Genesis of clone size heterogeneity in megakaryocytic and other hemopoietic colonies: the stochastic model revisited.

OBJECTIVE: We previously showed that the distributions of the numbers of doublings (NbD) undergone by individual megakaryocyte progenitors before commitment to polyploidization are markedly skewed and can consistently be fitted to straight lines when plotted on semilogarithmic coordinates. The slope of such lines, which yields the probability of polyploidization per doubling, is made less steep by stimulators of megakaryocyte colony formation and is less steep in mixed erythroid-megakaryocyte than in pure megakaryocyte colonies. Therefore, megakaryocytopoiesis provides a unique model for the study of clonal heterogeneity in a hemopoietic lineage, which is the subject of this review. DATA SOURCES: Articles relevant to the interpretation of these data were selected from the authors' and public databases. DATA SYNTHESIS: Exponential NbD distributions were first explained by postulating that following the assembly of thrombopoiesis-specific regulators, megakaryocyte progenitors require only a single random event to arrest proliferation and commit to polyploidization. However, this stochastic model was refuted by data indicating that intrinsic properties of individual progenitors affect the NbD they achieve. We suggest that the unequal repartition of critical compounds (including receptors, signaling molecules, and gene regulators) inherent in the stem cell-progenitor transition causes a heritable heterogeneity in megakaryocyte progenitor responsiveness to polyploidization inducers. This model would be compatible with 1) the evidence for intraclonal synchronization in megakaryocyte and other hemopoietic clones generated by committed progenitors; 2) the low probability of polyploidization of the relatively insensitive bipotent megakaryocyte progenitors; and 3) the thesis that stimulators act in part by recruiting megakaryocyte progenitor cells endowed with lesser responsiveness to polyploidization inducers and higher proliferative potential. CONCLUSION: The responsiveness of individual megakaryocyte progenitors to polyploidization inducers may be a major determinant of the exponential shape of NbD distributions.

Abnormal splenic megakaryopoiesis in MPSV-induced myeloproliferative disease.

The myeloproliferative sarcoma virus (MPSV) induces a murine myeloproliferative syndrome characterized by an erythromyelemia, an anemia, a thrombocytopenia associated with a myeloproliferation in the spleen and a splenic and medullar fibrosis. We have used the in-vitro plasma clot technique to measure megakaryocytic precursors in the spleen and bone-marrow of MPSV-infected mice. We report that megakaryocytic colonies are increased, in number (X75), in concentration (X9) and in size, in the spleen but not in the bone-marrow of neoplastic mice. Furthermore, these splenic precursors are hypersensitive to growth factors present in the anemic mouse serum used in the culture system. These data show that the thrombocytopenia observed in the MPSV-induced neoplasia does not result from a lack of megakaryocyte precursors, but rather from an excess of megakaryocyte destruction. This ineffective splenic megakaryopoiesis associated with the presence of a massive splenic fibrosis make the MPS-induced neoplasia a suitable model for studying the perturbation of megakaryopoiesis in myeloproliferative syndrome associated with fibrosis.

Myeloperoxidase and elastase as markers of leukocyte activation during cardiopulmonary bypass in humans.

To assess leukocyte activation during cardiopulmonary bypass, we measured white blood cell and neutrophil counts and lysosomal enzyme release, especially myeloperoxidase and elastase, throughout the operation and for 5 days postoperatively. A newly developed double antibody radioimmunoassay of myeloperoxidase and an enzyme-linked immunosorbent assay for detection of the polymorphonuclear elastase-alpha 1-proteinase inhibitor complex were used to determine their plasma levels in 15 patients undergoing elective aorta-coronary bypass grafting. Preoperatively white blood cell counts and plasmatic levels of myeloperoxidase and elastase-alpha 1-proteinase inhibitor were normal. Because no correlation has yet been established between levels of myeloperoxidase and elastase-alpha 1-proteinase inhibitor, the aim of this prospective study was to evaluate the use of these enzyme levels as markers for leukocyte activation in vivo. We addressed the clinical situation of cardiopulmonary bypass because it offered the possibility of monitoring the comparative evolution of blood levels of these enzymes in parallel to white blood cell counts through well-defined steps corresponding to known events. We document the advantages of myeloperoxidase blood levels over elastase measurement as reflecting more rapidly the in vivo activation of leukocytes. The time course kinetics of these three measurements were not parallel. White blood cell counts remained stable at the beginning of bypass, whereas myeloperoxidase levels increased sharply and continuously as soon as bypass was instituted until the end of bypass. Elastase levels also increased, but later than myeloperoxidase, beginning when the patients was rewarmed. High elastase plasma levels persisted later than myeloperoxidase after bypass, in parallel with white blood cell counts. It thus clearly appears that changes in myeloperoxidase levels more rapidly reflect the activation state of leukocytes induced by cardiopulmonary bypass and surgery, whereas peak levels of elastase were delayed and parallel to white blood cell counts. From this model, in which the evolution of leukocyte numbers could be followed in relation with known steps of stimulation, it appears that myeloperoxidase is a sensitive marker for monitoring in vivo activation of white blood cells.

Circadian and seasonal variations of hematopoiesis in cord blood.

Cord blood hematopoietic progenitors undergo circadian and seasonal variations. The lowest values are obtained between 4:00 and 12:00, as well as between May and August. This represents the first observation of such rhythms before birth.

A four-parameter index of marrow dysplasia has predictive value for survival in myelodysplastic syndromes.

Marrow dysplasia is a major characteristic of patients with myelodysplastic syndrome (MDS), along with marrow blastosis, cytopenia and cytogenetic anomalies. However, the impact of the degree of marrow dysplasia on survival has not been fully assessed. In this retrospective analysis of 111 patients selected according to the IPSS criteria of MDS diagnosis, the presence or absence of 21 dysplasia characteristics recognizable in bone marrow smears stained by the May-Grünwald-Giemsa method was correlated with patient survival. Using Cox proportional hazards regression analysis, megaloblastosis (MEGALO), neutrophil agranularity (AGRAN) and hypogranularity (HYPOGRAN) were highly significant predictors (p < 0.005), and Pelger-Huët anomaly (PELGHUET) a significant predictor (p = 0.05), of patient survival. The regression analysis yielded a dysplasia-based risk index (DI) where DI = 1.26 MEGALO + 0.82 AGRAN - 1.08 HYPOGRAN + 0.45 PELGHUET. The two subgroups of 60 and 47 patients with DI < or = 0 and > 0 showed highly significant differences in median survivals (2.6 vs 1.1 yrs; p <0.0001). Multivariate analysis further showed that DI offered additional predictive power that was independent of that provided by the IPSS (p=0.002 and 0.001 respectively). Analysis of survival curves stratified for IPSS and DI showed that the additional predictive power offered by inclusion of the DI essentially concerned the IPSS low/INT-1 risk categories. Further stratification for age did not improve survival prediction. The data indicate that a set of 4 dysplasia parameters can offer some prediction for survival of MDS patients in addition to that provided by the IPSS. Further multicenter studies should aim at including some form of evaluation of the degree of dysplasia in prognostic systems.

Influence of laminin or fibroblasts upon colony formation in the mouse by B16 melanoma cell spheroids: a morphometric analysis.

By microscopical observation and using an original morphometric method, we analyzed on histological sections the rate of lung colony formation after the intravenous injection into the mouse of B16 melanoma cells previously cultivated in vitro as aggregates. After the injection of B16 pure spheroids, superficial lung colonies were more numerous than internal lung colonies. After the injection of mixed spheroids (B16 + 3T3 fibroblasts), the size of colony sections was increased. Addition of laminin to pure or mixed spheroids decreased the size of colony sections but increased the number of internal lung colonies.

[Hyperdense red blood cells and spherocytosis]. / Hématies hyperdenses et sphérocytose.

A 12-year old female, suffering from recurring episodes of icterus and abdominal pain, is hospitalized in emergency. She is not anemic but her hemogram reveals a high level of hyperdense red blood cells (32%; controls 0-2.5%) and an abnormal reticulocyte count (201 x 10(3)/microL; controls 29-84 x 10(3)/microL), indicating a 3.5 fold increase in RBC production. The same abnormalities are found in the patient's mother. The blood smear shows few spherocytes. RBC osmotic fragility is increased only after incubation. Hereditary spherocytosis is diagnosed following electrophoresis of membrane proteins which reveals a deficiency in band 3, a protein which links the lipid bilayer to the cytoskeleton. This case of hemolytic anemia-illustrates the physiopathologic and diagnostic significance of hyperdense RBC, which reflect the cell dehydration associated with the membrane disorder.

[Myelodysplastic syndromes: preleukemic syndromes]. / Les syndromes myélodysplasiques: syndromes préleucémiques.

The myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by peripheral blood cytopenias with a hypercellular bone marrow exhibiting dyspoiesis. The predominant in elderly patients are associated with a high risk of progression to acute myelogenous leukemia. The etiology of MDS is unknown in most cases. About 10% of MDSs are secondary. MDS are classified by the French American British (FAB) classification into five subgroups. The incidence of the disorders is difficult to estimate but it seems to be increasing. Clonal cytogenetic aberrations are found in 30 to 50% of de novo MDS. The only currative treatment for MDS is allogeneic bone marrow transplantation.

[Conventional IVF versus ICSI in sibling oocytes: a French experience analysis for BLEFCO]. / FIV conventionnelle versus ICSI sur une même cohorte ovocytaire: analyse de l'expérience française pour les BLEFCO.

Assisted Reproductive Techniques (ART) separating oocytes in sibling oocytes treated either by conventional IVF or ICSI is called mid-IVF/ICSI. We sum up here 487 attempts of this kind from six French ART centers. The mid-IVF/ICSI technique was performed in 5.6% of cases. The fertilization rate by micro-injected oocytes was significantly higher (P<0.01) than oocytes inseminated conventionally, 72.6% versus 53.4%. A failure of fertilization was observed only in mid-IVF in 21.6% of cases, which prevented a complete fertilization failure when we decided to propose to the couples concerned the mid-IVF/ICSI technique. Conversely, in 75.2% of cases, fertilization was found for the two batches of oocytes. The overall pregnancy rate has improved since the use of the mid-IVF/ICSI technique (33.1% versus 28.9%, P=0.013) and the fertilization failures decreased (10.4% versus 14.3%, P=0. 019). The pregnancy rate in only mid-IVF/ICSI cases is very high at 39.8% but for a selected population. The indications for mid-IVF/ICSI remain to be clarified especially with regard to male and idiopathic indications.

Platelet size in man.

The shape and parameters of platelet size distributions were studied in 50 normal persons and 97 patients in order to test the proposed thesis that platelet size heterogeneity results mainly from aging in the circulation. This thesis was contradicted (1) by size distributions of age-homogeneous, newly-born cell populations which were lognormal with increased (instead of decreased) dispersion of volumes and (2) by the macrothrombocytosis found in some populations with normal age distribution. For these reasons, thrombocytopoiesis appeared to play the major role in determining platelet size. A model was built in which the volume variation of platelet territories due to megakaryocyte growth and membrane demarcation at each step of maturation was a random proportion of the previous value of the volume. This model explains the lognormal shape of both newborn and circulating platelet size distributions. It also implies that (1) the mean and standard deviation of platelet logvolumes depend on the rates of volume change of the individual platelet territories (growth rate minus demarcation rate) as well as on megakaryocyte maturation time; (2) platelet hyperdestruction causes an increase in the mean and dispersion of the rates of territory volume change; (3) Mediterranean macrothrombocytosis and some hereditary macrothrombocytotic thrombocytopenias or dysthrombocytopoieses reflect a diminished rate of territory demarcation, and (4) platelet size heterogeneity is caused mainly by the variations in territory growth and demarcation and not by aging in the circulation.

Platelet formation in Mediterranean macrothrombocytosis.

Platelet size distribution and survival parameters were studied in control subjects and ten subjects presenting Mediterranean macrothrombocytosis. The comparison showed that the latter group maintains a normal platelet circulating mass (thrombocytocrit) by combining normal platelet survival, increased splenic pooling and production of a reduced number of greatly enlarged cells. In addition to offering new insight into the megakaryocytic mechanisms that determine platelet size, the study of Mediterranean macrothrombocytosis indicates that platelet size distributions and survival parameters should be integrated in order to provide a meaningful picture of human platelet kinetics.

[Secretion of acetylcholinesterase by megakaryocytes in mice]. / La sécrétion d' acétylcholinestérase par les mégaryocytes de souris.

Acetylcholinesterase, an early and nearly specific marker of Rodent thrombocytic cells, undergoes a typical secretory cycle leading to the release of the enzyme in the demarcation system and the outer cellular space of bone marrow megakaryocytes. This secretion process suggests that megakaryocytes may adapt the amplification of their own precursors by modulating the concentration in cholinergic compounds in hematopoietic tissues.

Mouse megakaryocytes secrete acetylcholinesterase.

Acetylcholinesterase (AchE), an essentially specific and early marker of rodent thrombocytic series, in several tissues acts both as a constituent of the cellular membrane and as a secretory enzyme. This study presents the ultrastructural transport and fate of this substance in the megakaryocytes of mouse bone marrow, using Tranum-Jensen and Behnke's adaptation of the indirect thiocholine method. It is shown that megakaryoblasts and megakaryocytes undergo a typical secretory cycle consisting of (1) enzyme synthesis and segregation on the endoplasmic reticulum and nuclear envelope, (2) enzyme concentration in AchE-vesicles and AchE-granules formed from the cisternae of the Golgi apparatus, and (3) discharge in the demarcation membrane system and extracellular space. The AchE-vesicles and granules appear to be hitherto unrecognized megakaryocytic organelles as they do not resemble alpha nor the dense granules, and their mode of formation and fate differ from those of primary lysosomes and peroxidase granules. Released platelets reveal AchE activity in the open canalicular system. The data are compatible with the hypothesis that by controlling acetylcholine concentration in hematopoietic tissues, the secretion of AchE by megakaryocytes can modulate the proliferative activity of megakaryocytes progenitors.

The determination of megakaryocyte ploidy.

Methods which have been used to determine megakaryocyte ploidy in animals and humans are reviewed. Although the number of megakaryocyte nuclear units counted in bone marrow squashes is roughly proportional to ploidy, accurate determinations of DNA content require the use of microphotometry or flow cytometry. New techniques should make it possible to distinguish polyploidizing megakaryoblasts from promegakaryocytes and mature megakaryocytes which have arrested polyploidization. Only the latter should be included in histograms of the number of endoduplications, since only those have expressed their full polyploidization potential. Statistical techniques are available for analysis and comparison of both raw ploidy distributions or histograms of endoduplication numbers.

A strategy for multiple immunophenotyping by image cytometry: model studies using latex microbeads labeled with seven streptavidin-bound fluorochromes.

Multiple immunophenotyping is aimed at identifying several cell populations in a single labeling procedure by their ability to bind combinations of specific labeled antibodies. The present work demonstrates the simultaneous discrimination by using image cytometry of aminomethylcoumarin acetate (AMCA), Lucifer yellow (LY), fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), PE-Texas red tandem (Red613), peridinin-chlorophyll protein (PerCP), and allophycocyanin (APC), which were all bound to latex beads as streptavidin-conjugated fluorochromes. This has been the result of a step-by-step optimization of the several factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. First, 14 streptavidin-conjugated fluorochromes were evaluated by using spectrofluorometry. A primary selection was then made of ten spectrally separable dyes that could be evaluated by using image cytometry. These dyes were bound to latex particles, and specific filter combinations were assembled to minimize crosstalk between fluorophores while preserving sufficient fluorescence intensity and counting statistics. Potential probe associations were then assessed by measuring the emissions of all fluorochromes that were detected by each filter combination. The resulting crosstalk matrix served as the basic tool both for final selection of the optimal filter combination and for dye set (composed, in this case, of the seven fluorochromes described above) and for mathematical correction of residual spectral overlap. Next, an image cytometry system was adapted to collect seven images of matched brightness with the selected combination of excitation/emission filters and dichroic mirrors. Finally, seven-parameter synthetic images were generated by digital image processing.