Flow cytometric method for enumeration and classification of reactive immature granulocyte populations.

We developed a flow cytometric method for the enumeration and classification of nonmalignant immature granulocytes (IG). In this study, IG are defined as most immature (IG stage 1: promyelocytes and myelocytes) and as more mature (IG stage 2: metamyelocytes). Blood specimens from 46 patients with documented infectious or inflammatory disease and known presence of IG (by routine manual microscopy) were analyzed. For a reference manual differential count, we used a 400 white blood cell (WBC) differential and separated granulocytes into promyelocytes and myelocytes combined, metamyelocytes, and included band cells in the mature, segmented neutrophil population. The flow cytometric method is based on three-color staining of whole, anticoagulated blood with CD45-PerCP, CD16-FITC, and CD11b-PE-labeled monoclonal antibodies and a three-step gating procedure. The flow cytometric results were confirmed by cell sorting and microscopic evaluation of the sorted cells. A total of 10,000 events, excluding debris, were recorded per specimen and IG stage 1 (CD16-/CD11b-), IG stage 2 (CD16-/CD11b+), and mature neutrophils (CD16+/CD11b+) were categorized. Regression and correlation between flow cytometric IG and the manual differential showed y = 1.34x + 0.95, r(2) = 0.86 for IG stages 1 and 2 combined versus promyelocytes, myelocytes, and metamyelocytes. For IG stage 1 versus microscopic counts of promyelocytes and myelocytes, the results were y = 1.53x + 1.24, r(2) = 0.76; for IG stage 2 versus manual metamyelocyte count, y = 0.77x + 0.21, r(2) = 0.58. Reproducibility of the flow cytometric method showed a coefficient of variation (CV) of 6.8% for all IG combined compared with a CV of 50.2% for manual differential IG count (based on a routine 100 WBC count). Samples were found stable at least 12 h at 25 degrees C and at least 48 h at 4 degrees C for flow cytometry. After staining and lysing, the sample was stable for at least 120 min at room temperature. We analyzed samples from patients with myelodysplastic and myeloproliferative disease separately. We found that CD16- mature neutrophils falsely elevated the flow cytometric IG count. Similar results were obtained in blood from patients treated with granulocyte-colony stimulating factor (G-CSF). Although this restricts the use of the method somewhat, we believe that this flow cytometric method is useful for enumerating reactive IG, as well as for evaluating automated methods for IG identification by hematology analyzers.

Reticulocyte maturity as an indicator for estimating qualitative abnormality of erythropoiesis.

AIMS: To determine the maturity of reticulocytes in patients with anaemia as a result of various haematological disorders including those with qualitative abnormalities such as ineffective erythropoiesis or dyserythropoiesis. METHODS: The number of mature reticulocytes was measured with flow cytometry in venous blood samples from 122 patients with haematological disorders and 100 healthy controls. Reticulocytes were classified into three categories by the fluorescence intensity of auramin O staining: low fluorescence ratio (LFR), medium fluorescence ratio (MFR), and high fluorescence ratio (HFR). Immature reticulocytes were determined as the aggregate of MFR and HFR (%). RESULTS: The mean (2SD) number of immature reticulocytes in 100 normal subjects was 9.0 (7.0)%. Significantly high mean values of immature reticulocytes with a normal or reduced reticulocyte count were shown in 90 patients with dyserythropoietic or ineffective erythropoietic conditions, such as acute myeloid leukaemia (AML) (n = 37), myelodysplastic syndrome (MDS) (n = 35), aplastic anaemia (AA) (n = 8), or megaloblastic anaemia (MA), (n = 6). Reticulocyte ratios returned to normal after successful treatment of patients with AML (n = 10) and MA (n = 3). However, high percentages of immature reticulocytes with increased reticulocyte counts were consistently observed in patients with enhanced erythropoiesis such as those with acquired autoimmune haemolytic anaemias (AIHA) (n = 4) or acute blood loss (ABL) (n = 4). Reticulocyte maturity was within the normal range in patients with reduced erythropoiesis such as occurs in chronic renal failure (CRF) (n = 11), or in iron deficiency anaemia (IDA) (n = 13). CONCLUSIONS: The evaluation of reticulocyte maturity with total reticulocyte count seems to be clinically useful for estimating the qualitative impairment of erythropoiesis, and so could help differentiate haematological disorders.

In vivo administration of recombinant interleukin-2 induces granulocyte-macrophage colony formation in a murine system.

The in vivo administration of recombinant interleukin-2 (rIL-2) in humans has led to anemia, thrombocytopenia, and eosinophilia. In an attempt to evaluate the effects of the in vivo administration of rIL-2 on murine bone marrow, we administered rIL-2 to C57BL/6 female mice i.p. three times a day at doses ranging from 10,000 to 100,000 U for 10 days; we then harvested blood and bone marrow from these animals every other day and performed the following analyses: White blood cell count, red blood cell count, hemoglobin concentration, histogram analysis of nucleated cell volume, manual differential counts, and in vitro colony-forming assays for granulocytes and monocytes (CFU-GM). The administration of rIL-2 induced an overall increase in the total white blood cell count that was dose-dependent for its appearance and overall number. This increase was secondary to an increase in monocytes and granulocytes but not to a change in lymphocyte number. Myeloid proliferative activity measured by CFU-GM revealed a biphasic pattern of activity. An early proliferation at 2 days was not followed by lymphocytosis. However, a second peak of proliferation at 6 days was associated with peripheral blood granulocytosis and monocytosis. After rIL-2 was discontinued on day 10, the CFU-GM activity returned to normal by days 16-18. These results suggest that the in vivo administration of rIL-2 may play an important role in the regulation of hematopoiesis.

Differentiation-linked expression of the plasminogen activator inhibitor type-2 gene in the human HL-60 promyelocytic cell line.

HL-60 is a human promyelocytic cell line which was found to be capable of differentiating toward a macrophage-like or granulocyte-like phenotype. Histochemical analysis demonstrated that incubation of cells in the presence of phorbol myristate acetate (PMA) or 1,25-dihydroxyvitamin D3 induced varying degrees of monocytic differentiation, while incubation in the presence of retinoic acid (RA) or dimethyl sulfoxide (DMSO) induced granulocytic differentiation. The differentiation induced by PMA, RA, and to a lesser extent DMSO, was accompanied by the induction of plasminogen activator inhibitor expression. mRNA analysis of control and PMA-induced cultures revealed the induction of a 2-kb message in treated cells which hybridized with a PAI-2-specific oligonucleotide probe. This is consistent with the literature concerning the expression of PAI by macrophages and granulocytes. No hybridization was detected with a PAI-1 specific probe. Expression of PAI by cells of hematopoietic origin appears to be associated with differentiation or stimulation of committed cells. Furthermore, PAI-2 expression by HL-60 cells is not restricted to one specific hematopoietic lineage. Since other cells of hematopoietic origin such as platelets express PAI-1, future studies using pluripotential cell lines could provide information on the initial events of lineage commitment and gene expression.

Treatment of advanced Hodgkin's disease with high dose melphalan and autologous bone marrow transplantation.

Twenty patients with Hodgkin's disease which had relapsed at least once after chemotherapy, were treated with melphalan 140-220 mg/m2 i.v. followed by reinfusion of non-cryopreserved autologous bone marrow. Four patients (20%) remain alive and disease-free 28, 45, 52, and 96 months after treatment respectively. There were no treatment-related deaths. This appears to be the only reported series of patients treated with a single agent in this situation. The results are comparable to those achieved by multi-agent regimens with autologous or allogeneic bone marrow transplantation.

Double autologous bone marrow transplantation for acute myelogenous leukemia in a patient treated for Hodgkin's disease.

A 30-year-old man developed acute myelogenous leukemia nearly 3 years after treatment of Hodgkin's disease with radiation and three chemotherapy combinations. Remission was induced with one cycle of high-dose Ara-C therapy. Three cycles of consolidation chemotherapy were given. The patient then had two autologous bone marrow transplants, the first after conditioning with 5 Gy total body irradiation, the second after Melphalan 140 mg/m2. The procedures were well tolerated, although hematological reconstitution was very slow after the second autotransplant. The patient has been disease-free for over 4 years. Such patients may be more vulnerable to transplant-related complications because of their previous exposure to chemotherapy and radiation, which may damage several organs including the bone marrow. This report demonstrates that patients with secondary acute myelogenous leukemia may tolerate a double autotransplant procedure and achieve durable remissions.

Hairy cell leukemia and anti-leukocyte common antigen.

Thirty-three bone marrow biopsies from 15 patients with hairy cell leukemia (HCL) were evaluated morphologically and immunohistochemically by use of the peroxidase-antiperoxidase technique to demonstrate reactivity for leukocyte common antigen (LCA). Hairy cells in all biopsies showed a distinctive and characteristic pattern for LCA, which decorated the periphery of the cytoplasm but that left most of the cytoplasm and the nucleus unstained. Anti-LCA was particularly helpful in highlighting focal or subtle leukemic infiltrates. Hairy cells in biopsies from three patients had, on routine morphologic examination, a spindled and sarcomatoid appearance, but these too were strongly LCA positive. Treatment regimens were variable: five patients had splenectomy and received chemotherapy; five patients had splenectomy alone; and four patients had chemotherapy alone. Seven patients received interferon, and one patient received no treatment. In those six patients who had multiple biopsies as part of follow-up examinations, hairy cells as identified by anti-LCA were continuously present. Often in significant numbers, and were usually underestimated or not identified by routine examination. In those patients who received chemotherapy, qualitative alterations in the LCA reaction of hairy cells were observed.

Bone marrow transplantation for acute monocytic leukemia following the treatment of Hodgkin's disease.

A 32-year-old woman developed acute monocytic leukemia within a year of treatment for Hodgkin's disease with chemotherapy and radiation. Residual leukemia was present in the bone marrow after two induction courses of high-dose Ara-C. She received a bone marrow transplant from an HLA- and DR-identical sister and remains in complete remission more than 2 years after transplantation. Only one other instance of a remission greater than 2 years after transplantation for secondary acute leukemia could be found in the literature. Although bone marrow transplantation may be carried out successfully in these patients, it is possible that they may be more vulnerable to transplant-related complications because of their previous exposure to chemotherapy and radiation. Only further study can clarify this matter and determine the best time for the procedure and which regimen should be used.

Serum beta 2-microglobulin: a real improvement in the management of multiple myeloma?

Beta 2-microglobulin (B2-m) determinations in serum have recently been introduced as a method of stratifying patients suffering from multiple myeloma. Conflicting results from several studies prompted us to study retrospectively the correlation of B2-m with presenting features and disease stage, as well as the prognostic value of B2-m, in 87 myeloma patients. Significant correlations were found between B2-m and presenting features such as haemoglobin level, serum calcium level and total body myeloma cell mass. The strongest correlation existed between B2-m and serum creatinine (r = 0.68). B2-m did not discriminate between the different disease stages as defined by Durie and Salmon, nor between myeloma Stage IA and monoclonal gammopathy of undetermined significance (MGUS). Considered alone, B2-m was found to have prognostic value in terms of survival. This correlation disappeared after correction for serum creatinine level and tumour load (multivariate analysis). Furthermore, changes in tumour load during therapy were not reflected in changes in B2-m levels, thereby rendering B2-m levels invalid as tumour marker. Our findings indicate no value for B2-m determinations in the staging and follow-up of myeloma patients.

Plasma spermidine concentrations as early indication of response to therapy in human myeloma.

Eighteen patients with melphalan refractory myeloma were treated with vindesine and prednisone. Plasma spermidine concentrations were measured by radioimmunoassay before and after a single vindesine injection. Seven patients showed a significant rise of plasma spermidine after vindesine and five of these showed a clinical response on further evaluation. Of the 11 patients who did not show raised spermidine concentrations, 10 did not respond to the therapy. The correlation between clinical response/rise of spermidine and between non-response/no rise of spermidine was statistically significant (p less than 0.05). Pretreatment spermidine concentrations did not distinguish those who responded to treatment nor did they differ in patients and controls.

Gastrointestinal involvement with myeloma.

A patient with IgA-k multiple myeloma is presented. There was initially a good response to chemotherapy but the patient later developed small intestinal obstruction. This was due to multiple polypoid plasmacytomas. The mesenteric nodes were also involved, and were found on immunofluorescence microscopy and flow cytometry with double fluorescent labelling to be composed of two separate populations of cells (IgA-k and IgG-k), one population having a diploid content of DNA and the other tetraploid. These two distinct cell populations differed in both proliferative characteristics and size. The findings are discussed and the literature relating to gastrointestinal involvement in plasma cell dyscrasias reviewed.

Enteral nutrition by nasogastric tube in adult patients treated with intensive chemotherapy for acute leukemia.

In this study, nutritional status 3 wk after starting 20 induction course of chemotherapy with enteral nasogastric tube feeding was compared to the nutritional status after 35 courses with a normal oral hospital diet. Tube feeding consisted of 2000 to 3000 cal daily of a hospital made pasteurized formula or sterile Nutrison RTS. In the group fed by nasogastric tube the mean weight loss was significantly smaller (p less than 0.01) and there were fewer patients with a severe weight loss of more than 5% during the first 3 wk (p less than 0.01) than in the hospital diet group. Serum albumin reduction of more than 10% was present in 4/20 and 23/35 for each group respectively (p less than 0.01). Bacterial contamination occurred in the pasteurized hospital-made formula which led to Pseudomonas septicemia in one patient. During a short-term catabolic state (3 wk) sterile feeding by nasogastric tube can prevent weight loss and hypoalbuminemia in most patients. Bacteriological control of the food and supply system is mandatory in granulocytopenic patients.

Combination chemotherapy for acute lymphocytic leukaemia in 25 adults.

Twenty-five consecutive adult patients with acute lymphocytic leukaemia (ALL) all achieved complete remission, twenty-two with vincristine-prednisone, while thirteen patients also received daunorubicin (DNR). Three patients obtained remission only after treatment with cytosine arabinoside (Ara-C), 6 thioguanine (6-TG) and adriamycin (ADM). Despite central nervous system (CNS) prophylaxis by intraventricular infections via an Ommaya reservoir, 2/22 patients had a CNS relapse. The median remission duration of the whole group was 19 months. Four patients could stop therapy after 3 years. The median survival duration of all patients was 38 months. The quality of life of the patients during this chemotherapeutic regimen was good.

Phytohemagglutin-induced lymphocyte cytotoxicity in hemodialysis patients with hepatitis B virus infection.

Phytohemagglutinin (PHA)-induced or primary cytotoxicity in vitro which is mediated by T lymphocytes, was studied during hepatitis B virus (HBV) infection in 45 hemodialysis patients, and related to liver cell damage and recovery. HBsAg positive patients with raised transaminases had increased primary cytotoxicity similar to nine otherwise healthy subjects with acute hepatitis B. HBsAg positive patients with normal transaminases showed decreased primary cytotoxicity and recovered patients showed normal values. Increased primary cytotoxicity could not be attributed to an increase in T lymphocytes, as all groups of hemodialysis patients had decreased lymphocyte and T-lymphocyte counts without significant differences between them. In the follow-up study none of the 13 HBsAg positive patients with normal transaminases recovered. However, five of the 18 patients with raised transaminases did recover from hepatitis B, accompanied by a decrease in cytotoxicity. These results show that an increased PHA-induced lymphocyte cytotoxicity corresponds with the occurrence of liver cell damage and subsequent recovery in hemodialysis patients with HBV infection. This suggests that cytotoxic T lymphocytes are involved in liver cell damage and recovery in HBV infection.