Assessment of response to therapy in multiple myeloma by multiparameter flow cytometry. Usefulness of an eight-color single tube with monoclonal antibodies in dried formulation.

OBJECTIVE: Multiparameter flow cytometry is considered the gold standard to evaluate minimal residual disease in multiple myeloma (MM) and patients in complete remission can achieve "Flow MRD-negative" status (i.e. immunophenotypically abnormal plasma cells not detectable). In the current study we report the usefulness of an eight-color flow cytometric method with a 10-5 sensitivity, using monoclonal antibodies in dried formulation. MATERIALS AND METHODS: Forty-six patients with MM were treated with bortezomib-based regimens and, when eligible, with autologous stem cell transplantation. Response to therapy was assessed according to the criteria validated by the International Myeloma Working Group. Multiparameter flow cytometry was carried out with an 8-color panel validated by the Euroflow Consortium. A commercially available single 8-color tube in dried formulation was used and almost 2,000,000 events were acquired, in order to obtain a 10-5 sensitivity. RESULT: Sixteen patients achieved stringent complete remission and another three patients achieved complete remission. In these groups of patients, the "Flow MRD-negative" status was achieved in sixteen cases. In patients who had a different degree of response (very good partial response, partial response, minimal response) immunophenotypically abnormal plasma cells were always detected. CONCLUSION: Using a single eight-color tube in dried formulation, and an acquisition strategy able to obtain a 10-5 sensitivity, not only is it possible to detect a deep response to modern therapy in patients who obtained at least complete remission, but it is also always possible to detect minimal residual disease in patients with either complete remission or stringent complete remission.

Cell clonality in hypereosinophilic syndrome: what pathogenetic role?

OBJECTIVE: Idiopathic hypereosinophilic syndrome (HES) is a heterogeneous disorder, including either a myeloproliferative or a lymphoproliferative variant (l-HES). In l-HES, T-lymphocytes could be involved in the pathogenesis through several cytokines, including IL5. METHODS: We assayed both TCR Beta- and delta-rearrangements by fluorescent PCR, characterizing 14 patients affected by HES. Lyn activation (a src-kinase involved in the IL5 pathway) was also tested in 6 cases. RESULTS: FIP1L1-PDGFRa was detected in 4 cases (28.6%); a clonal TCR was found in 10 cases (71.4%), including cases FIP1L1-PDGFRalpha-positive; four cases did not show any molecular marker. In this series, levels of IL5, IL4, IL2 and gammaIFN were measured, without any significant difference among different subgroups. All pathological samples tested did not show Lyn activation. Immunophenotype was also characterized: only one case showed an atypical CD3-/CD4+ population in the bone marrow. CONCLUSION: This study would suggest that a real distinction between m- and l-HES is not wholly convincing and that clonal T-cell expansion could not be the "primum movens" but an epiphenomenon in HES.

Association of B-chronic lymphocytic leukemia and acute myeloid leukemia.

A 62-year-old man presented with fatigue, pallor and mild weight loss. Laboratory studies showed Hb 7.6 g/dl, Hct 21.8%, WBC 108x10(9)/1, PLT 143x10(9)/1. At morphological examination, circulating cells appeared as 60% blasts and 40% lymphocytes, with smudge cells. A bone marrow aspirate showed infiltration by blasts (50%) and lymphocytes (40%); alpha-naphtyl-acetate esterase was positive in 90% of blasts, while myeloperoxidase was positive in 10%. The immunologic phenotype of blasts was characterized by the co-expression of CD13, CD33, CD14, CD4, CD15, CD64, CD117, HLA-DR, CD11b. Lymphocytes were characterized by a B-CLL immunophenotype: CD19+, CD5+, CD23+, CD20+(dim), FMC7+(dim), K light chain+(dim). Karyotype was normal and PCR assays for AML-ETO, CBFbeta-MYH11, PML-RARalpha, BCR-ABL and bcl-1/JH translocation were negative. Coexistence of CLL and AML with monoblastic features was diagnosed. Simultaneous appearance of CLL and AML has rarely been described and represents a peculiar biological phenomenon.

Recombinant human granulocyte colony-stimulating factor administration in a case of neutropenia due to increased neutrophil sequestration.

A 55-year-old female was admitted with fever which followed an episode of pseudomembranous colitis. Despite an accurate clinical investigation, there was no evidence for specific sites of infection. Remission of fever was not obtained with antibiotic therapy (gentamycin plus carbepenem) and progressive neutropenia was observed. Neutrophils fell to 0.3 x 10(9)/1. The diagnostic approach, including a bone marrow aspirate, excluded mechanisms leading to impaired neutrophil production, and in the suspect of increased neutrophil sequestration/destruction, whole-body scintigraphy with (99m)technetium-hexamethylpropyleneamineoxime ((99m)Tc-HMPAO)-labeled autologous leukocytes was performed. As a result, a site of leukocyte sequestration localized at the medium lobe of the right lung was detected. In an attempt to enhance neutrophil functions and achieve remission of infection, recombinant human granulocyte colony-stimulating factor (Filgrastim, Granulokine 30, Roche) at the dosage of 300 microg/day, subcutaneously, was added. As a results, fever disappeared in three days, but neutrophil recovery was slower, and normalization of the absolute neutrophil count (ANC) was obtained on day +7. The results obtained in this peculiar case of neutropenia, and the kinetics of both fever and ANC, suggest the possible combination of neutrophil function enhancement and an anti-inflammatory effect of rhG-CSF.

Glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factor differently modifies actin polymerization in neutrophils.

AIM: Several neutrophil functions can be modified by rhG-CSF administration. Neutrophil morphology changes in the course of treatment with Filgrastim (nonglycosylated rhG-CSF), along with impairment of chemotaxis. Both morphology and chemotaxis are not affected by treatment with Lenograstim (glycosylated rhG-CSF). Thus, we evaluated actin polymerization in neutrophils induced by treatment with the two forms of rhG-CSF. In fact, actin polymerization is crucial for neutrophil motility. MATERIALS AND METHODS: We evaluated twelve healthy subjects undergoing peripheral blood stem cells (PBSC) mobilization for allogeneic transplantation to HLA-identical siblings. Neutrophils were isolated by peripheral venous blood before and after administration of either Filgrastim (six PBSC donors) or Lenograstim (six PBSC donors). Actin polymerization was investigated by a flow cytometric assay, using FITC-phalloidin as a specific probe for F-actin, and two parameters were measured: spontaneous actin polymerization in resting neutrophils; fMLP-stimulated actin polymerization. Results were expressed as relative F-actin content. Fifteen blood donors were studied as a control group. RESULTS: Filgrastim administration induced an increased relative F-actin content in resting neutrophils; however, no further actin polymerization was observed after fMLP stimulation. Neutrophils from subjects treated with Lenograstim showed a normal behaviour in terms of both spontaneous and stimulated actin polymerization. CONCLUSIONS: Glycosylated and nonglycosylated rhG-CSF differently affect actin polymerization in newly generated neutrophils. Such effects may explain some previous findings concerning both morphology and chemotactic properties and may be due to different effects of the two forms of rhG-CSF on proteins involved in neutrophil motility regulation.

An image processing workstation for automatic evaluation of human granulocyte motility.

Polymorphonuclear (PMN) motility is currently determined by a method using chemotactic chambers with micropore filters. PMN locomotive capacity is measured by evaluating the maximum distance travelled by the cells, or by counting the number of cells which have moved through the filter. Although in recent years some attempts to improve the analysis of chemotaxis data by computer-assisted systems have been made no technique has been shown capable of both measurement and correlation of these parameters in real time. In the present paper we describe an automated technique, based on a workstation capable of exploring microporous filters employed in chemotactic chambers and of measuring PMN motility in a reliable, reproducible, rapid manner, independent of the subjectivity of the operator. The measurement is carried out by custom software capable of undertaking computerized image analysis of microscopic fields acquired by a TV camera and of driving the motorized microscopic table. Depth of migration, cell distribution at each plane, correlation index of the random behaviour with the model described by a gaussian distribution and data about the patient or assay under study, are computed, displayed, stored in a data base and printed in real time.

Actin polymerization in neutrophils from patients affected by myelodysplastic syndromes--a flow cytometric study.

In this study F-actin polymerization in neutrophils from 21 patients affected by myelodysplastic syndromes (MDS) was evaluated by means of a flow cytometric assay. Neutrophils were stimulated with formyl-methionyl-leucyl-phenylanaline (fMLP; 10(-8) M final concentration) for 15, 30, 60 and 120 sec, and F-actin content was determined using fluorescein-isothiocyanate phallacidin as a specific probe. Eight normal subjects were studied as controls. We found that F-actin polymerization was defective in ten patients, with very impaired values after 60 and 120 sec of stimulation with fMLP. The remaining 11 patients showed a prevalent neutrophil population with normal F-actin polymerization and neutrophil sub-populations with either defective or undetectable F-actin polymerization. In the first group, patients with very poor prognosis (refractory anemia with excess blasts, refractory anemia with excess blasts in leukemic transformation, trisomy 8, multiple karyotypic abnormalities) were present, although patients with aberrations of karyotype were present in the second group. It is possible that defects in neutrophil F-actin polymerization may be responsible for neutrophil dysfunction, which has frequently been observed in MDS.

FcRIII (CD16) expression on neutrophils from chronic myeloid leukemia. A flow cytometric study.

FcRIII (CD16) expression on neutrophils from 17 patients with chronic myeloid leukemia (CML) was studied by flow cytometry using monoclonal antibodies. A variable proportion of CD16-negative neutrophils were found both in CML patients in chronic phase (3 out of 8 patients) and in CML patients in hematological remission (3 out of 9 patients). Neutrophils with reduced FcRIII expression showed more defective chemiluminescence and phagocytosis than neutrophils with normal FcRIII expression. Circulating myeloid cells from three patients in chronic phase, showing a normal percentage of CD16-positive neutrophils, were isolated and fractionated by discontinuous Percoll gradients. This study showed that CD16 appears at the stage of metamyelocyte, that band cells and segmented neutrophils display an identical pattern of membrane FcRIII, and that the fluorescence intensity shown by metamyelocytes is different from that displayed by more mature cells. The association between low FcRIII expression and function abnormality could be suggestive of a defect in CML neutrophil maturation.

fMLP-induced CD11b/CD18 upregulation on neutrophils from patients with non-Hodgkin's lymphomas treated with recombinant human granulocyte colony-stimulating factor.

The effects of rhG-CSF administration on fMLP-induced neutrophil CD11b and CD18 upregulation were studied in nine patients suffering from intermediate and high grade non-Hodgkin's lymphomas. Blood samples were obtained before recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration and 24 hrs after rhGSF interruption. The growth factor was administered subcutaneously for five days in a dosage of 5 microg/Kg/day. Nine normal subjects were studied as controls. Five patients showed an impaired baseline CD11b and CD18 upregulation, which was corrected by rhG-CSF therapy. Four patients showed a normal baseline CD11b and CD18 upregulation, but this function was reduced by rhG-CSF therapy. All patients showed a normal baseline fMLP-induced luminol-enhanced chemiluminiscence and significantly increased chemiluminescence values after rhG-CSF administration. We conclude that, while in some patients rhg-CSF is able to improve neutrophil CD11b and CD18 upregulation in response to chemotactic agents, in other patients a decrease of this function can occur, maybe due to a relative immaturity of the circulating neutrophils induced by rhG-CSF.

Inhibition of leukocyte function by serum from patients with trichinellosis.

Modification of leukocytic function has been reported in only a few human parasitic diseases. In this study we evaluated the effects of the sera from patients infected with Trichinella on chemotactic and phagocytic responses in leukocytes. Leukocyte chemotaxis was tested by the agarose method and phagocytosis by the technique of Yamamura, modified for Saccharomyces cerevisiae. Sera were acquired from patients during a trichinellosis outbreak that occurred in northern Italy in 1986. The parasite was isolated from 1 patient and isoenzymatically typed as Trichinella sp. 3, a new taxon, previously considered Trichinella nelsoni. The results indicated that sera from Trichinella-infected humans inhibited both chemotaxis and phagocytic responses in leukocytes. These findings suggest the existence of serum factor(s) in trichinellosis patients that modify host leukocytic functions. The source and nature of active serum components and the mechanism by which they modulate leukocyte function remain to be clarified.

Lithium and rubidium activities on platelet aggregation.

Lithium (Li) has been shown to increase the platelet number after chemotherapy. The present paper suggests that Li may increase the platelet aggregation induced by sub-optimal concentrations of ADP. Conversely, rubidium does not modify platelet aggregation in the present experiments.

Effects of lysine-arginine association on immune functions in patients with recurrent infections.

Combined L-lysine-L-arginine therapy is capable of inducting recovery in age-related decline of thymic activity in mice and in elderly humans. The clinical usefulness of the association has also been shown in children with recurrent respiratory infections, while an increase in the number of CD3+ lymphocytes has been shown in patients with chronic lymphatic leukaemia. Recently, in vitro effects of the association on neutrophil function have been reported. In particular, the association was able to increase random migration, chemotaxis, phagocytosis-associated- and f-MLP-induced chemiluminescence. In this paper the authors evaluate the effects of L-lysine-L-arginine combination (lisargin) on several humoral and cell-mediated immunologic parameters in patients with recurrent infection. An increase of neutrophil random migration and chemotaxis (evaluated by a new technique, based on a computer assisted image processing system) was found. Furthermore an increase in the absolute number of lymphocytes involved in cytotoxic activity and IgG levels was observed.

Experimental studies on diacerhein: effects on the phagocytosis of neutrophil cells from subcutaneous carrageenan-induced exudate.

Diacerhein (DAR), a new drug which is particularly suitable for the treatment of osteoarthritis, was studied for its interference with the phagocytic capacity of cells coming from exudates of subcutaneous carrageenan oedema and from the peripheral blood of Sprague-Dawley rats. DAR was found to inhibit phagocytosis in both types of cells examined. This finding indicates that DAR may exert its action by means of a direct effect on the cells involved in the inflammatory process.

Chronic lymphocytic leukemia (B-cell) in the course of polycythemia vera. Description of a case with an unusual chromosomic anomaly.

The present paper describes the case of a patient who developed a B-cell chronic lymphocytic leukemia (B-CLL) 15 months after the diagnosis of polycythemia vera, which had been treated only with phlebotomies. In spite of lymphocytosis and the clinical signs and symptoms of leukemia, the patient exhibited at the same time presumptive elements of polycythemia (high LAP index levels, a high number of neutrophils). Cytogenetic investigations, carried out after the appearance of B-CLL, revealed the presence of an unusual abnormality (18 p+) both in bone marrow not stimulated by mitogens and in PWM-stimulated circulating lymphocytes. This case, which is the ninth of its kind described in the literature, offers some interesting observations about the association between myeloproliferative and lymphoproliferative syndromes.

Modifications in the concentrations of circulating myoglobin after treatment with low doses of adriamycin.

The modifications in the concentration of circulating myoglobin have been studied by means of a radioimmunoassay in 15 cancer patients undergoing polychemotherapy including adriamycin. In 8 patients significant increases in myoglobin levels were found after injection of low doses of the drug (25-50 mg/m2). Moreover, a disturbance of the normal biorhythm of the protein was evident in 12 patients. Creatine kinase-MB was evaluated by means of a radioimmunoassay, but there was no relation between an increase in the isoenzyme and an increase in myoglobin. No ECG modifications were detected. These data indicate that the measurement of myoglobin may offer an indication of myocardial or skeletal muscle damage caused by adriamycin.

Immunomodulators and enzymes of purine metabolism in human lymphocytes.

The enzymes ADA and PNP were evaluated in lymphocytic subpopulations in peripheral blood obtained from healthy subjects, elderly subjects and patients with immunoproliferative diseases. Some similar assessments were performed on lymphoid cells from cord blood. Preliminary studies indicate that Thymostimulin can in some cases correct enzymic defects.

Effects of lithium and rubidium on the differentiation of mononuclear cells.

Rubidium ions added at 0.5 mEq to liquid cultures of human mononuclear cells stimulated the differentiation of monocyte and particularly of granulocyte cell lines.

Lysine-arginine association and human neutrophil function: enhancement of functions related to microbicidal activity.

The effects of L-lysine, L-arginine, and lysine-arginine association on human neutrophil function were studied. The combination of the two agents, used at pharmacological concentrations, was able to increase both the phagocytosis-associated and the f-MLP-induced chemiluminescence, in the presence of luminol as amplifier agent. Neither superoxide anion production nor phytohaemagglutinin-induced aggregation were affected by the lysine-arginine association. The two amino-acids did not show any effect when used as single agents. L-canavanine (competitive inhibitor of nitric oxide synthesis starting from L-arginine) did not reduce chemiluminescence. These results show that the association lysine-arginine may increase the microbicidal potential of human neutrophils, probably by increasing the production of hypochlorous acid. In addition, a role for nitric oxide in these effects can be excluded.

[The role of flow cytometry in the study of physiopathology of neutrophilic granulocytes]. / Il ruolo della citometria a flusso nello studio della fisiopatologia dei granulociti neutrofili.

Flow cytometry represents an interesting methodological approach to human neutrophil function assessment and to the study of neutrophil biology. Several aspects of neutrophil activation can be evaluated by means of such technology: phagocytosis, respiratory burst, surface markers, modifications in intracellular pH, actin polymerization, membrane potential, aggregation. Neutrophil flow cytometric evaluation is suitable for whole blood evaluation and has been shown to be very specific and sensitive. In particular, whole blood methods have been developed in order to investigate phagocytosis, hydrogen peroxide production, and surface markers expression (integrins, receptors for the Fc fragment of IgG, selectins, etc.). Other aspects of neutrophil activation have to be investigated by using purified neutrophils.

Modulating effects by Trichinella spiralis sensu stricto excretory/secretory antigens of human neutrophil functions.

Many parasites are able to modify the host response through different mechanisms, for example host leukocyte function modification. Since granulocytes represent the most important effector cells against Trichinella spiralis, we evaluated the effects of supernatants of T. spiralis sensu stricto muscle larvae cultured for at least 18 hours (T. Sup.) on some human neutrophil functions, i.e. chemotaxis, phagocytosis and Nitroblue tetrazolium dye reduction. The cells from blood donors were tested in the presence of T. Sup. at different concentrations (1, 5, 10, 20%). The results show no effect of T. Sup. on neutrophil phagocytosis. On the contrary, random migration of cells is increased at concentrations of 10-20%, while stimulated migration is significantly decreased at concentrations of 5-10% but not at 20%. We found a significant decrease of Nitroblue tetrazolium reduction at 5-10% concentrations of T. Sup. It is concluded that T. spiralis culture supernatants contain some factors capable of modifying human neutrophil chemotaxis, oxidative metabolism, but not phagocytosis.