Lymphocyte T subsets and natural killer cells in Italian and Philippino blood donors.

BACKGROUND AND OBJECTIVES: The characterization of lymphocyte subsets in blood donors has been utilized to determine the normal ranges that can be related to race. A study was performed in blood donors from two racial groups - Caucasian (Italians) and Asian (Philippinos) - to define respective T-lymphocyte subsets and levels of cytokines. MATERIALS AND METHODS: Ninety-two blood donors (46 Italians and 46 Philippinos) were enrolled. Blood count and immunophenotyping of lymphocytes by flow cytometry were carried out, and cytokine production was tested in six blood donors of each group. RESULTS: Philippino blood donors showed a significantly higher mean value of leucocytes (P = 0.01) and lymphocytes (P < 0.001) than Italians. The mean absolute count of lymphocyte subsets CD3- CD16+ CD56+ and CD3+ CD8+ were both significantly higher in Philippino than in Italian subjects, respectively, P < 0.01 and P < 0.0001. Philippinos showed a statistically significant higher frequency of lymphocytes producing interferon-gamma (IFN-gamma) compared to Italians (P = 0.02). CONCLUSIONS: T-lymphocyte subsets in Italian and Philippino blood donors seem to be correlated to ethnic background. The higher levels of CD3+ CD8+ T cells, natural killer (NK) cells and IFN-gamma-producing cells found in Philippinos suggest leucoreduction in Asian blood donors.

CD5 negative lymphocytosis mimicking typical B-chronic lymphocytic leukaemia. Description of 26 cases.

We report 26 elderly patients (median age 68.3 years) who met diagnostic criteria for B-cell chronic lymphocytic leukaemia (B-CLL) but whose lymphocytes lacked CD5 expression. Haematological and clinical features of this CD5- series were compared with those of 333 CD5+ B-CLL patients from the same institute. No significant differences were observed regarding peripheral blood (PB) and bone marrow (BM) lymphocytosis, Hb level, platelet count, incidence of adenomegaly, hepatomegaly or splenomegaly or diffuse BM pattern. Due to an absence of nodal enlargements or to general clinical condition, lymph node biopsy was performed in only three patients, while spleen histology was examined in two cases following splenectomy. All histological results confirmed the clinical diagnosis of CLL. The distribution of the CD5- subjects according to the different staging categories proposed by Rai, Binet and Mandelli was similar to that of CD5+ subjects. Ten patients received standard chemotherapy with Chlorambucil (CHL) and Prednisone (PDN). All achieved partial remission, although one of these patients later died of disease progression; 80 months after diagnosis. We conclude that rare cases of CD5- lymphocytosis fulfilling all criteria for B-CLL may occur. Haematological and clinical features at presentation and the response to conventional treatment with Chlorambucil support our hypothesis of considering this disease as a less frequent subgroup of B-CLL.

Splenectomy outcome in a hemophilic patient with HIV-related immune thrombocytopenia.

We report the case of a young hemophilic patient with antibodies against the human immunodeficiency virus (HIV) who was affected by immune thrombocytopenic purpura (ITP). This condition did not respond to pharmacological therapy with steroids and alpha-2b-r-IFN, and the patient was splenectomized. Immune status evaluation was performed before and after surgery and during follow-up with CD4-CD8 monoclonal antibodies and cytofluorimetric analysis in order to explore possible correlations between splenectomy and the cytologic immune regulatory system. Splenectomy resulted in a resolution of ITP with consequent disappearance of the hemorrhagic diathesis related to thrombocytopenia. Moreover, at 30 months from splenectomy the patient is still in remission, his CD4 count is not decreased, and no progression to AIDS has been evidenced. These aspects are analyzed and briefly discussed.

Rearrangements of the RAR-alpha gene in acute promyelocytic leukaemia: correlations with morphology and immunophenotype.

We have used genomic probes which specifically recognize DNA rearrangements of the RAR-alpha locus on chromosome 17q21 in patients with acute promyelocytic leukaemia (APL) and acute myeloid leukaemia (AML) subtypes. Molecular data were examined in comparison with morphological and immunophenotypic characterization at diagnosis in 20 hypergranular FAB M3 cases, five microgranular APL (M3v), 51 non-M3 AML and 12 myeloid CML blast crises. Rearrangements of the RAR-alpha locus were only detected in 23/25 APL cases and in none of the other FAB subtypes analysed. Surface marker characterization showed a consistent immunophenotypic profile--HLADR negative, CD9 and CD13/33 positive--in all M3 and M3v cases. Neither HLADR negativity nor CD9 positivity were associated with RAR-alpha rearrangements in non M3 AML. Our data indicate that RAR-alpha gene rearrangements are relevant diagnostic features of both M3 and M3v, and may prove useful molecular marker for follow-up analysis in APL patients.

Prednisone versus deflazacort in the treatment of autoimmune thrombocytopenic purpura: evaluation of clinical response and immunological modifications.

We report the results of a randomized clinical trial of two different coricosteroids (prednisone versus deflazacort) in patients affected by autoimmune thrombocytopenic purpura (ATP). We have evaluated the efficacy of the two steroids on platelet count, antiplatelet antibodies, lymphocyte subsets and the occurrence of side effects. Twenty-seven patients were evaluable: 13 were treated with PDN and 14 with DFC. After 24 weeks of treatment, 4/12 (33%), subjects treated with PDN were refractory while complete responses were obtained in 2/12 (17%) and partial responses in 6/12 (50%). Among patients treated with DFC, 4/11 (36%) were considered as refractory, 2/11 (18%) had a complete response and 5/11 (46%) a partial response. A statistically significant decrease of antiplatelet antibodies was recorded in both groups after 4 weeks of therapy, but only in subjects receiving PDN did the reduction last until the 24th week. We observed an increase of T lymphocyte subsets (CD3, CD2, CD4, CD8) in absolute number, due to an increase in circulating lymphocytes, after 4 weeks. No substantial modifications were observed in these populations regarding the percentage or the CD4/CD8 ratio. After 24 weeks, 91% (10/11) of patients treated with PDN presented an increase of body weight and 1 had a stable increase in blood pressure. Among the subjects treated with DFC, 64% (7/11) showed an increase of body weight after the same follow-up. In conclusion, no difference was observed the two steroids studied.

Immunological definition of acute promyelocytic leukemia (FAB M3): a study of 39 cases.

Acute promyelocytic leukemia (FAB-M3) is a distinct entity among acute non-lymphoid leukemias (ANLL) with peculiar morphological, biological, clinical and prognostic features. An atypical form of M3 (M3v) could be confused with other FAB ANLL and therefore the diagnosis of this variant requires ultrastructural analysis and/or cytogenetic study and/or selective gene rearrangement studies. The immunological phenotype of blast cells in 39 APL patients was studied at diagnosis. The diagnosis of M3 FAB type was ascertained in 32 and the diagnosis of M3v in 7 cases. Using a large series of monoclonal antibodies (mAb), the APL blast cells were B and T cell antigens-negative, HLA-DR constantly negative, CD13- and/or CD33-positive, CD9-positive. Among ANLL this phenotype seems to be closely related to APL both in M3 type and M3v subtype. Because the diagnosis of APL (M3 or M3v) is important in order to establish the specific therapeutic approach, the discriminant capacity of the immunological typing to identify M3 and mainly M3v (hypogranular) could be determinant for a "quick" diagnosis.

CD7 positive acute myeloid leukaemia: a subtype associated with cell immaturity.

Seventeen patients with acute myeloid leukaemia (AML) whose blasts co-expressed the T-cell associated CD7 antibody were identified among 160 consecutive AML cases. Fourteen had FAB defined AML according to morphocytochemical criteria, whereas three patients were classified as 'MO' on the basis of immunophenotype. The incidence of CD7 positively was particularly significant in the less differentiated subtypes M0 and M1 compared with other FAB groups (P less than 0.001). In all cases the myeloid determinants CD13 and/or CD33 were associated with CD7 expression. Other B-lymphoid (CD10, CD19) or T-lymphoid (CD2, surface and cytoplasmic CD3) markers were analysed and found to be negative. Five out of 15 cases examined were TdT+. Clonal rearrangements of the immunoglobulin heavy chain (IgH) and/or T-cell receptor (TcR) beta chain genes were identified in only three out of 13 cases. Among these, one out of five co-expressing TdT showed IgH rearrangement when analysed at the DNA level. Clinical features at presentation and response to induction therapy did not allow us to consider CD7+ AML patients as a distinct subgroup with prognostic significance. Our data indicate that CD7 expression is a common finding in immature AML, being generally found in the absence of other T-cell features. Rather than suggesting the occurrence of 'mixed leukaemia', such cases confirm a broader spectrum of CD7 reactivity and its possible identification of a particular subset of myeloid progenitors.

Induction of immunotolerance in hemophilia for high titre inhibitor eradication: a long-term follow-up.

Three hemophiliacs with high titre inhibitor were treated with a medium-high FVIII dose schedule (100 IU/kg bw daily) with the aim of inducing the immunotolerance. These patients were followed-up extensively concerning their immunological status and HIV serology. In all of them the inhibitor disappeared and normal FVIII kinetics were obtained after 22, 15 and 29 months. After eradication of the inhibitor, no recurrence took place in any of the patients. All the patients were HIV Ab positive before the beginning of the treatment. In one of them CD4+ cells fell progressively 32 months after the treatment was started, a full-blown AIDS showed up, and the patient died 5 1/2 years after the beginning of the treatment. In the second and third patient the CD4+ cells varied widely but remained greater than 400/microliter during the whole immunotolerance treatment. The latter two patients are AIDS and ARC free so far, but patient No. 2 developed a mild-to-severe thrombocytopenia. Considering the high cost of the treatment and the possibility that such an intensive administration of FVIII concentrates might worsen the immunological status of patients, this therapeutic procedure should only be applied with caution.

Immunophenotyping of acute myeloid leukaemia: relevance of analysing different lineage-associated markers.

The immunophenotype of 135 previously untreated patients with FAB defined acute myeloid leukaemia (AML) was studied at diagnosis. The panel of reagents included monoclonal antibodies (MoAb) recognising myeloid-associated determinants (CD11, CD13, CD14, CD33 and others) as well as MoAb directed towards lymphoid antigens (CD7, CD10, CD19) and TdT. The results indicate that CD13 and/or CD33 are consistently expressed in AML and only rarely in ALL blasts (131/135 + ve cases, versus 4/130 in ALL). Lymphoid antigen expression was rarely detected when CD10 and CD19 were investigated in AML (0.9% and 2% + ve cases, respectively), whereas significant positivities were found for TdT and CD7 (20% and 10% respectively). Concerning FAB subtypes, two new MoAb (LAM3 and LAM7) proved very useful in the specific recognition of AML with monocytic features. The phenotype CD13+ and/or CD33+, CD9+, HLA-DR- was found to be almost exclusive for M3 AML. The response to induction chemotherapy was analysed in CD7+ and in TdT+ patients. In the latter group a statistically significant lower response rate was found with respect to TdT-ve-AML patients.

Spectrum of HIV infection and AIDS in a cohort of Italian hemophiliacs.

A group of 173 subjects affected by congenital clotting factor deficiencies was evaluated with regard to the impact of HIV infection. On the whole, 78 patients (45%) were found to be HIV Ab-positive. As of March, 1988, of the seropositive patients, 63 (80.8%) had an asymptomatic HIV infection (Group II/CDC), three (3.8%) had a persistent generalized lymphadenopathy (Group III/CDC). The 12 (15.4%) remaining patients could be classified in Group IV of the CDC classification due to their symptoms and signs; in particular, 10 came under surveillance case definition for AIDS. Assay for the HIV antigen was positive in 14 (17.9%) seropositive hemophiliacs. With regard to the immunological features, our data clearly show that a sharp decline in the number of CD4+ cells was associated with symptomatic forms of the disease. An evaluation of the time elapsed from seroconversion to the appearance of the symptomatic clinical condition showed an average incubation of 37 months.

Terminal transferase positive acute myeloid leukemia: immunophenotypic characterization and response to induction therapy.

A quantitative evaluation of terminal deoxynucleotidyl transferase (TdT) was performed using a highly sensitive enzyme immunoassay (EIA) in 72 previously untreated patients with acute myeloid leukemia (AML). Biological analysis of the leukemic cells included in all cases cytochemistry, search for Ph' chromosome and immunophenotyping with both anti-lymphoid and anti-myeloid monoclonal antibodies (MoAbs). Thirteen AML cases (18 per cent) were considered TdT+ by EIA. According to the FAB classification, almost all of them (12 out of 13) were within the M1 and M2 subgroups. A mixed lymphoid-myeloid phenotype was observed in one of the 13 TdT+ cases, while in none of the others were lymphoid features detected. Nine of the 10 EIA TdT+ cases studied in parallel were TdT positive with the conventional immunofluorescence assay. All patients received standard protocol chemotherapy and in 61 (13 TdT+, 48 TdT--) the response to induction treatment was analysable. Only 3/13 TdT+ patients (23 per cent) achieved a complete remission (CR), while in the TdT- group 38 patients had a CR (79 per cent) and 10 were resistant (p less than 0.01). It is suggested that the incidence, biological interest and prognostic significance of TdT+ AML should encourage the routine and more accurate search for this marker in all patients with AML.

Deficiency of lymphocyte lectin-dependent cytotoxicity in myelodysplastic syndromes.

We studied a group of patients with myelodysplastic syndromes (MDS) for surface markers and cytotoxic activities of peripheral blood mononuclear cells (PBMNC). The results indicate a significant increase in the total count of CD11b+, Leu7+ and CD16+ with a percent reduction in CD4+. A reduction in PHA-induced cellular cytotoxicity (PHA-ICC) and NK activity were found. A similar phenotype was found both in refractory anemia (RA) and (RA) with excess of blasts (RAEB/RAEB-t). However, the functional activities reached the normal level only in RA patients; while in RAEB/RAEB-t patients a significant reduction was detected in PHA-ICC and NK activity.

Molecular characterization of Ph' + hybrid acute leukemia.

The configuration of the immunoglobulin heavy chain (IgH), T-cell receptor (TcR) beta and gamma chain regions, and the major breakpoint cluster region (M-bcr) genes were analysed in four cases of Ph' + acute leukemia (AL). Monoclonal rearrangements of the IgH region were detected in three cases exhibiting two phenotypically distinct cell populations (i.e. one lymphoid and one myeloid. In one of these cases, identical genetic events were observed by molecular analysis of FACS separated blasts. Multi-lineage rearrangements involving also the TcR gamma gene were observed in a biphenotypic AL showing co-expression of markers. The lack of rearrangements within the M-bcr gene, together with demonstration in one case of the Ph' + AL specific p190 protein product, pointed against the occurrence of chronic myeloid leukemias presenting in blastic transformation. Our results imply that such cases are to be considered as true AL and should therefore be included in the definition of hybrid AL.

Two murine monoclonal antibodies to peripheral blood monocyte differentiation antigens discriminate within M5 acute non-lymphoid leukemia (ANLL) cells.

Two murine monoclonal antibodies (MoAbs), LAM3 and LAM7 of the IgG1 isotype, which were produced by immunization with normal peripheral blood monocytes (PBM), were assayed in their specificity by indirect immunofluorescence against a panel of normal as well as leukemic cells. Both LAM3 and LAM7 were reactive with PBM while LAM3 also recognized platelets. Neither MoAb showed reactivity with erythrocytes, granulocytes, or resting and mitogen activated B and T lymphocytes. The reactivity with bone marrow cells correlated with the degree of monocyte contamination. Among the 62 cases of leukemia tested, which included three cases of B-CLL, 19 cases of ALL, and 40 cases of ANLL, both MoAbs reacted highly homogenously only with M5b ANLL cells. These findings indicate that the two MoAbs, which recognize two distinct epitopes, represent useful markers in the differential diagnosis of M5b ANLL.

T-helper phenotype chronic lymphocytic leukemia (Thp-CLL): characterization of an Italian case with particular biological findings.

A patient with Chronic Lymphocytic Leukemia (CLL) characterized by an expansion of helper phenotype mature T lymphocytes is here described. The phenotype of these cells was OKT3+, OKT4+, Leu 9+, 5/9+, OKT8-, Tac- and functional studies showed a strong helper activity on B cell differentiation; an "in vivo" presence of an IgG-lambda paraproteinaemia has been demonstrated. Cytogenetic studies showed multiple clonal, numerical and structural rearrangements which included a tandem t(14;14) (q11;32) translocation. Hybridization showed HTLV I related specific bands indicating the presence of exogenous sequences related to prototype virus but derived from a different Retrovirus (HTLV 1c). The clinical course was aggressive and unsuccessful treatments with various polichemotherapeutic protocols, associated with multiple leukaphereses, were performed. The authors underline that despite the morphological, immunological, biological and virological heterogeneity, the common feature of T-helper CLL is the inexorable clinical course which needs a new therapeutic approach.

Increased susceptibility of peripheral mononuclear cells of leukemic patients to HTLV-I infection in vitro.

Peripheral mononuclear cells (MNC) collected from 12 healthy donors and 44 leukemic patients at various stages of the disease were tested for natural killer (NK) activity and for their susceptibility to HTLV-I infection in vitro, measured in terms of percentage of p19 positive cells. MNC from leukemic donors at any stage of leukemia (ie, onset or relapse, ON/REL; complete remission or off-therapy, CR/OT donors) were highly susceptible to HTLV-I infection. This was true for acute leukemias of lymphoblastic (ALL) or nonlymphoblastic (ANLL) type. MNC of ON/REL patients were more susceptible to HTLV-I than those of CR/OT donors. In addition, leukemic blasts were more rapidly infected (ie, within five to seven days) than the HTLV-I-susceptible normal cord-blood lymphocytes. However, the presence of circulating blasts was not essential to virus susceptibility, since CR/OT MNC, presumably free of leukemic blasts, were still more susceptible to HTLV-I than normal cells. Basal NK function of MNC from leukemic patients was significantly lower than that detectable in healthy controls. However, no correlation was found between susceptibility to HTLV-I infection and NK activity.

CALLA-negative, TdT- and CD7-positive acute lymphoblastic leukemia: a phenotype associated with poor prognosis.

Eight ALL patients displaying a CD7+, Tdt+, CD10-, T MoAbs-, myeloid MoAbs-, AP+ phenotype are described. Some patients showed well-known risk factors such as cytogenetic abnormalities, high WBC count, mediastinal mass, and/or organomegalies. The clinical behaviour was very poor and only one patient is in CR and off therapy. Therefore such a pre-T phenotype, although sometimes associated with the other risk factors, could be considered a poor prognosis phenotype.