the IRF, a simple, Vazquez L, del Canizo MC et al. Effect of pre-transplant reproducible, inexpensive and widely available test, can cumulative doses of chemotherapeutic drugs on early and long- predict myeloid recovery several days before circulating term hematological recovery after autologous bone-marrow X transplantation for lymphoma. Haematologica 2005; 90: ANC 100/ml in most patients undergoing autologous stem 78–85. cell transplantation. The IRF could potentially be useful in 15 Dalal BI, Stockford GR, Naiman SC, Spinelli JJ, Phillips GL. the management of neutropenic cancer patients and in the Criteria for marrow engraftment: comparison of reticulocyte investigation of antimicrobial therapies in this setting. maturity index with conventional parameters. Bone Marrow Prospective trials assessing the contribution of the IRF to Transplant 1996; 17: 91–92. the clinical management of ASCT patients are warranted. 16 Noronha JF, De Souza CA, Vigorito AC, Aranha FJ, Zulli R, Miranda EC et al. Immature reticulocytes as an early predictor of engraftment in autologous and allogeneic bone marrow transplantation. Clin Lab Haematol 2003; 25: References 47–54. 17 Greinix HT, Linkesch W, Keil F, Kalhs P, Schwarzinger I, 1 Offidani M, Corvatta L, Olivieri A, Rupoli S, Frayfer J, Mele Schneider B et al. Early detection of hematopoietic engraft- A et al. Infectious complications after autologous peripheral ment after bone marrow and peripheral blood stem cell blood progenitor cell transplantation followed by G-CSF. transplantation by highly fluorescent reticulocyte counts. Bone Bone Marrow Transplant 1999; 24: 1079–1087. Marrow Transplant 1994; 14: 307–313. 2 Rackoff WR, Gonin R, Robinson C, Kreissman SG, Breitfeld 18 Davies SV, Cavill I, Bentley N, Fegan CD, Poynton CH, PB. Predicting the risk of bacteremia in childen with fever and Whittaker JA. Evaluation of erythropoiesis after bone marrow neutropenia. J Clin Oncol 1996; 14: 919–924. transplantation: quantitative reticulocyte counting. Br J 3 Klaassen RJ, Goodman TR, Pham B, Doyle JJ. ‘Low-risk’ Haematol 1992; 81: 12–17. prediction rule for pediatric oncology patients presenting with 19 Spanish Multicentric Study Group for Hematopoietic fever and neutropenia. J Clin Oncol 2000; 18: 1012–1019. Recovery. Flow cytometric reticulocyte quantification in the 4 Hughes WT, Armstrong D, Bodey GP, Bow EJ, Brown AE, evaluation of hematologic recovery. Eur J Haematol 1994; Calandra T et al. 2002 guidelines for the use of antimicrobial 53: 293–297. agents in neutropenic patients with cancer. Clin Infect Dis 20 Grotto HZ, Vigoritto AC, Noronha JF, Lima GA. Immature 2002; 34: 730–751. reticulocyte fraction as a criterion for marrow engraftment. 5 Akpek G, Knight RD, Wright DG. Use of oral mucosal Evaluation of a semi-automated reticulocyte counting method. neutrophil counts to detect the onset and resolution of Clin Lab Haematol 1999; 21: 285–287.

Bone Marrow Transplantation Immature reticulocytes predict neutrophil recovery ML Grazziutti et al 409 21 Torres A, Sanchez J, Lakomsky D, Serrano J, Alvarez MA, ming immature reticulocyte fraction: comparison in Martin C et al. Assessment of hematologic progenitor diagnosis of bone marrow aplasia. Am J Clin Pathol 2002; engraftment by complete reticulocyte maturation parameters 117: 871–879. after autologous and allogeneic hematopoietic stem cell 24 Marr KA. Issues in the design of the ﬂuconazole pro- transplantation. Haematologica 2001; 86: 24–29. phylaxis trials in patients undergoing hematopoietic stem 22 Sica S, Salutari P, Laurenti L, Ortu La Barbera E, Zini G, cell transplantation. Clin Infect Dis 2004; 39 (Suppl 4): d’Onofrio G et al. Highly ﬂuorescent reticulocytes after S170–S175. CD34+ peripheral blood progenitor cell transplantation. Bone 25 Mahfouz T, Anaissie E. Prevention of fungal infections in the Marrow Transplant 1998; 21: 361–364. immunocompromised host. Curr Opin Invest Drugs 2003; 4: 23 Buttarello M, Bulian P, Farina G, Petris MG, Temporin V, 974–990. Toffolo L. Five fully automated methods for perfor-

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