Prediction of survival in gynecological cancer based on immunological tests.

Patients with advanced cancer have a depressed immunological function. We performed a battery of tests on peripheral blood samples from 42 patients with gynecological cancer to determine the extent to which this depression was due to abnormal lymphocyte function, as compared to changes in the number of lymphoid cells in the peripheral blood or in the efficiency of purification of cells in Ficoll:Hypaque gradients in preparation for testing. The percentage of lymphocytes in the gradient-derived cell suspension (% LG) and the absolute lymphocyte count were more informative than mitogen stimulation, mixed leukocyte culture, and T- and B-cell measurements. Both values decreased significantly with the advancing stage of cancer, and we were able to predict survival of patients with uniform stage of disease. The % LG correlated with survival better than did any other test when multivariate analyses of all test combinations were performed. Low values of % LG reflected both the depressed lymphocyte counts and the altered buoyant density of the leukocytes of many patients with advanced cancer. A large portion of the depression in other immune function tests was statistically attributed to changes in % LG and the lymphocyte counts. We concluded that these two simple measurements provide valuable information about patients with gynecological cancer.

Inflammatory meningeal masses of unexplained origin. An ultrastructural and immunological study.

Two patients with inflammatory meningeal masses were studied. Lesions in both patients showed varying proportions of meningothelial and inflammatory components. The non-neoplastic nature of the inflammation was confirmed in one case by lymphocyte surface marker study, which showed T and B cells in one to four ratio, and by immunohistochemistry, which revealed polyclonal plasma cells. The abundant histiocytes contained muramidase and often enclosed intact lymphocytes or plasma cells within their cytoplasm, i.e., emperipolesis. Their surfaces bore slender interdigitating pseudopodia, intercellular junctions, and subplasmalemmal linear densities. The derivation of these histiocytes is uncertain: mononuclear phagocytes, meningothelial cells, and multipotential meningeal cells are all possible progenitors. A comparison with eleven similar reported cases reveals a tendency for inflammatory meningeal masses to occur in the young, as well as a predilection for posterior fossa involvement. They resemble the extranodal lesions of sinus histiocytosis with massive lymphadenopathy, as well as plasma cell granulomas or inflammatory pseudotumors of lung and other tissues. However, it is possible that these lesions represent a variant of meningioma in which an unusual immunological response has been evoked.

A comparison of commercially available adjuvants for use in research.

We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.

Increased whole blood viscosity during coronary artery bypass surgery. Studies to evaluate the effects of soluble fibrin and poloxamer 188.

This study was designed to test the hypothesis that soluble fibrin complexes resulting from the trauma of surgery could produce elevated blood viscosity, to characterize the soluble fibrin polymers, and to evaluate in vitro the effect of a new hemorheologic agent, poloxamer 188, on viscosity in these abnormal situations. Ten patients undergoing aortocoronary bypass surgery were studied before and at various times after surgery. By 6 h after surgery, the mean hematocrit decreased by 23%, fibrinogen decreased 48%, and erythrocyte sedimentation rate decreased 33%, whole blood viscosity at a low shear rate rose on average of 69% and soluble fibrin rose 118%. Over the 6-day observation period, the concentrations of soluble fibrin paralleled the changes in viscosity, whereas the concentrations of fibrinogen varied nearly inversely with viscosity. The effects of various forms of fibrinogen and fibrin were tested by additions to normal blood. Soluble fibrin polymers, but not fibrin monomers, increased blood viscosity two to three fold. Poloxamer 188 reduced the viscosity of all patient samples to the normal range. These data support the hypothesis that increased whole blood viscosity at low shear rates is caused by hydrophobic adhesion of fibrin polymers to red cells and that poloxamer 188 normalizes viscosity by effectively disrupting the weak hydrophobic bonds.

Breakthrough varicella infection in a healthcare worker despite immunity after varicella vaccination.

Although varicella vaccination is recommended for varicella-susceptible healthcare workers (HCWs), breakthrough infection after vaccination is not unusual, especially following household exposures. We report breakthrough varicella in a vaccinated HCW and review the data on breakthrough infection and concerns for the healthcare setting.

IgG subclasses in children with nephrotic syndrome.

To determine whether the hypogammaglobulinemia of childhood nephrotic syndrome is characterized by symmetric depression of the IgG subclasses, the authors compared the IgG subclass concentrations in nephrotic patients in relapse versus remission. The authors used a highly sensitive monoclonal antibody-based enzyme immunoassay that allows quantitation with comparable precision of all four subclasses. They analyzed 28 sera obtained from 22 nephrotic patients during relapse (n = 16) and/or remission (n = 12). The mean ages of the two groups were similar. IgG1 and IgG2 were significantly decreased during relapse compared with remission, whereas IgG3 and IgG4 were not significantly different. This pattern of asymmetric depression of IgG subclasses supports a cause other than urinary losses in the pathogenesis of this abnormality.

Diclonal gammopathy in chronic cold agglutinin disease.

Serum from a patient with chronic cold agglutinin disease (CCAD) contained two paraproteins identified as IgM kappa and IgM lambda by immunoelectrophoresis and confirmed by immunofixation on high resolution agarose gel electrophoresis. The IgM kappa band was completely removed by incubation of the serum at 4 degrees with ficin-treated O Rh-positive red blood cells, and partially removed by incubation with cord cells, suggesting that it reacted with I antigen. The IgM lambda band was unaffected by these absorptions. Previous investigators have shown CCAD to be a monoclonal gammopathy, usually IgM kappa. The presence of both IgM kappa and IgM lambda paraproteins in this patient with the IgM kappa showing cold agglutinin activity is unique. The patient has a lymphoplasmacytic infiltrate in the bone marrow. These data suggest that CCAD may be part of a spectrum of disorders in which paraproteins are produced and are stimulated by factors unrelated to the target red blood cell antigens.

An improved method for determining the microbial inhibitory activity of serum and its application to the study of patients with leukemia.

Numerous investigators have demonstrated that derangements in serum transferrin and iron can contribute to susceptibility to infection, but the complexity and imprecision of assays have impeded both research and development of clinical testing in this area. This article describes an automated assay for measuring the microbial inhibitory activity of transferrin in serum and its use in patients with acute myelogenous leukemia (AML) and in normal controls. The assay measured the ability of heat-inactivated serum to inhibit the growth of an antibiotic-resistant strain of Pseudomonas aeruginosa. The serum dilutions were prepared in a special low iron chemically defined broth. An inhibition index, the reciprocal of the serum dilution producing 50% inhibition of bacterial growth when compared with the growth in broth alone, was determined. The results showed the serum from the patients with leukemia had a significantly lower inhibition index than that of controls (16 +/- 11 vs. 35 +/- 13, P less than 0.01). In addition, they had higher serum iron levels (162 +/- 65 vs. 75 +/- 27, P less than 0.01), lower serum transferrin levels (231 +/- 65 vs. 309 +/- 71, P less than 0.01), and higher percentage saturation of transferrin with iron (59 +/- 21 vs. 20 +/- 8, P less than 0.01) than did controls. Because the assay uses equipment available in many clinical laboratories, it could be developed for routine use as an index of susceptibility to infection in selected patients.

Densitometric quantitation of high resolution agarose gel protein electrophoresis.

The authors developed a cost-effective procedure for high-resolution protein electrophoresis in agarose gel which results in electrophoretic patterns that can be both visually interpreted and densitometrically quantitated. Unique features include the development of special molds for the preparation of several gels simultaneously and the combination of chemical with densitometric procedures to accurately quantitate the protein fractions in both normal and pathologic sera. Since the albumin concentrations obtained by densitometric scan of either cellulose acetate or agarose gel were erratic with respect to colorimetric (ACA) or immunochemical (ICS) measurements, the authors incorporated colorimetric measurements of albumin and total protein with densitometric quantitation of the electrophoretic bands into an algorithm for calculating protein concentrations. The densitometric quantitation of the gamma globulin fractions compared well with the sum of the immunochemically determined IgG and IgM concentrations (r = 0.98). Alpha and beta globulins were calculated using mathematically adjusted densitometric scan percentages and the total alpha and beta globulin protein concentration. The agarose gel procedure was more sensitive than cellulose acetate in the detection of paraprotein bands in a comparison of 93 sera containing paraproteins.

T helper-suppressor cell imbalance in pyoderma gangrenosum, with relapsing polychondritis and corneal keratolysis.

We found decreased T helper/inducer and increased T suppressor/cytotoxic cells in a 45-year-old woman with pyoderma gangrenosum. Serum immunoglobulin levels were normal, suggesting that these T suppressor cells did not function primarily to regulate antibody synthesis. The patient had diminished cutaneous delayed hypersensitivity responses, reacting to only one of six antigens tested, but responded in vitro to three of three antigens. Because of their various regulatory functions, excess T suppressor cells, or a lack of T helper cells, could be a common factor underlying many of the humoral and cell-mediated immune derangements, as well as the neutrophil abnormalities, that have been found in pyoderma gangrenosum. Our patient also had relapsing polychondritis and corneal keratolysis, consistent with the systemic nature of the disorder. The T-cell imbalance persisted even as the ulcer improved. Since monoclonal antibodies against T cell subpopulations are readily available, measuring these cell types as part of the immunological workup of patients with pyoderma gangrenosum might yield valuable clues concerning the pathogenesis of this disorder.

Agarose electrophoresis and immunonephelometric quantitation of cerebrospinal fluid immunoglobulins: criteria for application in the diagnosis of neurologic disease.

The cerebrospinal fluid (CSF) IgG index, the CSF to serum albumin ratio, and electrophoresis on agarose gel of CSF and serum were evaluated retrospectively for their usefulness in the differential diagnosis of multiple sclerosis (MS). Standardized procedures were adopted for grading the intensity of oligoclonal banding and for the certainty of diagnosis of MS. One hundred and forty-nine patients were studied, including 23 with definite multiple sclerosis (MS), 12 with probable MS, 20 with possible MS, 20 with inflammatory neurologic disease, 65 with noninflammatory neurologic disease, and nine with no neurologic disease. The CSF IgG index and the CSF to serum albumin ratio were calculated from nephelometric measurements of serum and CSF IgG and albumin. The intensity of oligoclonal banding was graded relative to the density of the prealbumin band. Eighty-eight per cent of cases of definite MS had distinct oligoclonal bands, and an equal number had an elevated IgG index. These tests were not specific for MS, however, since 50% of cases of inflammatory neurologic disease and 5% of those with noninflammatory neurologic disease had an elevated IgG index. Similarly, 48% of cases with inflammatory disease and 25% with noninflammatory disease had oligoclonal bands. However, only patients with definite MS (21%) or possible MS (4%) had prominent oligoclonal bands whose density was greater than or equal to that of prealbumin, together with a CSF IgG index greater than 1.50. This combination of findings therefore may enhance the level of suspicion of MS. By contrast, an isolated increase in the CSF to serum albumin ratio may suggest a diagnosis other than MS.

T-cell imbalance in neutropenia of uncertain etiology.

The authors studied 16 consecutive patients with chronic neutropenia of uncertain etiology in whom bone marrow biopsies were performed. Using monoclonal antibodies, they measured the levels of the two major subclasses of T lymphocytes, cells with the T-suppressor/cytotoxic cell phenotype (Ts), and cells with the T helper/inducer cell phenotype (Th) in the blood of eight of these patients. Five patients had Ts lymphocytosis or increased proportions of Ts cells and Th lymphocytopenia. All five patients had greatly increased proportions of large lymphocytes with prominent cytoplasmic azurophilic granules. Since cells with similar morphology from normal individuals have been shown to express cytotoxic activities, the authors measured the cytotoxic function of cells from three of the patients with Ts lymphocytosis. All three possessed antibody-dependent cell-mediated cytotoxicity (ADCC) but not natural killer (NK) cell activity. Many patients had evidence of autoimmune disorders, conditions in which Ts cells usually are decreased. None had hypogammaglobulinemia, a disorder that has been associated with increased Ts activity. The high incidence of an imbalance in T lymphocyte subpopulations in patients with neutropenia suggests that disturbed immunoregulation involving T cells may play an important role in the pathogenesis of this disorder.

Immunophenotyping large B-cell lymphomas. Flow cytometric pitfalls and pathologic correlation.

Large cell lymphomas often challenge the diagnostic flow cytometrist. The purposes of this study were to improve our protocols for diagnosing large cell lymphomas and to correlate flow cytometric (FC) data with demographic and histologic features. We identified 63 cases of large B-cell lymphoma between January 1, 1995, and July 30, 1999, and reviewed the diagnostic slides and FC light scatter and staining patterns. The 51 lymphomas with adequate material for systemic review fell into 2 light scatter patterns: "clear cut," with large abnormal cells (high forward scatter relative to normal lymphocytes), 17 cases (33%); and "complex," 34 cases (67%). Clear-cut cases were more mitotically active (average of 42 vs 25 per 10 high-power fields), with higher cellularity. Apoptosis, geographic necrosis, and sclerosis were present histologically in many cases, regardless of FC findings. We conclude that morphologic features of large cell lymphomas do not predict which cases will be difficult to diagnose by FC. Gating strategies can be critical to improve the diagnostic yield.

Bronchoalveolar lavage cell differential in the diagnosis of sarcoid interstitial lung disease. Likelihood ratios based on computerized data base.

With the use of a microcomputer, the authors prospectively stored the bronchoalveolar lavage (BAL) cell differential counts from 208 patients undergoing initial evaluation for chronic interstitial lung disease. Sarcoidosis subsequently was diagnosed in 46% of these patients; the remainder had several other etiologies of their lung disease. The BAL lymphocyte count did not allow for clear separation between these two groups. To facilitate the interpretation of the BAL percent lymphocyte data, the authors applied a modification of Bayes' theorem to calculate likelihood ratios for the differential diagnosis of sarcoidosis versus other interstitial lung disease. These values ranged from 0.15 for patients with less than 6% lymphocytes to 3.5 when more than 40% lymphocytes were found. Similar results were obtained with a subsequent, independently analyzed group of 46 patients. To provide a more meaningful interpretation of the BAL fluid lymphocyte count, the authors designed a reporting scheme that includes both a written description and a graphic representation of the likelihood ratio. Prospective collection of data using microcomputers should extend this useful form of reporting to additional laboratory studies.

Immunohistochemical progesterone receptor assay. Measurement by image analysis.

To determine the efficiency of image analysis in immunohistochemical progesterone receptor (PgR) measurement, 94 primary breast carcinoma tissue samples were evaluated for PgR by biochemical dextran-coated charcoal assay (DCC) and an immunohistochemical method. Frozen sections immunostained for PgR with a monoclonal antibody (Abbott PgR-ICA, Chicago, IL) and the peroxidase-antiperoxidase technique were scored semiquantitatively histologic score by microscopy and quantitatively (percentage nuclear area immunopositivity [PNA] using the CAS 200 image analyzer (Cell Analysis Systems, Elmhurst, IL). There was a positive correlation between dextran-coated charcoal assay and both histologic score (r = 0.82) and PNA (r = 0.69). Selected cutoff points of 60 histologic score and 6.5% PNA based on sensitivity/specificity calculations yielded a predictive value of a negative test of 73% and 80%, respectively, and a positive predictive value of 100% for both; ranges of fmol/mg protein PgR correspond to ranges of histologic score and PNA. The use of an image analyzer to measure PNA in PgR-immunostained sections is a viable alternative to dextran-coated charcoal assay, especially when insufficient fresh tissue is available.

Prolymphocytic leukemia of helper cell phenotype: report of a case and review of the scientific literature.

A case of prolymphocytic leukemia was studied by using light and electron microscopy, cytochemistry and multiple immunological markers for T and B lymphocytes. Cytochemical and immunological studies showed that the cells were T-lymphocytes. By using monoclonal antibodies to human T-cell subsets, we found that these cells had the T-helper cell phenotype. The usefulness of multiparameter studies in subclassifying T-cell malignancies is discussed and the literature on T-prolymphocytic leukemia reviewed.

Immunologic abnormalities in two patients with pulmonary hyalinizing granuloma.

Pulmonary hyalinizing granuloma (PHG) is a disease of slowly enlarging pulmonary nodules made up of dense bundles of collagen accompanied by an infiltrate of chronic inflammatory cells. The etiology is unknown. Although it has been suggested that the lesions represent an exaggerated immune response to unidentified agents, results of a detailed immunologic work-up of these patients have not been published. This report presents the laboratory findings of two patients with biopsy-proven PHG who have been followed four and eighteen years. Autoantibodies were detected (antinuclear antibody, rheumatoid factor, and positive antiglobulin tests), although clinically there was no evidence of a specific collagen-vascular disorder. Both patients had elevated levels of circulating immune complexes. These data suggest that immune complex mechanisms may be important in the pathogenesis of PHG.

Systemic and lung protein changes in sarcoidosis. Lymphocyte counts, gallium uptake values, and serum angiotensin-converting enzyme levels may reflect different aspects of disease activity.

BAL lymphocyte percentages, quantitated gallium-67 lung uptake, and SACE levels have all been proposed as measures of disease activity in sarcoidosis. We analyzed 32 paired sera and BAL fluids from sarcoidosis patients by high-resolution agarose electrophoresis to look for protein changes characteristic of systemic or local inflammation and compared the results with those from the above tests. Nine patients (group 1) had serum inflammatory protein changes and increased total protein, albumin, beta 1-globulin (transferrin), and gamma-globulin levels in fluid recovered by BAL. Thirteen patients (group 2) had normal protein levels in sera but abnormal protein levels in BAL specimens. Ten patients (group 3) had normal protein levels in sera and in BAL specimens. Patients in groups 1 and 2 had a disproportionate increase in beta 1-globulin (transferrin) and gamma-globulin levels in their BAL specimens. The BAL lymphocyte percentage changes paralleled the BAL protein level changes, suggesting relationships among the immunoregulatory role of these cells, increased local immunoglobulin synthesis, and the pathogenesis of altered alveolar permeability. Gallium-67 uptake was highest in patients with serum inflammatory protein changes. Thus, systemic inflammation may facilitate pulmonary gallium-67 uptake, possibly by changes in BAL fluid or serum transferrin saturation and/or kinetics. SACE levels showed no relationship to changes in the levels of serum or BAL proteins. These data suggest that the various proposed measures of disease activity reflect different aspects of inflammation in sarcoidosis.

Lymphocyte stimulation by yeast phase Histoplasma capsulatum in presumed ocular histoplasmosis syndrome.

We measured skin reactions serum antibody, and lymphocyte stimulation to Histoplasma antigens in a series of patients with presumed ocular histoplasmosis syndrome and in controls. The most sensitive test, lymphocyte stimulation to H. capsulatum sonicate, also correlated with severity of the disease. Lymphocyte stimulation to Histoplasma may be a useful adjunct to the monitoring of presumed ocular histoplasmosis syndrome.

Prediction of survival in head and neck cancer based on leukocyte sedimentation in Ficoll-Hypaque gradients.

A battery of immunologic tests were performed on 42 patients with recurrent cancer of the head and neck whose median survival time was 6.8 months. Two tests correlated with survival: total white blood cell (WBC) count and the percentage of lymphoid cells (%LG) in the Ficoll-Hypaque gradient separated leukocyte fraction of blood. Patients with abnormally low values of %LB had a poor prognosis, as did patients who had greater than 8000 WBC count. Of the variation in %LG, 43% was due to the differences in differential WBC counts among the patients, with the remaining variation attributed to changes in the buoyant densities of the leukocytes. These data suggest that further study of the factors effecting buoyant density of cells may be clinically useful.