Blood-derived stem cell collection in acute nonlymphoblastic leukemia: predictive factors for a good yield.

Blood-derived stem cell (BDSC) autografting represents an interesting theoretical approach to the treatment of acute nonlymphoblastic leukemia (ANLL). The feasibility and safety of this procedure have now been established by several observations of rapid hematopoietic recovery following high-dose chemotherapy and autologous reinfusion of BDSCs harvested during remission. Using clonogenic assays for granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte erythrocyte monocyte colony-forming units (CFU-GEM), we tested peripheral blood samples from 20 consecutive patients recovering from induction chemotherapy for ANLL. Results were correlated with other clinical and hematological factors in order to define optimal criteria for successful BDSC harvesting. Two different patterns of increment were observed in the number of peripheral blood progenitor cells during early recovery from chemotherapy in our patients, suggesting that a total period of 10-12 days should be covered for ideal BDSC harvesting by cytapheresis. Associated clinical factors that appear predictive for a better yield are: 1) chemotherapy with daunorubicin, cytosine arabinoside, and thioguanine (DAT), 2) synchronous recovery of monocytes and platelets, and 3) a good overall performance status of the patient. Complete remission status and absence of ongoing infection should also be considered when selecting patients for BDSC harvesting. No correlation was found between the number of circulating CFUs, as indicated by the clonogenic assays, and that of either My-10- or HLA-Dr-positive cells identified by immunofluorescence on the same blood samples. In this study, less than 50% of patients with newly diagnosed ANLL would have been considered good candidates for a BDSC harvesting program.

Granulocyte recovery after sequential transfusion of mobilized blood stem cell concentrates in syngeneic recipients.

Three patients received sequential transfusions of G-CSF-mobilized peripheral blood stem cells from their identical twin in an attempt to abrogate neutropenia. Blood stem cells were harvested by leukapheresis in the healthy donor twins following mobilization with rhG-CSF at 5 micrograms/kg/day subcutaneously for at least 5 days. An average of 2.2 x 10(7) CFU-GM (range: 1.4-3.3) were collected and transfused without further manipulation. One patient, transfused with a CFU-GM dose of 3 x 10(7) on day +6 after a syngeneic marrow transplant, experienced near-complete elimination of absolute neutropenia until spontaneous engraftment occurred on day +11. In the other two patients, we unexpectedly observed a transient granulopoietic inhibition, possibly related to the high T cell content of the blood stem cell transfusions.

Evaluation of a simplified filtration technique for the routine measurement of erythrocyte deformability.

The purpose of this study was to assess the value of a widely-used simplified filtration technique (mini-test) by comparing it, in parallel experiments, with a constant-flow rate method currently used in our laboratory for the measurement of erythrocyte deformability. Both methods employed 3 micrometer pore-diameter Nucleopore membranes and were performed under different flow conditions, using washed erythrocyte suspensions. In a total of 49 evaluable test pairs obtained from blood samples of 55 patients with either diabetes mellitus [36], post-splenectomy status [13] or haemolytic anaemia [6], we observed a sensitivity of 40% and a specificity of 75.5% for the mini-test versus our reference method. A near 20-fold lower cell/pore ratio appears as the single most important technical difference explaining the apparent lack of sensitivity of the mini-test. Suggestions are given as to how to modify the mini-test in order to obtain maximum sensitivity in the future.

Calculation of surface area and volume of human erythrocytes from scanning electron micrographs.

This article demonstrates that surface and volume measurements of individual human erythrocytes with varying shapes can be obtained from scanning electron micrographic stereopairs using an approach based on established principles of photogrammetry. Instead of calculating the coordinates of several hundred points plotted for each cell, a procedure that proves tedious and time-consuming, we show that a reasonable approximation of cell surface and volume can be achieved from simple geometrical models constructed with a small number of carefully measured points and angles, using a stereocomparator. The values obtained for two normal erythrocytes and for two distorted red cells from a patient with congenital pyruvate kinase deficiency haemolytic anaemia are consistent with available information on the geometry of these cells. Because scanning electron microscopy requires extensive manipulation of the cells, the values obtained cannot be applied to fresh living material, but appear fairly accurate for the purpose of comparison between cells prepared in the same manner.

The measurement of erythrocyte deformability using micropore membranes. A sensitive technique with clinical applications.

This article describes a positive-pressure filtration technique using low pore-density, 3 microns pore-diameter polycarbonate membranes employed to evaluate erythrocyte deformability in a clinically oriented hematology laboratory. Mean erythrocyte resistance to filtration was expressed by a numerical index which takes into account both the initial resistance of dilute red cell suspensions to passage across the membrane and the relative pressure rise observed after filtration of 30 ml of the same suspension (3 . 10(8) cells). The resistance index of 99 blood samples obtained from 88 healthy adult volunteers ranged between 1.4 and 2.9, with normal Gaussian distribution and mean of 2.04. Values obtained on similarly prepared samples from 20 patients with various hemolytic anemias always fell outside this range, indicating a reduced deformability in every case. The existence of a strong positive correlation was found between the resistance index and the degree of reticulocytosis in these patients. This method appears more sensitive than previously described filtration techniques in detecting the presence of small numbers of poorly deformable erythrocytes in vivo, while being more practical and statistically more significant than the micropipette elastimetry technique. Our results, however, raise a new question concerning the role of reticulocytes in the evaluation of red cell deformability on blood samples from patients with hemolytic anemia.

[Morphology and deformability of erythrocytes in muscular dystrophy]. / Morphologie et déformabilité des érythrocytes dans la dystrophie musculaire.

In recent years, the presence of red cell morphological abnormalities in patients with Muscular Dystrophy has made the object of numerous, often contradictory reports. A possible source of such confusion may lie in the fact that human erythrocytes are extremely sensitive to morphologic transformations resulting from various manipulations or environmental conditions in vitro. We have examined the morphology and deformability of erythrocytes from 7 patients with Duchenne and 9 patients with Steinert (myotonic) Muscular Dystrophy. To avoid preparation artifacts, fresh, unwashed red cells suspended in their own plasma were examined under phase contrast microscopy for the presence of either echinocytes and stomatocytes. Deformability was measured by filtration of dilute cell suspensions at constant flow rate through nucleopore membranes (nominal pore diameter = 3 micrometer). No significant difference was found between the patients' cells and those of 22 healthy volunteer controls. We conclude that previously reported abnormalities may have been the result of preparation artifacts. It appears possible, however, that erythrocytes from Muscular Dystrophy patients may be more sensitive than normal ones to certain stimuli originating from red cell manipulations in vitro.

[Splenectomy for idiopathic thrombopenic purpura. A retrospective study of 40 cases]. / La splénectomie dans le purpura thrombocytopénique idiopathique. Etude rétrospective de 40 cas.

The authors analysed retrospectively the files of 40 patients who underwent splenectomy for idiopathic thrombocytopenic purpura and who were totally or partially (relapse) resistant to a therapeutic trial of corticosteroids. There was no operative mortality but morbidity, especially respiratory, was substantial, being 40%. Drainage of the splenic bed should be avoided because this is clearly associated with a greater frequency of pulmonary complications. In 37 patients with a mean follow-up of 30.3 months, there was complete remission in 89.2% after splenectomy. This confirms the therapeutic value of splenectomy in idiopathic thrombocytopenic purpura, especially in those who showed a partial (transitory) response to corticosteroids.

Erythrocyte populations in pyruvate kinase deficiency anaemia following splenectomy. II. Cell deformability.

The deformability of three previously described morphological populations of erythrocytes from splenectomized pyruvate kinase (PK) deficient patients was assessed using both Nucleopore filtration and micropipette techniques. Mean deformability was decreased in fresh cells from all patients studied. Selective measurements indicated that immature lobulated reticulocytes in each patient were the least deformable cells, followed by bizarre echinocytes characterized by a hyperviscous behaviour, and by thin macrocytic discocytes whose values were closest to those of control erythrocytes. We conclude that poorly deformable reticulocytes in these patients, after initial trapping in the spleen, are unable to return to the circulation because of their inordinate dependence on mitochondrial oxydative phosphorylation for maintenance of a sufficient intracellular concentration of adenosine triphosphate (ATP); hence their rapid and selective destruction. Splenectomy therefore may be beneficial by allowing such cells to mature more slowly in the circulation of these patients until they develop crenation and hyperviscosity, two manifestations of profound ATP depletion possibly leading to destruction by the liver.

Erythrocyte populations in pyruvate kinase deficiency anaemia following splenectomy. I. Cell morphology.

Light and scanning electron microscopic examination of fresh erythrocytes from the blood of four patients splenectomized for severe pyruvate kinase (PK) deficiency haemolytic anaemia has revealed three morphologically different cell populations. Most striking in these patients was the presence of crenated red cells (30-50% of total) which fit current criteria for definition as echinocytes, but also with unusual surface retraction compatible with the existence of intracellular shrinkage, a phenomenon known to occur after incubation of PK deficient red cells in vitro. Additional cell types include immature reticulocytes (7-8% of total) and thin macrocytic discocytes (45-65% of total) none of which are considered specific for the disease. These morphological findings are in sharp contrast with the paucity of red cell shape abnormalities usually described in PK deficient patients before splenectomy and may provide insight into the mechanisms leading to accelerated erythrocyte destruction in this disease.

[Morphology and deformability of erythrocytes in muscular dystrophy]. / Morphologie et déformabilité des érythrocytes dans la dystrophie musculaire.

In recent years, the presence of red cell morphological abnormalities in patients with Muscular Dystrophy has made the object of numerous, often contradictory reports. A possible source of such confusion may lie in the fact that human erythrocytes are extremely sensitive to morphologic transformations resulting from various manipulations or environmental conditions in vitro. We have examined the morphology and deformability of erythrocytes from 7 patients with Duchenne and 9 patients with Steinert (myotonic) Muscular Dystrophy. To avoid preparation artifacts, fresh, unwashed red cells suspended in their own plasma were examined under phase contrast microscopy for the presence of either echinocytes and stomatocytes. Deformability was measured by filtration of dilute cell suspensions at constant flow rate through nucleopore membranes (nominal pore diameter = 3 micron). No significant difference was found between the patients' cells and those of 22 healthy volunteer controls. We conclude that previously reported abnormalities may have been the result of preparation artifacts. It appears possible, however, that erythrocytes from Muscular Dystrophy patients may be more sensitive than normal ones to certain stimuli originating from red cell manipulations in vitro.

The use of plasma as a replacement fluid in plasma exchange. Canadian Apheresis Group.

BACKGROUND: The Canadian Apheresis Group has maintained a national registry of apheresis activities for the past 16 years. Since 1991, the use of plasma as a replacement fluid in plasma exchange has been recorded. STUDY DESIGN AND METHODS: Six years of data from the registry on the use of plasma as a replacement fluid were analyzed. RESULTS: Plasma was used in more than 25 percent of all plasma exchange procedures. Of 41,519 plasma exchange procedures reported, 11,970 used plasma alone or in combination with albumin. In 1991, 1026 (78%) of these procedures used plasma appropriately for either thrombotic thrombocytopenic purpura (TTP) or adult hemolytic uremic syndrome (HUS); between 1992 and 1996, these numbers were 1043 (81%), 1570 (86%), 1171 (87%), 2192 (92%), and 2741 (90%), respectively. In the remaining procedures, frozen or cryosupernatant plasma was administered to 326 patients for a total of 40 diseases other than TTP or HUS. CONCLUSIONS: In those diseases for which plasma was administered as the sole replacement fluid, no disease appears to justify such treatment without the existence of an associated condition requiring specific replenishment of some plasma component. Further evaluation of the specific indications for the use of plasma as a replacement fluid in plasma exchange is required for diseases other than TTP or HUS.

Acute Heinz-body anemia due to severe cresol poisoning: successful treatment with erythrocytapheresis.

A patient with massive intravascular Heinz-body hemolytic anemia associated with the presence of bizarre-looking erythrocytes following the oral ingestion of approximately 100 mL of "penetrating oil", a petroleum distillate containing 85% kerosene, 12% cresol and 2% surfactant, is described. He was treated successfully with immediate erythrocytapheresis and forced diuresis.

Observation of echinocytosis in eight patients: a phase contrast and SEM study.

We report eight cases of echinocytosis discovered after screening of stained smears. The presence of echinocytes was exceptional in adults and children but was more frequent in neonates. In all cases we confirmed the presence of abnormal red cells by careful examination of the blood in wet preparations observed in phase contrast and after glutaraldehyde fixation and processing for SEM. No discernible common denominator was found to explain the presence of echinocytes in the blood of our patients. No echinocytogenic factor could be demonstrated in the fresh plasma. Echinocytes appeared quickly in patients who received exchange transfusion or transfusion of normal red cells. By contrast, in vitro incubation in the presence of substrates allowing constant ATP level never resulted in the formation of echinocytes when normal red cells were suspended in patient's plasma.

Contrasting splenic mechanisms in the blood clearance of red blood cells and colloidal particles.

We have studied the blood clearance and organ uptake of colloidal particles and of antibody-coated and chemically treated Na2 51Cr O4-labeled erythrocytes (RBC) in mice. Hepatic and splenic uptake of both colloidal particles and autologous RBC coated with rabbit antibody were reduced significantly following pretreatment of animals with cortisone acetate. Hepatic removal of RBC previously treated in vitro with N-ethyl-maleimide (NEM) or phenylhydrazine-HCl (PHZ) was similarly depressed by pretreatment with cortisone. In contrast, the splenic uptake of NEM- and PHZ-altered erythrocytes was unaffected by cortisone. Scanning and transmission electron microscopic examination of perfused spleens from PHZ-injected animals demonstrated extensive mechanical trapping of Heinz body-containing RBC in sinus wall apertures, whereas little erythrophagocytosis was observed. These studies suggested that, while clearance of inert particulate matter and of antibody-coated RBC from the blood occurred primarily by a cortisone-suppressible, presumably phagocytic process in the spleen, chemically altered RBC were removed primarily by a cortisone-insensitive filtration process in the splenic microvasculature.

Persistent polyclonal B-cell lymphocytosis: further evidence for a genetic disorder associated with B-cell abnormalities.

Persistent polyclonal B-cell lymphocytosis (PPBL) is an intriguing disorder diagnosed predominantly in women, usually cigarette smokers, characterized by an increase in the number of polyclonal B lymphocytes. Abnormality of the B-cell population is also evidenced by the presence of multiple bcl-2/Ig gene rearrangements and the finding of an additional long arm chromosome 3q+ (i3)(q10) within a significant proportion of B cells. The physiopathology of PPBL is unknown but its association with the HLA DR7 phenotype suggests a possible genetic disorder. To further determine whether PPBL has a genetic predisposition, we have undertaken an extensive study in a large family of a patient diagnosed with PPBL. Three individuals among the first-degree relatives presented all the criteria for a diagnosis of PPBL. A slight increase in serum IgM without evidence of B-cell proliferation was shown in two additional siblings. Multiple bcl-2/Ig gene rearrangements, a typical feature of PPBL, were identified in 8/10 individuals among first-degree relatives. A statistically significant association was found between the presence of these rearrangements and of a paternal HLA haplotype. We conclude that PPBL has a familial occurrence suggesting an underlying genetic defect. The development of the complete syndrome probably relies on unidentified additional co-factors.

Hematopoietic engraftment from a minimal number of apheresis procedures after mobilization of peripheral blood stem cells with chemotherapy and rhG-CSF.

In a cohort of 13 patients, peripheral blood stem cells (PBSC) were harvested by apheresis after mobilization with chemotherapy and rhG-CSF. Nine patients who had excellent mobilization were transplanted with PBSC concentrates from a minimal number of apheresis procedures (mean of 1.5, range = 1-3). During collection, the number of circulating progenitors was on average 50 times higher than those observed at the steady state in the peripheral blood of healthy unstimulated individuals. The mean number of CFU-GM/kg reinfused per patient was 28.1 x 10(4) (range = 18.0-50 x 10(4)). The use of rhG-CSF, at either 1 or 5 micrograms/kg/day, resulted in a significantly greater yield of CFU-GM per mononuclear cells than that observed previously in a comparable group of patients receiving chemotherapy alone. Prompt and durable engraftment occurred after myeloablative chemotherapy. The average duration of absolute neutropenia was 9 days. Transfusion requirements were low with an average of four packed red cell units and two platelet transfusions per patient. The shortest follow-up is 5 months and the longest is 20+ months. The convenience of this new approach to support myeloablative therapy offers new possibilities for the administration of a higher dose-intensity of chemotherapeutic agents. A limited number of apheresis procedures timely harvested will improve the cost effectiveness of transplant programs.