Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory.

Worldwide, mass spectrometry is widely used to detect and quantify food allergens, especially in complex and processed food products. Yet, the absence of a regulatory framework for the developed methods has led to a lack of harmonization between laboratories. In this study, ten allergens were analyzed in eight food products by UHPLC-MS/MS, in order to establish criteria for the retention time, variation tolerance, the ion ratio deviation, and the signal-to-noise ratio for allergen detection. The set of criteria should help laboratories to compare results and avoid false positives and negatives. Furthermore, a strategy combining standard addition and labeled peptide correction was used to quantify milk, soy, peanut, and egg allergens in eight food products. This strategy is particularly interesting for routine laboratories, which receive hundreds of samples and cannot use an external calibration curve for each sample.

Peptide influences on lipids.

The extent to which the length of the membrane-spanning part of intrinsic membrane proteins matches the hydrophobic thickness of the lipid bilayer may be an important factor in determining membrane structure and function. To gain insight into the consequences of hydrophobic mismatch on a molecular level, we have carried out systematic studies on well-defined peptide-lipid complexes. As model peptides we have chosen gramicidin A and a series of artificial hydrophobic alpha-helical transmembrane peptides that resemble the gramicidin channel. These peptides consist of a hydrophobic stretch of alternating leucine and alanine residues with variable length, flanked by tryptophan residues. Using wide-line NMR techniques, we have investigated the interaction of these peptides with the bilayer-forming diacyl phosphatidylcholines and with phospholipids which by themselves have a tendency to form non-bilayer structures. We have shown that hydrophobic mismatch leads to systematic changes of the bilayer thickness and that it can even change the macroscopic organization of the lipids. The type of lipid organization induced by the peptides and the efficiency of the various processes depend on the properties of the lipids and on the precise extent of mismatch.

Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane alpha-helical peptides.

Specific interactions of membrane proteins with the membrane interfacial region potentially define protein position with respect to the lipid environment. We investigated the proposed roles of tryptophan and lysine side chains as "anchoring" residues of transmembrane proteins. Model systems were employed, consisting of phosphatidylcholine lipids and hydrophobic alpha-helical peptides, flanked either by tryptophans or lysines. Peptides were incorporated in bilayers of different thickness, and effects on lipid structure were analyzed. Induction of nonbilayer phases and also increases in bilayer thickness were observed that could be explained by a tendency of Trp as well as Lys residues to maintain interactions with the interfacial region. However, effects of the two peptides were remarkably different, indicating affinities of Trp and Lys for different sites at the interface. Our data support a model in which the Trp side chain has a specific affinity for a well defined site near the lipid carbonyl region, while the lysine side chain prefers to be located closer to the aqueous phase, near the lipid phosphate group. The information obtained in this study may further our understanding of the architecture of transmembrane proteins and may prove useful for refining prediction methods for transmembrane segments.

Influence of lipid/peptide hydrophobic mismatch on the thickness of diacylphosphatidylcholine bilayers. A 2H NMR and ESR study using designed transmembrane alpha-helical peptides and gramicidin A.

We have investigated the effect of a series of hydrophobic polypeptides (WALP peptides) on the mean hydrophobic thickness of (chain-perdeuterated) phosphatidylcholines (PCs) with different acyl chain length, using 2H NMR and ESR techniques. The WALP peptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked, e.g., FmAW2(LA)nW2AEtn. 2H NMR measurements showed that the shortest peptide with a total length of 16 amino acids (WALP16) causes an increase of 0.6 A in bilayer thickness in di-C12-PC, a smaller increase in di-C14-PC, no effect in di-C16-PC, and a decrease of 0.4 A in di-C18-PC, which was the largest decrease observed in any of the peptide/lipid systems. The longest peptide, WALP19, in di-C12-PC caused the largest increase in thickness of the series (+1.4 A), which decreased again for longer lipids toward di-C18-PC, in which no effect was noticed. WALP17 displayed an influence intermediate between that of WALP16 and WALP19. Altogether, incorporation of the WALP peptides was found to result in small but very systematic changes in bilayer thickness and area per lipid molecule, depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. ESR measurements with spin-labeled lipid probes confirmed this result. Because thickness is expected to be influenced most at the lipids directly adjacent to the peptides, also the maximal adaptation of these first-shell lipids was estimated. The calculation was based on the assumption that there is little or no aggregation of the WALP peptides, as was supported by ESR, and that lipid exchange is rapid on the 2H NMR time scale. It was found that even the maximal possible changes in first-shell lipid length were relatively small and represented only a partial response to mismatch. The synthetic WALP peptides are structurally related to the gramicidin channel, which was therefore used for comparison. In most lipid systems, gramicidin proved to be a stronger perturber of bilayer thickness than WALP19, although its length should approximate that of the shorter WALP16. The effects of gramicidin and WALP peptides on bilayer thickness were evaluated with respect to previous 31P NMR studies on the effects of these peptides on macroscopic lipid phase behavior. Both approaches indicate that, in addition to the effective hydrophobic length, also the physical nature of the peptide surface is a modulator of lipid order.

Induction of nonbilayer structures in diacylphosphatidylcholine model membranes by transmembrane alpha-helical peptides: importance of hydrophobic mismatch and proposed role of tryptophans.

We have investigated the effect of several hydrophobic polypeptides on the phase behavior of diacylphosphatidylcholines with different acyl chain length. The polypeptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked. First it was demonstrated by circular dichroism measurements that these peptides adopt an alpha-helical conformation with a transmembrane orientation in bilayers of dimyristoylphosphatidylcholine. Subsequent 31P NMR measurements showed that the peptides can affect lipid organization depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. When a 17 amino acid residue long peptide (WALP17) was incorporated in a 1/10 molar ratio of peptide to lipid, a bilayer was maintained in saturated phospholipids containing acyl chains of 12 and 14 C atoms, an isotropic phase was formed at 16 C atoms, and an inverted hexagonal (HII) phase at 18 and 20 C atoms. For a 19 amino acid residue long peptide (WALP19) similar changes in lipid phase behavior were observed, but at acyl chain lengths of 2 C-atoms longer. Also in several cis-unsaturated phosphatidylcholine model membranes it was found that these peptides and a shorter analog (WALP16) induce the formation of nonbilayer structures as a consequence of hydrophobic mismatch. It is proposed that this unique ability of the peptides to induce nonbilayer structures in phosphatidylcholine model membranes is due to the presence of two tryptophans at both sides of the membrane/water interface, which prevent the peptide from aggregating when the mismatch is increased. Comparison of the hydrophobic length of the bilayers with the length of the different peptides showed that it is the precise extent of mismatch that determines whether the preferred lipid organization is a bilayer, isotropic phase, or HII phase. The peptide-containing bilayer and HII phase were further characterized after sucrose density gradient centrifugation of mixtures of WALP16 and dioleoylphosphatidylcholine. 31P NMR measurements of the isolated fractions showed that a complete separation of both components was obtained. Chemical analysis of these fractions in samples with varying peptide concentration indicated that the HII phase is highly enriched in peptide (peptide/lipid molar ratio of 1/6), while the maximum solubility of the peptide in the lipid bilayer is about 1/24 (peptide/lipid, molar). A molecular model of the peptide-induced HII phase is presented that is consistent with the results obtained thus far.

Graft failure in children receiving HLA-mismatched marrow transplants with busulfan-containing regimens.

Identifying risk factors that lead to graft failure may reduce morbidity and mortality after bone marrow transplantation (BMT) for hematologic malignancies. We evaluated engraftment of all patients with acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) and myelodysplastic syndrome (MDS) receiving an unmanipulated marrow allogeneic BMT at the Detroit Medical Center from 1987 to 1992 using a busulfan, cyclophosphamide +/- cytarabine preparative regimen. Three of 118 patients had graft failure (2.5%; (95% confidence interval (CI) 0.7%, 6.4%). Graft failure was high in patients < or = 15 years with 3 of 12 patients with failure (25.0%) compared with 0 of 106 patients > 15 years (p = 0.002). Failure to engraft was not seen in HLA-identical (related or unrelated) donor transplants (0 of 103) whereas 3 of 15 HLA-mismatched donors failed (p = 0.003). Patient diagnosis, locus of HLA-mismatch, cytarabine in the preparative regimen, marrow cell dose and the relative reactive index (RRI) were not significant factors. Altered busulfan kinetics secondary to young age was probably not a major factor since 8 of 8 HLA-identical donor transplants engrafted in children. These findings demonstrate that patients receiving an unmanipulated marrow graft using busulfan-containing regimens were at a high risk for graft failure only if they were < or = 15 years of age and had an HLA-mismatched donor. More immunosuppressive preparative regimens, possibly including total body irradiation, should be considered to prevent potential graft failure in children.

Busulfan-based regimens and allogeneic bone marrow transplantation in patients with myelodysplastic syndromes.

Preparative regimens containing busulfan (BU) followed by allogeneic bone marrow transplantation (BMT) were used in 27 consecutive patients with myelodysplastic syndromes (MDS). The median age was 33 years (range, 4 to 54). Ten were female and 17 male. Sixteen patients had primary MDS, 11 other patients had antecedent hematologic diseases or developed MDS after cytotoxic and/or radiation therapy. Six patients had leukemic transformation and received antileukemic therapy before BMT. Pre-BMT cytogenetic studies showed complex chromosomal abnormalities in 13 patients, a simple abnormality in 5 patients, and normal chromosome in 8 patients. Three BU-based preparative regimens were used: 1 patient received BU 4 mg/kg orally (PO) daily for 4 days and cyclophosphamide (CY) 50 mg/kg intravenously (IV) daily for 4 days (BUCY-4); 24 patients received BU 4 mg/kg PO daily for 4 days, cytosine arabinoside (ara-C) 2 g/m2 IV every 12 hours for 4 doses, and CY 60 mg/kg IV daily for 2 days (BAC); and 2 patients with preceding Fanconi anemia received BU 2 mg/kg PO daily for 4 days followed by total lymphoid irradiation of 5 Gy. Seventeen of 27 patients are alive with no evidence of disease. Ten patients have died: 2 from hepatic veno-occlusive disease, 3 from sepsis, 1 from a cerebral bleed, 1 from a massive gastrointestinal (GI) bleed associated with acute graft-versus-host disease, 1 from hemolytic uremic syndrome with adult respiratory distress syndrome, 1 from bronchiolitis obliterans, and the only patient who did not engraft died from acute myeloid leukemia. Regimen-related toxicities (RRT) include GI tract (diarrhea, 14; stomatitis, 11), liver (9), cardiac (1), and skin (5). Patients who received a genotypically matched marrow graft had a significantly better disease-free survival (DFS) than patients who received a nongenotypic marrow graft (P = .02). The Kaplan-Meier analysis projects an overall DFS of 56% +/- 13% and 78% +/- 10% for patients who received a genotypically matched marrow graft. With the exception of a child who did not engraft, there was no relapse of MDS or leukemia. Excellent DFS, acceptable RRT, and the ease of administration are advantages of this regimen.

Allogeneic bone marrow transplantation in high-risk myeloid disorders using busulfan, cytosine arabinoside and cyclophosphamide (BAC).

Twenty-one patients (median age = 34, range = 10-49; F:M = 7:14) received a preparative regimen consisting of busulfan 4 mg/kg/day x 4, cytosine arabinoside 2 g/m2/12 h x 4 and cyclophosphamide 60 mg/kg/day x 2 ('BAC' regimen) for allogeneic bone marrow transplantation. Out of 12 patients with acute myeloid leukemia (AML), two were in first remission, six were in second remission and four had resistant, relapsed disease or prolonged marrow aplasia after induction chemotherapy. Five of the 12 patients with AML had secondary AML. Four patients had transfusion-dependent myelodysplastic syndrome. Three patients with chronic myeloid leukemia were in the accelerated phase and two were in the blastic phase. Organ toxicities related to the preparative regimen were graded. Liver toxicity occurred in 11 patients, two of these were fatal veno-occlusive disease (VOD) (10%). Nineteen of the 21 patients had grade 2 or less diarrhea, and 13 also had mucositis. One patient developed grade 3 cardiac toxicity, and one other patient had grade 1 skin toxicity. Four patients had gross hematuria related to treatment (19%). No renal, pulmonary or CNS toxicities were encountered. Ten patients have died, two from regimen-related hepatic VOD. Of the remaining eight deaths, four were from respiratory failure in four patients (one case each of Pneumocystis pneumonia, CMV pneumonia, bronchiolitis obliterans associated with chronic graft-versus-host disease, and interstitial pneumonitis complicated pulmonary emboli), and one patient each from GI bleeding, cardiac arrhythmia, sepsis and CNS bleeding. Thus far, only one patient transplanted for secondary AML in second remission relapsed at day 230.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for clonal disease by magnetic resonance imaging in patients with hypoplastic marrow disorders.

Some patients with hypoplastic marrow disorders, including aplastic anemia (AA), are at risk for clonal evolution to myelodysplastic syndromes (MDS) and leukemia. Magnetic resonance imaging (MRI) of marrow of the spine, pelvis, and femurs was performed in 24 patients with hypoplastic marrow disorders. In 12 patients (three AA, nine MDS) MRI was compatible with the clinical and biopsy diagnoses and served to define the spectrum of marrow patterns in these disorders. In eight patients with hypocellular marrow biopsies and a clinical diagnosis of AA, MRI showed an unexpected inhomogeneous or diffuse cellular pattern. Concurrent or subsequent marrow or cytogenetic studies have led to diagnoses of hypoplastic MDS in seven of these patients. In four patients with prolonged hypoplasia after bone marrow transplantation for lymphoma, a speckled pattern superimposed on a fatty background appeared in serial MRI studies. One case evolved to AML, two developed megaloblastic foci, and one remains hypoplastic at 19 months. This study suggests that MRI is able to detect early clonal disease in patients with AA, and can distinguish AA from hypoplastic MDS.

Acquired severe aplastic anaemia in adults--a single centre study with 13 years follow-up.

Between 1974 and July 1987 the diagnosis of severe aplastic anaemia (SAA) was confirmed in 82 patients. Overall actuarial survival was 57% at 7 yr. Four patients recovered while receiving conventional therapy, and four died before treatment with antithymocyte globulin (ATG) or bone marrow transplantation (BMT) could be initiated. Nineteen patients (median age 19.6 yr) were treated with allogeneic BMT (11 as initial therapy, eight after ATG). Incidence of acute and chronic graft versus host disease was high, occurring in 14/16 and 4/11 patients at risk, respectively. Survival of BMT patients (18/19 transfused) was 32% at 7 yr. Of 63 patients treated with ATG, survival was 63% at 7 yr but decreased to 43% at 11 yr. The 2.5 yr survival following ATG was influenced by pretreatment disease severity (defined by percentage reticulocytes, granulocyte and platelet counts), age and--in patients under 45 yr of age--by sex. However, pretreatment disease severity was less in patients aged between 20 and 45 yr and in females. Concomitant androgen therapy, animal source of ATG, interval diagnosis--ATG (which was in general rather short) and aetiology did not influence survival. Thirty-four patients became transfusion independent for up to 26 months after ATG. A gradual increase in granulocyte and platelet counts could be observed over a period of many years, and 26 patients recovered to show a normal haemoglobin level, granulocytes greater than or equal to 1.0 X 10(9)/l and platelets greater than or equal to 100 X 10(9)/l). Late complications (paroxysmal nocturnal haemoglobinuria, myelodysplastic syndrome/acute leukaemia, hepatocellular carcinoma) were observed in nine patients who survived with autologous marrow function. Five died within 12 yr of initial therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Autologous bone marrow transplantation in acute myeloid leukemia in first remission: results of a Dutch prospective study.

Recent investigations have suggested a role for marrow ablative chemotherapy and radiotherapy given with autologous bone marrow transplantation (auto-BMT) in the treatment of acute myeloid leukemia (AML), but prospective studies have not been reported. We assessed the comparative values of auto-BMT and allogeneic marrow transplantation (allo-BMT) in 117 15- to 60-year-old consecutive patients (median, 43 years) with AML following remission-induction therapy. In 32 cases of the 90 (77%) complete responders, auto-BMT (nonpurged) was undertaken at a median of 3.8 months and in 23 eligible cases human leukocyte antigen (HLA)-matched allo-BMT occurred at 3.0 months after attainment of remission. Thus, nearly 60% of complete responders had access to transplantation, the others being withdrawn because of relapse, refusal, or other causes. Median time of regeneration to neutrophils 0.5 x 10(9)/L and platelets 20 x 10(9)/L were 39 and 63 days following auto-BMT versus 21 and 19 days after allo-BMT, respectively. AML relapse was the predominant cause of failure after auto-BMT (17 of 32) and procedure-related death was seen in three of 32 patients. The actuarial rates of relapse at 3 years are 60% (auto-BMT) and 34% (allo-BMT) (log-rank, P = .03). Patients treated with auto-BMT and allo-BMT have an overall survival of 37% and 66% at 3 years posttransplant, respectively (P = .05). Relapse-free 3-year survival rates are 35% and 51%, respectively (P = .12). Survival of the nongrafted complete responders is less than 10%. This study shows that allo-BMT in adult patients with AML in first complete remission (CR) results in more rapid hematopoietic reconstitution, is followed by fewer recurrences, and provides better survival than auto-BMT.

Allogeneic bone marrow transplantation (BMT) for acquired severe aplastic anaemia (SAA) in children.

The SAA Registry of the EBMT now contains data on 171 children younger than 15 years of age with acquired SAA and undergoing BMT between 1970 and 1988. The overall actuarial survival is 63% at 10 years. In a multivariate Cox analysis, the year of transplant was the most important prognostic factor with a significant advantage for children grafted in 1984-88 (81%) vs 1981-83 (67%) and 1970-80 (41%) (p = 0.02). Cyclosporine A given for GVHD prophylaxis, no treatment before transplant and an interval less than 90 days from diagnosis to BMT were all favourable variables in univariate analysis. As regard to transplant procedures, the better results were obtained using Cyclophosphamide and Cyclosporine A (78%) followed by Cyclophosphamide plus irradiation plus Cyclosporine A (77%). Sex, etiology and the severity of the aplasia had no impact on survival in both uni and multivariate analysis.

Long-term follow-up of severe aplastic anaemia patients treated with antithymocyte globulin. Severe Aplastic Anaemia Working Party of the European Cooperative Group for Bone Marrow Transplantation (EBMT).

468 severe aplastic anaemia (SAA) patients registered in the EBMT-SAA registry who did not undergo bone marrow transplantation and were treated with immunosuppressive therapy (IS; 96% of patients received ATG) were evaluated. Their median age was 23 years (range 1-73) at initial IS therapy, 59% were males; in 69% the aetiology of SAA was idiopathic. Of these 468 patients, 245 had a follow-up of less than 2 years after IS 166/245 died, 71/245 are still alive, 8/245 are lost to follow-up. Of 223 patients who survived greater than or equal to 2 years (LTS long-term survivors), 191 are alive, 21 died greater than 2 years and 11 are lost. Median follow-up of 223 LTS was 4.1 years (range 2.0-10.9). Comparison of 166 patients who died less than 2 years and 223 LTS revealed no difference at time of initial IS therapy as regards sex, duration of AA, or its aetiology, but the age distribution and, in particular, severity of SAA differed significantly: more LTS were between 21 and 40 years old (44% v. 32%, P less than 0.02), less LTS had reticulocytes less than 20 x 10(9)/l (63% v. 80%, P less than 0.001), polymorphonuclear granulocytes (PMN) less than 0.2 x 10(9)/l (30% v. 57%, P less than 0.001), haemorrhages (58% v. 79%, P less than 0.002) and infection (30% v. 49%, P less than 0.005) at time of IS. A gradual improvement of blood counts was seen in patients alive greater than or equal to 2 years after IS. At 2 years after IS 80% had a normal haemoglobin and PMN greater than 0.5 x 10(9)/l, but only after 5 years 80% of cases had platelets greater than 50 x 10(9)/l. Development of clonal disease was reported of 31 LTS: 19 developed paroxysmal nocturnal haemoglobinuria (PNH), one acute leukaemia, 11 myelodysplastic syndromes and of these 11 five subsequently acute leukaemia. The majority of these patients (23/31) are still alive. Actuarial mortality of LTS is 22% at 8 years, but so far no plateau was achieved. It is concluded that SAA patients who become LTS following IS, show an improvement in haematological status but are probably not cured and are prone to develop clonal (malignant) disease.

Bone marrow histopathology of patients with severe aplastic anaemia before treatment and at follow-up.

Pretreatment bone marrow biopsies of 63 patients with severe aplastic anaemia (SAA) who were not transplanted and of whom 55 received ATG, were evaluated according to the amount and character of residual haematopoiesis ('genuine' aplasia/intermediate/hypoplastic myelodysplasia (MD], inflammatory infiltrate (Te Velde & Haak, 1977, grade I/II/III), and number of mast cells (normal or slightly increased/increased). Of 61 evaluable biopsies, 47 were 'genuine' aplastic, 11 intermediate and three hypoplastic MD. Inflammatory infiltrates were graded as III in 36/60 evaluable biopsies, as II in 21 and I in three. A moderate to marked increase of mast cells was seen in 19/61. Of grade III patients, 86% had a less than 90 d interval between diagnosis and administration of ATG, versus 50% of grade I/II patients (P less than 0.01). No other correlations with pretreatment characteristics were found. No significant prognostic value for survival or response to ATG of any of these three criteria has been identified. More patients with grade III inflammation tended to show adequate recovery at 4 and 6 months after ATG. Stem cell damage, not identifiable morphologically, and/or impairment of accessory cells might play a major role in eventual outcome of SAA patients. Thirty-five patients are currently alive, median 3.8 years (up to 12.4) after ATG. Follow-up bone marrow aspirates and biopsies of 32 patients were evaluable and none showed normal haematopoiesis. One patient revealed persistent aplasia. Of the remaining 31, haematopoiesis was decreased in 14 and increased in eight. All had dyserythropoiesis, 28 dysplastic myelopoiesis and in 16/29 with evaluable megakaryocytes, dysmegakaryopoiesis was found. Sixteen patients had normo- to hypercellular bone marrows with two dysplastic cell lines (consistent with myelodysplastic syndrome (MDS) according to the FAB-group). The prognostic impact of the dysplastic abnormalities found in these patients needs longer follow-up. Close observation is indicated in view of the previously recognized, albeit uncommon, evolution of SAA to MDS/acute non-lymphocytic leukaemia.

Failure to diagnose fatal disseminated toxoplasmosis in a bone marrow transplant recipient: the possible significance of declining antibody titres.

A 46-year-old patient with acute myelogenous leukaemia developed lethal disseminated toxoplasmosis 8 weeks after allogeneic bone marrow transplantation. Clinical features included pulmonary infiltrates, respiratory insufficiency and neurological signs. Post-transplantation toxoplasma serological tests were characterised by declining IgG titres and failure to detect IgM, whereas titres of IgG against the various herpes viruses remained constant and even increased over the same period. Circulating toxoplasma antigen could not be detected. Post mortem, specific immune complexes were identified in serum. Autopsy revealed widely disseminated toxoplasmosis with several foci in the brain, lungs and various other organs as well as concomitant infection with cytomegalovirus.

Haematopoietic and immunologic abnormalities in severe aplastic anaemia patients treated with anti-thymocyte globulin.

Thirty-five patients with severe aplastic anaemia (SAA) were extensively evaluated 0.3-12.4 years (median 3.8) after anti-thymocyte globulin (ATG) treatment. All but one were transfusion independent. Most patients revealed a normal Hb level and a granulocyte count over 1.5 x 10(9)/l but were still thrombocytopenic due to decreased platelet production. Lymphocytopenia and/or monocytopenia was found in about 30%. Two patients had a monocytosis. Although there was a great range in degree of recovery at various time intervals after ATG, patients tested more than 4 years after ATG tended to have higher cell counts. Lymphocyte counts correlated with the interval between ATG and evaluation, and with haematopoietic recovery. Qualitative abnormalities were found in all cell lines. Most patients showed a homogeneous macrocytic RBC population, and almost 50% a positive sucrose lysis test; only three patients showed evidence of haemolysis and only two of these showed a positive Ham test. Mean platelet volumes were reduced out of proportion to their number. Platelet function, determined by bleeding time and aggregometry, was impaired in over 30%. The granulocytic series showed a shift to the left in about 30%. Hypersegmentation and pseudo Pelger-Huet anomaly were seen in some patients. Lymphocyte subset distribution in blood and bone marrow was within the normal range but absolute blood levels of CD4 cells in particular were slightly decreased, and tended to increase gradually with time after ATG. IgG and IgA levels were significantly decreased. In only one patient cytogenetic analysis of unstimulated bone marrow cells revealed an abnormal karyotype, but in eight of eight patients an increased sensitivity of lymphocytes to X-rays was found. These data suggest impairment at the level of the very early haematopoietic progenitor cell in all patients up to 10 years after ATG. Since similar findings have been reported in clonal (pre-)malignant disease, SAA, improved after ATG treatment, might be prone to clonal (malignant) evolution.

Bone marrow transplantation for severe aplastic anemia from donors other than HLA identical siblings: a report of the BMT Working Party.

Data were obtained from 46 patients with severe aplastic anemia (SAA) who received bone marrow transplants (BMT) from donors other than genotypically HLA-identical siblings. The data were collected in the SAA Registry of the European Bone Marrow Transplant Group. The donors were non-HLA-identical siblings in six cases, parents in 28 cases, a son in one case and unrelated individuals in 11 cases. Fifteen donor-recipient pairs were HLA-A, -B and -DR identical and mutually non-reactive in mixed lymphocyte culture; nine were mismatched at one locus, 17 were mismatched at two or more loci and in five cases data were not available for D/DR determinants. Actuarial survival was predicted by the degree of mismatch. It was 45% for phenotypically HLA-identical grafts, 25% for grafts mismatched at one locus and 11% for graft mismatched at more than one locus. Whether the graft was derived from a family member or an unrelated donor seemed to be less important and results were comparable. Age, patient sex and year of transplant had no significant influence on survival. The use of cyclosporine (CSA) for graft-versus-host disease (GVHD) prophylaxis (n = 21, survival 34%) appeared superior to both methotrexate (n = 9, survival 11%) and to CSA with T cell depletion of donor marrow (n = 13, survival 14%). The causes of death were rejection (n = 15), GVHD (n = 13), pneumonitis (n = 5) and infection (n = 1). Twelve patients are alive at 16-84 months post-BMT.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone marrow transplantation (BMT) versus immunosuppression for the treatment of severe aplastic anaemia (SAA): a report of the EBMT SAA working party.

This is an analysis of 509 patients with severe aplastic anaemia (SAA) treated in Europe between 1981 and 1986; 218 patients were treated by allogeneic bone marrow transplantation (BMT) from HLA identical sibling donors and 291 with immunosuppressive therapy (IS) with antilymphocyte globulin (ALG). The overall actuarial survival was 63% after BMT and 61% after IS therapy at 6 years. All patients fulfilled the criteria of SAA; however, most patients with a neutrophil count of less than 0.2 x 10(9)/l also had infections and haemorrhages. Therefore a further subclassification was defined by pretreatment peripheral blood neutrophil count: very severe aplastic anaemia (vSAA) (less than 0.2 x 10(9)/l neutrophils) and moderately severe aplastic anaemia (mSAA) (0.2-0.5 x 10(9)/l neutrophils). A Cox regression analysis showed that the only significant pre-treatment variables were a low neutrophil count (P = 0.001) and increasing age (P = 0.05). Thus it seemed reasonable to analyse survival data after combined stratification for neutrophils (vSAA versus mSAA) and age (cut off at 20 years). BMT was superior to IS in patients with vSAA under 20 years of age (64% v. 38%; P = 0.01). IS was superior to BMT in patients with mSAA aged 20 or more (82% v. 62%; P = 0.002). The two treatments gave comparable results in young patients with mSAA (BMT = 58%, IS = 62%; P = 0.1), and in older patients with vSAA (BMT = 44%, IS = 43%; P = 0.06). Overall 75/218 and 87/291 patients, given BMT or IS respectively, died. The major cause of failure in BMT patients was graft rejection (n = 22) or problems associated with graft-versus-host disease. For ALG patients the major problem was persistence of the aplasia with haemorrhage (n = 32) or infections (n = 46). This study indicates that over 60% of patients with SAA can be successfully treated with either BMT or IS. Overall survival does not differ in the two groups, though significant differences emerge after stratification for severity of the aplasia and age.

Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults.

Myelodysplasia (MDS) and leukaemia following acquired aplastic anaemia has been reported as a rare event occurring in about 5% of patients. Improved results in survival of patients with severe aplastic anaemia (SAA) and subsequent prolonged follow-up created the possibility of evaluating the occurrence of MDS and leukaemia in 38 adult patients with acquired SAA surviving two or more years without bone marrow transplantation. Five patients, age 22, 35, 47, 56, 72 years, two females, three males, all with idiopathic SAA and normal cytogenetic analysis developed a refractory anaemia (RA) 7, 30, 48, 56, 142 months after diagnosis of SAA. In 3/5 RA evolved into an acute myeloid leukaemia (AML) either via a chronic myelomonocytic leukaemia (CMML) (2/3) or via RA with excess of blasts (RAEB) (1/3). Three patients revealed a monosomy 7 during MDS and/or leukaemic phase. One patient died during RA phase without cytogenetic abnormalities. A pattern of evolution could be identified in these patients revealing well-documented SAA - improvement of bone marrow haematopoiesis - dyshaematopoietic features of one or more cell lines with predominance of dyserythropoiesis - RA - RAEB or CMML - AML. These five patients represent more than 10% of all patients surviving at least 2 years. This implies that the risk of developing MDS and leukaemia in SAA patients surviving with autologous marrow, might increase with longer follow-up.

Testosterone reduces complement sensitivity of precursor cells in aplastic anaemia patients treated with antilymphocyte globulin.

Of 53 consecutive patients with aplastic anaemia who were re-examined at various intervals after treatment with antilymphocyte globulin, 30 had sufficient bone marrow colony forming capacity to permit evaluation of androgen effects in vitro. In 22 patients, precursor cells of the myeloid and erythroid line were abnormally sensitive to a preincubation in isosmolar sucrose with 5% fresh autologous serum compared to heat-inactivated autologous serum. This phenomenon was interpreted as excess complement sensitivity. This inhibitory effect of fresh serum in the bone marrow sucrose test was abrogated by addition of 10(-6) M testosterone to the preincubation phase in 15 of the 22 patients. In six of these 15, 10(-7) M dexamethasone had a similar effect; in the other nine patients only testosterone rendered the bone marrow sucrose test negative. This effect of testosterone on colony growth was indirect, since addition of 10(-9)-10(-5) M testosterone to primary bone marrow cultures from the same patients had no effect. We propose that testosterone in these experiments interacted with the complement system. In patients who have complement sensitive precursor cells, androgens might thus prevent complement mediated lysis of haemopoietic cells to some extent. The test described could help identification of patients in autologous bone marrow remission who are likely to benefit from androgen treatment.