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Evaluation of Leu-M5 (CD11c) in hairy cell leukemia by the alkaline phosphatase anti-alkaline phosphatase technique.

The concomitant presence of B antigens and of the antigen recognized by the monoclonal antibody Leu-M5 (CD11c) on neoplastic lymphoid cells has been reported to be largely restricted to hairy cell leukemia (HCL). The authors studied Leu-M5 reactivity of neoplastic cells from 59 patients whose specimens were referred with a stated diagnosis of HCL by using the alkaline phosphatase anti-alkaline phosphatase technique on peripheral blood (PB) and bone marrow (BM) specimens. Tartrate-resistant acid phosphatase (AcP-T) activity was also studied. In 49 patients, HCL had been confirmed previously by BM biopsy, and specimens were evaluated for disease status during or after therapy with interferon (IFN) or 2'-deoxycoformycin. The remaining ten patients were newly referred for confirmation of the diagnosis of HCL before therapy. In all 55 patients in whom the BM biopsy demonstrated HCL, virtually every leukemic cell was Leu-M5 reactive, and the reaction proved, in some cases, to be helpful in the detection of small numbers of hairy cells in PB or BM preparations. AcP-T reactivity was demonstrated in the neoplastic cells of 52 of these 55 patients, including all but 3 of those receiving IFN, and was helpful in confirming persistent leukemia when interpretation of BM biopsy sections was difficult because the numbers of hairy cells were small. However, in four of the ten newly referred patients, BM biopsy showed features of splenic lymphoma with villous lymphocytes, rather than HCL. The neoplastic cells of these four patients were of B-cell origin and in three were Leu-M5 reactive. The authors' study indicates that Leu-M5 is present in nearly all hairy cells, but its presence in conjunction with other B-cell markers is not specific for HCL.

Acid Phosphatase↗

Metastatic workup of patients with prostate cancer employing alkaline phosphatase and skeletal alkaline phosphatase.

PURPOSE: To compare the efficacy of two tests, alkaline phosphatase (AP) and skeletal alkaline phosphatase (SAP) as staging markers to discriminate patients with cancer of the prostate (CaP) with bone metastases (M+) from those without bone metastases (Mo). MATERIALS AND METHODS: Patients with previously untreated CaP were entered in the retrospective analysis. Serum concentrations of AP (n = 215) and SAP (n = 73) were available. After staging the patients could be divided into 2 groups: Group I: patients with CaP and bone metastases (cT2-4 NxMoss AP: n = 40; SAP: n = 21) Group II: patients with CaP without bone metastases (cT3-4 Nx Mo; pT1-3 No Mo; AP: n = 175; SAP: n = 52). RESULTS: None of the Mo patients but 71% of the M+ patients exhibited a SAP value above the reference range (< 19 ng/ml). This difference is statistically significant (p < 0.001) and resulted in a sensitivity and specificity of 71% and 100%, respectively. The Youden-index is 0.7. In contrast 7% of the Mo patients and only 13% of the M+ patients exhibited a AP value above the reference range (< 170 U/l). This difference is statistically not significant (p = 0.71) and resulted in a sensitivity and specificity of 13% and 93%, respectively. The Youden-index is 0.06. CONCLUSION: SAP could become a useful marker in the evaluation of patients with newly diagnosed CaP as it provides more information than AP concerning the skeletal status of these patients.

Adult↗

Monoclonal antibody study of Philadelphia chromosome-positive blastic leukemias using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technic.

The leukemic cells from 15 cases of Philadelphia chromosome-positive blastic leukemia were immunophenotyped by the alkaline phosphatase anti-alkaline phosphatase (APAAP) immunocytochemical technic using nine monoclonal antibodies (MoAb) reactive with various myeloid or lymphoid antigens. On the basis of morphology, cytochemistry, terminal deoxynucleotidyl transferase (TdT) reactivity, and electron microscopy, five of the cases had been classified as lymphoid; eight, myeloid; one, mixed myeloid-lymphoid; and one, undifferentiated. The blasts from all five lymphoid cases were reactive with lymphocyte differentiation antigen MoAb, and four of five reacted with MoAb to anti-common acute lymphoblastic leukemia-associated antigen (CALLA) (BA3). The blasts from all eight myeloid cases were reactive with MY7, a marker of myelomonocytic differentiation. Some of the blasts from three of the eight myeloid cases reacted with HP1-1D and AP3, markers of megakaryocytic differentiation; megakaryocyte differentiation was confirmed by electron microscopy. In the case classified as mixed myeloid-lymphoid, the blasts showed morphologic and immunophenotypic heterogeneity; ultrastructural studies demonstrated lymphoid, basophil, and erythroid differentiation. The blasts from the case classified as undifferentiated were immunophenotypically heterogeneous. In all cases in which the leukemic cells were also immunophenotyped by flow cytometry, the results correlated well with those obtained by the APAAP technic. The APAAP technic is a reliable method for immunophenotyping leukemias. Advantages of this method include its applicability to routinely prepared blood and bone marrow smears and cytocentrifuge preparations, lack of endogenous peroxidase background staining, and a permanent record.

Adult↗

Leukocyte alkaline phosphatase and serum alkaline phosphatase in patients with metastatic breast and colon cancer.

Peripheral blood leukocyte alkaline phosphatase (LAP) scores and serum alkaline phosphatase (SAP) levels in 70 patients with metastatic breast and colorectal cancer (metastases to the liver, lung, bone and abdomen) and in 18 healthy controls were measured. The mean LAP score in the metastatic cancer patients was significantly higher than in the control group (244 vs. 61) and there was no overlap between the 95% confidence level intervals of the two groups. The mean (SAP) level in the metastatic patients was also higher than in the controls (249 u/l vs. 162 u/l) but the 95% confidence level interval of the controls was inside the 95% interval of the metastatic patients meaning that considerable percentage of the metastatic patients will have a SAP level within the normal range. We conclude that of the two markers, the LAP is the better one for detection of metastatic tumors.

Abdominal Neoplasms↗

Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex.

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.

Alkaline Phosphatase↗

Immunocytochemical typing of fine needle aspirates by means of alkaline phosphatase anti-alkaline phosphatase method. A study of 16 selected patients.

The immunocytochemical characterization of neoplastic cells recognized by a large panel of monoclonal antibodies in fine needle aspirates obtained from 16 subjects with superficial or profound tumoral masses of unknown origin was carried out using alkaline phosphatase anti-alkaline phosphatase (APAAP) immunoenzymatic method. The patients were selected on the basis of particular clinical criteria or since a correct cytohistological diagnosis was not available. APAAP immunophenotyping allowed to establish the existence of non-Hodgkin lymphoma in 6 cases, hairy cell leukemia in 1 case and carcinoma in 5 cases, while in other 2 patients the existence of Hodgkin's disease was suspected and subsequently confirmed; only in 2 subjects diagnostic informations were lacking. Furthermore, the evaluation of growth fraction by means of Ki 67 monoclonal antibody revealed a high degree of malignancy in all cases of lymphoma. Although some cautions have to be considered in the interpretation of results, APAAP immunocytochemical typing of fine needle aspirates appears a simple and reliable noninvasive diagnostic tool in selected patients.

Adult↗

Relationship between prostate-specific antigen, clinical stage, and degree of bone metastasis in patients with prostate cancer: comparison with prostatic acid phosphatase and alkaline phosphatase.

BACKGROUND: The study was designed to examine the relation of the levels of prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), and alkaline phosphatase (ALP) to clinical stage and bone metastasis in prostate cancer patients. METHODS: Serum PSA, PAP, and ALP levels were evaluated in 272 patients with prostate cancer. The relation of the level of PSA, PAP, and ALP to clinical stage and to degree of bone metastasis were examined by a multiple comparison method using ranks. The superiority of a marker in the rate of detection of bone metastasis was evaluated with receiver operating characteristic (ROC) curves. The correlation coefficients of the order of the extent of bone metastasis with PSA, PAP, and ALP were examined with Spearman's rank order correlation coefficient test. RESULTS: The levels of PSA showed significant differences among 8 pairs of clinical stages. In contrast, the levels of PAP showed significant differences among 6 pairs, and the levels of ALP showed significant differences among only 4 pairs. The area under the ROC curves of PSA, PAP, and ALP for revealing bone metastasis was 84.9%, 81.4%, and 77.3%, respectively. The correlation coefficients of the order of extent of disease (EOD) with log (PSA), log (PAP), and log (ALP) were 0.346, 0.394, and 0.618, respectively, and the levels of ALP showed the most significant differences regarding the extent of bone metastasis. CONCLUSION: PSA was the best marker for differentiating clinical stages, but showed limited reliability for stratifying the extent of bone metastasis.

Acid Phosphatase↗

Phosphocholine phosphatase and alkaline phosphatase are different enzymes in hamster heart.

The CDP-choline pathway is the major pathway for the synthesis of phosphatidylcholine in the hamster heart. The formation of phosphocholine from choline was regarded as the first committed reaction in this pathway. We demonstrated earlier that the phosphocholine pool in the heart was substantially less than that found in other tissues, and we observed that a substantial amount of the phosphocholine was hydrolyzed back to choline by a phosphatase. This phosphatase was located in the microsomal fraction of the heart, and unlike alkaline phosphatase, it was not inhibited by amino acids. The pH optima and heat sensitivity of phosphocholine phosphatase were also found to differ from alkaline phosphatase. Phosphocholine did not inhibit the hydrolysis of p-nitrophenylphosphate, but a "mixed type" inhibition of the hydrolysis of phosphocholine was observed in the presence of p-nitrophenylphosphate. Our data support the hypothesis that these two activities originate from separate and distinct enzymes, and we postulate that the cardiac phosphocholine phosphatase may play a role in the regulation of the phosphocholine pool size in the hamster heart.

Alanine↗

The alkaline phosphatase anti-alkaline phosphatase technique in dermatopathology.

The APAAP technique is an unlabelled antibody bridge technique which can be used on either frozen or paraffinembedded sections. One applies first a monoclonal antibody, then a polyclonal bridge antibody, and finally a soluble complex of alkaline phosphatase and monoclonal mouse anti-alkaline phosphatase. Subsequently, the enzyme label is developed with a naphthol salt and new fuchsin as a dye. This technique was used in our laboratory on frozen and/or paraffin embedded sections by using 15 different monoclonal antibodies, which are commercially available. The reaction product was bright red and could easily be distinguished from the brown color of melanin, which makes the APAAP technique particularly suitable for dermatopathology.

Alkaline Phosphatase↗

Preoperative serum acid phosphatase and alkaline phosphatase are not predictors of pathological stage and prostate-specific antigen failure after radical prostatectomy.

OBJECTIVE: To examine the utility and prognostic significance of enzymatic serum acid phosphatase (total acid phosphatase, TAP, and prostatic fraction of acid phosphatase, PFAP) and alkaline phosphatase (ALP) for staging, grading and outcome of patients who underwent radical retropubic prostatectomy (RRP) after the introduction of prostate-specific antigen (PSA) testing. PATIENTS AND METHODS: In all, 180 consecutive patients with clinically localized prostate cancer who underwent RRP with standard obturator lymph-node dissection between 1 January 1990 and 31 December 1995 were evaluated. Levels of TAP of > 5.4 IU/L, PFAP of > 1.2 IU/L and ALP of > 120 IU/L were classified as abnormally high. The relationship between abnormally high values and prostate cancer stage, grade and time to recurrence after RRP were calculated. The median follow-up was 86 months (approximately 7 years). RESULTS: Of the 180 patients, information about preoperative TAP, PFAP and ALP were available in 164, 163 and 154, respectively; TAP was abnormal in seven (4%), PFAP in 33 (20%) and ALP in only 13 (8%). None of the markers examined was associated with any variables of disease severity, as measured by pathological stage, Gleason score, perineural invasion, capsular penetration, positive margins, seminal vesicle involvement, and lymph node involvement. Abnormal TAP, PFAP or ALP were not associated with recurrence (P = 0.96, 0.45 and 0.41, respectively). In contrast, a PSA level of > 4 ng/mL was predictive of recurrence after RRP (P < 0.001). In the sample overall, 25 (14%) of the patients had recurrence and only one died from prostate cancer. CONCLUSIONS: Preoperative enzymatic serum TAP, PFAP and ALP levels are not predictors of the severity of disease or PSA disease-free recurrence after RRP.

Acid Phosphatase↗

Improved double immunohistochemical staining method for cryostat and paraffin wax sections, combining alkaline phosphatase anti-alkaline phosphatase and indirect immunofluorescence.

AIMS: To develop and immunohistochemical staining method for cryostat and paraffin wax sections so that two different antigens in the same section of tissues could be detected by combining immunoenzyme and immunofluorescence techniques. METHODS: This double immunohistochemical staining method combines alkaline phosphatase-anti-alkaline phosphatase (APAAP) using New Fuchsin as a chromogen and indirect immunofluorescence. RESULTS: APAAP staining for one antigen of this double immunohistochemical staining method was observed under bright field conditions alternating with immunofluorescence for another antigen under ultraviolet light. The double exposed photograph of both easily identified the two signals within the same cell. CONCLUSIONS: This double immunohistochemical staining method can overcome the disadvantages of any masking effect of the double immunoenzymatic methods and the background problems of double immunofluorescence method especially when applied to paraffin wax sections. It also permits good morphological identification of the doubly stained cells which may be of crucial importance in studies on pathology specimens.

Alkaline Phosphatase↗

Placental alkaline phosphatase (PLAP)/PLAP-like alkaline phosphatase as tumour marker in relation to CA 125 and TPA for ovarian epithelial tumours.

The significance of the PLAP (Placental alkaline phosphatase)/PLAP-like isozyme as tumour marker in relation to CA 125 and TPA for the monitoring of patients with malignant ovarian epithelial tumours was evaluated. Of all patients (n = 85), 40% had all three markers elevated. CA 125 being the most sensitive (60%), and the PLAP/PLAP-like isozyme and TPA both 40%. A tendency to certain tumour marker patterns of these three antigens in serum can be seen with regard to histopathology. Serous and anaplastic adenocarcinomas usually have all three markers moderately elevated, mucinous and mesonephric adenocarcinomas both have low incidences and low average levels of all three markers. Endometrioid and non-mucinous adenocarcinomas are often associated with high levels of the PLAP/PLAP-like isozyme and CA 125, while TPA shows moderate elevation. The PLAP/PLAP-like isozyme is positively correlated to tumour burden and the outcome of the disease. It may provide additional information on CA 125 in the monitoring of patients with ovarian cancer.

Alkaline Phosphatase↗

Alkaline phosphatase possessing alkaline phosphodiesterase activity and other phosphodiesterases in Bacillus subtilis.

In Bacillus subtilis Marburg strain, single-point mutations in the phoP locus brought about simultaneous losses of the major activities of alkaline phosphatase (APase) and alkaline phosphodiesterase (APDase). Revertants recovered the two activities. APases with APDase activity were purified from the membrane fraction of B. subtilis 6160-BC6 and from the culture fluid of an APase-secreting B. subtilis mutant strain, RAN 1. In addition to these major APases with APDase activity, at least two kinds of phosphodiesterase (PDase) without phosphatase activity were found in the cytoplasmic supernatants of RAN 1 and an APase-less B. subtilis mutant strain, SP25. Another minor APase with a molecular weight of about 80,000, which had almost no PDase activity, was isolated from the membrane fraction of strain 6160-BC6. Enzyme distribution in subcellular fractions from various strains cultured in high- and low-phosphate media was analyzed. The PDases did not cross-react with rabbit antiserum against the RAN 1 APase with APDase activity. The main component of the PDases had a molecular weight of about 80,000 and was most active at pH 8.0. These results suggest that APase with APDase activity is different from PDases detected in cytoplasmic supernatants and that phoP is the structural gene for the phosphate-repressible APase with APDase activity.

Alkaline Phosphatase↗

Participation of a proton-translocating plasma membrane ATPase, acid phosphatase and alkaline phosphatase in ATP degradation by Aspergillus niger extracts.

Extracts of A. niger could catalyze sequential hydrolysis of the three phosphate moieties of the ATP molecule optimally at pH 2 and at pH 8. At pH 2 the hydrolysis was effected by an ATPase followed by acid phosphatase while at pH 8 alkaline phosphatase was the only involved enzyme. Separation of these three phosphate-hydrolyzing enzymes was achieved by Sephadex G-100 column chromatography. The A. niger ATPase seems to have two unique features. First, it was easily solubilized in distilled water and second it had optimum activity at pH 2. The activity of this enzyme was not affected on addition of Na+, K+ or Ca2+ to the assay reaction mixture. It was neither inhibited by sodium azide nor by potassium nitrate but inhibited by orthovanadate, DES, DCCD, Mg2+ and Pi. The substrate concentration-activity relationship was of the hyperbolic type. The enzyme had high specificity for ATP, was inert with ADP and its activity with GTP represented about 6% only of that obtained with equimolar amount of ATP.

Acid Phosphatase↗

Modification of DNA topoisomerase I enzymatic activity with phosphotyrosyl protein phosphatase and alkaline phosphatase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea:Decapoda).

DNA topoisomerase I was partially purified from the hepatopancreas of the shrimp Penaeus japonicus. The specific activity of the final preparation was 7,000,000 units/mg of protein with SV40 viral DNA as substrate. SDD-polyacrylamide gel electrophoresis of the final preparation yielded two major bands of proteins with M(r) 70,000 and M(r) 67,000, as well as less intense bands of proteins with M, 64,000 and M(r) 56,000. Incubation of the partially purified enzyme fraction with rabbit antiserum against human DNA topoisomerase I, allowed all these proteins except that of M(r) 56,000, to be positively reacted. Treatment of the partially purified DNA topoisomerase I with tyrosine kinase p43v-abl resulted in phosphorylation of only the two major subunits. Phosphorylation by tyrosine kinase p43v-abl or dephosphorylation by phosphotyrosyl protein phosphatase resulted in a decrease of the enzymatic activity. The treatment with shrimp alkaline phosphatase abolished the enzymatic activity of the purified DNA topoisomerase I in a dose-dependent manner. Thus, the DNA topoisomerase I was apparently isolated from the hepatopancreas of the shrimp P. japonicus in a phosphorylated form, and this phosphorylation was essential for expression of enzymatic activity in vitro. The activity of DNA topoisomerase I is inhibited by ZnCl2, CuCl2 and Pb(NH3)3 at millimolar concentrations, but less inhibition was observed with CaCl2.

Alkaline Phosphatase↗

Tissue-non-specific alkaline phosphatase mRNA expression and alkaline phosphatase activity following application of retinoic acid in cultured human dental pulp cells.

Retinoic acid is a potent inducer of tissue-non-specific alkaline phosphatase (TNSALP) expression in various osteoblastic and fibroblastic cells, and may be involved in morphogenesis, cellular growth and differentiation. This study investigates the effects of retinoic acid on alkaline phosphatase activity and TNSALP gene expression in human dental pulp cells. Cultured cells were treated with various concentrations of retinoic acid (0, 10(-7), 10(- 6), 10 (-5) M) in 0.5% bovine serum albumin without serum. Alkaline phosphatase activity was determined by the rate of p-nitrophenyl phosphate hydrolysis and was also assayed in the presence of various inhibitors and under thermal inactivation. A set of specific oligonucleotide primers was selected, based on the nucleotide sequences of two human TNSALP mRNA (bone and liver) types, and reverse transcription-polymerase chain reaction (RT-PCR) performed. Inhibitory and thermal inactivation experiments revealed that the elevated alkaline phosphatase activity had properties of the TNSALP type. RT-PCR showed that retinoic acid enhanced the expression of bone-type TNSALP mRNA in pulp cells. However, the liver-type TNSALP mRNA was not detected. These findings suggest that the high alkaline phosphatase activity of retinoic acid-treated dental pulp cells is associated with increased transcription of the bone-type mRNA of the TNSALP gene and not with liver-type.

Alkaline Phosphatase↗