Jean-Michel Paulus

Consequences of Total and Subtotal Myeloperoxidase Deficiency: Risk or Benefit ?

A group of 100 totally or subtotally myeloperoxidase (MPO)-deficient individuals was compared to a reference population of 118 probands selected at random. Data for a protective effect of the deficiency against cardiovascular damage are presented. On the other hand, a significantly higher occurrence of severe infections and chronic inflammatory processes was noted among the deficient patients. An increased incidence of cancer among the MPO-deficient individuals was not demonstrated.

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.

Hematopoietic recovery in cancer patients after transplantation of autologous peripheral blood CD34+ cells or unmanipulated peripheral blood stem and progenitor cells

BACKGROUND: A study of CD34+ cell selection and transplantation was carried out with particular emphasis on characteristics of short‐ and long‐term hematopoietic recovery. STUDY DESIGN AND METHODS: Peripheral blood stem and progenitor cells (PBPCs) were collected from 32 patients, and 17 CD34+ cell‐selection procedures were carried out in 15 of the 32. One patient in whom two procedures failed to provide 1 × 10(6) CD34+ cells per kg was excluded from further analysis. After conditioning, patients received CD34+ cells (n = 10, CD34 group) or unmanipulated (n = 17, PBPC group) PBPCs containing equivalent amounts of CD34+ cells or progenitors. RESULTS: The yield of CD34+ cells was 53 percent (18–100) with a purity of 63 percent (49–82). The CD34+ fraction contained 66 percent of colony‐forming units‐granulocyte‐ macrophage (CFU‐GM) and 58 percent of CFU of mixed lineages, but only 33 percent of burst‐forming units‐erythroid (BFU‐E) (p < 0.05). Early recovery of neutrophils and reticulocytes was identical in the two groups, although a slight delay in platelet recovery may be seen with CD34+ cell selection. Late hematopoietic reconstitution, up to 1.5 years after transplant, was also similar. The two groups were thus combined for analyses of dose effects. A dose of 40 × 10(4) CFU‐GM per kg ensured recovery of neutrophils to a level of 1 × 10(9) per L within 11 days, 15 × 10(4) CFU of mixed lineages per kg was associated with platelet independence within 11 days, and 100 × 10(4) BFU‐E per kg predicted red cell independence within 13 days. However, a continuous effect of cell dose well beyond these thresholds was apparent, at least for neutrophil recovery. CONCLUSION: CD34+ cell selection, despite lower efficiency in collecting BFU‐E, provides a suitable graft with hematopoietic capacity comparable to that of unmanipulated PBPCs. In both groups, all patients will eventually show hematopoietic recovery of all three lineages with 1 × 10(6) CD34+ cells per kg or 5 × 10(4) CFU‐GM per kg, but a dose of 5 × 10(6) CD34+ cells or 40 × 10(4) CFU‐GM per kg is critical to ensure rapid recovery.

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-Griinwald-Giemsa method was correlated with patient survival. Using Cox proportional hazards regression analysis, megaloblastosis (MEGALO), neutrophil agran-ularity (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 (D1) where DI = 1.26 MEGALO + 0.82 AGRAN - 1.08 HYPOGRAN + 0.45 PELGHUET. The two subgroups of 60 and 47 patients with DI ≤ 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 mul-ticenter studies should aim at including some form of evaluation of the degree of dysplasia in prognostic systems.

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.

Circadian and circannual variations in cord blood hematopoietic cell composition

Several previous studies have demonstrated that cord blood unit composition is an important factor that may predict outcomes after cord blood transplantation, with higher doses of transplanted nucleated cells and hematopoietic stem and progenitor cells being associated with faster engraftment and better overall survival. In the setting of this study involving 3 University centers, we analyzed factors potentially influencing cord blood cell composition. In accordance with the results of several previous publications, we observed that gestational age, birth weight and babys gender impacted concentrations of nucleated and hematopoietic progenitor cells in cord blood. We also showed that uses of epidural anesthesia and of oxytocin were associated with higher concentrations of hematopoietic progenitor cells. Interestingly, we observed that nucleated cell and progenitor cell concentrations were also determined by time of day and month of delivery. Recent studies have suggested chronological rhythmic egress of hematopoietic stem and progenitor cells from the bone marrow to the peripheral blood in adult individuals. Our findings suggest that such physiological rhythm may not be restricted to post-natal life. We think our study may have practical implications for cord blood banking strategies and also raises questions about chronological rhythm in hematopoietic cell trafficking during fetal life.

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.

Multiple immunophenotyping at the single-cell level

There is a growing interest for flow and image cytometry analytes permitting the simultaneous discrimination of 6-10 fluorochromes. We have derived a strategy to optimize the factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. Following a spectrophotometric analysis of 14 fluorochromes conjugated to streptavidin (SA), a set of 7 spectrally separable SA- dyes and appropriate filter combinations were selected for evaluation in image cytometry. The SA-dyes were bound to latex particles labeled with biotinilated mIgG1 and the emissions of all fluorochromes detected by each filter combination were measured. The resulting crosstalk matrix served as the basic tool for final selection of dyes, design of optimal trichroic beamsplitter and filter combination, modulation of illumination and mathematical correction of residual spectral overlap. Using this strategy we demonstrated that latex-bound SA conjugates of Cascade Blue, Lucifer Yellow, FITC, R-PE, Red613, PerCP and APC could be discriminated. More recently we extended the applicability of the technique by analyzing blood cells bound to glass slides. The same field could be initially measured for autofluorescence and non-specific IgG binding and then remeasured for specific binding of lineage markers. The ability to use paired measurements of background and total fluorescence is a significant advantage of image over flow cytometry.

The Genesis of Platelet Volume and Density Distributions

Distributions of platelet volumes have been considered to be a meaningful index of platelet heterogeneity since they can be determined with high resolution (McDonald et al. 1964; von Behrens 1975; Paulus 1975; McDonald 1976; Mundschenk et al. 1976; Haynes 1980; Paulus et al. 1986), they are correlated with platelet function (Karpatkin 1969, 1978a; Thompson et al. 1982, 1983a, 1984; Thompson and Jakubowski 1988) and they have clinical significance (Kraytman 1973; Godwin and Ginsburg 1974; Paulus 1975; von Behrens 1975; Paulus and Casals 1978; Zeigler et al. 1978; Levin and Bessman 1983; Bury et al. 1983; Thompson and Jakubowski 1988). However, they are also instructive from the biological point of view because they result from an effective randomizing process which appears in evolution with the emergence of higher orders. Lognormal size distributions may be generated by a limited number of plausible mechanisms (Aitchison and Brown 1957) and they therefore provide insight into the physiology of megakaryocyte terminal maturation. This chapter will summarize the main features of platelet size distributions and discuss the cytological mechanisms which explain their genesis.