Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay.

We have adapted an assay for the direct detection of Mycobacterium tuberculosis using a prototype automated instrument platform in which probes are amplified with Q-beta replicase. The assay was based on amplification of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. The assay signal was the time required for fluorescence to exceed background levels (response time [RT]). RT was inversely related to the number of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing > or = 10 CFU of M. tuberculosis responded in the assay (average RT, 13.91 min), while most (83%) samples containing as many as 10(7) CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The samples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. After resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 45 negative samples responded within 14 min. Assay sensitivity, specificity, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.

Real-time fluorescence detection of RNA amplified by Q beta replicase.

Amplification of RNA probes by Q beta replicase can be used to detect a wide range of analytes with a potential sensitivity of a single molecule. A system has been developed in which Q beta amplification of midivariant-(MDV)-based RNA is measured in real time by fluorescence. This was accomplished by including a fluorescent intercalating dye, propidium iodide, in the reactions and monitoring the fluorescence change using a custom fluorometer. The time at which fluorescence is detectable above background is referred to as the "response time" and is calculated using curve-fitting algorithms. A response time is inversely and linearly proportional to the logarithm of the number of template RNA molecules which initiated the reaction. Therefore, this system permits an unknown amount of input RNA probe to be quantified through 11 orders of magnitude when compared to a standard curve. Under the described conditions with MDV RNA, the response time occurs when about 3 x 10(11) RNA molecules are synthesized and occurs within the exponential phase of the reaction, before the number of active enzyme molecules are saturated with RNA templates. This system has been used to determine the replication properties of MDV RNA reporter molecules bearing specific probe sequences and to develop hybridization assays for the clinical diagnostic field.

Polymerase chain reaction and Q beta replicase amplification.

The polymerase chain reaction (PCR) and Q beta replicase are two methods in which nucleic acid polymerases are used for amplification. Although these approaches share many similar problems concerning target contamination and probe specificity, they differ dramatically in their mechanisms of action and modes of application. The PCR method amplifies target sequences between two priming oligonucleotides and in essence amplifies a portion of the analyte. Q beta replicase, on the other hand, amplifies a specific template molecule hybridized to target sequences and therefore amplifies a signal component of the system. For this reason, Q beta replicase amplification has applications in areas other than for the detection of nucleic acid sequences. The requirements for application and the advantages of both PCR and Q beta replicase amplification are reviewed.

Phase change enzyme immunoassay.

A novel enzyme-linked immunoassay employing a partitioning chromophore was developed. The assay system consisted of an aqueous phase and an immiscible organic solvent. Antigen-antibody interaction was indicated by transfer of a chromogenic indicator from the aqueous phase to an organic layer. The indicator employed was a water-soluble phosphate ester of phenylazophenol. Hydrolysis of the ester by acid or alkaline phosphatase produced a water-insoluble phenol that partitioned into toluene. The enzyme employed in this assay format can be covalently linked to antibody or a specific antibody for the phosphatase can be used. Phase change immunoassays were developed for the measurement of alkaline phosphatase, human IgG in whole blood, and the human tumor marker prostatic acid phosphatase. Solid supports of small polystyrene latex particles and Sephadex were employed.

Radioimmunodetection of prostatic cancer. In vivo use of radioactive antibodies against prostatic acid phosphatase for diagnosis and detection of prostatic cancer by nuclear imaging.

Radioimmunodetection (RAID) of prostatic cancer is done by injecting 131I-labeled rabbit antibody IgG against prostatic acid phosphatase (PAP) and performing total-body photoscans with a gamma scintillation camera. Of two patients tested, the PAP RAID scintiscans located the primary or recurrent prostatic cancers in both and showed no disease in the lungs of the patient shown subsequently to have lung cancer. The lung tumor nodules showing anti-PAP IgG accretion were assumed to be of prostatic cancer origin, since one of the original tumors removed from this patient's other lung a year earlier stained for PAP by immunohistochemistry. This study showed that PAP RAID can locate primary and metastatic tumors of prostatic origin.

Bone marrow acid phosphatase by radioimmunoassay: 3 years of experience.

Clinical followup of 112 patients staged by the immunochemical determination of prostatic acid phosphatase from bone marrow aspirates is presented. This represents a 94 per cent (112 of 118) retrieval rate of a group studied more than 2 years previously. Of the 11 patients judged to be at high risk 4 (36 per cent) have suffered bony metastases, whereas only 3 of 86 patients (3 per cent) with normal bone marrow acid phosphatase by radioimmunoassay have done so. An additional 184 patients with carcinoma and 77 controls have been studied. Although radioimmunoassay greatly improves specificity in bone marrow aspirates a few falsely positive results can occur. This finding may be secondary to cross reaction from leukocyte acid phosphatase and/or interference from lipid.

The significance of prostatic acid phosphatase in adenocarcinoma of the prostate.

Our radioimmunoassay for prostatic acid phosphatase was compared to commercial radioimmunoassay kits. A close correlation among all 3 assays was found in control groups, and in patients with benign prostatic hyperplasia and adenocarcinoma of the prostate. These results also were compared to recent reports from other centers using similar methodologies. In 7 to 15 per cent of the patients with bone metastasis normal levels of serum prostatic acid phosphatase were found. Variability in prostatic acid phosphatase production by the tumor may account for this finding. Elevated levels of prostatic acid phosphatase were associated more commonly with less differentiated primary tumors. A low percentage of prostatic acid phosphatase elevations in patients with early localized and incidental adenocarcinoma was found for the 3 assays evaluated. These factors, along with the falsely positive rates in patients with benign disease, limit severely the application of these assays to the screening of male patients at risk for adenocarcinoma of the prostate.

Immunohistochemical evaluation of prostatic carcinoma before and after radiotherapy.

Immunohistochemical procedures were applied to the examination of human tissues for prostatic acid phosphatase. With antisera against purified human prostatic acid phosphatase 173 normal and neoplastic tissues were tested. Samples of 45 non-prostatic carcinomas and their respective normal tissues were negative. Of 4 seminal vesicles studied 2 showed weak reactivity. The epithelial cells of normal prostatic acini were uniformly positive in 25 patients studied. In contrast to normal prostatic tissue the malignant acini in 53 of 55 patients with prostatic carcinoma had variable but positive reactivity. Of 27 patients receiving radiotherapy for adenocarcinoma of the prostate variable staining was observed in the neoplastic cells of 24, 8 to 52 months after treatment. The continued production of prostatic acid phosphatase in the malignant cells after radiotherapy suggests that they also may maintain metabolic activities necessary for growth and metastasis.

The role of the radioimmunoassay for prostatic acid phosphatase in prostatic carcinoma.

Considerable controversy exists as to the exact role of prostatic acid phosphatase in the investigation of patients with adenocarcinoma of the prostate. In order to compare different clinical trials, consistent standards of patient selection and staging criteria must be applied on a prospective basis. Other variables which can lead to discrepancies in the results include sources and purification of antigen as well as the analytic accuracy of the immunologic method used. The statistical approach has illustrated the futile outcome of screening an unselected population. On the other hand, if appropriate criteria must be stressed that these estimates have been based on the best possible data available; we have not been able to reproduce either the high sensitivity or specificity reported using three different radioimmunoassays. Therefore, despite the advantages of immunologic methods, we cannot recommend the application of the radioimmunoassay for prostatic acid phosphatase for screening of males for adenocarcinoma of the prostate. At present, no clear explanation can be offered for the elevation of serum prostatic acid phosphatase in patients with apparently localized adenocarcinoma, but it may be of prognostic significance. We have found marked variability in the production of prostatic acid phosphatase by neoplastic acini, and normal levels of prostatic acid phosphatase in patients with metastatic carcinoma may be associated with tumors of limited secretory capacity. Clearly, both the physiologic and methodologic limitations of current radioimmunoassays should be recognized.

Prostatic trauma and release of acid phosphatase. Radioimmunochemical and enzymatic comparison.

Radioimmunoassay for prostatic acid phosphatase and a conventional enzymatic method using alpha-naphthyl phosphate were employed to document the changes in serum levels of this enzyme following transurethral prostatectomy and prostatic massage. Thirty-four patients with histologically proved benign prostatic hyperplasia and 120 controls were studied. Consistent parallel elevations were noted after surgical trauma. A rapid clearance was observed with normal levels returning at twenty-four hours. Prostatic massage did not elicit a change by either method.

Radioimmunoassay of bone marrow prostatic acid phosphatase.

The clinical value of prostate acid phosphatase (PAP) measurements in the bone marrow aspirate of patients with prostatic adenocarcinoma has been unclear. Using a radioimmunoassay (RIA) to measure PAP, we have evaluated this potential indicator of occult metastases in 127 controls and in 300 patients with prostatic adenocarcinoma. Elevations of the tumor marker were found in 9%, 10%, 19%, and 82% of patients with stages B, C, D1, and D2 adenocarcinoma respectively. Clinical follow-up ranging from 7 to 43 months (average 23 months) was available for 97 patients without any initial indication of metastasis by bone scan. In this group 11 patients had elevated levels of bone marrow acid phosphatase (BMAP) by RIA and four developed radiological evidence of bone metastasis 21-25 months following initial staging. However, only three of the 86 patients with normal BMAP levels have developed bone metastasis. Our results indicate that measurement of bone marrow PAP by immunological methods has prognostic significance. Dilution of the bone marrow aspirate by peripheral blood, however, may limit the application of this technique.

Radiation therapy for adenocarcinoma of the prostate.

Forty-eight patients with different stages of adenocarcinoma of the prostate were treated by external beam therapy. Biopsy results are correlated with stage of disease, original grade of tumour and with the findings on examination. The authors discuss the significance of histologic progression of the disease particularly in relation to prognosis. They review the place of staging lymphadenectomy before radiotherapy and the overall morbidity associated with treatment.

An objective look at acid phosphatase determinations: a comparison of biochemical and immunological methods.

Measurements of serum and bone marrow acid phosphatase were made by 3 enzymatic methods, alpha-naphthyl phosphate, beta-glycerol phosphate, and thymolphthalein monophosphate, and ocmpared to a double antibody radioimmunoassay. Serum and bone marrow acid phosphatase levels were studied in 46 controls with histologically proven benign prostatic hyperplasia and in 135 patients with various stages of prostatic carcinoma. In the control group the upper limit for bone marrow acid phosphatase was found to be significantly higher than the corresponding serum limit with respect to the enzymatic assays studied. The radioimmunoassay was the only method suitable for the analysis of the prostatic acid phosphatase content of bone marrow. A larger number of elevations were noted in patients with extracapsular and metastatic disease when prostatic acid phosphatase measurement was carried out by radioimmunoassay as compared to enzymatic methods. However, only 8% of the patients with intracapsular disease had elevations of prostatic acid phosphatase as measured by radioimmunoassay. Additional standardisation of immunological methods and clinical trials is required before comparison can be made of results from various centres using immunological methods for the measurement of prostatic acid phosphatase and a true assessment made of the usefulness of this procedure.

Prostatic acid phosphatase by radioimmunoassay tumor marker in bone marrow.

Bone marrow acid phosphatase was determined by radioimmunoassay and enzymatic analysis in 95 patients with benign prostatic hypertrophy, 50 patients with disseminated prostatic carcinoma and 36 patients with non-prostatic malignancy. The results indicate superior specificity of the radioimmunoassay. A brief review of the topic and the clinical implications are discussed.

A radioimmune assay for human prostatic acid phosphatase-levels in prostatic disease.

Prostatic acid phosphatase from human seminal fluid was purified to homogeneity. The enzyme was characterized as to its purity, molecular weight and amino acid composition. Analytical isoelectric focusing of purified enzyme on polyacrylamide gels resolved the enzyme activity into eleven discrete bands, apparently due to various amounts of sialic acid associated with the glycoprotein. Antisera raised against the purified enzyme produced only one precipitan arc on immunoelectrophoresis. A double antibody radioimmune assay was developed and used to evaluate serum prostatic acid phosphatase in 226 patients without prostatic disease, in 186 patients with benign prostatic hyperplasia and in 93 patients with prostatic carcinoma. No statistical difference was noted in serum prostatic acid phosphatase between patients with benign prostatic hyperplasia and in those without prostatic disease Serum prostatic acid phosphatase was elevated in 94% of the patients with metastatic prostatic carcinoma. Significant elevations were also found in carcinoma patients without metastases.

Adenocarcinoma of the prostate in perspective.

Adenocarcinoma of the prostate is responsible for one of every nine deaths from cancer in Canada. In this review epidemiologic factors are considered and current staging systems are outlined. The American Urological System is recommended for staging because of its ability to reflect changes in the understanding of the biologic behaviour of this neoplasm. The adoption of a quantitative grading scheme is suggested to complement the information obtained from the staging assessment. The routes of spread of this disease, along with the procedures used to assess metastatic involvement, are described. Immunologic methods for the analysis of prostatic acid phosphatase have been shown to be superior to the enzymatic methods previously used, and the role of the new techniques is discussed. Emphasis is placed on radiotherapy and endocrine therapy for the treatment of this neoplasm, and the concept of withholding endocrine therapy until symptoms appear is discussed. Potential future developments in this field are considered.

Bone marrow acid phosphatase by radioimmunoassay.

A double-antibody radioimmunoassay was developed and utilized to measure prostatic acid phosphatase in bone marrow aspirates. One hundred-eighteen patients with carcinoma of the prostate in various clinical stages, and fifty with benign prostatic hyperplasia were studied. In patients with carcinoma, levels of prostatic acid phosphatase in bone marrow aspirates were found to correlate well with increasing clinical stage of the disease. Determination of bone marrow prostatic acid phosphatase by radioimmunoassay may be a valuable adjunct to clinicopathologic staging of prostatic carcinoma.

A method using toluene-2,4-diisocyanate and glutaraldehyde to stabilize and conjugate antigens to erythrocytes for use in passive hemagglutination tests.

Erythrocytes were stabilized and activated for conjugation by simultaneous treatment with toluene-2,4-diisocyanate and glutaraldehyde. Following conjugation with an antigen, these cells were suitable for use in passive hemagglutination tests and could be stored in either hypo- or hyper-tonic solutions at 4 degrees C for at least 3 months without hemolysis or loss of agglutinating properties. Furthermore, these cells can be lyophilized and reconstituted with minimal loss of conjugation ability or of agglutination titer.