Significance of DNA ploidy and cell proliferation in juvenile respiratory papillomatosis.

The clinical course of recurrent respiratory papillomatosis (RRP) in children is variable and unpredictable. At present there is no way to identify patients at risk for aggressive disease. The objective of this study was to evaluate whether DNA ploidy and cell proliferation analyses can predict the clinical course in children with RRP. Two different methods of estimating proliferation activity were compared. Nonembedded papilloma biopsy specimens from 18 pediatric patients were analyzed by flow cytometry providing DNA content with cell cycle analysis. The expression of the proliferative marker Ki-67 in papilloma tissue was quantified by immunohistochemistry. The patients were prospectively observed for 12 to 18 months. DNA content analysis and Ki-67 expression were compared to clinical information regarding number of disease sites, distal tracheobronchial spread, number of recurrences, need for tracheostomy, and disease remission. High S-phase fraction, proliferative index, and Ki-67 expression correlated with an aggressive clinical course. DNA ploidy analysis and immunodetection of proliferative markers may assist in predicting prognosis in children with RRP.

CD8-positive B-cell chronic lymphocytic leukemia. A report of two cases.

Most B-cell chronic lymphocytic leukemias and a small normal subset of B lymphocytes express the T-cell-associated CD5 antigen; expression of other T-cell antigens has been reported only rarely. The authors report two cases of typical B-cell chronic lymphocytic leukemia, seen during 1 year, in which two-color flow cytometric analysis documented expression of the T-cell-associated CD8 antigen by the monoclonal B cells. Genotypic studies showing immunoglobulin but not T-cell-receptor gene rearrangements confirmed the B-cell origin of the neoplastic cells. The true frequency of the CD8-positive B-cell chronic lymphocytic leukemia, any clinical implications, and the possibility of a normal subset of CD5-positive CD8-positive B cells remain to be determined.

Pharmacokinetics of zidovudine phosphorylation in patients infected with the human immunodeficiency virus.

The relationship between zidovudine phosphorylation inside mononuclear cells and plasma zidovudine pharmacokinetic was assessed in six subjects. Plasma and intracellular concentrations were measured by radioimmunoassay over an 8-h period after administration of 100 or 200 mg of zidovudine. Plasma pharmacokinetics followed expected patterns, with considerable interpatient variability in area under the concentration-versus-time curve (AUC), and a terminal half-life of 1.5 h. Intracellular AUC was even more variable than plasma AUC, but the data suggested a crude linear relationship between these parameters. The intracellular half-life of 3.5 h was consistently longer than the plasma half-life, and varied little between patients. The prolonged intracellular half-life suggested that total phosphorylated zidovudine, as measured by the method described, is not greatly dominated by the 5'-monophosphate as predicted from the in vitro studies reported in the literature. Plasma concentrations of zidovudine have shown little correlation with clinical effect. Study of the relationship between phosphorylated zidovudine and clinical outcome could lead to a more effective management of therapy.

Clinical applications of cytometry: 6th annual meeting.

The Sixth Annual Clinical Applications of Cytometry Meeting was held September 11-14, 1991, in Charleston, SC. Attendance reached a record 470. The meeting provides a forum for interactions among investigators who utilize cytometry as a tool in their clinical immunology, cell biology, hematology, and cancer investigations. Clinical laboratory directors and their technical staff find the meeting of practical value because of the presentation of new applications that they can take home to their own laboratories. The emphasis of the meeting is on advances in the application of cytometry to clinical problems. Often, advances result from new dyes or reagents or improved instrumentation. Sometimes they result from advances in biology that make the studies possible. Occasionally a new way of looking at the same data provides a useful answer. In every case, the effort is to provide a reliable, straightforward way to quantitate biologic information in order to provide improved diagnosis or treatment of human disease.

Immunoglobulin gene rearrangement in abnormal lymph node hyperplasia.

The histologic designation "abnormal lymphoid hyperplasia" is applied to lymph nodes demonstrating varying degrees of architectural effacement and/or cytologic atypia. Although some of these cases may be suggestive of non-Hodgkin's lymphoma, a definitive diagnosis is not possible despite careful morphologic and immunophenotypic studies. Because the demonstration of immunoglobulin and T-cell receptor gene rearrangements by Southern blot analysis provides a sensitive marker of lineage and clonality in lymphoid malignant conditions, the frequency with which such gene rearrangements could be identified in abnormal hyperplasia and their significance were studied. DNA samples from lymph node biopsy samples of 11 patients with abnormal lymphoid hyperplasia were analyzed for rearrangements of immunoglobulin and T-cell receptor genes by Southern blot hybridization. Six of these patients had monoclonal B-cell populations identified by immunoglobulin gene rearrangements; all were found subsequently to have non-Hodgkin's lymphoma by repeated biopsy from 8 days to 46 months later. Two patients with negative Southern blot studies also developed lymphoma, one a T-cell non-Hodgkin's lymphoma and one a cutaneous B-cell non-Hodgkin's lymphoma. Three patients without detectable gene rearrangements showed no evidence of malignant lymphoma at 36-, 45-, and 60-month follow-up evaluations. Southern blot analysis thus identified monoclonal B-cell lymphoid populations in a subset of patients with abnormal lymphoid hyperplasia; the presence of clonal immunoglobulin gene rearrangement predicted progression to overt non-Hodgkin's lymphoma.

Concentrations of phosphorylated zidovudine (ZDV) in patient leukocytes do not correlate with ZDV dose or plasma concentrations.

Zidovudine (ZDV) elicits its antiviral effect through intracellular metabolism to the 5'-triphosphate, which interferes with viral replication. Monitoring of the active metabolites of ZDV in cells could lead to an intracellular therapeutic range. This study was performed to determine whether a radioimmunoassay, previously used for in vitro quantitation of total phosphorylated ZDV inside peripheral blood leukocytes, could be used for similar determinations in patient samples. The relationship between ZDV dose, plasma concentrations, and intracellular metabolite concentrations was also examined. Ten-milliliter blood samples were drawn from each of 13 human immunodeficiency virus-infected patients and were assayed. Intracellular concentrations of phosphorylated ZDV ranged from 0.33 to 3.54 pmol/10(6) cells, similar to those observed in vitro. Phosphorylated ZDV was independent of dose, and did not correlate with plasma concentrations. Intracellular concentration in the patient population as a whole did not change during the 4-h dosing interval, while plasma concentration decayed normally. Later determinations in the same patients gave intracellular values within 31% of earlier values. Intraassay variability was less than 10%. Thus, the method is valid for measurement of phosphorylated ZDV in patient cells. Although individual concentrations showed no clear change during the 3-month study period, intracellular concentrations decreased with increasing length of therapy (up to 3 years) in the population as a whole. This suggests a decreased cellular ability to phosphorylate ZDV after prolonged exposure to drug. The lack of intracellular decay implies a half-life longer than the 1-h half-life of plasma ZDV. These data suggest that smaller doses or longer dosing intervals might maintain intracellular concentrations once steady state is achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

Multilobated lymphoma of B cell type: a multiparameter investigation.

Multilobated lymphomas were originally described as T-cell neoplasms, but many of B-cell type have subsequently been reported. A case of B-cell origin is reported in which both immunophenotypic and genotypic studies performed on a cell suspension of the lymphoma gave inconclusive and potentially misleading information, while paraffin and frozen section immunohistologic studies, as well as genotypic studies performed on DNA obtained from snap-frozen tissue, were definitive. Thus, this case illustrates some of the problems that may be encountered using cell suspensions as a source for immunophenotypic, and even the much more sensitive genotypic, studies.

In vitro measurement of phosphorylated zidovudine in peripheral blood leucocytes.

A method has been developed to measure the concentration of total phosphorylated zidovudine (ZDV) inside peripheral blood leucocytes (PBLs) using a modified commercial radioimmunoassay (RIA) specific for ZDV. ZDV 5'-monophosphate was readily synthesized and used as a procedural control for RIA modification. PBLs were isolated from healthy volunteers and incubated with ZDV for 24 h to allow metabolic phosphorylation. Viable cells were counted, washed, and extracted overnight with 60% methanol. After evaporation, the extract was reconstituted in Tris buffer, pH 9.5. Because of minimal RIA antibody cross reactivity with phosphorylated ZDV, samples were split into two fractions, one of which was treated with alkaline phosphatase (AP) to liberate phosphate groups. Each fraction was then assayed for ZDV. Concentrations of phosphorylated ZDV were determined by subtracting the concentration of the non-AP-treated fraction from that of the treated fraction. Recovery of phosphorylated ZDV from cell extracts was approximately 90%, and reproducibility was acceptable (coefficients of variation less than 15% for concentrations greater than or equal to 0.25 ng/ml). Intracellular concentrations (0.1-1.4 pmoles/10(6) cells) followed a nonlinear dose-response relationship over the range 0-50 microM extracellular ZDV, with concentration-dependent saturation. These results demonstrate the feasibility of determining concentrations of phosphorylated ZDV in HIV-infected patients, a potentially key step in establishing a therapeutic range and optimal dosing regimen for these patients.

Prenatal screening for anticardiolipin antibody.

Anticardiolipin antibodies, immunoglobulin G, and M (IgG, IgM) have been associated with recurrent abortion and with maternal death. This study tested whether anticardiolipin titers would be a useful prenatal screening test to determine high-risk pregnancies. Titers were obtained at the first clinic visit in 686 patients, mean gestation, 20 weeks. The outcome variables were taken from a medical records computer data base. IgG anticardiolipin correlated inversely with birthweight (p less than 0.025), but not with gestation. IgM anticardiolipin correlated strongly with the inverse of patient age (p less than 0.0002) and with chronic hypertension (p less than 0.01), but not with preeclampsia. There was a weak correlation with the 1-minute Apgar score (p less than 0.05). Thirty-seven patients had titers of IgG or IgM greater than 3 standard deviations above the mean for nonpregnant patients. Sixteen of these patients were studied for antinuclear antibody and coagulopathy (prothrombin time, partial thromboplastin time, viper venom time) and all were normal. Six of eight patients tested had low range elevated antibody titers to double-stranded DNA. Ten placentas were examined and showed no infarctions. None of the correlations were of practical clinical utility. The biologic basis of the correlations found is of further interest. The value of anticardiolipin titers with lupus erythematosus, or with coagulopathy, was not tested.

Flow-cytometric DNA analysis of hematopoietic and lymphoid proliferations: a comparison of fresh, formalin-fixed and B5-fixed tissues.

Flow-cytometric DNA analysis of human tumors using paraffin-embedded tissue samples is becoming an increasingly popular method of determining ploidy and proliferative rate. Particularly with hematopoietic/lymphoid proliferations, little is known about how these data compare with data from fresh suspension studies, or how B5-fixed tissues compare with those fixed in formalin. For these reasons, flow-cytometric DNA ploidy and cell cycle analysis was performed on 16 hyperplastic tonsils and 28 lymphoid/hematopoietic neoplasms using fresh (or fresh-frozen) cell suspensions (FR), B5-fixed paraffin-embedded (B5), and formalin-fixed paraffin-embedded (FO) tissue. Ploidy analysis showed all tonsil specimens to be diploid regardless of preparative method; however, for the neoplastic cases the FO and B5 preparations agreed with the FR preparations in 50% and 61% of the cases, respectively. Complete agreement between all three tissue preparation methods was present in only 36% of the neoplastic cases. Percent S or S + G2M phase fractions showed relatively poor correlation between the three preparative methods, with the best correlations found between the FR and B5 samples (S phase, r = 0.44; S + G2M, r = 0.43). In conclusion, while potentially useful data can be obtained from flow-cytometric DNA analysis of fixed, paraffin-embedded lymphoid/hematopoietic tissues, the specific limitations of such analyses must be recognized. Ploidy and proliferative phase data from fixed tissue preparations are not equivalent to information obtained from fresh suspension studies. B5-fixed tissue provides an acceptable alternative to formalin-fixed tissue for such analyses.

Failure of orthoclone OKT3 retreatment in a pancreas transplant recipient with antimurine antibodies.

Orthoclone OKT3 is a murine monoclonal antibody that blocks the generation and function of T lymphocytes. It has been shown to be effective in reversing acute cellular rejection in solid organ transplants. However, potential development of antimurine antibodies restricts the duration that the drug can be used and the ability to reuse the drug. The case reported in this article illustrates the failure of retreatment with OKT3 when high titer (1:3200) antimurine antibodies are present. Lack of efficacy of the drug was documented by virtually undetectable circulating OKT3 levels in plasma, no decrease in T3 lymphocytes, and organ rejection. OKT3 should only be reused when immune monitoring (antimurine antibody status, lymphocyte subsets, and OKT3 plasma levels) is performed before, during, and after its use. Patients with high-titer anti-OKT3 antibody should not be retreated with OKT3.

Antimurine antibody formation following OKT3 therapy.

OKT3 is an IgG2a murine monoclonal antibody directed against the CD3 antigen receptor of human T lymphocytes. A major concern with OKT3 treatment in solid organ transplant recipients is the development of antimouse antibody, which may preclude retreatment with this agent. We have administered OKT3 on 215 occasions (150 renal, 34 hepatic, 26 cardiac, 5 pancreatic) in 179 patients between April 1982 and December 1988. The mean duration of treatment was 10.5 days (range, 2-22 days). Antimouse antibody data were analyzed on the most recent 133 treatment courses where the antibody status was available pretreatment. Determination of antimouse antibody production was elicited by ELISA technology at days 0, 7, 14, and 28 of OKT3 treatment. Patients were categorized according to the antibody response as follows: (a) absence of antibody; (b) low titer (1:100); or (c) high titer (greater than or equal to 1:1000). Our earlier experience has demonstrated that retreatment with OKT3 is successful in groups a and b. The development of antimurine antibodies was analyzed with regard to the following parameters: (1) The duration of OKT3 treatment; (2) treatment type (prophylactic, primary, or secondary); (3) primary treatment or retreatment; (4) concomitant immunosuppressive regimen (double or triple therapy); (5) dosage of concomitant immunosuppressive drugs; and (6) transplant organ type. The following results were obtained. (1) Duration of treatment had no effect on antibody production (11.0 days in antibody negative and 10.0 days in antibody positive). (2) There was no difference in antibody formation rates for the first treatment of OKT3 when it was used as prophylaxis (26%), primary (19%), or secondary (27%) therapy. (3) Antibody formation rate with first treatment was 29%; with retreatment, patients who were antibody negative following first treatment became positive in 28% of cases, and retreated patients who were low titer positive following first treatment converted to high titer in 57% of cases. (4) Antibody formation was higher in patients receiving double immunosuppressive therapy (36%) than in those receiving triple immunosuppressive therapy (21%) during OKT3 treatment. (5) Concomitant immunosuppression was lower in the antibody-positive group during OKT3 therapy: steroids, 61 mg/day vs. 52 mg/day; azathioprine, 89 mg/day vs. 66 mg/day; CsA, 317 mg/day vs. 186 mg/day. (6) Antibody formation rates were lower in non-renal transplants following first treatment with OKT3 (liver 17%, heart 17%, kidney 28%); this reflects the higher doses of concomitant immunosuppressive therapy used in nonrenal transplants.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of histologic nodal reactive patterns, cell suspension immunophenotypic data, and HIV status.

While cell suspension immunophenotypic studies are widely used as an aid in the diagnosis and classification of lymphomas and leukemias, much less attention has been directed toward interpretation of the results in reactive lymphoid proliferations. Cell suspension immunophenotypic data were therefore analyzed for 119 lymph nodes with reactive lymphoid proliferations which were divided into five major histologic categories: follicular hyperplasia, marked (FH,M), or moderate (FH); dermatopathic lymphadenopathy (DL); diffuse hyperplasia (DH); or "other." With the aid of a computer-assisted morphometer, the following were also measured and calculated: proportion of node occupied by follicles, mean relative follicle size, and mean follicle shape factor. Finally, in 57 cases, the influence of human immunodeficiency (HIV) status on the findings was analyzed. Although individual cases varied widely, cases of DL had significantly more CD3+ (T) cells, higher CD4:CD8 ratios, and fewer CD19+ (B) cells than other categories. Cases of FH,M had significantly lower CD4:CD8 ratios and more CD19+, CD10+, and transferrin receptor positive cells. Cases of FH,M and FH known to be HIV-negative had higher CD4:CD8 ratios than the HIV-positive cases. Peripheral blood CD4:CD8 ratios performed in 38 patients showed a strong correlation with nodal ratios. Morphometric data supported the correlation between follicular hyperplasia and increased proportions of CD19+, CD10+, and transferrin receptor-positive cells. Rare cases had CD5:CD2 or CD3 ratios of greater than 1 or "monoclonal" kappa to lambda ratios. CD4:CD8 ratios varied widely, but aberrant T cell phenotypes were not identified. These studies demonstrate that, although great variation exists, there are certain associations between types of reactive lymphoid hyperplasia and cell suspension immunophenotypic findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic monitoring with Orthoclone OKT3 therapy.

Muromonab-CD3 monoclonal antibody (Orthoclone OKT3) was used 146 times in 123 transplant recipients to treat or prevent rejection. Reversal and prevention of rejection were evaluated 1 week and 1 year after OKT3 therapy. Eighty-one percent (73 of 90) of the rejection episodes in kidney transplant patients were reversed with 67% of these grafts functioning at 1 year. Eighteen of 20 (90%) rejection episodes in liver transplant recipients were reversed, as were 11 of 13 (85%) heart transplant rejection episodes. Only one of five pancreas transplant episodes were reversed. OKT3 was used prophylactically in 18 transplant recipients (13 kidney, four heart, one liver). Immunologic monitoring (lymphocyte subsets, serum OKT3 levels, and antimurine antibodies) was performed during and after OKT3 therapy. Antimurine antibody formation rate was 28% (26 of 94 patients monitored). OKT3 therapy resulted in a rapid depletion of CD3+ cells from the peripheral circulation (less than 20/mm3) and trough serum OKT3 levels of greater than 800 ng/ml by the third day of therapy in all transplant types. Twenty-three patients (14 kidney, five liver, three heart, and one pancreas) were retreated with OKT3; reversal of rejection occurred in 87% of patients (13 of 15) with no antimurine antibodies and in 83% of patients (five of six) with a low antibody titer but did not occur in the two patients with a high antibody titer. Retreatment of patients with no anti-OKT3 antibody resulted in a depletion of CD3+ cells from the peripheral blood, but it took longer than in patients treated with OKT3 for the first time. Similarly, serum OKT3 levels increased slower in retreated patients compared with first treatment. In retreatment patients with a low titer antimurine antibody, often it was necessary to increase the dose of OKT3 to achieve adequate serum OKT3 levels and to deplete CD3+ cells. Antimurine antibody developed de novo in four of the 15 antibody negative patients (27%) who were retreated. Overall, OKT3 was an effective agent in reversing and preventing rejection in solid organ transplantation with few severe side effects and a low mortality. Retreatment with OKT3 should not be considered unless the antibody status of the patient is known. Development of low titer antibodies does not preclude successful retreatment with OKT3. Alternate antirejection therapy, however, should be used in patients with high titer antimurine responses.

A monoclonal immunoglobulin A (kappa) factor VIII:C inhibitor associated with primary amyloidosis: identification and characterization.

Factor VIII:C inhibitors associated with primary amyloidosis have not been previously reported. A patient with autopsy-proved primary amyloidosis developed renal failure requiring peritoneal dialysis. Purpura and a prolonged partial thromboplastin time (PTT) were first observed 3 years later, after treatment was changed from peritoneal dialysis to hemodialysis. Plasma contained a time-dependent factor VIII:C inhibitor. The inhibitor on isoelectric focusing showed two peaks of activity, one with an isoelectric point (pl) of approximately 4 and the second larger, with a pl of approximately 8. Both were neutralized only by antisera to IgA and kappa light chain. A monoclonal antibody prepared in Balb/c mice against the variable region of the kappa light chain also blocked the inhibitor. The delayed onset of the coagulopathy could be explained by the change from peritoneal to hemodialysis, because in the former, significant amounts of the paraprotein, indicated by an "M-spike," were recovered in the dialysate. N-terminal amino acid sequencing of the first 20 amino acids of the variable region of the kappa light chain from the urinary protein and the splenic amyloid subunit showed identity.

Successful retreatment of allograft rejection with OKT3.

OKT3 is a murine monoclonal antibody to the CD3 antigen of human T lymphocytes. The production of human antimurine antibodies after treatment with OKT3 has been perceived as a major limitation to its extended use and reuse. Treatment of 142 patients with 168 courses of OKT3 resulted in the development of antimouse antibody in 28% of the patients. Twenty-six patients (16 kidney, 6 liver, 3 heart, 1 pancreas) have been retreated with 27 courses of OKT3. Eighteen patients had no antimurine antibodies present, and the rejection reversal rate was 83% (15/18). Six patients had a low-titer antimurine antibody present, and rejection reversal occurred in 5 (83%). Rejection was not reversed in 2 patients with a high-titer antibody. Development of antimurine antibody was more frequent in renal transplant recipients (33%) than in hepatic (12%) or cardiac transplant recipients (18%). We believe that this reflects the fact that concomitant immunosuppressive therapy is more likely to be reduced during OKT3 therapy in renal transplant recipients than in hepatic or cardiac transplant recipients. Retreatment of patients with no anti-OKT3 antibody resulted in depletion of CD3+ cells from the peripheral blood, but it took longer than in patients being treated with OKT3 for the first time. Similarly, serum OKT3 levels rose more slowly in retreated patients compared to first treatment. In retreating patients with a low-titer antimurine antibody, it often was necessary to increase the dose of OKT3 in order to achieve adequate serum OKT3 levels and to deplete CD3+ cells. De novo antimurine antibody developed in 4 of the 18 (22%) antibody-negative patients who were retreated. In conclusion, retreatment with OKT3 should not be considered unless the antibody status of the patient is known. Development of low-titer antibodies does not preclude successful retreatment with OKT3; however, alternate antirejection therapy should be used in patients with high-titer antimurine responses.

Immunohistochemical classification of acute leukemias using peripheral blood smears.

Immunophenotypic classification of the acute leukemias (AcL) is of well documented value in those of lymphoid or uncertain origin and of increasing importance in those of nonlymphoid origin. Most of these studies have been performed on viable cell suspensions. To study the efficacy of a simpler immunohistochemical approach to the classification of the acute leukemias requiring only peripheral blood smears, 15 AcL (including three CGL-BC) were studied using an immunoalkaline phosphatase method and a panel of anti-lymphoid and anti-myeloid monoclonal antibodies. Routine cytochemistries were also performed (Sudan black, PAS). Using immunohistochemistry, five cases marked as common ALL (four were undifferentiated by cytochemistry, one ALL), eight cases as ANLL (all ANLL by cytochemistry) and two cases marked only with anti-HLA-DR (AUL by cytochemistry). These results show that immunophenotypic analysis of AUL, ALL and ANLL can be successfully performed even when only air dried peripheral blood smears are available.

T-cell morphology in nodular follicular center cell lymphomas.

T cells are known to be frequently numerous in follicular lymphomas but are generally considered to be a homogeneous population of small, round, "resting" lymphocytes. Although immunologic studies have suggested the presence of some "activated" T cells in these B-cell neoplasms, virtually no attention has been paid to the T-cell morphology. Wright-stained cytocentrifuged sheep erythrocyte (SRBC) rosette preparations from 20 cases of nodular follicular center cell lymphomas were therefore examined and the morphologic appearance of the SRBC rosetting cells was categorized. A mean of 19% (range 11-33%) of the SRBC-positive cells present were medium or large in size (diameter greater than two SRBCs, approximately 10 micron). A mean of 62% of SRBC-positive cells had round nuclei; 18% had indented nuclei and 20% had irregularly shaped nuclei. These data provide supportive morphologic evidence for some T-cell "activation" in certain follicular lymphomas. In addition to having possible functional and biologic implications, the previously undescribed presence of enlarged and sometimes "atypical" T cells in uncomplicated follicular lymphomas has implications for interpreting the histopathology of these B-cell neoplasms and the reported occurrence of composite T-cell/follicular center-cell lymphomas.