Atypical micromegakaryocytes, promegakaryoblasts and megakaryoblasts: a critical evaluation by immunohistochemistry, cytochemistry and morphometry of bone marrow trephines in chronic myeloid leukemia and myelodysplastic syndromes.

A morphometric analysis of bone marrow trephine biopsies has been performed to study the frequency and planimetric characteristics of so-called atypical micromegakaryocytes in chronic myeloid leukemia (CML) and myelodysplastic syndromes (MDS). In addition, an attempt was made to discriminate this particular cell population from small immature elements of megakaryocytopoiesis, such as promegakaryoblasts and megakaryoblasts. The staining reactions employed included periodic acid-Schiff (PAS), alpha-naphthyl acetate esterase (ANAE) and immunohistochemistry with a monoclonal antibody against platelet glycoprotein IIIa (Y2/51-CD61). Comparison of the various staining reactions applied to the different megakaryocytic elements together with morphometric measurements resulted in a clearcut identification of promegakaryoblasts. These were defined as the earliest immature and exclusively CD61-positive precursors. Atypical micromegakaryocytes were characterized by their dysplastic features and strong ANAE reactivity in addition to their positive CD61 staining. When stringent diagnostic criteria (diameter ranging between 10 to 15 microns, mean size about 12 microns) were applied, this abnormal cell population comprised less than 10% of total megakaryocytopoiesis in CML and MDS. It may be assumed that dysmegakaryocytic features in the latter disorders are partially generated by small to medium-sized megakaryocytes (diameter less than 30 microns). In conclusion, the relative frequency of promegakaryoblasts in the normal bone marrow (range 6-8%) is confirmed by evaluation of the immunohistochemical and cytochemical staining methods (CD61 and ANAE). Furthermore, the ANAE reaction facilitates the recognition of atypical micromegakaryocytes as well as small megakaryocytes. Thus cytochemistry provides a better insight into alterations of these cell lineages in various pathological conditions.     

Atypical micromegakaryocytes,promegakaryoblasts and megakaryoblasts: a critical evaluation by immunohistochemistry,cytochemistry and morphometry of bone marrow trephines in chronic myeloid leukemia and myelodysplastic syndromes

A morphometric analysis of bone marrow trephine biopsies has been performed to study the frequency and planimetric characteristics of so-called atypical micromegakaryocytes in chronic myeloid leukemia (CML) and myelodysplastic syndromes (MDS). In addition, an attempt was made to discriminate this particular cell population from small immature elements of megakaryocytopoiesis, such as promegakaryoblasts and megakaryoblasts. The staining reactions employed included periodic acid-Schiff (PAS), alpha-naphthyl acetate esterase (ANAE) and immunohistochemistry with a monoclonal antibody against platelet glycoprotein IIIa (Y₂/ 51-CD61). Comparison of the various staining reactions applied to the different megakaryocytic elements together with morphometric measurements resulted in a clearcut identification of promegakaryoblasts. These were defined as the earliest immature and exclusively CD61-positive precursors. Atypical micromegakaryocytes were characterized by their dysplastic features and strong ANAE reactivity in addition to their positive CD61 staining. When stringent diagnostic criteria (diameter ranging between 10 to 15 μm, mean size about 12 urn) were applied, this abnormal cell population comprised less than 10% of total megakaryocytopoiesis in CML and MDS. It may be assumed that dysmegakaryocytic features in the latter disorders are partially generated by small to medium-sized megakaryocytes (diameter less than 30 μm). In conclusion, the relative frequency of promegakaryoblasts in the normal bone marrow (range 6–8%) is confirmed by evaluation of the immunohistochemical and cytochemical staining methods (CD61 and ANAE). Furthermore, the ANAE reaction facilitates the recognition of atypical micromegakaryocytes as well as small megakaryocytes. Thus cytochemistry provides a better insight into alterations of these cell lineages in various pathological conditions. This work was supported by the Deutsche Forschungsgemeinschaft (DFG-Th 390/1–2)     

Bone marrow angiogenesis and proliferation in B-cell chronic lymphocytic leukemia

OBJECTIVE: To assess angiogenesis and the proliferative activity of bone marrow in patients with chronic lymphocytic leukemia (CLL) in relation to the bone marrow infiltration pattern. STUDY DESIGN: Bone marrow samples were obtained by trephine biopsy from 46 patients with B-cell CLL (B-CLL). Infiltration pattern was diffuse in 20 patients and nondiffuse--i.e., nodular, interstitial or mixed--in the remaining 26 patients. Ten normal bone marrow samples were used as a control group. Studies were carried out by immunohistochemical staining of paraffin-embedded bone marrow samples. Angiogenesis was assessed in the zones of highest vascular density (hot spots), visualized by the expression of endothelial antigen CD34 and expressed as a number of microvessels per high-powerfield (hpf) (final magnification, 400x). Proliferative activity was estimated by the expression of nuclear protein Ki-67, cyclin A and mean number of nucleolar organizer regions (AgNORs). RESULTS: Microvessel density was higher in B-CLL marrow than in normal marrow (30.1 and 16.44 per hpf, respectively) and was higher in the diffuse than nondiffuse pattern (33.6 and 27.5 per hpf, respectively). B-CLL bone marrow also showed higher proliferative activity, as assessed by mean number of AgNORs, than did normal marrow (1.52 and 1.25, respectively) and a higher mean percentage of cyclin A-positive cells (7.5 and 6.8, respectively). In contrast, mean Ki-67 expression was similar in B-CLL and the control group. Mean AgNORs number, Ki-67 and cyclin A-positive cell percentage were significantly higher in B-CLL marrow with a diffuse as compared to nondiffuse involvement pattern (AgNORs, 1.75 and 1.35; cyclin A, 9.27% and 3.95%; Ki-67, 34.9% and 23.3%, respectively). CONCLUSION: Our results indicate enhancement of bone marrow angiogenesis in B-CLL and a relationship between microvessel density and the bone marrow infiltration pattern. The study points also to a possible relationship between the bone marrow infiltration pattern and proliferative activity of bone marrow cells.     

Correlations between cytogenetics and morphology in myelodysplastic syndromes

In order to detect possible relationships between cytogenetic abnormalities and morphologic features in myelodysplastic syndromes (MDS), 48 patients with MDS were investigated. Clonal cytogenetic abnormalities were present in bone marrow cells from 27 patients (56%). The most frequent single anomaly was del (5 q) (10 cases), followed by monosomy 7 (3 cases), trisomy 8 (3 cases) and del (20 q) (2 cases). Complex anomalies were present in 6 patients. Morphologically, according to the French-American-British (FAB) classification: 17 cases were considered as refractory anemia (RA), 17 as RA with excess of blasts (RAEB), 2 as RAEB in transformation, 2 as acquired idiopathic sideroblastic anemia and 10 as chronic myelomonocytic leukemia. With regard to the FAB classification, del (5 q) was often associated with RA and complex cytogenetic anomalies with RAEB. When myelodysplasia was studied in individual myeloid lineages, del (5 q) was associated with hypolobulated megakaryocytes, monosomy 7 with micromegakaryocytes and complex chromosomal anomalies with the association of two or more features of dysmegakaryocytopoiesis. Del (11 q) was associated with increased iron storage and del (20 q) with marked dyserythropoiesis. No correlation was observed between cytogenetic anomalies and features of dysgranulocytopoiesis.     

Morphometric classification of hairy cell leukemia in bone marrow trephine biopsy

OBJECTIVE: To analyze cytologic and histologic parameters in bone marrow trephine biopsy in an attempt to define heterogeneity of hairy cell leukemia cells. STUDY DESIGN: The study group consisted of 28 trephine biopsies. Immunohistochemistry for CD20 antigen was used. Image processing and measurements were performed with AnalySIS 3.0 image analysis system (Soft Imaging System GmbH, Germany) and custom built programs. For planimetric measurements of nuclei, automatic segmentation was implemented. The measured parameters were: surface area, perimeter, minimum, mean and maximum diameter, and a set of form factors. Relative volumes of bone trabeculae, adipose tissue, hematopoietic tissue and neoplastic infiltrate were assessed by the point counting method. Nuclear volume was measured by the point sampled intercept method. Bone marrow fibrosis was assessed using a curvilinear line test system. RESULTS: Significant variability of cell nuclei was found, and their classification into 3 types was possible. The relative frequency of those types was different in various cases and allowed subdivision of cases into 3 groups that differed in some clinical and histologic manifestations. CONCLUSION: The present study demonstrated the heterogeneity of cell populations of hairy cell leukemia.     

Disparate effects of interleukin 11 and thrombopoietin on megakaryocytopoiesis in vitro

The effects of interleukin-11 (IL-11) and thrombopoietin (TPO) on murine megakaryocytopoiesis were studied using a serum-free culture system. Acting alone, both IL-11 and TPO increased the number of acetylcholinesterase (AchE)(+)cells (megakaryocytes), the latter being more potent than the former. TPO, but not IL-11, increased the mean AchE activity per megakaryocyte (AchE activity/megakaryocyte). TPO increased both the number of megakaryocytes with high ploidy, and of those with low ploidy. In contrast, IL-11 increased only the number of megakaryocytes with high ploidy. The effect of TPO on megakaryocyte ploidy was stronger than that of IL-11. Both IL-11 and TPO increased the proportion of large megakaryocytes, but the latter was more potent than the former. While the stimulatory effects of IL-11 and TPO on the number of megakaryocytes were enhanced by IL-3 or stem cell factor (SCF), synergism of IL-11 or TPO with IL-3 or SCF in stimulating AchE activity/megakaryocyte was inconsistent. IL-11 and TPO stimulated the formation of colony-forming units of megakaryocyte in the presence of IL-3, but not alone, with similar maximum colony numbers for both cytokines. Our findings thus demonstrate that IL-11 principally stimulates megakaryocyte maturation rather than the proliferation of megakaryocytes, whereas TPO stimulates both.     

Splenic haematopoiesis in primary (idiopathic) osteomyelofibrosis: immunohistochemical and morphometric evaluation of proliferative activity of erythro- and endoreduplicative capacity of megakaryopoiesis (PCNA- and Ki-67 staining)

Using monoclonal antibodies against proliferating cell nuclear antigen or PCNA (PC10) and the Ki-67 antigen (MIB1), an immunohistological and morphometric study was performed on routinely processed splenic tissue from ten patients with primary (idiopathic) osteomyelofibrosis (OMF). To determine the proliferation capacity of erythroid precursors and the endoreduplicative activity of megakaryocytes, corresponding antibodies (Ret40f and CD61) were applied in combination with the cell-cycle markers (sequential double-immunostaining). Morphometric analysis revealed no significant differences in PCNA or Ki-67 reactivity in either cell lineages. In comparison with previous studies on normal bone marrow, in splenic tissue showing myeloid metaplasia, the numbers of PCNA-labelled proerythroblasts, erythroblasts and megakaryocytes were conspicuously increased. Considering the ineffective erythropoiesis in OMF, there seemed to be a disproportional enhancement in PCNA and Ki-67 immunostaining of the red cell lineage. Similarly, the small size of megakaryocytes in advanced, OMF-associated myeloid metaplasia was in keeping with an impairment of endoreduplicative activity. In addition to various other contributory factors, anaemia in OMF may be partially caused by secondary folate (haematinic) deficiency. From experimental studies this defect is known to cause an abnormal arrest in the S-phase of the cell-cycle, comparable to that characterising pernicious anaemia. As a sequel of this pathomechanism, an undue overexpression of PCNA and Ki-67 has to be assumed, that is not necessarily associated with DNA synthesis or cell cycling. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG-Th 390/1-3)     

Abnormalities of megakaryocytes in myelitis and chronic myeloproliferative diseases

A planimetric study of megakaryopoiesis in various chronic myeloproliferative diseases (CMPD) was performed and the results compared with those from controls and myelitis of rheumatic origin. Morphometric measurements included at least 200 megakaryocytes in each case observed in Giemsa-stained semithin sections of resin-embedded core biopsies. Twenty specimens were evaluated from the controls and inflammatory disorders and from each of the following CMPD: 1, chronic granulocytic leukaemia (CGL); 2, polycythaemia vera (P. vera); 3, chronic megakaryocytic-granulocytic myelosis without or with minimal increase in reticulin fibre content (CMGM); 4, myelofibrosis or osteomyelosclerosis (MF/OMS). Megakaryocytes were classified as follows: 1, normal megakaryocytes at all stages of maturation; 2, giant forms; 3, microforms; 4, intussusceptions; 5, a-nuclear cytoplasmic fragments; 6, naked nuclei or necrotic forms. The results of this study demonstrate obvious abnormalities of megakaryopoiesis in addition to the increase in absolute numbers of megakaryocytes per marrow area and their different sizes as reported earlier (Thiele et al. 1982). Aberrations are particularly conspicuous when pure granulocytic proliferation or neoplasia of CGL is compared with the so-called mixed cellularity of megakaryocytes and granulocytes in CMGM including MF/OMS. Abnormalities of the giant forms of megakaryocytes are especially evident and comprise irregular cellular and nuclear perimeters (as calculated by a modified shape factor) in the two latter entities (CMGM-MF/OMS). This remarkable feature is associated with a disorganization of nuclear development and/or a disproportionate nuclear-cytoplasmic ratio which has never been observed in CGL previously. In combination with this striking cellular anomaly, which is compatible with an extreme amoeboid shape of giant forms in CMGM and MF, intussuceptions and a-nuclear cytoplasmic fragments are frequently encountered. The final stage of megakaryopoiesis, i.e. naked nuclei, are increased in number in all CMPD, probably because of enhanced proliferation and platelet shedding. Naked nuclei are often small in CGL (as remnants of the frequent micromegakaryocytes) and large in P. vera and CMGM/MF (depending on the high incidence of giant megakaryocytes in these latter disorders).     

Erythropoietin attenuates lipopolysaccharide-induced splenic and thymic apoptosis in rats

Apoptosis of lymphoid tissues during sepsis is well documented and linked to the pathobiology of organ failure and death. In this study, we evaluated the effect of a single dose of recombinant erythropoietin (EPO) on thymic and splenic apoptosis in an endotoxic sepsis model. Young male Wistar rats were divided into 3 groups and administered intraperitoneally (IP) either normal saline; lipopolysaccharide (LPS) 10 mg/kg; or EPO (5000 U/kg) 30 min before lipopolysaccharide. Six hours following LPS administration animals were sacrificed. Apoptosis was assessed by hematoxylin-eosin staining, terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick end labeling (TUNEL), and caspase-3 immunostaining. When compared with animals given LPS, animals pretreated with EPO displayed reduced splenic and thymic TUNEL positivity of 44+/-3 (p<0.05) and 143+/-4 (p<0.05) nuclei per high power field (hpf), respectively. Caspase-3 positivity was also significantly reduced in the spleen and thymus, with 31+/-4 (p<0.05) and 93+/-3 (p<0.05) positive stained nuclei per hpf, respectively. Serum nitrite levels were elevated in animals given lipopolysaccharide. Pretreatment with EPO attenuated the increase in nitrite levels; however, this did not reach statistical significance. We conclude that a single dose of recombinant erythropoietin can reduce thymic and splenic apoptosis associated with lipopolysaccharide administration.     

Effects of the tyrosine kinase inhibitor imatinib mesylate (STI571) on bone marrow features in patients with chronic myelogenous leukemia

Preliminary data are available about bone marrow (BM) changes in patients with chronic myeloid leukemia (CML) who received the molecularly targeted and highly effective tyrosine kinase inhibitor Imatinib mesylate (STI571). This review is focused on a systematic assessment of BM features detectable at different stages of CML (stable, accelerated, blastic) following long-term (more than 10 months) treatment. By applying enzyme- and immunohistochemistry including monoclonal antibodies visualizing proliferating cell nuclear antigen (PCNA) and apoptosis (anti-apostatin), a more elaborate insight into alterations affecting hematopoiesis and the stroma compartment was gained. In patients with stable-phase CML therapy resulted in a significant reduction in cellularity, neutrophil granulopoiesis and number of megakaryocytes, accompanied by a retrieval of erythroid precursors. In patients with Imatinib as the only treatment morphometric analysis of CD61+ megakaryopoiesis was in keeping with a significant decrease in maturation defects implying a lesser amount of atypical micromegakaryocytes almost consistent with normalization. Moreover, a reduction of the initially enhanced (CD34+) microvessel density was detectable associated with a decrease in luminal distension. Regression of marked to moderate myelofibrosis was recognizable in about 70% of patients especially in the accelerated and blastic phases. The amount of myeloblasts, CD34+ progenitor cells and lysozyme-expressing immature myelomonocytic cells declined with treatment, but recurred in about 19% of patients that developed a leukemic relapse after 21+/-6 months of therapy. Data on proliferative activity and apoptosis in general supported in vitro findings concerning the inhibitory effect of this agent on growth associated with a tendency for stimulated apoptosis, at least in responding patients.     

Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells

Abstract. The kinetics of megakaryocyte formation from mouse bone marrow cells in semi-solid medium was studied directly in the culture dish by staining the cells for acetylcholinesterase after drying the cultures. A WEHI-3 cell-conditioned medium (WEHI-3 CM) was used as a general source of stimulus for megakaryocyte colony formation. The addition of peritoneal exudate supernatant as well as WEHI-3 CM increased the frequency of megakaryocyte colonies detected. Colonies containing acetylcholinesterase-positive cells were first detected at day 3. Maximum numbers of 25–40 megakaryocyte colonies per 10⁵ nucleaet mouse bone marrow cells were observed from days 7 to 11. The mean number of cells within each colony increased progressively with time of culture, and a modal range of 11–20 cells was obtained by day 7. Between 3 and 200 cells per colony were generally detected. A continuous distribution of the number of megakaryocytes per colony suggests that the clonable precursor cells are not synchronized either with respect to maturation stage or with respect to their capability to undergo nuclear endoreduplication. The addition of peritoneal exudate supernatant to the cell cultures increased the DNA levels of megakaryocytes grown in the presence of WEHI-3 CM but did not affect the number of cells per colony. The DNA content of colony megakaryocytes was measured after staining the cells with Feulgen reagent. A modal DNA value of 8 N was observed between days 4 and 7 for megakaryocytes stimulated with WEHI-3 CM. In the presence of both WEHI-3 CM and peritoneal exudate supernatant, the DNA content of megakaryocytes increased with the time of cell culture. Modal DNA values increased from 8 N at days 4 and 5, to 16 N by day 6. In these optimally stimulated cultures, 44% of colony megakaryocytes were 32 N or greater, a proportion higher than in normal bone marrow, but similar to that seen in the marrow of acutely thrombocytopenic animals. It is concluded that megakaryocytopoiesis in cell cultures is not a strictly controlled process with respect to cell division and endomitosis and that when certain culture conditions are employed, megakaryocyte development in vitro might reflect that seen in a stressed animal condition.     

The activity of alpha1,6-fucosyltransferase during human megakaryocytic differentiation

alpha1,6-Fucosyltransferase (6FucT, E.C. 2.4.1.68) is one of the enzymes involved in the synthesis of N-linked glycans of the GpIIb/IIIa complex (CD41a) which is present on megakaryocytes (MKs) and platelets. In this study, we examined 6FucT activity in ex vivo cultures of immunoselected cord blood CD34(+) cells grown in a medium promoting megakaryocytopoiesis. Our results show that the activity of 6FucT increased ahead of, and thereafter concomitantly with, cells expressing the CD41a antigen. When the CD41a(+) subpopulation of cells was immunoselected (using anti-CD61 i.e. anti-GpIIIa antibodies), its 6FucT activity increased proportionally to the yield of CD61(+)(+)(+) cells. Taking into account the heavy load of 6FucT in platelets and megakaryocytes, we regard this enzyme as a candidate for the earliest marker of MK-commitment in cultured hematopoietic stem cells. Such a marker should allow an earlier detection and earlier transplantation of patients' own, ex vivo expanded, Mk progenitors.     

Immunophenotypic characterization of myelopoiesis in early and late myelodysplastic syndromes: use of CD44 as an aid in early diagnosis

We investigated the possibility that myeloid cells from the bone marrow (BM) of myelodysplastic patients differ in their expression of CD44 antigen compared with expression of the antigen in normal controls. In addition, two triple-surface marker assays incorporating, respectively, CD44/CD33/CD66 and CD33/CD34/HLA-DR were used to evaluate the degree of myeloid maturation and assess the number of blasts in BM by flow cytometry. Patients with early-stage myelodysplastic syndrome (MDS; RA [FAB classification]) have significantly decreased expression of CD44 on gated myeloid cells. In contrast, patients with late-stage MDS (RAEB and RAEB-T [FAB classification]) showed an elevated expression of CD44 and an increased number of CD34 blasts compared with early-stage MDS patients and normal controls. Late-stage MDS patients also had an increase in the immature myeloid compartment (CD66 weak expression) compared with early-stage MDS patients and normal controls. We have already included this assay as part of our MDS evaluation protocol alongside BM morphology and cytogenetics.     

The use of the anti-factor VIII method on trephine biopsies of the bone marrow for the identification of immature and atypical megakaryocytes in myeloproliferative diseases and allied disorders. A morphometric study

A morphometric analysis was performed on trephine biopsies of the bone marrow to identify atypical megakaryocyte proliferation following PAS staining and the immunohistological demonstration of factor VIII. This study includes nine patients with a megakaryoblastic crisis in chronic myeloid leukemia (CML), four with acute megakaryoblastic leukemia (AM) and three with myeloid dysplasia later evolving into overt acute leukemia. Comparison and statistical evaluation of the PAS reaction with anti-factor VIII staining reveals that the latter technique not only facilitates the recognition of immature and abnormal megakaryocytes, but leads to a significantly increased count for all megakaryocytic elements in the bone marrow. Thus our retrospective investigation of routinely processed and paraffin-embedded trephine biopsies shows that the diagnosis of a megakaryoblastic crisis in CML as well as AM may be easily established with the aid of the anti-factor VIII method. In all cases of megakaryoblastic proliferation in CML and AM, the appearance of blasts was associated with moderate to pronounced myelofibrosis which could be also determined by morphometry.     

Megakaryocyte precursors (pro- and megakaryoblasts) in bone marrow tissue from patients with reactive thrombocytosis, polycythemia vera and primary (essential) thrombocythemia. An immunomorphometric study

To determine the number of megakaryocyte precursors (pro- and megakaryoblasts), an immunomorphometric study was performed on paraffin-embedded trephine biopsies of the bone marrow using a monoclonal antibody against platelet glycoprotein IIIa. Eighteen control specimens from patients with no evidence of any hematological disorder and a normal platelet count were selected and assessed together with the same number of specimens from patients with reactive thrombocytosis, polycythemia vera rubra (P. vera) or primary (essential) thrombocythemia (PTH). A strikingly proportionate increase in early megakaryocytes occurred in all patients enrolled in this study, compared with the controls. Moreover, there were no significant correlations between counts for precursors or total megakaryocytes per square millimeter of bone marrow with the corresponding values for platelets. This indicates that despite an orderly increase in immature forms in the bone marrow, the number of platelets circulating in the blood is influenced by other additional factors, such as the expanded platelet pool in the enlarged spleen. The non-disproportionate expansion of megakaryocyte precursors extends previous findings on progenitor cells of this lineage in vitro, particularly in PTH. Histological evaluation of the bone marrow of patients with P. vera and PTH indicated that megakaryopoiesis proceeded to the production of appropriate mature forms with no obvious excess of very small or blastic elements.     

Number of megakaryocytic progenitors and adhesion molecule expression of stem cells predict platelet engraftment after allogeneic hematopoietic stem cell transplantation

BACKGROUND: The mechanism of platelet recovery after hematopoietic stem cell transplantation and the factors that influence its time-course are not fully understood. Rapid hematopoietic recovery results in a reduction of transplantation-related complications. In the present study, we questioned and analyzed whether there were important factors predicting the speed of platelet engraftment. METHODS: Thirty-seven patients with various hematologic diseases transplanted with allogeneic BM between January 2002 and December 2005 were included. We investigated the differences in mononuclear cell counts (MNC), numbers of infused CD34(+), CD34(+) CD41(+) and CD34(+) CD61(+) cells and phenotypic analysis of homing-associated cell adhesion molecules (CXCR4, CD49d and CD49e). The number of megakaryocytes formed in vitro (colony-forming unit-megakaryocytes; CFU-Mk) was also measured. RESULTS: Median days of ANC >/=0.5x10(9)/L and platelet count >/=20x10(9)/L were 14.8 and 17.3, respectively. The number of infused CD34(+) CD41(+) and CD34(+) CD61(+) cells correlated much better with the time to platelet engraftment than that of infused CD34(+)cells (P<0.05 each). Rapid platelet recovery also occurred in patients receiving both higher homing-associated cell adhesion molecule doses and CFU-Mk (P<0.05 each). DISCUSSION: Rapid platelet recovery has several advantages, including reducing the cost of supportive therapy and reducing the risk of fatal bleeding as a result of severe thrombocytopenia. Our findings suggest that phenotypic and clonogenic assessment of infused progenitor cells can identify patients in whom platelet engraftment is likely to be significantly delayed, and new strategies to overcome related problems might be employed in the very near future.     

Regeneration of megakaryocytopoiesis and thrombopoiesis in vitro from X-irradiated human hematopoietic stem cells

In the present study, we investigated whether X-irradiated hematopoietic stem cells can be induced to undergo megakaryocytopoiesis and thrombopoiesis in vitro using cytokine combinations that have been demonstrated to be effective for conferring increased survival on irradiated human CD34(+) megakaryocytic progenitor cells (colony-forming unit megakaryocytes; CFU-Meg), such as thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor and FLT3 ligand. Culture of nonirradiated CD34(+) cells in serum-free medium supplemented with multiple cytokine combinations led to an approximately 200- to 600-fold increase in the total cell numbers by day 14 of culture. In contrast, the growth of X-irradiated cells was observed to be one-sixth to one-tenth that of the nonirradiated cultures. Similarly, total megakaryocytes were increased by 50- to 130-fold, while culture of X-irradiated cells yielded one-fourth to one-eighth of the control numbers. At this time, CD41(+) particles, which appeared to be platelets, were produced in the medium harvested from nonirradiated and irradiated cultures. Although radiation suppressed cell growth and megakaryocytopoiesis, there were no significant differences in thrombopoiesis between the two types of culture. These results suggest that X-irradiated CD34(+) cells can be induced to undergo nearly normal terminal maturation through megakaryocytopoiesis and thrombopoiesis by stimulation with appropriate cytokine combinations.