Bone Marrow Transplantation (2006) 37, 403–409 & 2006 Nature Publishing Group All rights reserved 0268-3369/06 $30.00 www.nature.com/bmt 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 con- ducted 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 between time to IRF-Dto IRF-D and among to patients with ANC levels lower than Hgb the levels, other the groups.Mel-ASCT. period As This a latter result of groupderivation the had delayed set significantly time higher of Hgb D the overall 75 and within patientslevels each were undergoing group associated their with of significantly patients second each earlier except group time for to of IRF- the patientstime to and IRF-D overall by (Table dividingWe patients 3).