The use of adjusted ideal body weight for overweight patients undergoing HPC mobilisation for autologous transplantation.

Generally, patients' actual body weight (ABW) is used to calculate the number of CD34⁺ cells to be harvested for autologous haematopoietic progenitor cell (HPC) transplantation. In our institution, 'overweight' patients weighing at least 25% more than their ideal body weight (IBW) have their adjusted ideal body weight (AdjIBW) used for determination of blood volume to be processed to achieve a minimum target of CD34⁺ cells per kilogram, as well as CD34⁺ cell dosage calculation at transplant. AdjIBW is calculated as follows: AdjIBW = IBW + 0.25 × (actual weight - IBW). We have used AdjIBW for 65/153 patients who have had autologous HPC harvests, with a median AdjIBW of 69 kg (range, 50-110 kg). Median actual weight was 90 kg (range, 62-175 kg). Median volume of peripheral blood processed to achieve a minimum 2 × 106 CD34⁺ cells/kg for these patients was 13.2 L (range, 5-35 L), and the median CD34⁺ cells × 106/kg collected for AdjIBW was 6.3 (range, 1.7-33). For normal-weight patients (n = 88; median ABW, 75 kg; range, 49-98 kg), the corresponding median apheresis volume was 16 L (range, 7-24 L), and median CD34⁺ cells × 106/kg harvested was 4.5 (range, 1.4-15.9). In total, 35 in a total transplant cohort of 82 patients had AdjIBW used to determine CD34⁺ cell dose at time of transplant, with a median of 4.5 × 106/kg, (if their ABW was used in the calculation; 3.1 × 106/kg), compared to median dose of 3.2 × 106/kg ABW for the normal-weight patient cohort. All patients engrafted with no significant difference between median times to neutrophil and platelet engraftment for the overweight (13 and 15 days, respectively) compared with normal-weight (12 and 14 days, respectively) patient cohorts. We conclude that the use of AdjIBW is a useful tool for successful harvest and subsequent transplant for overweight patients, with no adverse effect on engraftment times.

Electrical stimulation of acupuncture points and blood pressure responses to postural changes: a pilot study.

BACKGROUND: Application of transcutaneous electrical stimulation over acupuncture points (Acu-TENS) facilitates heart rate recovery after exercise and restores hemodynamic stability after open heart surgery. The role of Acu-TENS on cardiovascular parameters in response to postural changes has not been reported. OBJECTIVE: To investigate (1) the effect of Acu-TENS on blood pressure responses to -10º head-down postural change and (2) whether such effects were associated with modulation by the autonomic nervous system. METHOD: Sixteen healthy volunteers, mean age 22.8 (SD, 3.1) years, were subjected to a -10º head-down tilt from the supine position on 3 separate occasions and received in random order the following 3 intervention protocols for 40 minutes before the postural change: Acu-TENS (over bilateral acupuncture points, PC6), sham-TENS (TENS applied to the skin over the patellae), and control (no electrical output from the TENS device applied at PC6). Mean arterial pressure, large artery elasticity index, cardiac output, and heart rate were recorded and compared at different stimulation protocols in the supine and -10º head-down tilt positions. Spectral analysis of heart rate variability was used to determine any modulation by the autonomic nervous system. RESULTS: Change in large artery elasticity index was observed only in the Acu-TENS group (P < .05) and mean arterial pressure appeared most stable during Acu-TENS. Autonomic nervous system modulation was not apparent with spectral analysis, irrespective of intervention. Sympathetic activity predominated in all positions. CONCLUSION: Acu-TENS seems to reduce blood pressure changes with -10º head-down tilt with concomitant changes in arterial vessel tone.

Viable CD34+/CD133+ blood progenitor cell dose as a predictor of haematopoietic engraftment in multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation.

Both CD34 (cluster of differentiation 34) and the more recently described CD133 are markers of primitive stem cells with haematopoietic repopulating ability. Most transplanting centres use a minimum number of CD34+ cells as the requirement for a transplant and consider this a predictor of haematopoietic engraftment. However, transplanted CD34+ cell dose does not always give a close correlation with time to engraftment nor explain delayed engraftment in some patients. We have retrospectively evaluated the potential of measuring viable CD133+ cell numbers in the autograft as an alternative predictor of haematological engraftment after autologous stem-cell transplantation in a cohort of patients with multiple myeloma (MM). We found an average 32% loss of viability of CD34+ cells in the post-thaw sample compared with the fresh sample. Of the original estimated CD34+ cell numbers transplanted per kg, 43% of the thawed samples were double positive for CD34+/CD133+. In this patient group, the CD34+/CD133+ subset gave the closest statistical correlation with time to neutrophil engraftment (p < 0.05), particularly for patients given above median (1.8 x 10(6)/kg) dose of the double-positive cells. The CD34+/CD133+ population was the only parameter to give a significant correlation with white cell engraftment in this patient cohort (p < 0.05). There was no significant correlation between CD34+, viable CD34+ or viable CD34+/CD133+ cells/kilogram with platelet engraftment. Determination of viable CD34+/CD133+ progenitor cell dose in the autograft may be a useful tool to predict neutrophil recovery after autologous transplantation than conventional assessment of CD34+ numbers. These results warrant further investigation of the role of CD133 in haematopoietic engraftment.

The expansion of megakaryocyte progenitors from CD34+-enriched mobilized peripheral blood stem cells is inhibited by Flt3-L.

This study aimed to determine the optimal growth factor combination for expansion of megakaryocyte (Mk) progenitors with clonogenic potential from CD34+-enriched mobilized peripheral blood stem cells (PBSC). Mobilized PBSC were monocyte depleted and CD34+ enriched, then cultured with various combinations of interleukin-3 (IL-3), IL-6, IL-11, Flt3 ligand (Flt3-L), stem cell factor (SCF), granulocyte-macrophage colonystimulating factor (GM-CSF), and erythropoietin (EPO), using a 2(7-3) IV fractional factorial design. Expansion of Mk committed progenitors (CD41+) and primitive precursors (CD61+ CD34+) was determined using FACS and colony-forming assays. Amplification of Mk progenitor production was attributed to IL-3 (p < 0.002), SCF (p < 0.001), and GM-CSF (p < 0.05). Flt3-L inhibited the production of total CD61+ cells (p < 0.05), CD61+CD34+ cells (p < 0.03), and total CD41a+ cells (p < 0.01). Addition of Flt3-L to the optimum growth factor combination of megakaryocyte growth and development factor (MGDF), SCF, IL-3, and GM-CSF caused the greatest increase in total nucleated cells but reduced Mk progenitor expansion. There was also a 20% reduction in Mk+ colonies from cells expanded in the presence of Flt3-L. Factorial analysis identified the optimal combination of growth factors required to expand Mk precursors with clonogenic potential. The addition of Flt3-L to the optimal combination of MGDF, SCF, IL-3, and GM-CSF reduced both the fold expansion of Mk progenitors and Mk colony numbers.

Successful peripheral blood stem cell mobilisation with filgrastim in patients with chronic myeloid leukaemia achieving complete cytogenetic response with imatinib, without increasing disease burden as measured by quantitative real-time PCR.

Imatinib mesylate (Glivec) is a selective inhibitor of bcr-abl tyrosine kinase, the product of the Philadelphia chromosome, which is the hallmark of chronic myeloid leukaemia (CML). With imatinib, complete cytogenetic response (CCR) can be achieved in over 70% of newly diagnosed patients with CML. However, the optimal long-term management of patients who achieve CCR after imatinib is unknown. With longer follow-up, it is anticipated that some patients are likely to progress and become candidates for autologous transplantation. We studied filgrastim (r-metHuG-CSF) mobilisation of peripheral blood stem cells (PBSC) in 32 patients who have achieved CCR with imatinib. Our data demonstrate that (1) the target CD34(+) cell yields of >/=2.0 x 10(6)/kg were attained with filgrastim 10 microg/kg/day, in 9/18 (50%) of patients during uninterrupted imatinib therapy, and in 10/14 (70%) when imatinib was temporarily withheld. The median CD34(+) cell yield per aphaeresis was 0.70 x 10(6)/kg (range 0.14-2.18) and 2.90 x 10(6)/kg (range 0.15-8.71) in the two groups, respectively (P&<0.005). (2) The cell yields did not correlate with the duration of imatinib administration. (3) There was no impact of the mobilisation procedure on the level of leukaemia as measured by serial blood bcr-abl levels using real-time quantitative PCR with either protocol. (4) bcr-abl remained detectable at low levels in the harvests in most but not all patients. In conclusion, filgrastim can safely be used to mobilise PBSC in patients who have achieved CCR with imatinib, but CD34(+) cell yields are significantly improved when imatinib is temporarily withheld.

Platelet activation in the hypertensive disorders of pregnancy.

OBJECTIVE: The purpose of this study was to determine whether platelet activation occurs only in preeclampsia or also in normal pregnancy. STUDY DESIGN: Thirty women with preeclampsia, 30 women with gestational hypertension, 20 women with essential hypertension, 30 pregnant women with normotension, and 30 nonpregnant women were recruited at St George Hospital, Sydney, Australia. Platelet activation was determined by flow cytometry on whole blood samples. RESULTS: Platelet activation was similar in all groups, except the group with preeclampsia. Compared with normal pregnant women, women with preeclampsia had significantly greater CD62 expression (1.35% vs 0.61%; P =.002), CD63 expression (1.73% vs 0.95%; P <.0001) and annexin V binding (1.03% vs 0.66%;P =.03) and significantly fewer circulating platelet microparticles (33 vs 49 x10(9)/L; P =.001). This was unrelated to other parameters that included platelet counts. Women with gestational hypertension in whom preeclampsia developed did not have enhanced platelet activation profiles. CONCLUSION: Platelet activation is increased in preeclampsia but not in other hypertensive disorders or in normal pregnancy. This may be part of the pathophysiologic factors of preeclampsia complications but is not predictable by the platelet count and is not apparent in all women with preeclampsia.

Accurate calculation of blood volume to be processed by apheresis to achieve target CD34+ cell numbers for PBPC transplantation.

BACKGROUND: In recent years there have been clear improvements in the procurement of peripheral blood progenitor cells (PBPC) for autologous transplantation, such as the description of more effective mobilization regimens and the use of CD34 monitoring to determine the appropriate time to start collection. However, currently there is no accurate method of predicting the volume of blood required to be processed by apheresis to yield the target number of CD34(+) progenitor cells. METHODS: This study was performed to determine whether there is a correlation between the harvested number of CD34(+) cells per kilogram body weight and the 'CD34 prediction score' calculated from the concentration of CD34(+) cells in the blood prior to harvest, the blood volume processed, and the patient's weight. RESULTS: A strong correlation between the CD34 prediction score and the quantity of CD34(+) cells harvested was found. This facilitated the construction of an algorithm for calculating the minimum volume of blood required to be processed by apheresis to yield the target number of CD34(+) cells. Subsequent validation of the algorithm facilitated successful tailoring of the apheresis time. DISCUSSION: The ability to accurately calculate the minimum volume of blood to be processed by apheresis to yield a target number of PBPC produces significant benefits in patient management, cost savings and equipment utilization.

Heparin-induced thrombocytopenia: cross-reactivity between standard heparin, low molecular weight heparin, dalteparin (Fragmin) and heparinoid, danaparoid (Orgaran).

The incidence of cross-reactivity between unfractionated heparin and LMWH, fragmin, in patients with HIT is significantly lower (6/15, 40%) than hitherto reported in the literature. 7/9 patients with a negative cross-reactivity test were treated with dalteparin sodium (Fragmin) without any untoward events.

Pulmonary embolism in patients with intermediate probability lung scans: diagnosis with Doppler venous US and D-dimer measurement.

PURPOSE: To test the usefulness of lower limb Doppler venous compression ultrasound (US) and serum D-dimer measurements in diagnosis of pulmonary embolism in patients in whom ventilation-perfusion (V-P) scans indicate intermediate probability of pulmonary embolism. MATERIALS AND METHODS: V-P scanning, pulmonary angiography, US, and D-dimer measurements were performed in 36 patients without known deep venous thrombosis but with intermediate probability of having a pulmonary embolism. RESULTS: Pulmonary angiography demonstrated pulmonary embolism in 15 (41%) of 36 patients. US demonstrated deep venous thrombosis in only two patients, both with pulmonary embolism. Sensitivity of US was only 13%, but specificity was 100%. Five (14%) of the 36 patients had normal (< 220 micrograms/L) D-dimer levels; none of the five had pulmonary embolism. Sensitivity and specificity of D-dimer values were 100% and 16%, respectively, with a negative predictive value of 100%. CONCLUSION: Combined D-dimer measurement and US were helpful in correctly diagnosing pulmonary embolism in only seven (20%) of 36 patients. Pulmonary angiography is still required to diagnose pulmonary embolism in the majority of patients.

Lomustine, etoposide, methotrexate and prednisone (LEMP) therapy for relapsed and refractory non-Hodgkin's lymphoma.

Twenty-two patients with non-Hodgkin's lymphoma (NHL) were treated with a regimen aimed at administering 6-9 cycles at 6-weekly intervals of oral lomustine (CCNU) 50 mg/m2 on day 2, i.v. etoposide 50 mg/m2 d 1 and 8, methotrexate 30 mg/m2 d 1 and 8, prednisone 60 mg/m2 d 1-10 (LEMP). The patients had a median age of 65 years (range 34-81) at diagnosis and comprised 14 males and 8 females. Eighteen patients had had prior chemotherapy (5 patients received more than two combinations). Seven had also received prior radiotherapy. Five patients achieved a complete remission (CR) with a median duration of 18 months (range 4-37). Twelve patients achieved a partial remission with a median survival of 8 months (range 2-45). Responses were seen in 6/6 nodular small cleaved cell, 1/1 diffuse small cleaved cell, 0/2 nodular mixed small and large cell, 3/4 diffuse mixed small and large cell, 7/9 diffuse large cell. One patient with diffuse large cell histology was treated initially with LEMP, achieving and maintaining CR for 18 months. Five patients did not respond to LEMP. This regimen was well tolerated requiring only 2 injections every 6 wk, there was minimal toxicity, no alopecia or cardiotoxicity, and little myelosuppression. There were no treatment-related deaths. This regimen has a useful role in inducing partial or complete remission in patients who have relapsed or progressed following previous intensive chemotherapy. It may also be used as first-line therapy in patients who may not tolerate more intensive regimens. These results are encouraging and warrant further investigation.

A sensitive method for the detection of poly-A tails of mRNA using a biotin-labelled heteropolymer of dT:rA.

We have developed a highly sensitive non-radioactive in situ hybridization technique that enables us to study the production of mRNAs in tissues. As part of the validation procedure of our methods, we examined various methods of detecting poly-A RNA tails of mRNA. We have used three types of biotin-labelled probes complementary to poly-A sequences: a 25-mer poly-dT oligonucleotide, a polymer of dT, and a heteropolymer of dT:rA. All the probes had the same specificity of reactivity but the heteropolymer of dT:rA gave the strongest signals as visualized histochemically by the use of alkaline phosphatase as the detection enzyme. All the probes tested for poly-A detection showed reactivity. The poly-dT oligonucleotide showed a strength of signal comparable to published results. The biotinylated polymer of dT gave a stronger signal than that of the oligonucleotide, and the heteropolymer was the strongest of all. The strong signal seen with the heteropolymer probe is due to probe complexing during hybridization, in which additional binding between sense and antisense strands of the probe (i.e. poly-rA and poly-dT) amplifies the number of biotin molecules at the hybridization site; this strategy has been exploited by us as a means of visualizing low copy numbers of specific mRNAs.

An improved method of alkaline phosphatase--antialkaline phosphatase immunophenotyping using microwave irradiation.

The application of microwave irradiation from a domestic microwave oven to the alkaline phosphatase-antialkaline phosphatase immunophenotyping has resulted in substantial time saving of the procedure. This has been achieved without compromising the quality of the final preparation. It can be carried out on peripheral blood smears, bone marrow smears and cytocentrifuge preparations on fresh slides, slides that have been stored at -20 degrees C wrapped in aluminium foil and unfixed smears left at room temperature for up to 8 days.

Trisomy 4 in a case of acute biphenotypic leukemia.

Trisomy 4 was the sole chromosome anomaly in a 5-year-old girl with acute leukemia. Morphologically, there appeared to be distinct myeloid and lymphoid blast cells on presentation. Immunophenotyping, however, showed extensive overlap of myeloid and lymphoid markers, confirming the leukemia to be biphenotypic rather than true "bilineal." She attained remission only after lymphoid-cell-specific induction was added to the initial "myeloid type" induction. She relapsed 4 years later with morphologically acute lymphocytic leukemia (French-American-British L2 type) despite still retaining the original immunophenotypic characteristics. She was successfully reinduced and subsequently received an autologous bone marrow transplant. Second relapse, morphologically and immunophenotypically similar to the first, occurred 5 months after transplant.

Hemoglobin Randwick or beta 15 (A12)Trp----Gly: a new unstable beta-chain hemoglobin variant.

A new beta-chain hemoglobin variant, Hb Randwick [beta 15(A12)Trp----Gly] was detected in a 43-year-old female of Northern Italian parentage. During investigation for possible diabetes, mild red cell changes were noted and hemoglobin electrophoresis studies were requested. Independently, her sister's assessment had resulted in similar investigations. The most prominent findings were numerous "Hb H"-like inclusions and a positive isopropanol stability test. The hemoglobin variant separated poorly towards the anode at pH 9.2 and the level was estimated to be between 48-50% of the total hemoglobin. The variant beta-chain was partially purified by column chromatography, and its tryptic peptides fractionated by high performance liquid chromatography. Amino acid analysis and sequence data indicated that the tryptophan at residue 15 (A12) had been substituted by a glycine residue. Further study has indicated that eight other family members are heterozygous for the variant; they are clinically normal with no evidence of splenomegaly or history of jaundice, although four of them showed a mild reticulocytosis.

Familial splenomegaly syndrome with reduced circulating T helper cells and splenic germinal centre hypoplasia.

Familial splenomegaly is a rare occurrence and is occasionally associated with immune abnormalities. We report three members of a family with massive splenomegaly associated with a reduction in circulating T helper cells, a reversed T4/T8 ratio and cutaneous anergy. The spleen and lymph nodes were shown in one family member to have germinal centre hypoplasia with T helper cells being present in normal numbers and distribution in these tissues. Various abnormalities of immunoglobulins were also noted. Despite the demonstrated immune abnormalities, the affected subjects showed few serious consequences. The pathogenesis of this disorder is unclear but we postulate it may involve a functional defect of the T helper cell. This defect probably is responsible both for the diminished circulating capacity of T helper cells as well as a reduced ability to aid in the formation of germinal centres.

Incidence and clinical significance of protein-bound vitamin B12 malabsorption.

Patient records from January 1975 to December 1984 were analysed to assess the possible incidence of protein-bound vitamin B12 malabsorption. This condition is characterised by a low serum vitamin B12 level and a normal Schilling test but impaired absorption of vitamin B12 bound to protein. We found that 48 (25%) patients with a low serum cobalamin level unexplained by other causes had a normal Schilling test. Megaloblastic haemopoiesis was found in 25 of these. From this group, all 10 patients who had a test of protein-bound vitamin B12 absorption showed impaired absorption. Protein-bound vitamin B12 malabsorption may represent an early phase of pernicious anaemia when hypochlorhydria precedes intrinsic factor deficiency and should be tested for when the serum vitamin B12 level is decreased and the Schilling test is normal.

Bone-marrow transplantation for haematological malignancy in childhood.

Twenty-three children with haematological malignancies and a poor prognosis underwent bone-marrow transplantation. Thirteen children had acute lymphoblastic leukaemia, eight had acute nonlymphoblastic leukaemia, one had chronic myeloid leukaemia and one had malignant histiocytosis. One child was in relapse at the time of transplant and 22 were in first or subsequent remission. Before transplantation all patients received cyclophosphamide (60 mg/kg) on two consecutive days followed by total body irradiation given as a single dose of 10 Gy at 0.18 Gy/min (one patient) or 0.07 Gy/min (three patients), or as a fractionated dose of 10-12 Gy at 0.07-0.1 Gy/min (19 patients). One child with malignant histiocytosis also received two doses of etoposide (5 mg/kg). Methotrexate was given after transplantation to prevent or modify graft-versus-host disease (GVHD). One patient who received a transplant in relapse died early from overwhelming bacterial sepsis. Twenty-two patients engrafted, and of these 11 developed acute GVHD; five developed chronic GVHD; seven developed interstitial pneumonitis, with four deaths; and five relapsed between three and 12 months after transplantation, with three deaths. Fifty-nine per cent (13/22) of patients who received a transplant during remission remain in continuous complete remission and 68% (15/22) have survived for a median of 18 months (range, four to 73 months). Bone-marrow transplantation that is undertaken during remission of disease offers a prolonged disease-free survival in selected childhood malignancies.