HLA-haploidentical hematopoietic stem cells transplantation with regulatory and conventional T-cell adoptive immunotherapy in pediatric patients with very high-risk acute leukemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is still needed for many children with very high-risk acute leukemia. An HLA-haploidentical family donor is a suitable option for those without an HLA-matched donor. Here we present outcomes of a novel HLA-haploidentical HSCT (haplo-HSCT) strategy with adoptive immunotherapy with thymic-derived CD4+CD25+ FoxP3+ regulatory T cells (Tregs) and conventional T cells (Tcons) performed between January 2017 and July 2021 in 20 children with high-risk leukemia. Median age was 14.5 years (range, 4-21), 15 had acute lymphoblastic leukemia, 5 acute myeloid leukemia. The conditioning regimen included total body irradiation (TBI), thiotepa, fludarabine, cyclophosphamide. Grafts contained a megadose of CD34+ cells (mean 12.4 × 106/Kg), Tregs (2 × 106/Kg) and Tcons (0.5-1 × 106/Kg). All patients achieved primary, sustained full-donor engraftment. Only one patient relapsed (5%). The incidence of non-relapse mortality was 15% (3/20 patients). Five/20 patients developed ≥ grade 2 acute Graft versus Host Disease (aGvHD). It resolved in 4 who are alive and disease-free; 1 patient developed chronic GvHD (cGvHD). The probability of GRFS was 60 ± 0.5% (95% CI: 2.1-4.2) (Fig. 6), CRFS was 79 ± 0.9% (95% CI: 3.2-4.9) as 16/20 patients are alive and leukemia-free. The median follow-up was 2.1 years (range 0.5 months-5.1 years). This innovative approach was associated with very promising outcomes of HSCT strategy in pediatric patients.

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications.

Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.

FIP1L1-PDGFRA in chronic eosinophilic leukemia and BCR-ABL1 in chronic myeloid leukemia affect different leukemic cells.

We investigated genetically affected leukemic cells in FIP1L1-PDGFRA+ chronic eosinophilic leukemia (CEL) and in BCR-ABL1+ chronic myeloid leukemia (CML), two myeloproliferative disorders responsive to imatinib. Fluorescence in situ hybridization specific for BCR-ABL1 and for FIP1L1-PDGFRA was combined with cytomorphology or with lineage-restricted monoclonal antibodies and applied in CML and CEL, respectively. In CEL the amount of FIP1L1-PDGFRA+ cells among CD34+ and CD133+ cells, B and T lymphocytes, and megakaryocytes were within normal ranges. Positivity was found in eosinophils, granulo-monocytes and varying percentages of erythrocytes. In vitro assays with imatinib showed reduced survival of peripheral blood mononuclear cells but no reduction in colony-forming unit growth medium (CFU-GM) growth. In CML the BCR-ABL1 fusion gene was detected in CD34+/CD133+ cells, granulo-monocytes, eosinophils, erythrocytes, megakaryocytes and B-lymphocytes. Growth of both peripheral blood mononuclear cells and CFU-GM was inhibited by imatinib. This study provided evidence for marked differences in the leukemic masses which are targeted by imatinib in CEL or CML, as harboring FIP1L1-PDGFRA or BCR-ABL1.

Tolerance induction by 'megadose' transplants of CD34+ stem cells: a new option for leukemia patients without an HLA-matched donor.

Early studies in murine models and more recent clinical data in heavily pre-treated leukemia patients have shown that escalation of the dose of hematopoietic progenitor cells can overcome major genetic barriers and enable rapid and durable engraftment of haploidentical, three-locus-mismatched transplants without graft-versus-host disease. In vitro studies suggest that veto cells within the progenitor population most probably mediate this facilitating effect.

Genomic gain at 6p21: a new cryptic molecular rearrangement in secondary myelodysplastic syndrome and acute myeloid leukemia.

Fluorescence in situ hybridization and comparative genomic hybridization characterized 6p rearrangements in eight primary and in 10 secondary myeloid disorders (including one patient with Fanconi anemia) and found different molecular lesions in each group. In primary disorders, 6p abnormalities, isolated in six patients, were highly heterogeneous with different breakpoints along the 6p arm. Reciprocal translocations were found in seven. In the 10 patients with secondary acute myeloid leukemia/myelodysplastic syndrome (AML/MDS), the short arm of chromosome 6 was involved in unbalanced translocations in 7. The other three patients showed full or partial trisomy of the 6p arm, that is, i(6)(p10) (one patient) and dup(6)(p) (two patients). In 5/7 patients with unbalanced translocations, DNA sequences were overrepresented at band 6p21 as either cryptic duplications (three patients) or cryptic low-copy gains (two patients). In the eight patients with cytogenetic or cryptic 6p gains, we identified a common overrepresented region extending for 5-6 megabases from the TNF gene to the ETV-7 gene. 6p abnormalities were isolated karyotype changes in four patients. Consequently, in secondary AML/MDS, we hypothesize that 6p gains are major pathogenetic events arising from acquired and/or congenital genomic instability.

Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.

Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed >30 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: <100 copies; five cases: 580-5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.

Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin.

We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

Anaplastic large-cell lymphoma: clinical and prognostic evaluation of 90 adult patients.

PURPOSE: During the last few years, the application of CD30 monoclonal antibodies has led to the identification of a new lymphoma entity, termed anaplastic large cell lymphoma (ALCL). This tumor includes four distinct histologic subtypes, among which the Hodgkin's-like/Hodgkin's-related one (ALCL-HL) shares morphologic and phenotypic features with Hodgkin's disease (HD). PATIENTS AND METHODS: From September 1988 to October 1993, 90 ALCL patients were treated with third-generation chemotherapy regimens (either vincristine, cyclophosphamide, fluorouracil, cytarabine, doxorubicin, methotrexate with leucovorin, and prednisone [F-MACHOP] or methotrexate with leucovorin, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin [MACOP-B]) during the course of an Italian multicentric randomized trial on high-grade non-Hodgkin's lymphomas (HG-NHL). In particular, 47 patients had ALCL of the common type (ALCL-CT) and 43 ALCL-HL. Null phenotype was the most common (39.8%), while T-cell, B-cell, and hybrid forms accounted for 35.5%, 22.2%, and 2.5%, respectively. RESULTS: Complete remission (CR) was achieved in 66 of 90 (73.5%) patients (33 of 47 [70%] with ALCL-CT and 33 of 43 [77%] with ALCL-HL). The majority of the patients in CR (56.5%) were alive and well at a median follow-up time of 38 months; no significant differences were observed between the two histologic groups, with the rate of complete responders being 49% and 65% in ALCL-CT and ALCL-HL, respectively. The probability of relapse-free survival (RFS), projected at 63 months, was 67% for ALCL-CT and 82% for ALCL-HL. The risk of lower CR and RFS rates was associated with the presence of bulky disease, advanced stage, and B symptoms. CONCLUSION: The data of the present study confirm that ALCL responds to third-generation chemotherapy regimens similarly to other aggressive malignant lymphomas in terms of both CR and RFS rates.

The in vivo effect of thymic factor (thymostimulin) administration on circulating immune complexes and serum lysozyme levels in untreated Hodgkin's disease patients.

The in vivo effect of a calf thymus extract, thymostimulin, on the levels of circulating immune complexes (CIC) and serum lysozyme was evaluated in 32 patients with untreated Hodgkin's disease. Using the platelet aggregation test for detecting CICs, 12 patients (37%) had positive titers before thymostimulin treatment; 3 patients (10%) remained positive following therapy. Serum levels of Clq-binding immune complexes were evaluated (greater than 24.5 micrograms/ml) in 8 patients prior to thymostimulin therapy (mean value: 42.3 micrograms/ml); 3 patients continued to have elevated levels after treatment. Serum lysozyme levels for Hodgkin's patients was similar to control values (10.6 vs. 8.3 micrograms/ml); however, the Hodgkin's patients with initially elevated CICs had a lower serum lysozyme level than patients with initially normal CICs (12.9 vs. 7.3, p less than 0.02). Thymostimulin increased serum lysozyme levels in the Hodgkin's patients in whom the CICs were initially elevated (7.3 vs. 10.4 micrograms/ml, p less than 0.05). These data suggest that thymostimulin exerts an effect on the nonspecific immune system of Hodgkin's disease patients.

High-dose chemotherapy followed by autologous bone marrow transplantation versus dexamethasone, cisplatin, and cytarabine in aggressive non-Hodgkin's lymphoma with partial response to front-line chemotherapy: a prospective randomized italian multicenter study.

PURPOSE: To evaluate, in a prospective multicentric study, the efficacy of a conventional salvage chemotherapy (dexamethasone, cisplatin, and cytarabine [DHAP]) versus high-dose chemotherapy (carmustine, etoposide, cytarabine, and cyclophosphamide [BEAC]) followed by autologous bone marrow transplantation (ABMT) in patients with aggressive non-Hodgkin's lymphoma (NHL) in clinical partial response (PR) after two thirds of a conventional front-line therapy. PATIENTS AND METHODS: From August 1988 to August 1991, 286 patients with aggressive NHL were randomized in seven Italian institutions to receive fluorouracil, methotrexate, cytarabine, cyclophosphamide, doxorubicin, vincristine, and prednisone (F-MACHOP) or methotrexate with leucovorin, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B) as front-line therapy. Of the 286 patients enrolled onto the trial, 77 (27%) were considered in PR after two thirds of the front-line therapy, and 49 of 77 (64%) were randomized: 27 to receive DHAP chemotherapy and 22 to receive BEAC followed by ABMT. RESULTS: The response after second-line treatment was as follows: in the DHAP group, four patients (15%) achieved a complete remission (CR), 12 (44%) remained in stable PR, and 11 (41%) showed progressive disease; in the ABMT group, three patients (14%) obtained a CR, 18 (82%) obtained a stable PR, and one (4%) progressed, with an overall response (CR + stable PR) of 59% and 96% (P < .001) in the DHAP and ABMT groups, respectively. The overall survival was 59% versus 73% and the progression-free survival (PFS) was 52% versus 73% in the DHAP and ABMT groups, respectively (P, not significant). The toxicity was mild, particularly in the ABMT group, and no treatment-related deaths occurred in either group. CONCLUSION: Because of the small number of patients randomized, we were unable to determine whether ABMT or a standard salvage regimen (DHAP) is superior for PR patients. However, we confirmed that myeloablative treatment is a safe and well-tolerated procedure in this category of patients and this may enable us to evaluate its role as part of a front-line treatment in poor-risk NHL patients.

Improved outcome with T-cell-depleted bone marrow transplantation for acute leukemia.

PURPOSE: To eliminate the risk of rejection and lower the risk of relapse after T-cell-depleted bone marrow transplants in acute leukemia patients, we enhanced pretransplant immunosuppression and myeloablation. PATIENTS AND METHODS: Antithymocyte globulin and thiotepa were added to standard total-body irradiation/cyclophosphamide conditioning. Donor bone marrows were depleted ex vivo of T lymphocytes by soybean agglutination and E-rosetting. This approach was tested in 54 consecutive patients with acute leukemia who received transplants from HLA-identical sibling donors or, in two cases, from family donors mismatched at D-DR. No posttransplant immunosuppressive treatment was given as graft-versus-host disease (GVHD) prophylaxis. RESULTS: Neither graft rejection nor GVHD occurred. Transplant-related deaths occurred in six (16.6%) of 36 patients in remission and in seven (38.8%) of 18 patients in relapse at the time of transplantation. The probability of relapse was .12 (95% confidence interval [CI], 0 to .19) for patients with acute myeloid leukemia and .28 (95% CI, .05 to .51) for patients with acute lymphoblastic leukemia who received transplants at the first or second remission. At a median follow-up of 6.9 years (minimum follow-up, 4.9 years), event-free survival for patients who received transplants while in remission was .74 (95% CI, .54 to .93) for acute myeloid leukemia patients and .59 (95% CI, .35 to .82) for acute lymphoblastic leukemia patients. All surviving patients have 100% performance status. CONCLUSION: Adding antithymocyte globulin and thiotepa to the conditioning regimen prevents rejection of extensively T-cell-depleted bone marrow. Even in the complete absence of GVHD, the leukemia relapse rate is not higher than in unmanipulated transplants.

Transplantation tolerance induced by "mega dose" CD34+ cell transplants.

Early studies in murine models and more recent clinical data in heavily pretreated leukemia patients have shown that escalation of hematopoietic progenitor cells can overcome major genetic barriers and enable rapid and durable engraftment of haploidentical 3-loci mismatched transplants without graft-versus-host disease. In vitro studies suggest that veto cells within the progenitors population most likely mediate this facilitating effect. Leukemia relapse is relatively low in patients with acute myeloid leukemia (AML) but is greater in adults with acute lymphoblastic leukemia (ALL). Donor NK cells most likely mediate the resistance to relapse in patients with AML who are recipients of haploidentical transplants. Immune reconstitution in adults but not in children is slow as in adult recipients of HLA matched unrelated bone marrow transplants. The "mega dose" concept was also shown recently to be useful for tolerance induction in sublethally irradiated mice, so as to effectively overcome the marked resistance presented by the large number of lymphocytes surviving the sublethal conditioning. Thus, allogeneic chimeras generated by transplantation of large doses of Sca1+Lin- cells, permanently accept allogeneic donor type skin grafts. However, the numbers required to attain this desirable goal may not be easily collected from human donors. Nonalloreactive T cells synergize with murine Sca1+Lin- cells and might, therefore, enable achievement of engraftment of haploidentical transplants in sublethally conditioned patients.

Haploidentical hematopoietic stem cell transplantation: state of art.

For patients with hematologic malignancies at high risk of relapse who do not have matched donors, a suitable alternative stem cell source is the HLA-haploidentical 2- or 3-loci mismatched family donor who is readily available for nearly all patients. Transplantation across the major HLA barrier is associated with strong T-cell alloreactions, which were originally manifested as a high incidence of severe GVHD and graft rejection. The present overview of the 7th symposium on haplidentical transplantation that took place at the Weizmann Institute on February 2014, shows how these obstacles to successful transplantation can now be overcome. The review also discusses the advantages and drawbacks of current options for full haplotype-mismatched transplantation and highlights innovative approaches for rebuilding immunity, reducing leukemia relapse and improving survival after transplantation. In addition, new modalities for immune tolerance induction following nonmyeloablative conditioning are discussed, showing new options for treatment of elderly patients who cannot tolerate myeloablative conditioning protocols, as well as novel strategies for immune tolerance and chimerism induction as a platform for cell therapy and organ transplantation.

Next generation HLA-haploidentical HSCT.

Relapse is still the major cause of failure of allogeneic stem cell transplantation in high-risk acute leukemia patients. Indeed, whoever the donor and whatever the transplantation strategy, post-transplant relapse rates are ~30%, which is hardly satisfactory. The present phase 2 study analyzed the impact of adoptive immunotherapy with naturally occurring FoxP3+ T-regulatory cells (2 × 10(6) per kg) and conventional T lymphocytes (1 × 10(6) per kg) on prevention of GvHD and leukemia relapse in 43 high-risk adults undergoing full-haplotype mismatched transplantation without any post-transplant immunosuppression. Ninety-five percent of patients achieved full-donor type engraftment. Only 6/41 patients (15%) developed ⩾ grade II acute GvHD. Specific CD4(+) and CD8(+) for opportunistic pathogens emerged significantly earlier than after standard T-cell-depleted haplo-transplantation. The probability of disease-free survival was 0.56. At a median follow-up of 46 months (range 18-65 months), only 2/41 evaluable patients have relapsed. The cumulative incidence of relapse was significantly lower than in historical controls (0.05 vs 0.21; P = 0.03). These results demonstrate that the immunosuppressive potential of Tregs can be used to suppress GvHD without loss of the benefits of GvL activity. Humanized murine models provided insights into the mechanisms underlying separation of GvL from GvHD.

Haploidentical stem cell transplantation in leukemia.

In acute leukemia patients, infusing a megadose of extensively T-cell-depleted hematopoietic stem cells after an immuno-myeloablative conditioning regimen ensures sustained engraftment of full-haplotype mismatched transplants without graft-vs-host disease. Besides the conditioning regimen and the megadose of stem cells donor natural killer cell alloreactivity also plays a role in facilitating engraftment and in preventing relapse. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in 112 very high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.

Histological and immunohistological analysis of human lymphomas.

Morphological and immunological characteristics of lymphoproliferative diseases are reviewed. In particular, a basic distinction is made between non-Hodgkin's lymphomas and Hodgkin's disease. As to the non-Hodgkin's lymphomas, emphasis is given to the problems of classification, technical approach, histogenesis, and prognosis. The authors adopt a version of the Kiel Classification modified to take account of new knowledge regarding T-cell lymphomas. The value of immunophenotyping in making an accurate distinction between the various categories is stressed; immunocytochemical detection of the growth fraction is also discussed and then proposed as a new prognostic tool. Finally, the criteria for differential diagnosis between non-Hodgkin's lymphomas, malignant histiocytosis, non-lymphoid large cell tumors, and atypical immune reactions are outlined.

T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection.

Generation of an efficient graft-versus-leukemia (GVL) effect in patients with hematological malignancies who relapse after allogeneic bone marrow transplantation depends in part upon the number of infused T lymphocytes. Currently, a GVL reaction cannot be achieved without inducing concomitant graft-versus-host disease (GVHD); thus, one strategy is to try to modulate this GVL/GVHD ratio. We engineered human T lymphocytes with herpes simplex virus-thymidine kinase and neomycin resistance genes, with an LXSN-derived vector that confers a ganciclovir-specific sensitivity to the transduced T cells. We analyzed proliferation, interleukin-2 production, alloreactivity in a mixed lymphocyte culture, and clonogenicity during the different stages of retroviral infection and G418 selection. Our results confirm that a sufficient number of transduced T lymphocytes can be obtained after selection for clinical studies. Their proliferative activity, alloresponsiveness, and ability to produce and respond to interleukin-2 were retained. Compared with control populations, their clonogenicity, as assessed by limiting dilution assays, was reduced after retroviral infection and G418 selection by 1.6 and 2.9 logs, respectively, with both viral supernatant incubation and coculture procedures. This study shows that infection and selection with the thymidine kinase-neomycin resistance gene retroviral vector significantly reduces the number of functional T lymphocytes. This finding should be taken into account when establishing the dose of T lymphocytes necessary to trigger a modulated GVL/GVHD effect.

Protein A-peroxidase conjugates for two-stage immunoenzyme staining of intracellular antigens in paraffin-embedded tissues.

Staphylococcal protein A conjugated to horseradish peroxidase was employed in an indirect immuno-staining technique to identify intracellular antigens in paraffin-embedded tissues. The sections were incubated with specific antisera and the antigen-IgG complexes demonstrated with protein A-peroxidase conjugate. Immunoglobulins, lysozyme and insulin were satisfactorily detected by this technique. A comparison of this method with the PAP, "labelled antigen" and peroxidase-labelled antibody sandwich techniques was made.

Description of a sequential staining procedure for double immunoenzymatic staining of pairs of antigens using monoclonal antibodies.

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H2O2 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.

Interstitial pneumonitis in acute leukemia patients submitted to T-depleted matched and mismatched bone marrow transplantation.

PURPOSE: To identify factors that could contribute to interstitial pneumonitis (IP), which remains one of the major causes of morbidity and mortality after both matched and mismatched bone marrow transplantation (BMT). METHODS AND PATIENTS: Ninety acute leukemia patients received an allogeneic T-depleted matched (n = 54) or mismatched (n = 36) BMT. They were preconditioned with total body irradiation (TBI), thiotepa, rabbit anti-thymocyte globulin, and cyclophosphamide. The TBI scheme was hyperfractionated in matched, and a single dose in mismatched patients. The dose to the lungs was reduced in both groups. RESULTS: Five of the 54 matched patients developed IP. All cases were fatal. There were 16 cases of IP, 13 fatal, in the mismatched group. The probability of developing IP was 11.3 +/- 4.9% and 48.6 +/- 9.0%, respectively. The between-group difference was statistically significant (p < 0.0001). The type of transplant and the TBI scheme were the most important parameters for IP development in univariate analysis, whereas acute graft-versus-host disease, disease stage and sex were nonsignificant. Median follow-up was 342 days (range 17-2900). CONCLUSIONS: The low incidence of IP in matched patients and the lack of idiopathic cases are evidence for the validity of the TBI schedule. In contrast, the incidence in mismatched patients remains too high; therefore, new strategies should be studied in an attempt to lower it.