Choosing sample sizes for various blood parameters of broiler chickens with normal and non-normal observations.

Experimental power is a measure of the ability of an experiment to detect differences between treatment means. Researchers design experiments and then calculate the probability that differences are simply due to chance, the null hypothesis. The objective of the analyses reported here was to determine the appropriate number of samples to demonstrate significant differences of various magnitudes from broiler chicken blood constituents. Over 800 samples were taken for a study of the effects of sample storage time, serum vs. plasma, light intensity, and fed vs. fasted birds on blood cholesterol, triglycerides, uric acid, glucose, total protein (TP), albumin, globulin, alanine aminotransferase (ALT), aspartate aminotransferase, gammaGT, creatinine, alkaline phosphatase, Ca and P. Various transformations increased the QQ plot R2 values from 0.000 to 0.149 or 0.00 to 17.62%. Most of the QQ plot R2 values were at or above 0.90. The 1/x2 transformation of blood P data showed the biggest increase in QQ plot R2 (0.846 to 0.995). The different standard deviations and coefficients of variation (CVs) found for each variable resulted in widely different numbers of replicates needed to detect differences in 2 treatment means. The extremes were glucose with a CV of 6.9% and ALT with a CV of 39.7%. For glucose, 15 replicates are needed to find a 10% difference in 97% of experiments; for ALT, 15 replicates would detect a 50% difference 91% of the time. The use of parameters such as cholesterol, glucose, TP, albumin, and globulin showed low CVs, indicating they may be considered as stable parameters. The lower CVs make it possible to find differences with a smaller number of replicates used in studies. As reported, the phosphorus values did not have a normal distribution of the data, so a transformation of these data could be an alternative to better discuss the results found.

Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Automated immunostaining of cell smears: an alternative to flow cytometry.

AIMS: To assess the applicability of an automated tissue immunostainer machine for the phenotypic analysis of peripheral blood and bone marrow aspirate smears. METHODS: Air-dried smears of peripheral blood and bone marrow from normal individuals and 14 patients with haematological malignancies were stained, following fixation, with a range of antibodies to haemopoietic antigens using both immunoperoxidase and immuno-alkaline phosphatase methods on the Bond-maX (Leica Microsystems) automated immunostaining machine. RESULTS: Automated protocols used for staining formalin-fixed paraffin-embedded tissue could be applied to cell smears, giving high quality staining with excellent cytological detail and antigenic preservation for an extensive antibody panel. Optimal quality (morphological preservation and antigen detection without background staining) was obtained with an alkaline phosphatase method and Fast Red chromogenic substrate. Both cell surface and intracellular (cytoplasmic and nuclear) antigens could be detected and gave the expected reactivity. CONCLUSIONS: Automated immunostaining is possible for haematological smears. It generates consistent high quality staining, with the exception of surface immunoglobulin, and is applicable to haematological and, potentially, cytological smears. It provides a practical alternative to flow cytometry and will have particular application when smears are available and there is no suitable sample for flow cytometry.

Lymphocyte apoptosis in acute respiratory syncytial virus bronchiolitis.

Respiratory syncytial virus (RSV) infection may have an effect on the development of T cell memory responses. RSV bronchiolitis in infants is associated with a transient decline in circulating lymphocytes. We hypothesized that the mechanism underlying this lymphopenia is apoptosis. Blood was taken from 32 infants during primary RSV bronchiolitis and three months later. Using flow cytometry, we found that absolute numbers of both CD3+/CD4+ T-helper lymphocytes (P = 0.029) and CD3+/CD8+ cytotoxic lymphocytes (CTL) (P = 0.043) were significantly reduced during acute infection. Up-regulated expression both of Fas (P < 0.001) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor (P < 0.001) was found during acute illness on both CD3+/CD4+ and CD3+/CD8+ lymphocytes, when compared with convalescent samples. Expression of Fas on CD4+ lymphocytes was inversely related to CD4+ number (P = 0.03). Plasma levels of soluble Fas ligand (P = 0.028) and caspase-1 (P = 0.037), determined by enzyme-linked immunosorbent assay, were increased during bronchiolitis. Plasma interleukin-18, a product of caspase-1 activity, was not raised. Taken together, these data suggest that in acute RSV infection, CD4+ helper lymphocytes and CD8+ cytotoxic lymphocytes are primed to undergo apoptosis. This is a mechanism through which lymphopenia may occur and T cell memory may be altered.

Fluorescence in situ hybridization detection of two telomeres on the short arm of a derived chromosome 16 in an infant with thrombocytopenia.

We report a case of severe thrombocytopenia with an abnormal bone marrow karyotype described by G-banding analysis as t(16;21)(p?13;q11). Using fluorescence in situ hybridization (FISH) analysis with whole chromosome paints, the chromosome rearrangement was shown to be more complex, with the additional cryptic involvement of the long arm of chromosome 3. The chromosome rearrangement involved the breakpoints 3q26, 16p13.3, and 21q11; this rearrangement has not been previously described. The size of genomic material translocated from the chromosome 16 homologue was too small to be detected by chromosome paint. A 16p-specific telomeric probe was hybridized to locate the translocated 16p material. The 16p telomeric unique sequence DNA was retained on the der(16) chromosome, indicating a more distal breakpoint. This study demonstrates that telomeric translocations can occur that would be undetected by telomeric-specific FISH probes.

Phosphatidylserine-dependent adhesion of T cells to endothelial cells.

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.

A comparison of CD34+ cell selected and unselected autologous peripheral blood stem cell transplantation for multiple myeloma: a case controlled analysis.

Following ASCT for multiple myeloma, it is unclear whether relapse is due solely to the presence of residual myeloma cells after myeloablation, or whether it is in part attributable to contamination of the stem cell harvest with viable malignant cells. Positive selection of CD34+ cells markedly reduces plasma cell contamination. We performed a case controlled analysis in which 15 patients with myeloma who underwent autologous PBSCT with CD34+cell selection using the Ceprate System (index group), were compared with 15 matched controls. All subjects received an identical preparative regimen. The median times to neutrophils >/=0.5 x 10(9)/l and unsupported platelets >/=50 x 10(9)/l were 14 and 23 days for the CD34+cell selected group and 11 (P = 0.03) and 14 (P = 0.029) for the case controls. Median follow-up of purged patients from autologous PBSCT was 32 months (range 18-43). At 36 months, the probability of PFS was 47 +/- 14% and 46 +/- 14% in the index and control groups (P = 0.44). The 3 year probability of OS was 69 +/- 13% for the CD34+ cell selected arm and 66 +/- 12.4% in unpurged patients (P = 0.91). Median PFS for the cell selected group is 24 months (CI 19.1-36.0), and 29 months for controls (CI 7.1-50.9). Eleven patients undergoing cell selection remain alive, seven of whom are progression free. At the same time-point after unpurged autologous PBSCT, the corresponding figures are 12 patients alive, with seven remaining progression free. Autologous PBSCT with CD34+ cell selection is both feasible and safe, but results in delayed engraftment as compared to case controls. The 3 year probability of PFS and OS in the cell selected arm was similar to that of the unpurged controls. Our findings indicate that autologous PBSCT with CD34+ cell selection appears not to have any favourable effect on disease progression. However, the results of this case controlled analysis should be cautiously interpreted, and the role of CD34+ selection in autologous PBSCT should be further investigated by large randomised trials.

The minimum CD34 threshold depends on prior chemotherapy in autologous peripheral blood stem cell recipients.

We analysed 57 patients with non-myeloid malignancies who received a non-purged autologous PBSCT. All had similar mobilisation and conditioning regimens. A high prior chemotherapy score and the number of chemotherapy lines used (P = 0.015 and P = 0.01, respectively) were adverse predictors of CD34 cell yields. Lower CD34 values (P = 0.002) were seen in patients treated with potent stem cell toxins (BCNU, melphalan, CCNU and mustine), designated toxicity factor 4 agents (TF4). All patients infused with grafts containing CD34 cell doses between 1.0 and 2.0 x 10(6)/kg (range 1.25-1.90) engrafted by day 51. The only variable associated with slow platelet recovery was exposure to TF4 (P = 0.007). The majority of patients with CD34 >1.0 x 10(6)/kg achieved rapid and sustained engraftment and the only predictive factor of delayed recovery is prior exposure to stem cell toxins. Potential PBSCT candidates should if possible avoid first line and salvage chemotherapy containing TF4 drugs. We therefore advocate a minimum CD34 threshold of >1.0 x 10(6)/kg in patients without extensive prior chemoradiotherapy, and > or = 2.0 x 10(6)/kg in all other patients.

Biological properties of peripheral blood progenitor cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor.

Patients transplanted with mobilized blood progenitor cells (PBPC) recover their neutrophil counts more rapidly than patients transplanted with bone marrow even when they receive the same dose/kg of granulocyte-macrophage colony-forming cells (CFU-GM). Here we have sought a biological explanation for this phenomenon. Most CD34-positive PBPC are quiescent (<1% in S phase) when they are collected from the bloodstream of patients treated with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), but we have shown that they are able to resume proliferation rapidly in vitro by measuring the kinetics of CFU-GM production by primitive plastic-adherent (Pdelta) cells. Also, Pdelta cells in PBPC harvests, unlike normal marrow Pdelta cells, were insensitive to cell-cycle restraint imposed by contact with marrow-derived stromal cells. We found that Pdelta cells in PBPC collections produce relatively more CFU-GM and relatively fewer BFU-E than Pdelta cells in bone marrow, indicating that granulopoiesis might occur at the expense of erythropoiesis, but we were unable to find any differences in the kinetics of granulocytic maturation between PBPC and bone marrow. Our interpretation of these findings is that transplanted PBPC rapidly enter the cell cycle and contact with stromal cells in the marrow does not reduce the proportion of progenitors participating in neutrophil production. Consequently. neutrophil recovery after PBPC infusion is more rapid than neutrophil recovery after marrow infusion. Granulopoiesis at the expense of erythropoiesis may also contribute to this effect.

Improved yield of peripheral blood progenitor cells on the Cobe Spectra.

The efficiency of leucapheresis using the Cobe Spectra depends on the observation of the operator to monitor the haematocrit (Hct) in the collection line at the recommended Hct of 1%. Cells were harvested by processing 3 x total blood volume (TBV) over 3-4 h. During the harvest the manual Hct and the colony-forming units granulocyte-macrophage (CFU-GM) concentrations were analysed in the collection line in the observed Hct range of 1-7.5%. There was a correlation between the manual and observed Hct (r = 0.97). The CFU-GM concentration was normalised to allow comparison between patients. The increase was statistically significant between 1-2% (P = 0.04) and 1-3% Hct (P = 0.05). The increase in CFU-GM concentration remained at the termination of harvest, indicating that not all available CFU-GM were harvested. The optimum concentration of progenitor cells was found to be at 3% Hct. We postulate that this may permit the collection of sufficient cells on one occasion to allow PBPC autografting in the majority of patients who respond to mobilisation.

The inhibition of antigen-induced eosinophilia and bronchoconstriction by CDP840, a novel stereo-selective inhibitor of phosphodiesterase type 4.

1. The novel tri-aryl ethane CDP840, is a potent and selective inhibitor of cyclic AMP phosphodiesterase type 4 (PDE 4) extracted from tissues or recombinant PDE 4 isoforms expressed in yeast (IC50S: 4-45 nM). CDP840 is stereo-selective since its S enantiomer (CT 1731) is 10-50 times less active against all forms of PDE 4 tested while both enantiomers are inactive (IC50S: > 100 microM) against PDE types 1, 2, 3 and 5. 2. Oral administration of CDP840 caused a dose-dependent reduction of interleukin-5 (IL-5)-induced pleural eosinophilia in rats (ED50 = 0.03 mg kg-1). The eosinophils in pleural exudates from CDP840-treated animals contained higher levels of eosinophil peroxidase (EPO) than cells from control animals, suggesting a stabilizing effect on eosinophil degranulation. CDP840 was approximately equi-active with the steroid dexamethasone in this model and was 10-100 times more potent than the known PDE 4-selective inhibitors rolipram and RP73401. The activity of CDP840 was not influenced by adrenalectomy, beta-sympathomimetics or beta-sympatholytics. 3. Antigen-induced pulmonary eosinophilia in sensitized guinea-pigs was reduced dose-dependently by CDP840 (0.01-1 mg kg-1, i.p.) and intracellular EPO levels were significantly higher. CDP840 was more potent in these activities than CT1731 or rolipram and comparable in potency to RP73401. 4. Rolipram or CDP840 were less active than dexamethasone in preventing neutrophil accumulation, or exudate formation in carrageenan-induced pleurisy in rats and thus do not exhibit general anti-inflammatory activity. 5. In sensitized guinea-pigs, aerosols of the antigen ovalbumin caused a dose-dependent bronchoconstriction demonstrated by an increase in pulmonary inflation pressure. Administration of CDP840 (0.001-1.0 mg kg-1, i.p.), 1 h before antigen challenge, resulted in dose-dependent reduction in response to antigen. This activity was not due to bronchodilatation since higher doses of CDP840 (3 mg kg-1) did not significantly change the bronchoconstrictor response to histamine. Rolipram was approximately 10 times less active than CDP840 in preventing antigen-induced bronchoconstriction. 6. These results confirm the observations that selective PDE 4 inhibitors reduce antigen-induced bronchoconstriction and pulmonary eosinophilic inflammation. CDP840 is more potent than rolipram in inhibiting native or recombinant PDE 4. Unlike the recently described potent PDE 4 inhibitor RP73401, CDP840 is more active than rolipram in the rat IL-5 model following oral administration. The novel series of tri-aryl ethanes, of which CDP840 is the lead compound, could be the basis of an orally active prophylactic treatment for human asthma.

High-dose therapy and autologous stem cell rescue for poor risk and refractory lymphoma: a single centre experience of 123 patients.

Over an 8-year period we autografted 123 patient with poor-risk lymphoma. Sixty-three patients had Hodgkin's disease (HD) and 60 non-Hodgkin's lymphoma (NHL). Of the patients with HD, 45 had responsive and 18 resistant disease prior to high-dose therapy. Fifty-three patients with NHL had responsive and seven had resistant disease at the time of transplantation. Seventy-seven patients received autologous bone marrow (BM) rescue, 39 autologous peripheral blood progenitor cell (PBPC) rescue, and seven combined BM and PBPC rescue. High-dose chemotherapy was BEM in 67, BEAM in 39, TBI and cyclophosphamide or etoposide or BCNU in 10, etoposide/mitozantrone in six and etoposide/melphalan in one. There was eight (6.5%) deaths due to treatment-related toxicity, within the first 100 days post-transplantation. Of the patients with HD 41 (65%) are alive at a median follow-up of 39 months (range 2-94). Thirty-three (52%) patients remain in CR. The median DFS of the 63 patients with HD is 34 months (95% CI 7-61). The median DFS for patients transplanted with responsive disease was significantly better than for those transplanted with refractory disease (61 vs 21 months P < 0001). Thirty-five (58%) of the patients with NHL are alive, and 20 (33%) remain in CR. The median DFS for patients transplanted with responsive and refractory disease was 11 months (95% CI 3-19) and 4 months (95% CI 0-9; P = NS) respectively. The median DFS for patients transplanted with HD was significantly better than for patients transplanted with NHL (34 vs 8 months, P < 0.002). In both groups there was no significant difference in DFS in patients receiving one, two, three or more lines of therapy prior to transplantation. In summary, in patients with poor-risk lymphoma who have responsive disease high-dose therapy may result in durable CRs. Conversely, only a small proportion of patients with HD or NHL with resistant disease achieve CR after autologous stem cell rescue.

Peripheral blood progenitor cell transplantation: a single centre experience comparing two mobilisation regimens in 67 patients.

Between June 1991 and January 1995 we performed 67 peripheral blood progenitor cell transplants (PBPCT). Ten patients (group 1) were mobilised with 7 gm/m2 of cyclophosphamide followed by daily G-CSF injections (5 micrograms/kg, subcutaneously). When the white cell count reached 1 x 10(9)/1 they were leukapheresed for 5 days. After stem cell infusion they received G-CSF (10 micrograms/kg/day) until the neutrophil count reached 1.5 x 10(9)/1. Fifty-six patients had PBPCs mobilised with 3 gm/m2 of cyclophosphamide followed by daily subcutaneous G-CSF (5 micrograms/kg) and PBPCs were harvested on 2 consecutive days, when the white cell count rose to 4 x 10(9)/1. After stem cell infusion this group did not receive G-CSF. In 47 of the 56 patients (group 2) adequate MNC (> or = 4 x 10(8)/kg) and/or CFU-GM (> or = 10 x 10(4)/kg) were obtained. Insufficient MNC and/or CFU-GM were obtained in 10 patients. They were therefore transplanted using a combination of bone marrow and peripheral blood progenitor cells (group 3). Overall 64 patients successfully engrafted. Median days to neutrophils > or = 0.5 x 10(9)/1 were 9 (range 8-13), 12 (range 8-25) and 11 (range 9-16) and to platelets > or = 50 x 10(9)/1 were 11 (range 9-23), 13 (range 9-90) and 16 (range 13-99) in groups 1, 2 and 3 respectively. Patients in group 1 had a faster neutrophil recovery than patients in group 2 (P = 0.0002). The three patients who failed to engraft all received a combination of autologous peripheral blood and bone marrow cells.

Plastic-adherent progenitor cells in mobilized peripheral blood progenitor cell collections.

The use of peripheral blood progenitor cells (PBPC) to reconstitute hematopoiesis after high-dose chemoradiotherapy is now commonplace in the treatment of malignancies. Attempts to characterize these cells have concentrated primarily on their phenotype and their content of clonogenic colony-forming cells (CFC). We have used a plastic-adherent delta (P delta) assay system to evaluate the quantity and quality of more primitive cells in addition to the conventional measurements of CFC and CD34-positive cells. The leukapheresis products from 20 patients mobilized using cyclophosphamide (Cy) and granulocyte colony-stimulating factor (G-CSF) were examined for progenitor cell content. The mean number of mononuclear cells (MNC), colony-forming units-granulocyte/macrophage (CFU-GM), and CD34-positive cells from two leukaphereses per patients were 7.9 x 10(8)/kg, 47.3 x 10(4)/kg, and 10.5 x 10(6)/kg, respectively. The mean number of P delta progenitors was 9.3 x 10(4)/kg. Limiting dilution analyses showed the frequency of P delta progenitors in PBPC to be between 1 and 5.3 per 10(5) MNC and that each P delta progenitor has the proliferative capability to generate an overall mean of 4.5 CFU-GM. Of the 20 patients, 16 underwent autografting with PBPC alone. Fifteen patients engrafted neutrophils and platelets within 16 days. One patient had delayed engraftment associated with inadequate etoposide clearance. Statistical analysis showed a strong correlation between numbers of CFU-GM and CD34 positivity. The numbers of plastic-adherent P delta progenitor cells did not correlate with CFU-GM or CD34-positive cells. We conclude that the plastic-adherent P delta progenitor cell assay is capable of measuring primitive hematopoietic cells and that it may be useful for the investigation of primitive progenitors in PBPC harvests.

Peripheral blood progenitor cell harvesting in multiple myeloma and malignant lymphoma.

Peripheral blood progenitor cells are being used increasingly as part of the treatment protocol for a variety of haematological malignancies. The most appropriate mobilisation therapy and the optimum collection procedures have yet to be fully elucidated. 28 patients with myeloma (9), NHL (11) and HD (8) underwent PBSC mobilisation and harvesting between November 1992 and October 1993. Two protocols were used; the myeloma group received high-dose cyclophosphamide, 7 g/m2 + G-CSF and were leucapheresed on 5 consecutive days during the recovery period using the Haemonetics V50 and the lymphoma group a lower dose of cyclophosphamide, 3 g/m2 + G-CSF followed by leucapheresis on 2 or 3 occasions using a Cobe Spectra. Median time to achieve a WBC of 1 x 10(9)/l during the recovery phase, was 14 days (11-16) and 10 days (9-15) respectively. Median numbers of MNC and CFU-GM collected for the myeloma group were 5.9 x 10(8)/kg (2.5-13.5) and 69.4 x 10(4)/kg (9.9-268.1) and for the lymphoma group. 5.1 x 10(8)/kg (1.2-11.1) and 35.4 x 10(4)/kg (1.2-129.7). Three patients with lymphoma had a low yield of CFU-GM, two of which did not proceed to autograft. The third patient failed to engraft and died despite receiving bone marrow backup. For the remaining 25 patients, median time to neuts > 0.5 x 10(9)/l and platelets > 50 x 10(9)/l was 9 (8-13) and 11 (9-23) days for the myeloma group and 12 (9-15) and 13 (9-180) days for the lymphomas. We found a strong correlation between CD34+ cells and CFU-GM from the last 9 patients. There is a correlation between CFU-GM infused and speed of engraftment. All patients who received > 10 x 10(4) CFU-GM/kg showed a rapid engraftment for neutrophils and platelets. In all cases, when > 4 x 10(8)/kg MNC were harvested, > 10 x 10(4) CFU-GM/kg were obtained. Sufficient cells for a rapid engraftment can be obtained from 2 leucaphereses in the majority of patients. The recovering peripheral blood WBC provides a good indicator of when to harvest. The target value of CFU-GM can be predicted by the number of cells harvested and by the number of CD34 positive cells in the leucapheresis product.

Two unusual neurological presentations of granulocytic sarcoma in Philadelphia positive chronic myeloid leukaemia.

Granulocytic sarcoma (GS) is a rare extramedullary tumour consisting of immature myeloid precursors. It occurs most commonly in association with myeloid leukaemias and myeloproliferative disorders. Rarely there may be no evidence of haematological malignancy. We describe neurological presentations of GS in two patients with Philadelphia (Ph) positive chronic myeloid leukaemia (CML). In both cases the bone marrow was in chronic phase at the time of presentation of the GS, but there was rapid subsequent transformation into the blastic phase.

Molecular detection of clonally rearranged cells in peripheral blood progenitor cell harvests from multiple myeloma patients.

Peripheral blood progenitor cells (PBPC) are increasingly used for autologous reconstitution following high-dose chemotherapy in multiple myeloma but it is unclear whether these cells are less likely to be contaminated with malignant cells than bone marrow (BM). We have investigated this using immunoglobulin heavy-chain (IgH) gene fingerprinting, a polymerase chain reaction based technique with a sensitivity of 0.1-0.01% (10(-3)-10(-4)). We have looked for patient-specific IgH rearrangements in leukapheresis samples from eight myeloma patients undergoing PBPC harvest. Seven were in first remission (six partial, one complete) and one in second complete remission. Mobilization of PBPC was accomplished using cyclophosphamide (4 or 7 mg/m2) and rhG- or GM-CSF. Between two and five leukaphereses were performed in each patient. Patient-specific IgH rearrangements were identified in diagnostic BM in all patients and bands of identical size were found in one or more leukaphereses from 6/8 patients. Overall, 14/32 leukaphereses were shown to be contaminated. Two patients who showed contamination of at least one PBPC harvest had BM harvests in which contaminating cells were not detectable, suggesting that PBPC are not necessarily less likely to be contaminated than marrow stem cells. These results indicate that PBPC harvests from the majority of myeloma patients are likely to contain contaminating cells. Further studies are needed to determine whether these cells are clonogenic and whether they contribute to relapse.