Flow cytometric reticulocyte quantification in the evaluation of hematologic recover

Abstract: The role of flow cytometric reticulocyte (RET) counting and the immature RET fractions (IRF) in the evaluation of hematopoietic recovery following chemoradiotherapy-induced aplasia was studied. RET counts and IRF were studied using an automated flow cytometric reticulocyte counter (Sysmex R-2000) in three groups of patients: 58 patients undergoing an autologous bone marrow transplantation (ABMT group), 28 of whom received granulocyte colony-stimulating factor (G-CSF); 28 patients undergoing an allogeneic bone marrow transplantation (BMT group); and 28 patients receiving remission-induction chemotherapy for acute leukemia (CHEMO group). To evaluate the IRF the percentages of RET fractions with middle and high fluorescence reticulocyte (MFR and HFR, respectively) were used. A rising IRF (expressed as the percentage of MFR ± HFR) was the first sign of hematopoietic recovery (ABMT group, IRF 9 days versus 18 days for the absolute neutrophil count (ANC); BMT group, 15 versus 18 days; CHEMO group, 9 versus 11 days). When recovery of the ANC (>0.5 times 109/1) was compared with that of the IRF (MFR ± HFR > 5%), statistically significant differences were found in all three groups. Additionally, 93.1% of the ABMT, 92% of the BMT and 91.2% of the CHEMO recovered the IRF before the ANC. In conclusion, an elevation in the percentage of IRF is the first sign of hematologic recovery in the majority of patients receiving remission-induction chemotherapy and the first sign of engraftment in those submitted to ABMT or BMT. Serial automated flow cytometric quantitative reticulocyte counting provides a useful and early measure of erythropoiesis indicative of hematopoietic reconstitution or successful bone marrow engraftment following marrow transplantation.     

采用未成熟网织红细胞指数监测恶性肿瘤患者放疗前、后骨髓功能恢复的临床价值

目的　探讨采用未成熟网织红细胞指数 (IRF)监测恶性肿瘤患者放疗前、后骨髓功能恢复的临床价值。方法　采用XE 2 10 0型血液细胞分析仪检测未成熟网织红细胞指数 (IRF)、网织红细胞比率 (RET)和嗜中性粒细胞计数 (NEUT )。结果　患者组放疗后第 10天以上 3项指标均显著低于对照组 (P <0 .0 1) ,放疗后第 15天IRF开始增高 ,并显著高于第 10天的水平 (P <0 .0 1) ,而RET、NEUT未发生显著改变 (P >0 .0 5 ) ,患者组放疗后第 2 0天IRF继续升高 ,并显著高于第 15天的水平 (P <0 .0 1) ,RET和NEUT也开始增高 ,但与第 15天比较 ,无显著性差异 (P >0 .0 5 )。患者组放疗后第 2 5天 ,IRF已经基本恢复至放疗前水平 ,RET和NEUT也开始显著升高 ,但仍低于放疗前水平 (P <0 .0 1)。结论　肿瘤患者IRF升高较RET和NEUT要早。未成熟网织红细胞指数是放、化疗时反映骨髓抑制和恢复较敏感的指标     

Long-lasting decrease of marrow and circulating long-term culture initiating cells after allogeneic bone marrow transplant.

We investigated bone marrow (BM) and circulating (PB) hematopoietic progenitor cells in 37 normal donors and in 25 patients 1 to 8 years after successful allogeneic bone marrow transplant. At the time of testing, transplanted patients had normal blood counts and bone marrow cellularity. By flow cytometry, BM CD34+ cells were found to be three- to four-fold decreased in transplanted patients compared to normal donors, while the number of PB CD34+ cells was the same as in normal donors. Using a methylcellulose colony assay, primary BM colony-forming cells (CFU-GM) were decreased 2.1-fold, whereas PB CFU-GM were only marginally decreased. In a long-term culture initiating cell (LTC-IC) assay, an eight-fold decrease of early progenitor cells was observed in the marrow of transplanted patients compared to normal donors, and a five-fold decrease was documented in peripheral blood. We found that the BM LTC-IC cell number correlated with concurrently determined BM CD34+ cells and committed progenitor cell number (measured as CFU-GM) and with PB LTC-IC number, but not with PB CFU-GM and CD34+ cells. We conclude that marrow and circulating early stem cell compartments, as measured by the LTC-IC assay, are greatly and permanently depressed following bone marrow transplant. The correlation between BM and PB LTC-IC indicates that the enumeration of circulating LTC-IC can be used as a measure of the stem cell compartment in the bone marrow after transplant. It seems that the deficiency of the most immature progenitor cells persists forever after successful bone marrow transplant; this means that a complete hematopoietic reconstitution can be sustained by a reduced stem cell pool.     

Erroneously elevated immature reticulocyte counts in leukemic patients determined using a Sysmex XE-2100 hematology analyzer

The immature reticulocyte fraction (IRF) in peripheral blood, as determined by automated reticulocyte analysis, is calculated using the sum of medium and highly fluorescent reticulocyte numbers and provides information about erythropoietic activity in bone marrow. The purpose of this study was to investigate erroneously elevated IRF in leukemic patients, as determined using a Sysmex XE-2100 hematology analyzer (Sysmex, Kobe, Japan). Normal reticulocyte scattergram patterns show regions corresponding to reticulocytes located between matured RBCs and an upper particle (UPP) region, which show a continuum of non-separated fraction. The UPP represents erythroblasts and some immature reticulocytes. As a control group, peripheral blood was taken from patients with benign hematologic diseases, and their reticulocyte scattergrams all showed a normal pattern; UPP values were all less than 100. However, the reticulocyte scattergrams of 5 of 11 leukemia patients showed abnormal patterns and displayed a gap between RBC and reticulocyte regions. Three patients showed a flag with a message such as “RET Abn Scattergram”. IRF results were elevated in these five patients, and their UPP values were above 100. For the remaining six leukemia patients with a normal reticulocyte scattergram pattern, immature reticulocytes were not markedly increased, and UPP values were less than 100. The findings of the present study demonstrate that IRF results may be erroneously elevated in leukemia patients and indicate that hematologists should examine reticulocyte scattergrams and UPP values carefully.     

Hematopoietic growth factors in the pathogenesis and for the treatment of aplastic anemia

The production and release of hematopoietic growth factors from bone marrow stromas established in vitro from patients with aplastic anemia is normal or increased. Addition of hematopoietic growth factors to aplastic anemia bone marrow cells results in only modest increases in colony growth, with the exception of granulocyte colony-stimulating factor (G-CSF), which corrects their impaired cloning efficiency to normal. Most clinical data on the use of hematopoietic growth factors in aplastic anemia have derived from uncontrolled and small single-arm studies or case reports. Sustained trilineage hematologic responses have not been observed when hematopoietic growth factors have been used alone or in combination. Serious side effects have been reported for most of the hematopoietic growth factors in patients with aplastic anemia, with the exception of G-CSF. There is a major concern that they may further increase the risk of clonal disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Hematopoietic growth factors should not be used alone in newly diagnosed patients as specific treatment for aplastic anemia, and their use in combination with immunosuppressive therapy should be confined to multicenter, prospective randomized studies.     

Ineffective hematopoiesis linked with a mitochondrial tRNA mutation (G3242A) in a patient with myelodysplastic syndrome

In a patient with refractory anemia with excess blasts (RAEB), a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly, the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However, PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays, CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis, thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient.     

Sustained long-term hematologic recovery despite a marked quantitative defect in the stem cell compartment of patients with aplastic anemia after immunosuppressive therapy

Previously, we reported that patients with aplastic anemia (AA) have profoundly decreased numbers of hematopoietic progenitor and stem cells as measured in the long-term culture initiating cell (LTC-IC) assay (Blood 1996;88:1983-1991). We now present results of a long-term prospective study of LTC-IC numbers in peripheral blood (PB) and bone marrow (BM) of patients treated with antithymocyte globulin and cyclosporin A. Numbers of secondary colony forming cells (secondary CFC) in long-term bone marrow culture (LTBMC) were used to quantitate LTC-IC. BM (N = 35) and PB (N = 41) secondary CFC from both untreated severe AA patients and responders to immunosuppressive therapy who were sampled up to 6 years after initial treatment were compared. Normal controls showed 148 +/- 38 (N = 17) and 16 +/- 3 (N= 14) secondary CFC per 10(6) in BM and PB, respectively. In cross-sectional analysis, prior to therapy, AA patients showed 2.6 +/- 1 (mean +/- SD) secondary CFC/10(6) BM MNC; within the first year after initial treatment (N = 14), secondary CFC number rose modestly to 8.2 +/- 2.2/10(6) MNC, and further increased to 15.8 +/- 7 (N = 17) at 2 years and 16.2 +/- 7/10(6) MNC (N = 25) 3 years after treatment. There was no further improvement in the secondary CFC numbers at 4, 5, and > or =6 years (N = 37). Thus, while BM secondary CFC increased about 6-fold at 3 years post-therapy compared to presentation, they remained about only 10% of normal despite hematologic recovery. Similar data were obtained for PB, with approximately 4-fold increase in secondary CFC numbers within 2 years of therapy, to about 15% of normal values. We confirmed these observations in patients studied serially over a period of 4 years: initial secondary CFC were 2.35 +/- 1/10(6) BM MNC and 0.11 +/- 0.1/10(6) PB MNC improving to an average of 6 +/- 1. 2 (BM; N = 12) and 2.4 +/- 1/10(6) MNC (PB; N = 14). In many cases of partial recovery, PB counts improve but do not normalize. When we studied secondary CFC numbers only in patients who achieved complete normalization of PB counts (ANC >1,500/mm(3); platelets >10(5)/mm(3) and absolute reticulocytes >5 x 10(4)/mm(3)), BM secondary CFC were significantly higher than in patients with partial recovery; the PB secondary CFC number was modestly increased but remained below the normal values. Within the group of patients with complete recovery, there was no correlation between the secondary CFC and time after initial treatment. In addition, there also was no correlation between the secondary CFC number at presentation and the quality of hematopoietic recovery. Despite a limited expansion potential of a severely reduced stem cell pool, their numbers are sufficient to provide a long-term supply of mature blood cells. Am. J. Hematol. 65:123-131, 2000. Published 2000 Wiley-Liss, Inc.     

Immune dysregulation and dyserythropoiesis in the myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are clonal disorders characterised by ineffective haematopoiesis with high risk of leukaemia progression. The relevance of immune-dysregulation for emergence, dominance and progression of dysplastic clones has been suggested, but valuable criteria to obtain insight into these connections are lacking. This study showed significant increase of CD8 lymphocytes and mature B cells in the bone marrow (BM) compared to peripheral blood (PB) of low risk MDS patients. Different BM levels of Regulatory T cells (Treg) identified two sub-groups in these patients; only the sub-group with lower Treg percentage showed BM recruitment of CD8 lymphocytes. Different levels of CD54 on BM CD8 cells revealed two sub-groups of Intermediate-1 (Int-1) patients. The sub-group with higher CD54 expression on BM CD8 showed high levels of this molecule also on CD4 cells. BM recruitment of CD8 lymphocytes in the low risk group and/or the presence of high CD54 expression on BM CD8 in Int-1 patients were associated with more pronounced dyserythropoiesis and erythropoietin treatment. Our data shed light on the involvement of immune-mediated mechanisms in Low and Int-1 risk MDS patients and suggest that BM versus PB levels of immune effectors could represent useful criteria for a more homogeneous grouping of MDS patients.     

骨髓小粒对AA、MDS诊断及非典型再障、MDS、增生性贫血鉴别诊断的意义

目的 :探讨骨髓小粒对再生障碍性贫血 (AA )、骨髓增生异常综合征 (MDS)、增生性贫血 (简称增贫 )的鉴别诊断价值。方法 :对 10例 AA、5例 MDS、5例增贫及 10例正常人的骨髓小粒进行研究。结果 :AA患者的骨髓小粒中非造血细胞所占比例明显高于 MDS、增贫和正常人 ,差异有显著性 (P <0 .0 1)。 MDS患者的骨髓小粒中的原粒 早幼粒比例明显高于其他三组 (P <0 .0 1) ,并出现多个系统的病态造血。增生性贫血患者的骨髓小粒仅表现为红系的增生和红系的病态造血。结论 :骨髓小粒的观察对 AA、MDS、增贫的鉴别诊断有着重要的意义。     

Analysis of CD34-positive cells in bone marrow from patients with myelodysplastic syndromes and acute myeloid leukemia and in normal individuals: a comparison between FACS analysis and immunohistochemistry

Abstract: In patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), expression of the hematopoietic stem cell marker CD34 has been associated with a poorer prognosis. CD34 is usually analyzed by flow cytometry (FC), but may also be analyzed using immunohistochemistry (IH). The present study was undertaken to compare these 2 methods. Bone marrow from 16 patients with MDS and 12 with AML and from 12 healthy young volunteers was studied. The expression of CD34 was analyzed with FC on fresh bone marrow cells and with IH on sections of paraffin-embedded bone marrow. The correlation between FC and IH was good both for patients with MDS (p<0.0001) and AML (p<0.01). However, in patients with a high number of CD34-positive cells, the FC method seemed to result in a higher percentage of positive cells compared to the IH method. In normal bone marrow, the ratio between the percentage of CD34-positive cells and the percentage of bone marrow blasts was approximately 0.8. In the whole group of MDS patients, this ratio was 1:1, while in patients with refractory anemia (RA) and ring sideroblastic anemia (RAS) it was 1.6. Patients with MDS differed significantly from patients with de novo AML, who showed a ratio of only 0.23 (p<0.01). We conclude that the FC and IH methods for measuring expression of CD34 are well-correlated in MDS and reasonably well correlated in AML. A stem cell phenotype is more commonly expressed on precursor cells from patients with MDS than from patients with AML.     

Cell cycling stress in the monocyte line as a risk factor for progression of the aplastic anaemia/paroxysmal nocturnal haemoglobinuria syndrome to myelodysplastic syndrome

Severe aplastic anaemia (SAA) causes permanent stem cell damage from which patients do not recover after treatment with antilymphocyte globulin (ALG). To produce peripheral blood values compatible with life, the few remaining stem and precursor cells are put under stress. We defined a 'stress factor' (SF) for various haematopoietic lines as the ratio of the corresponding peripheral blood (PB) value to the total colony number in short-term bone marrow cultures from 86 patients with different outcomes. Both values are expressed as percentage of normal, hence SF averages 1 in normal steady-state haematopoiesis. SF was elevated in all patients, from 2-to 40-fold, with wide variations in different patient groups and striking differences between haematopoietic lineages. In long-term disease-free survivors after ALG (group 1) the mean total colony count was 19% of normal, with a significantly higher proportion of erythroid burst-forming units compared to normal. They had ineffective erythropoiesis with haemoglobin (Hb) values below, and reticulocyte counts above normal; platelet counts were 67% of normal. In contrast, monocyte counts were in the high normal range, resulting in a high SF (18.7 +/- 1.9) for monocytes. In patients who developed paroxysmal nocturnal haemoglobinuria (PNH) after ALG (group 2), ineffective erythropoiesis, reflecting haemolysis, was more pronounced and they had striking relative monocytosis, resulting in a significantly higher SF for monocytes (33.7 +/- 5.7) compared with group 1 (p < 0.0001). High monocyte counts most likely reflect the relative resistance of nucleated cells to complement, compared with red cells and platelets. Patients who developed myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML) after ALG, with or without PNH (group 3), had the highest SF for monocytes (39 +/- 10). They also had neutrophil counts in the upper range, or above normal, resulting in a high SF for neutrophils: 32 +/- 19. In patients with persisting or relapsing-remitting pancytopenia without a clinically detectable clonal disorder (group 4), all values were strikingly similar to those of the PNH group. In patients who achieved normal PB values after uncomplicated bone marrow transplantation (group 5), the SF averaged 3, but they also had ineffective erythropoiesis and mild relative monocytosis, a possible sign of occult PNH. We conclude that all patients after treatment of SAA have ineffective erythropoiesis and relative monocytosis, and that these abnormalities probably reflect PNH. We suggest that the resulting high SF for the leukocyte - particularly the monocyte line - predisposes to the development of MDS/AML. We discuss how these results may provide some of the missing pieces in the puzzle of SAA/PNH.     

Effects of recombinant human interleukin-3 on human hematopoietic progenitor and precursor cells in vivo

DNA-synthesis rates and concentrations of bone marrow (BM) and peripheral blood (PB) progenitor cells were studied in 22 patients treated with recombinant human interleukin-3 (rhIL3) as part of a clinical phase I/II study. Recombinant hIL3 at doses of 60 to 500 micrograms/m2 was administered by subcutaneous bolus injection for 15 days to 13 patients with solid tumors and preserved hematopoietic function and to nine patients with bone marrow failure, including five with myelodysplastic syndromes. Following treatment with rhIL3, the percentage of actively cycling BM erythroid (BFU-E) and multilineage (CFU-GEMM) progenitors in patients with preserved hematopoietic function increased from 16% to 36% (P less than .05) and from 10% to 40% (P less than .01), respectively. The DNA-synthesis rates of early and late granulocyte macrophage progenitor cells increased from 11% to 26% (CFU-GM day 14; P less than .02) and from 13% to 30% (CFU-GM day 7; P less than .05). There was an increase in BM cellularity from 37% to 58%, and of the myeloid to erythroid ratio from 1.4 to 3.2, while the concentration of marrow progenitors on a per cell basis was unchanged or slightly decreased. The frequencies of blast cells in the BM were unchanged. Mean levels of PB CFU-GM day 14 and CFU-GEMM were 100% and 72% above baseline values after 7 days of rhIL3 but only 25% and 28% above initial levels at the end of treatment. Peripheral blood BFU-E were reduced in the majority of patients with normal marrow after both 7 and 15 days of rhIL3. No augmentation of circulating BFU-E and CFU-GEMM was seen in 5 patients with MDS who had few or no PB BFU-E or CFU-GEMM initially. Total leukocyte, neutrophil, and eosinophil counts increased significantly (P less than .01) in 21 of 22 patients with a peak response after a median of 13 days of rhIL3. While a small increase in reticulocytes was not accompanied by an elevation of the hemoglobin or hematocrit, platelet counts increased by 50% in patients with preserved marrow function. Thus, rhIL3 induces a multilineage response in vivo, apparently by stimulating proliferation of multipotential and lineage-restricted progenitors. It remains to be determined whether this is due to direct or indirect effects on the progenitor cells.     

Progenitor content of autologous grafts: mobilized bone marrow vs mobilized blood

The progenitor content of autologous peripheral blood progenitor and stem cell collections is a major determinant of prompt hematopoietic recovery following autologous stem cell transplantation. We analyzed unstimulated bone marrow (BM) and peripheral blood (PB) apheresis products in comparison to those collected following G-CSF or GM-CSF stimulation. We quantitated their committed (CFU-GM) and primitive (long-term culture-initiating cells, LTC-IC) progenitors in relation to hematologic recovery in 63 patients undergoing autografting for lymphoid malignancies. G-CSF, but not GM-CSF, substantially enriched the committed progenitor content (2.5-3.6-fold) of both PB and BM grafts. G-CSF also enriched the LTC-IC content of BM and PB compared to control grafts. GM-CSF augmented (11.5-fold) the LTC-IC content of stimulated BM, but not GM-CSF-mobilized PB. Neutrophil recovery was substantially quicker in recipients of BM or PB mobilized with G-CSF or GM-CSF. In contrast, red cell and platelet recovery was accelerated in recipients of GM-CSF-stimulated BM (but not PB) and G-CSF-stimulated PB (but not BM). No direct correlation between progenitor dose and hematopoietic recovery for neutrophils, platelets or red cells was observed. Cytokine stimulation can augment the committed and more primitive multilineage progenitor content of BM and PB grafts, to a differing extent. The uncertain relationship with multilineage myeloid recovery emphasizes the limitations in using clonogenic progenitor analyses to assess the adequacy of an autologous graft prior to transplantation.     

Hematologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study [see comments]

We analyzed data from 388 subjects with Fanconi anemia reported to the International Fanconi Anemia Registry (IFAR). Of those, 332 developed hematologic abnormalities at a median age of 7 years (range, birth to 31 years). Actuarial risk of developing hematopoietic abnormalities was 98% (95% confidence interval, 93% to 99%) by 40 years of age. Common hematologic abnormalities were thrombocytopenia and pancytopenia. These were often associated with decreased bone marrow (BM) cellularity (75% of cases studied). Clonal cytogenetic abnormalities developed in 23 of 68 persons with BM failure who had adequate studies. Actuarial risk of clonal cytogenetic abnormalities during BM failure was 67% (47% to 87%) by 30 years of age. Fifty-nine subjects developed myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). Actuarial risk of MDS or AML was 52% (37% to 67%) by 40 years of age. Risk was higher in persons with than in those without a prior clonal cytogenetic abnormality (3% [0% to 9%] v 35% [0% to 79%]; P = .006). One hundred twenty persons died of hematologic causes including BM failure, MDS or AML and treatment related complications. Actuarial risk of death from hematologic causes was 81% (67% to 90%) by 40 years of age.     

Prognostic parameters in myelodysplastic syndromes: a multiple regression analysis

The influence on survival of 21 basic clinical and hematologic parameters was evaluated in 72 patients with previously untreated myelodysplastic syndromes (MDS). Only five parameters were significant by both survival curves and multiple regression analyses: hemoglobin level, bone marrow (BM) cellularity (estimated from trephine BM biopsies), BM blast percentage, age and BM erythro/myeloid (E/M) ratio. Using these parameters, multiple regression analysis enabled us to predict 34% of the survival of all MDS patients (p less than 0.002), 38% of that of patients who had stable disease (p less than 0.04) and over 80% of that of patients who developed acute leukemia (p less than 0.02). High BM cellularity was the most predictive factor for the development of leukemia. No factor was predictive for patients who died of cytopenic or other complications.     

再生障碍性贫血和骨髓增生异常综合征患者骨髓CD34+细胞及其粒细胞集落刺激因子受体的研究

目的:检测再生障碍性贫血(AA)和骨髓增生异常综合征(MDS)患者CD34 细胞占骨髓单个核细胞(BMMNC)的比率及其表面粒细胞集落刺激因子受体(G-CSFR)的表达率。方法:用流式细胞术(FCM)检测13例AA、22例MDS及12例非血液病患者CD34 细胞占BMMNC的比率及其表面G-CSFR的表达率。结果:AA组与对照组、AA组与MDS组、MDS-难治性贫血(RA)组与难治性贫血伴原始细胞增多(RAEB)组BMMNC中CD34 细胞的比率比较差异有统计学意义(P<0.05),但G-CSFR的表达率差异无统计学意义(P>0.05)。多数重型AA(SAA)患者(3/4)及少数慢性AA(CAA)患者(1/9)BMMNC中的CD34 细胞少于0.1%。多数G-CSFR表达率低(<14%)的患者(7/9)外周血中性粒细胞减少;而表达率正常(14.0%~28.9%)的患者(1/6)很少见;表达率高(>28.9%)的患者(3/7)也可存在中性粒细胞减少。结论:造血干细胞减少是AA的主要发病机制之一,其表面G-CSFR的表达率不是影响AA的主要因素;MDS患者CD34 细胞比率升高是一个预后不良的指标,G-CSFR的检测可部分解释MDS患者外周血中性粒细胞减少的原因。     

Juvenile chronic myelogenous leukemia: characterization of the disease using cell cultures

To characterize juvenile chronic myelogenous leukemia (JCML), the proliferative properties of bone marrow (BM) and peripheral blood (PB) cells from nine patients were studied using assays for CFU-C and CFU- GEMM and liquid cultures. All specimens showed two reproducible abnormalities: impaired growth of normal hematopoietic progenitors and excessive proliferation of monocyte-macrophage colonies in the absence of exogenous colony-stimulating activity (CSA). Cytogenetic studies in one patient indicated that the CFU-C were malignant because BM cells at diagnosis and monocyte-macrophage colonies showed an abnormal karyotype, whereas PB lymphocytes did not. In contrast to JCML, PB from six adults with Philadelphia (Ph1) chromosome-positive chronic myelogenous leukemia (Ph1 + CML) yielded CSA-dependent CFU-C colonies which were composed of granulocytes, macrophages, or both, as well as exuberant growth of BFU-E colonies. Co-cultures of JCML BM adherent or nonadherent cells with normal BM resulted in suppression of normal hematopoietic colony formation. Supernatant from JCML adherent cells in liquid culture or plasma from newly diagnosed untreated JCML patients also suppressed control BM colony growth in a dose-dependent manner. These findings confirm that JCML is a malignant disorder of monocytic lineage and suggest that the mechanism of hematopoietic failure in JCML is mediated by an inhibitory monokine secreted by malignant JCML cells.     

Characterization of CD34+ peripheral blood cells from healthy adults mobilized by recombinant human granulocyte colony-stimulating factor

Primed peripheral blood hematopoietic stem cells (PBSC) generate and sustain lymphohematopoiesis in myeloablated animals, and recent reports indicate that allogeneic transplantation using PBSC grafts may be feasible in humans. A major concern with the use of PBSC transplants is that permanent engraftment may be limited because of lack of sufficient numbers of primitive progenitor cells in the graft. In the present study, in vitro colony formation and immunophenotype of CD34+ cells in PB of healthy adults during short-term granulocyte colony-stimulating factor (G-CSF) administration were compared with that of CD34+ cells in normal bone marrow (BM). The number of CD34+ cells mobilized to PB peaked at day 4 or 5 of G-CSF administration. The phenotypic profile of CD34+ PB cells showed a substantial increase in the percentage of CD34+CD13+ and CD34+CD33+ cells (myeloid progenitors) and a corresponding decrease in the percentage of CD34+CD10+ and CD34+CD19+ cells (B lymphoid progenitors) compared with CD34+ BM cells. The other subsets studied, including CD34+CD38- and CD34+HLA-DR- cells, were present in both compartments in similar proportions. Furthermore, primed CD34+ PB cells were enriched for colony-forming cells (CFC) and displayed an increased clonogenicity when compared with their counterparts in BM. A comparison between a postulated PBSC graft and an average BM graft is presented, showing that such PBSC grafts will be enriched for CD34+ cells as a whole, CD34+CD33+ cells, and colony- forming cells (CFC), factors which have been shown to correlate to acceleration of hematologic reconstitution and reduction in requirements for supportive care in autografting. Hence, we predict that allogeneic transplantation using G-CSF-primed PBSC grafts will result in a more rapid hematologic reconstitution after myeloablative conditioning than BM grafting. The question of whether PBSC allografting will result in permanent engraftment and clinical benefits as observed in autografting has to be determined in prospective clinical studies.     

Utility of reticulocyte maturation parameters in the differential diagnosis of macrocytic anemias

The aim of this study was to test the clinical utility of reticulocyte maturation parameters in the differential diagnosis of macrocytic anemias. Using an automated reticulocyte counter, we analyzed immature reticulocyte fraction (IRF), mean reticulocyte volume (MRV) and mean fluorescence index (MFI) in peripheral blood samples from healthy donors (n = 30), patients diagnosed with myelodysplastic syndromes (MDS, n = 35), with megaloblastic anemia (MA, n = 10) and with non-megaloblastic macrocytic anemias (NMMA, n = 30). Macrocytic anemias due to ineffective erythropoiesis (MA and MDS) showed reticulocytes skewed to a more immature fraction. Therefore, they have a larger volume and a greater RNA content than healthy controls. Interestingly, reticulocytes in both low and high risk MDS are significantly larger (127.3 vs. 118.3 fl, P < 0.01) and have a greater RNA content (MFI 20.5 vs. 12.9, P < 0.01 and IRF 22.5 vs. 9.1%, P < 0.01) than NMMA patients. We conclude that measurement of reticulocyte maturation parameters may be a very useful tool in the differential diagnosis of macrocytic anemia. The presence of extremely high values of IRF (>16%), MFI (>18) and MRV (>129 fl), makes the diagnosis of NMMA very unlikely. An underlying MDS should, therefore, be sought.     

Reticulocyte counts in the aged

Reticulocytes were measured on an automated reticulocyte counter in five groups; healthy adults, non-elderly patient with low hematopoiesis, adults with anemia, healthy elderly persons and elderly patients with anemia. The reticulocyte percent, absolute reticulocyte count, and reticulocyte composition as classified by fluorescence intensity (highly, moderately, and slightly fluorescent cells) were calculated. The healthy adults had a reticulocyte count of 0.70 +/- 0.55%, an absolute reticulocyte count of 4.36 +/- 1.90 X 10(4)/microliters, 2.33 +/- 1.95% highly fluorescent cells, 18.73 +/- 5.07% of moderately fluorescent cells, and 78.82 +/- 6.55% of slightly fluorescent cells. Patients with low hematopoiesis had lower counts except for the percentage of slightly fluorescent cell. Aged persons without anemia showed no differences in reticulocytes from healthy adults. However, elderly patients with anemia had a low reticulocyte count; there was no tendency towards an increase in the percentage of highly fluorescent cells or a decrease in the percentage of slightly fluorescent cells. Their reticulocyte percent was not significantly higher than healthy controls, suggesting that anemia observed in the aged arises from low hematopoietic activity in the bone marrow.