Acute non-hemolytic transfusion reactions and HLA class I antibody: advantages of solid phase assay compared with conventional complement-dependent assay.

To evaluate the specific reactivity of HLA Class I antibodies (HLA-I Abs) in acute non-hemolytic transfusion reactions (ANHTRs) using solid phase assays (SPAs) and conventional complement-dependent lymphocyte cytotoxicity test (LCT). ANHTRs are major issues in transfusion medicine. Anti-leukocyte antibodies have been implicated as one of the causative agents of transfusion-related acute lung injury (TRALI) and febrile reaction. Antibodies to HLA Class I and/or Class II (HLA Abs) have been intensively studied using SPAs for TRALI, but not for febrile reaction. About 107 patients and 186 donors associated with ANHTRs were screened for HLA Abs by SPAs such as enzyme-linked immunosorbent assay (ELISA) and the Luminex method. When HLA-I Ab was detected, its specific reactivity was evaluated by comparing its specificity identified by the Luminex method using recombinant HLA molecules and cognate HLA antigens (Ags), as well as LCT with or without anti-human globulin (AHG). The incidences of HLA Abs were as high as 32.7% of patients' serum samples and 16% of donors' serum samples. The incidence of HLA-I Abs did not differ significantly between cases of febrile and allergic reactions. However, HLA-I Abs associated with febrile reaction showed a significantly higher rate of possessing specific reactivity to cognate HLA Ags than those associated with allergic reactions. In addition, the Luminex method enabled the detection of HLA-I Abs much earlier than AHG-LCT in serum samples from a patient with febrile reaction and platelet transfusion refractoriness (PTR). SPAs seem more useful than AHG-LCT for evaluating reactivity of antibodies in ANHTR cases.

Predominance of a distinct subtype of hairy cell leukemia in Japan.

Forty Japanese patients with hairy cell leukemia (HCL) were reviewed. Nine cases were diagnosed as typical HCL, and two cases had the features of HCL variant (prolymphocytic variant). The remaining 29 cases (72.5%) differed morphologically and hematologically from the other two groups in that they usually had a moderately high leukocyte count (average 27.9 x 10(3)/microliters), and abnormal cells showing a densely stained round nucleus and an inconspicuous nucleolus. Tartrate-resistant acid phosphatase reaction was weak, and their cells exhibited generally smooth or slightly irregular, cellular outlines in smears. The cells showed weak expression of surface immunoglobulin G (IgG) with kappa-chain predominance. CD25 antigen was not detected. Some of these findings resemble those of B-cell chronic lymphocytic leukemia, but the patients also had several important features of HCL. They had splenomegaly without significant lymphadenopathy. The abnormal cells were CD20+, CD11c+ and showed typical 'hairy morphology' under phase-contrast and scanning electron microscopy. Furthermore, spleen sections revealed diffuse infiltration by the abnormal cells in the red pulp. From these findings, we speculated that this group of patients constitute a distinct subtype of HCL which is commonly seen in Japan. We propose to term the disease as HCL Japanese variant.

Association of protein tyrosine phosphorylation with B cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).

We have assessed whether tyrosine protein kinase (TPK) is involved in B cell differentiation. In vitro phosphorylation of an endogenous substrate in B cell leukemias showed that leukemic B cells at different stages of differentiation had specific endogenous substrates in tyrosine phosphorylation as well as distinct TPK activity. To clarify the relationship between TPK and the process of B cell differentiation, we studied protein tyrosine phosphorylation in two kinds of leukemic B cells, which showed distinct responses to TPA (12-O-tetradecanoylphorbol-13-acetate) in B cell differentiation. TPA-treated leukemic B cells from patients with B cell chronic lymphocytic leukemia (B-CLL) differentiated into cytoplasmic immunoglobulin (clg)+ plasmacytoid cells, while TPA-treated leukemic B cells from patients with hairy cell leukemia (HCL) did not differentiate into clg+ cells, but showed a peculiar morphological change, spreading. Untreated B-CLL cells and HCL cells showed similar TPK activities and tyrosine protein phosphorylation. When treated with TPA, enhanced phosphorylation was seen in B-CLL cells, while a clear reduction in phosphorylation was found in HCL cells. However, using 4-hydroxycinnamide derivatives which reduce TPK activity, we found that only the reduction of TPK activity did not lead HCL cells to spreading. These data suggest that protein tyrosine phosphorylation and/or dephosphorylation might be involved in B cell differentiation, but only the change of TPK activity in HCL cells is not sufficient to induce effects.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on leukemic cells from chronic B cell leukemia.

It is not clear whether cells from various chronic B cell leukemias including B-chronic lymphocytic leukemia (CLL), CLL in prolymphocytoid transformation (CLL-PLT), B-prolymphocytic leukemia (PLL), and hairy cell leukemia (HCL) simply represent different stages of a single B cell differentiation pathway. Furthermore, it is not known whether cells from any given B cell leukemia are characterized by the same population during the differentiation process. Differentiation of various B cell leukemic cells was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and the resulting changes in their morphology, cytoplasmic immunoglobulin (clg), and cytochemistry were evaluated. With respect to peculiar morphological change, i.e. extending long thin processes (spreading) and the appearance of clg, each sample showed different responses. According to these two indices samples were classified into three groups; spread+ clg- samples (one case of CLL-PLT, all HCL), spread+ clg+ samples (one of CLL, one of CLL-PLT), and spread- clg+ samples (a majority of CLL, one of CLL-PLT, and all PLL). Unexpectedly, both CLL and CLL-PLT consisted of heterogenous populations as to the reactivity to TPA. In the process of TPA-induced differentiation in CLL cells, features similar to those of HCL cells were not found. Since three different TPA-induced response patterns were observed in each chronic B cell leukemia type, it was not possible to sequentially assign each of these leukemias along a single B cell differentiation pathway. In order to explain this result, we introduced the hypothesis that these groups might be divided into different lineages in B cell differentiation. Since TPA-induced spreading cells were present in the B cell fraction of normal peripheral blood mononuclear cells, this morphological change should not be associated with malignant transformation.

Immunophenotypic features and configuration of immunoglobulin genes in hairy cell leukemia-Japanese variant.

Immunophenotypes and Ig gene rearrangements were investigated in 12 patients with a variant form of hairy cell leukemia (HCL) termed HCL-Japanese variant (HCL-J), and in an HCL-J-derived cell line. The leukemic cells of HCL-J characteristically showed the phenotype of CD20+, CD5-, CD10-, CD11c+, CD22+, CD24- and CD25-. Ig light (L) chain was undetected in nine cases, and the remaining four cases expressed kappa chain. Expression of Ig heavy (H) chain was studied in nine cases. In addition to Igkappa+ cases showing expression of predominantly gamma H chain isotype, alpha chain was detected in one case without expression of L chain. Rearranged bands in Ig heavy chain (JH) genes were recognized in all 12 cases tested. Rearranged bands in kappa chain genes and germline configuration in chi chain genes were seen in all three Igkappa+ cases tested. Four of nine cases without expression of L chain had a rearranged chi chain gene. The other three cases had chi chain genes in the germline configuration and rearranged and/or deleted kappa chain genes. In the remaining two cases, no rearrangement in either kappa or chi chain genes was detected. The Ig gene configuration and expression in HCL-J, partially overlapping with those described for immature B cell leukemia, were dissociated from the cytological features and CD20+, membrane CD22+ phenotype characteristic of mature B cells.

Effects of interferon-alpha on L-BCGF- and TNF-alpha-induced proliferation of hairy cell leukemia in Japan.

Interferon-alpha (IFN-alpha) is very effective in patients with hairy cell leukemia (HCL), although its mechanism of action is still unknown. To investigate this issue, we studied the in vitro response to IFN-alpha of a variant type of HCL, recently reported by us as the Japanese variant. Their clinical response to IFN-alpha (remission rate 35.7% in the multicenter study in Japan) was inferior to that of typical HCL (remission rate 80%; mean of previous reports). We found that both low molecular weight B-cell growth factor (L-BCGF) and tumor necrosis factor-alpha (TNF-alpha) induced the proliferation of HC from patients with the Japanese variant, as well as those with typical HCL. While, in typical HCL, IFN-alpha strongly inhibited the in vitro proliferation of HC induced by L-BCGF and TNF-alpha, the inhibitory effect of IFN-alpha on L-BCGF and TNF-alpha-induced proliferation was low in most Japanese variant patients. These in vitro findings may be related to the extent of clinical efficacy of IFN-alpha in the Japanese variant, obtained in the multicenter study. Since the degree of inhibition was parallel in L-BCGF- and TNF-alpha-induced proliferation in three patients examined simultaneously, it appeared that the antiproliferative effect of IFN-alpha is not specific to individual growth factors. Rather, IFN-alpha might affect fundamental growth mechanisms triggered by these factors.

HTLV-I associated myelopathy (HAM) after blood transfusion in a patient with CD2+ hairy cell leukemia.

Hairy cell leukemia complicating hemolytic anemia developed in a 46-year-old woman. Morphologically and cytochemically typical hairy cells were found to express both CD20 and CD2 antigens. Expression of surface IgG of kappa-chain type and the rearrangement of Ig but not T-cell receptor beta genes confirmed a B-cell origin of the leukemia. Blood transfusion was followed by disappearance of the hemolysis and a marked improvement of the leukemia. However, the patient developed progressive spastic spinal paraplegia about seven months after transfusion and was diagnosed as having HTLV-I associated myelopathy (HAM) by the demonstration of HTLV-I antibodies in serum and cerebrospinal fluid. HTLV-I infection via the transfusion may have been involved in the hematologic improvement seen in this patient. Autopsy showed demyelination, vacuolar degeneration, gliosis, and perivascular cuffing in the white matter of spinal cord without evidence of leukemic infiltration.

Early age onset of hairy cell leukemia presenting with leukocytosis.

Hairy cell leukemia (HCL) is an uncommon type of chronic B cell leukemia mainly affecting middle-aged adults. HCL presenting with pancytopenia is rare in Japan and a distinct subtype of HCL termed HCL-Japanese variant is predominantly seen. We describe a HCL patient with unusual presentation. The patient was a 26-year-old male, such early onset of HCL being quite rare. The patient showed leukocytosis with many circulating hairy cells and cellular bone marrow. These findings were preferentially seen in HCL-Japanese variant, but, cytomorphologic, cytochemical and immunophenotypical studies on the pathologic cells were consistent with those of typical HCL seen in Western countries. Interferon-alpha therapy was very effective in this case. Differentiation of the subtype of HCL appears to be important for the choice of the treatment. The cytological findings were useful for the differential diagnosis of HCL presenting with leukocytosis.

Establishment of a new cell line from a patient with hairy cell leukemia-Japanese variant.

A cell line, JHC-2, was established from the peripheral blood of a patient with hairy cell leukemia (HCL)-Japanese variant. The JHC-2 cells have cytologic features similar to those of the original tumor cells. They displayed hairy cytoplasmic projections by phase contrast and scanning electron microscopy. The tartrate-resistant acid phosphatase reaction was weakly positive. The immunophenotype of the JHC-2 cells was CD5-, CD10-, CD11c+/-, CD19+, CD21+, CD23+, CD24-, CD25+/-, CD38- and FMC-7+. The expression of surface immunoglobulin (IgG, kappa) and the configuration of Ig gene rearrangements in the JHC-2 cells were identical to those in the original leukemic cells, and the JHC-2 cells displayed trisomy 9 on cytogenetic examination. Southern blot analysis for the Epstein-Barr virus (EBV) genome showed that the JHC-2 cells contained the EBV genome, although the freshly isolated leukemic cells did not. These results indicate that the JHC-2 cell line is an EBV spontaneously transformed B cell line originating from HCL cells.

Gastric T-cell lymphoma presenting with epithelioid granulomas mimicking tuberculosis in regional lymph nodes.

In patients with malignant lymphomas, a sarcoid reaction is occasionally observed. However, lymphoma-related granulomas with caseous necrosis are rare. We describe such a case of T-cell gastric lymphoma that was difficult to diagnose. A 50-year-old man was referred to our hospital because of abnormal gastric endoscopic findings: hypertrophic folds with narrowing of the gastric lumen and multiple ulcers in the body. Gastric biopsy specimens showed non-specific inflammation. An open biopsy of the enlarged gastric regional lymph nodes was performed. The sections revealed effacement of the normal architecture and replacement by numerous epithelioid granulomas accompanied by Langhan's type giant cells with or without central caseous necrosis, strongly suggesting tuberculosis. However, mycobacteria and other causative organisms were not detected, and an anti-tuberculous regimen was ineffective. Repeat gastric biopsies were performed and, finally, atypical lymphocytes were observed infiltrating the mucosa. The patient was diagnosed with gastric T-cell lymphoma based on the results of immunohistochemical stainings. After chemotherapy, a total gastrectomy was performed. The diagnosis of gastric T-cell lymphoma with a sarcoid reaction was confirmed by histological findings of the sections. Namely, the gastric wall was replaced by atypical lymphocytes showing the phenotype of helper T cells, admixed with epithelioid granulomas with Langhan's type giant cells. Thus, this case suggests that regional lymph nodes in gastric lymphomas may be present as epithelioid granulomas with caseous necrosis, mimicking tuberculosis.

[Hairy cell leukemia of European-American type with dual T and B-cell phenotype].

A 46-year-old woman was admitted because of palpitation and conjunctival jaundice. Physical examination revealed hepatosplenomegaly and purpura without lymphadenopathy. Blood count showed 4.7 g/dl hemoglobin with increased reticulocytosis. The platelet count was 1.5 X 10(4)/microliters and the leukocyte count was 6,000/microliters with 17% abnormal mononuclear cells (hairy cells). Hairy cells had nuclei of frequently folded shape and abundant cytoplasma with irregular edges on blood films. The hair-like cytoplasmic projections of the cells were clearly seen under the phase-contrast microscopy. Hairy cells were strongly positive for tartrate resistant acid phosphatase. Bone marrow aspiration was unsuccessful. The biopsy specimens showed small patchy and scattering infiltrations by hairy cells. Surface marker studies of hairy cells revealed that they were strongly positive for SmIg (IgG kappa). They also reacted with alpha B 1, alpha Tac, alpha Leu-M 5 monoclonal antibodies and a rabbit anti-hairy cell serum (alpha HC-M). 53% of hairy cells were shown to react with alpha B 1 and alpha OKT 11 simultaneously by double labelling. The southern blot analysis of peripheral blood mononuclear cells showed IgH chain genes rearrangement and germ line patterns of T-cell receptor genes. Hemolysis was promptly disappeared after blood transfusion. Moreover, the red blood cells, platelets and leukocytes have spontaneously returned to normal levels with disappearance of circulating hairy cells and palpable spleen one year after admission.

A unique variant of hairy cell leukemia in Japan.

We studied 25 Japanese patients with hairy cell leukemia (HCL) and found a manner in which HCL can be divided into two subtypes. In each patient, hairy cells (HC) showed striking surface hairs and reacted with HC-specific antibodies (alpha Leu-M5 and alpha HC-M). Twenty of the 25 patients had HC characterized by round nuclei with dense nuclear chromatin, weak tartrate-resistant acid phosphatase (TRAP) activity and the phenotype of low density surface immunoglobulin (SIg)+, Tac-. In this group of 20 patients, the male to female ratio was low, and there was frequent leukocytosis. On the other hand, the remaining 5 patients showed a high male to female ratio and a normal or decreased leukocyte count. HC had folded nuclei, strong TRAP activity and the phenotype of high density SIg+, Tac+. The features of the latter patients are consistent with those of HCL in Western countries, while those of the former group appear to indicate a unique variant of HCL.

Inhibition against CFU-C and CFU-E colony formation by soluble factor(s) derived from hairy cells.

The inhibitory effect by hairy cell conditioned medium (HCCM) on the growth of granulocyte and erythrocyte colony forming cells was studied in vitro. The percent inhibition of CFU-C formation by HCCM from four hairy cell leukemia (HCL) patients ranged from 36% to 76%, while no inhibition was observed with conditioned medium (CM) obtained from three B-cell chronic lymphocytic leukemia (B-CLL) patients nor from two normal controls. HCCM inhibited specially the growth of rG-CSF responding stem cells. The hairy cell-derived colony inhibitory factor from HCCM was nondialyzable, fairly stable to heat treatment, and trypsin sensitive. Its maximal inhibitory activity against granulopoiesis was observed in the fractions of 5,000 to 6,000 daltons. Moreover HCCM inhibited CFU-E colony formation but not BFU-E. These results indicate that hairy cells produce a factor that inhibits granulopoiesis and erythropoiesis in vitro. This factor may play a role in neutropenia and anemia observed in HCL.

Phenotypical heterogeneity of Japanese adult T-cell leukaemia.

The leukaemic cells of 11 patients with Japanese adult T-cell leukaemia (ATL) were characterized in terms of phenotype employing various monoclonal antibodies against T-cell surface antigens. The leukaemic cells of all ATL patients except patient no. 11 displayed the phenotype OKT4+/OKT8-. However, the % of OKT3+ cells varied from 3 to 98. In 1 case, cells with phenotype OKT4+/OKT8+, which is characteristic of T-cells at an early stage of differentiation, were found. These results suggest that some ATL cells may be in an immature differentiation stage. Among the 11 patients, 2 with unusual ATL cell phenotypes (1 case with OKT3- cells and 1 case with Ia+/EAC+ cells) showed spontaneous regression of leukaemic cells. Characterization of leukaemic cells from a larger number of ATL patients may demonstrate a significant relationship between cell phenotype and prognosis.

Serum immunosuppressive acidic protein in adult T-cell leukaemia (ATL).

The availability of serum immunosuppressive acidic protein (IAP) as a marker of subtypes of adult T-cell leukaemia (ATL) was examined. Serum IAP levels were measured in 34 patients with ATL (18 typical, 9 atypical and 7 smoldering), 7 healthy carriers of ATLA antibody and 53 healthy controls. The mean value of serum IAP was significantly higher in patients with typical ATL (897.8 +/- 502.4 micrograms/ml) than in those with atypical ATL (426.7 +/- 106.6 micrograms/l), smoldering ATL (310.0 +/- 51.3 micrograms/ml), healthy carriers of ATLA antibody (302.9 +/- 39.5 micrograms/ml) and normal controls (350.5 +/- 73.2 micrograms/ml). Serial determinations of IAP revealed that the level was correlated with the clinical course in patients with ATL; there was a difference in the prognosis of patients with high and normal levels of IAP (P less than 0.05). Thus, routine measurement of serum IAP seems useful in differentiating typical, atypical and smoldering ATL and also in evaluating the prognosis of patients.

Leu 11+ T gamma cell chronic lymphocytic leukemia with partially activated natural killer function and its further activation by recombinant IL2 in vitro.

A patient with T gamma cell chronic lymphocytic leukemia with the Leu 11+ phenotype and novel function of activated natural killer cells is reported. The peripheral blood mononuclear cells of this patient showed large granular lymphocytes by May-Giemsa staining and lamellipodia by scanning electron micrography. Tests on reactivity with monoclonal antibodies showed that most cells were Leu 11+, OKT3-/Leu 1-, OKT4-, OKT8-, Leu 7-, OKM1-, and Tac-. Freshly collected cells lysed not only K562, which is highly sensitive to natural killer cells, but also Raji cells and Daudi cells, which are not. Leu 11+ cells were triggered by recombinant interleukin 2 (rIL2) to proliferate, produce gamma-interferon (gamma IFN), and show enhanced HLA-DR antigen expression, and 30% of the Leu 11+ cells became positive for IL2 receptor antigen (Tac). The spectrum of cytotoxic activity of these cells against target cells was extended by rIL2; after treatment with rIL2, the cells also lysed HeLa cells and even fresh cancer cells. This stimulation also increased the activities of acid phosphatase and tartrate-resistant acid phosphatase of the cells and resulted in the appearance of nonspecific esterase activity. The expanded cell population may represent a neoplasm, but these findings provide information on a novel differentiation stage of activated NK cells.

OKM1-positive T-cell leukemias. Relationships among morphologic features, phenotype, and functional activities.

The morphologic features, phenotype, and functions of OKM1+ leukemic T-cells were studied. The leukemic T-cells in two patients with chronic lymphocytic leukemia (CLL) had specific features of large granular lymphocytes (LGL), and those in two patients with acute lymphocytic leukemia (ALL) had L2 morphologic characteristics. The phenotype of the leukemic cells from one patient with CLL was OKM1+, ER+, OKT3+, OKT4+, OKT8-, OKIa1-, IgGFc receptor (EA gamma)+, Leu-7+, Leu-11b+, and anti-Tac-. The cells had antibody-dependent cell-mediated cytotoxicity (ADCC), but no natural killer (NK) activity. They had a definitive helper effect on pokeweed mitogen-induced normal B-cell differentiation. The leukemic cells from the other patient with CLL were Leu-7-, and Leu-11b-, and lacked both ADCC and NK activity. The leukemic cells in the two patients with ALL were ER+, OKM1+, Leu-7-, and Leu-11-, and did not have any cytotoxicity. One was EA gamma +, and the other was EA gamma -. These findings suggest that OKM1+ leukemic T-cells consist of at least two subgroups: (1) T-cells with the morphologic features of LGL; and (2) those with a lymphoblastic morphologic type. In either case, the phenotype is novel and suggests the emergence of a small, distinct lymphocyte subset.