Circulating Cd34+ cell count differentiates primary myelofibrosis from other Philadelphia-negative myeloproliferative neoplasms: a pragmatic study.

A high number of circulating CD34+ cells has been advocated to distinguish primary myelofibrosis from other Philadelphia-negative myeloproliferative neoplasms. We re-evaluated the diagnostic interest of measuring circulating CD34+ cells in 26 healthy volunteers and 256 consecutive patients at diagnosis for whom a myeloproliferative neoplasm was suspected. The ROC curve analysis showed that a number of CD34+ <10/µl excludes the diagnosis of primary myelofibrosis with a sensitivity of 97 % and a specificity of 90 % (area under the curve: 0.93 [0.89-0.98]; p < 0.001). Patients with PMF harboring a CALR mutation had more circulating CD34+ cells than patients with either a JAK 2 or MPL mutation (p = 0.02 and p < 0.01, respectively). These results suggest that this fast, simple, non-invasive, and standardized test is of particular interest to exclude the diagnosis of primary myelofibrosis.

Comparison of four serum-free, cytokine-free media for analysis of endogenous erythroid colony growth in polycythemia vera and essential thrombocythemia.

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC), a characteristic of polycythemia vera and essential thrombocythemia, is not standardized. In this multicentric study, we tested four semisolid, serum-free, cytokine-free media based on either methylcellulose (M1, M2) or collagen (C1, C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26), essential thrombocythemia (19), secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without, or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific, as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia, nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria, respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo, the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay, now part of the new criteria of polycythemia vera.

Use of pathology-specific peripheral blood CD34 thresholds to predict leukapheresis CD34 content with optimal accuracy: a bicentric analysis of 299 leukaphereses.

CD34+ cell counts in peripheral blood (PB) and corresponding numbers of CD34+ cells and colony-forming units-granulocyte/macrophage (CFU-GM) in 299 leukapheresis products of 209 patients undergoing PB progenitor cell (PBPC) mobilization for autologous transplantation in two different centers were analyzed and compared according to diagnosis: non-Hodgkin lymphoma (NHL, 94 leukaphereses), multiple myeloma (MM, 75), Hodgkin's disease (HD, 37), solid tumors (35), and chronic myeloid leukemia (CML, 32). Without separating disease entities, correlations between PB CD34+ cell counts and leukapheresis content of CD34+ cells (r>0.83, P<0.01) and CFU-GM (r>0.81, P<0.01) were excellent. In both centers, a PB CD34 threshold ensuring a leukapheresis yield > 10(6) CD34/kg was determined. This threshold was higher in center 1 than in center 2, and its predictive accuracy (91.4%, i.e., prediction correct 91.4% of the time) was significantly lower than in center 2 (98.4%, P=0.02). When data were analyzed by pathology, PB CD34+ cell counts and leukapheresis content of CD34+ cells and CFU-GM remained well correlated, and in both centers PB CD34 thresholds predictive of a yield > 10(6) CD34/kg per leukapheresis could be determined for each pathology. For most patients, pathology-specific PB CD34 thresholds could be obtained directly from the equation of the PB CD34/leukapheresis CD34 correlation curve; they varied depending on both pathology and center (range: 7-20 x 10(6) CD34/l). Pathology-specific thresholds predicted a leukapheresis yield > or = 10(6) CD34/kg accurately 100% of the time for MM patients in center 2 and HD and solid tumor patients of both centers, resulting in overall rates of accurate prediction of sufficient graft CD34 content of 96.6% in center 1 and 98.9% in center 2.

Reproducible scoring of CFU-GM and BFU-E grown in collagen-based semisolid medium after a short (3 h) training.

Colony counting remains an important source of variation in colony-forming unit-granulocyte-macrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3 h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.

Endogenous erythroid and megakaryocytic colony formation in serum-free, cytokine-free collagen gels.

We studied the suitability of collagen-based semisolid medium for assay of endogenous erythroid colony formation performed in myeloproliferative disorders. Bone marrow (BM) mononuclear cells (MNC) from 103 patients suspected of having polycythemia vera (PV, 76 patients) or essential thrombocythemia (ET, 27 patients) were grown in collagen-based, serum-free, cytokine-free semisolid medium. Colony analysis at day 8 or 10 showed that this collagen assay is specific, as endogenous growth of erythroid colonies was never observed in cultures of 16 healthy donors and 6 chronic myelogenous leukemia (CML) patients. Endogenous erythroid colony formation was observed in 53.3% of patients suspected of PV, with only 15.4% of positive cultures for patients with 1 minor PV criterion and 72% (p = 0.009) of positive cultures for patients with > or =2 minor or 1 major PV criterion. Similarly, endogenous growth of erythroid colonies was found in 44.4% of patients suspected of ET, with 31.6% of positive cultures for patients with 1 ET criterion versus 75% for patients with > or =2 ET criteria. In addition, we found that in collagen gels, tests of erythropoietin (EPO) hypersensitivity in the presence of 0.01 or 0.05 U/ml of EPO and tests of endogenous colony-forming units-megakaryocyte (CFU-MK) formation cannot be used to detect PV or ET, as these tests were positive for, respectively, 21.4% and 50% of healthy donors and 83% and 50% of CML patients. A retrospective analysis suggests that collagen assays are more sensitive than methylcellulose assays to assess endogenous growth of erythroid colonies. In summary, serum-free collagen-based colony assays are simple and reliable assays of endogenous growth of erythroid colonies in myeloproliferative diseases. They also appear to be more sensitive than methylcellulose-based assays.

Use of collagen for standardization of PBSC graft quality evaluation: a multicenter comparative analysis of commercial collagen-based and methylcellulose-based colony-forming unit (CFU) assay kits.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay, an essential test in evaluation of the quality of autologous grafts of hematopoietic stem cells, has yet to be standardized. With this aim in view, we carried out a multicenter study of five commercially available culture kits for CFU-GM evaluation. Four kits were methylcellulose-based (H4431, H4434, H4435, StemBio1d) and one was collagen-based (EasyClone-Multi). Using fresh and frozen samples of PBSC grafts, we compared CFU-GM and burst-forming unit-erythrocytes (BFU-E) growth using the EasyClone kit to each of the methylcellulose kits. BFU-E and CFU-GM clonogenicity of both fresh and frozen PBSC was clearly inferior with the H4431 kit, which provides conditioned medium only. CFU-GM numbers obtained with fresh and frozen PBSC samples were significantly higher with the EasyClone kit than with the H4434 and StemBio kits. BFU-E numbers were also higher with the EasyClone kit, but only when colonies were scored after May-Grünwald-Giemsa (MGG) staining. Finally, although the H4435 kit provides higher doses of recombinant cytokines than the EasyClone kit, CFU-GM and BFU-E numbers obtained for fresh or frozen PBSC with both kits were similar. In addition, CFU-GM and BFU-E numbers correlated well with CD34+ cell numbers for all five kits for both fresh and frozen PBSC. In summary, our study shows that the EasyClone-Multi and H4435 kits provide the best CFU-GM growth. The collagen-based EasyClone kit has the additional advantage of allowing gel staining and storage, which facilitates colony identification and, more importantly, makes gel exchange possible for standardization of the CFU-GM assay.

Tissue concentrations and correlations of prostaglandins in healthy and inflamed human esophageal and jejunal mucosa.

The PGE2, PGF2 alpha, PGI2, and TXB2 content in biopsies of healthy esophageal mucosa and inflamed mucosa and from subjects with chronic esophagitis was measured and statistically analyzed. No significant differences were found between the tissue concentrations of prostaglandins in the inflamed and the healthy mucosa, except for elevated PGI2 content in the inflamed esophageal mucosa in comparison to healthy mucosa. The prostaglandin content of jejunal mucosa was unchanged in jejunitis and in atrophy compared to findings in healthy subjects. Regression analysis revealed a significant negative correlation between the PGF2 alpha and PGI2 content in both inflamed esophageal and inflamed jejunal mucosa. In healthy mucosa, no correlation was found between the tissue concentrations of these two prostaglandins, either in the esophagus or in the jejunum. These results suggest the redistribution of cyclic endoperoxide metabolism under certain pathological conditions.

Collagen matrix: an attractive alternative to agar and methylcellulose for the culture of hematopoietic progenitors in autologous transplantation products.

Autografts using untreated or in vitro manipulated bone marrow and peripheral blood stem cells represent promising approaches to the treatment of malignant diseases. In this work, the collagen gel culture technique was compared with agar and methylcellulose for its capacity to permit the growth of human granulomonocytic (day 14 CFU-GM; collagen vs agar or MTC) or erythroblastic (day 7 CFU-E and day 14 BFU-E; collagen versus methylcellulose) colonies in autologous transplantation products. Our results show that the collagen culture system always gave as many or more colonies than the other techniques. It also allowed harvesting of gels onto glass slides and subsequent May-Grünwald-Giemsa, cytochemical or immunocytochemical staining. We suggest that the collagen assay represents an interesting alternative to the widely used agar or methylcellulose systems for the culture of hematopoietic progenitors because of the equal or higher number of colonies detected, the easy phenotypical identification of colonies in stained gels, and the ability to store high-quality documentation. This technique is particularly attractive for use in the quality control of autologous bone marrow transplantation procedures.

Collagen gel cultures for detection of spared hematopoietic progenitors in Asta Z 7557 purged human marrows.

Asta-Z 7557, an in vitro active metabolite of cyclophosphamide, is used for human bone marrow purging in acute leukemias and solid tumors since it is more highly toxic to tumor than normal hematopoietic clonogenic cells. However, doses higher than 80 microM totally inhibit the growth of hematopoietic progenitors in semisolid assays, despite preservation of marrow repopulating ability in vivo. This discrepancy complicates assessment of the functional value of purged grafts. In 11 patients undergoing autologous bone marrow transplantation with 100 microM Asta-Z purged marrow, we investigated the advantages of the collagen gel culture system for detection of spared hematopoietic progenitors. This technique provides a suitable and convenient assay as compared with results with agar. In nine of 11 samples after 14 or 21 days of incubation, up to 100 colonies were observed, (64% monocytic, 27% granulocytic). Day-14 granulocytic colonies were mostly immature, suggesting that they derived from early CFU-GM or pre-CFU-GM. This development of early progenitors could account for the better sensitivity of this technique compared with agar or methylcellulose systems. The mechanism involved as well as the correlation between colony growth in collagen and clinical hematopoietic recovery is also considered.

[Tomato skin in the common bile duct after endoscopic sphincterotomy]. / Endoszkópos sphincterotomia után a közös epevezetékbe jutott paradicsomhéjakról.

Lithotomy and endoscopic sphincterotomy were carried out in a 83-year-old woman because of choledocholithiasis. One year later calculi and tomato skins were found in the common bile duct. Significance is attributed to the tomato skins in the development of calculi. The attention is drawn to the observation that operative interventions performed through the Vater's papilla, spontaneous or iatrogenic choledochoduodenal fistulas create favourable conditions for foreign material to get into the choledochus and this may cause lithogenesis.

[A simple isotope method for studying of gastric emptying of solid foods]. / Egyszerü izotóp módszer a szilárd táplálék gyomorürülésének vizsgálatára.

A simple isotope method is explained by the authors for noninvasive functional evaluation of the gastric emptying of solid meal. According to the results of in vitro measurements suitable solid phase labelling was provided by boiling the egg traced with injected Tc-99m-pertechnetate. The evaluation of gastric emptying by means of test meal containing the radiolabelled boiled egg is simple, inexpensive and the radiation hazard is minimal. In cases of intact stomach the gastric emptying of solids (T2 = 115.1 +/- 12.4 min) were significantly slower, than in cases of Billroth I (T2 : 66.7 +/- 6.9 min, p less than 0.01) and Billroth II (T2 = 28.8 +/- 3.3 min, p less than 0.001) resections. Similarly, there was a significant difference (p less than 0.001) between the mean half time values of the two operated groups.

Mucosal prostaglandin levels of the gastric stump.

The endogenous mucosal PG levels (PGE2, PGF2 alpha, PGI2, TXB2) of the gastric stump were investigated from biopsy materials, after partial gastrectomy made for ulcer disease. The mucosa of the stomach remnant were found to contain mainly PGE2 and PGI2. The PG contents of the mucosa of gastric stump were not influenced by the type of resection. Mucosal PG concentrations on the greater curvature were not dependent on the patients' age, the indication of the gastrectomy, the duration of postoperative interval, or the sex of the patient. There was no relation between the secretion capacity of the resected stomach and the mucosal PG contents of the greater curvature. After Billroth I and II gastrectomy procedures equivalently fair correlations have been established between the mucosal levels of PGE2 and PGF2 alpha, PGE2 and TXB2, PGF2 alpha and TXB2, PGI2 and TXB2 on the greater curvature, respectively. After both types of gastrectomy the mean PGI2 mucosal concentration of the greater curvature was significantly lower than those of the gastroenteroanastomosis and lesser curvature below the cardia, which in turn did not differ from each other. Biliary reflux does not cause characteristic alterations of the mucosal PG levels on the greater curvature. No definite relation between the histological findings of the mucosa and the PG concentrations was observed, which suggests a secondary role of the endogenous PGs in the pathogenesis of light microscopic mucosal alterations of the resected stomach.

On growth regulation of the rat promyelocytic leukemia (BNML): growth inhibition and eradication of clonogenic cells by cholera toxin.

Our recent establishment of several permanent in-vitro cell lines from Brown Norway rat leukemia (BNML) and the development of a clonogenic assay prompted us to undertake detailed studies on the growth control mechanism of a cell type which for several years has served as an animal model for human AML and preclinical studies. So far, these cells have no defined biological regulators but require intricate cellular interactions to sustain their growth. The effects on cell growth and clonogenicity, of agents known to modify the intracellular levels of cyclic nucleotides, were analysed. Here we report that CT binding strongly inhibited cell growth at a wide range of concentrations (10(-6)-10(-14) M) while beta chain pentameric subunits or alpha chain had no effects. Cell growth was inhibited in a dose-dependent manner. The ligand-receptor interactions mediated the alpha chain's transit through the membrane; the adenylate cyclase activation and the rise in c-AMP levels (60 min) resulted in DNA synthesis arrest (5 h), then finally ended in cell death (24-48 h). A significant decrease in the clonal ability of treated cultures was seen. A decrease of up to five logs in the clonogenic cell number was observed after 48 h of toxin treatment (10(-7) M). The growth inhibition of CT were reproduced by several agents (PGE, theophylline, isobutylmethylxanthine) known to raise intracellular c-AMP levels. Data are commented from a biochemical approach to intracellular events controlling the cell growth of this leukemia. The potential interests of c-AMP inducing agents on the eradication of this leukemia by ex-vivo marrow treatments are also considered.

Evaluation of covalent antisickling compounds by PO2 scan ektacytometry.

The ektacytometer, a device to measure erythrocyte flexibility, has been used to evaluate antisickling agents that covalently modify hemoglobin S (HbS). The instrument has been adapted to produce a continuous gradient of oxygen pressure in the measuring cuvette, which permitted the rapid determination of sickle cell rigidity over the complete oxygenation range. Inspection of curves allows classification of the compounds according to their mode of action: altering oxygen affinity or increasing deoxy-HbS solubility. Reagents that modify amino groups, thiols, and histidine, as well as a crosslinking agent, were examined. The method directly evaluates deoxygenated cell deformability rather than cell shape. Many of the compounds that are effective in preventing the morphological sickling of deoxygenated sickle cells do not necessarily restore cell deformability. The method also readily detects membrane damage brought about by covalent agents that nonspecifically derivatize membrane proteins. Cystamine and pyridoxal appear to improve deformability in deoxygenated SS cells at concentrations that do not damage the membrane. This method, which examines the intact cell, fills a gap in the available experimental techniques for drug evaluation, between studies of isolated hemoglobin and in vivo studies.