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We evaluated a new immunoselective enzyme assay of bone-specific alkaline phosphatase (ALP). The monoclonal antibody used in this assay was raised against purified bone-specific ALP obtained from SAOS-2 human osteosarcoma cell line. Calibration was based on the enzyme's own activity. The relative activity of the antibody was 100% with bone ALP, 8.7% with liver ALP, and 0% with placental and intestinal ALPs. Intra- and inter-assay coefficients of variation were less than 4%. The sensitivity of the assay was 0.7 U/L, and the linearity extended from 2 to 140 U/L. The recovery of bone-specific ALP standard added to serum was 94-106%. The correlation coefficient between this method and the polyacrylamide gel (PAG) electrophoretic method was 0.94. The mean value of bone-specific ALP in 89 healthy adults (mean age 29 years, SD 5 years) was 18.5 U/L (SD 4.1 U/L). Interestingly, mean bone-specific ALP activities in 60 premenopausal women (mean age 39 years, SD 8 years) and 70 postmenopausal women (mean age 57 years, SD 5 years) were 20.3 U/L (SD 6.5 U/L) and 31.1 U/L (SD 11.1 U/L), respectively. The age-related increase in bone-specific ALP was significant and more pronounced in women (P < 0.01). We conclude that this new immunoassay of bone-specific ALP would be useful for clinical investigation of patients with osteoporosis or other metabolic diseases of bone.

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Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor-suppressor genes. Investigation of genomic instability of microsatellites has indicated a new mechanism for human carcinogenesis in hereditary nonpolyposis colorectal cancer and sporadic cancer and this instability has been shown to be related to inherited predisposition to cancer. This study was conducted to determine whether such microsatellite instability is associated with the evolution of chronic myelogenous leukemia (CML) to the blast crisis. Nineteen CML patients clinically progressing from the chronic phase to accelerated phase or blast crisis and 20 other patients in the CML chronic phase were studied. By polymerase chain reaction assay, DNAs for genomic instability in five separate microsatellites in chromosome arms 5q (Mfd27), 17p (Mfd41), 18q (DCC), 3p (CI3-9), and 8p (LPL) were examined. Differences in unrelated microsatellites of chronic and blastic phase DNAs in 14 of 19 patients (73.7%) were demonstrated. Somatic instability in five microsatellites, Mfd27, Mfd41, DCC, CI3-9, and LPL, was detected in 2 of 19 (10.5%), 8 of 19 (42.1%), 11 of 19 (57.9%), 4 of 17 (23.5%), and 4 of 17 (23.5%) cases. In 10 of 19 cases (52.6%), genetic instability in at least two of five microsatellites was observed and was categorized as replication error (RER+) phenotype. CML evolution cases with myeloid, lymphoid, and mixed phenotypes and the blast crisis and accelerated phase showed somatic instability in a number of microsatellites. No alterations in leukemic cells at the chronic phase could be detected in any microsatellites. These data indicate instability of microsatellites (RER+) but not familial predisposition to possibly be a late genetic event in the evolution of CML to blast crisis. In the microsatellite of the DCC gene, complicated alterations in band patterns caused by instability as well as loss of heterozygosity (LOH) were observed in 13 of 19 cases (68.4%): instability in 9 cases, instability plus LOH in 2 cases, and only LOH in 2 cases. These highly frequent alterations in microsatellites, including instability and LOH, suggesting that secondary events due possibly to loss of fidelity in replication and repair machinery may be significantly associated with CML evolution.

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A 30-year-old woman was admitted in August 1984 with anemia. She had an initial hemolytic attack in 1977. Unstable hemoglobinopathy was suspected. Despite splenectomy in 1979, hemolytic attacks continued. On admission, she was anemic and cyanotic. Hb heat denaturation test was positive. However, the first structure of amino acid of hemoglobin was normal. A large amount of white powder was found in her belongings, and was later identified as phenacetin. N-acetyl-P-aminophenol, a metabolite of phenacetin was demonstrated in her urine. Hemolytic attacks disappeared completely after she stopped taking phenacetin.

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The frequencies of chromosomal breaks and sister chromatid exchanges (SCE) are influenced by pregnancy, oral hormonal contraceptives and the menstrual cycle. The changes in the number and sites of spontaneous and aphidicolin-induced breaks on chromosomes from peripheral blood lymphocytes during the menstrual cycle were examined in 8 healthy women. Menstrual cycle was determined by menstruation and the quantity of serum estrogen, progesterone and luteinizing hormone. The number of spontaneous breaks at the follicular phase, the interval phase (which includes ovulation) and the luteal phase were 3.1 +/- 1.1, 2.7 +/- 2.3 and 3.9 +/- 2.6 per 100 mitoses, respectively. The frequencies of aphidicolin-induced breaks in the same phases were 95.8 +/- 23.3, 90.6 +/- 14.3 and 122.7 +/- 20.1 per 100 mitoses, respectively. The higher frequency at the luteal phase was statistically significant compared with the other phases. In the luteal phase, bands 2q32, 3q27, 6q26 and 16q23 had higher frequencies of breaks (P less than 0.05); however, breaks at band 9q32 decreased significantly. SCE showed considerable variation, but with no statistical significance.

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The identification of a P-glycoprotein product of multidrug-resistant gene (mdr 1) was reported recently. To examine the expression of the P-glycoprotein in acute leukemias of various types, we have prepared leukemic blast cells from patients and measured their positivity of P-glycoprotein using monoclonal anti-P-glycoprotein antibody (C219) by flow cytometry. P-glycoprotein is expressed in 8 out of 44 cases including leukemic blast cells but not lymphocytes and monocytes. In these cases showed drug resistance was shown clinically. In addition, the expression of the P-glycoprotein was not observed in the drug-sensitive cases and at the time of initial chemotherapy. Our results suggest that measurement of P-glycoprotein in acute leukemias by flow cytometry may prove to be a valuable tool for the design of chemotherapy protocols.

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The fact that cancer cell acquires multidrug resistance to carcinostatics at cancer treatment is a very important subject clinically. The mode of multidrug-resistance is complicated, but the gene associated with multidrug resistance (MDR 1) has been isolated. It has become evident that MDR 1 gene carries membrane glycoprotein (P-glycoprotein) which occurs in the cell acquired drug-resistance. Assessment has been made this time regarding the occurrence of P-glycoprotein in the tumorous cells and tissues by the use of monoclonal antibody (C 219) to P-glycoprotein. Occurrence of P-glycoprotein in malignant lymphoma exhibited positivity in 9 cases out of 36 immunohistologically. 170 KD P-glycoprotein was detected in 4 cases out of 10 at Western blotting analysis of the protein isolated from the nuclear cell in the peripheral blood in the patients with leukemia. Further, P-glycoprotein positive cases were all progressive cases clinically and showed resistance to treatment. From these results, it has been clarified that occurrence of P-glycoprotein in haematological tumors is related to multidrug resistance.

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Bone marrow biopsy specimens obtained from 24 untreated patients with several types of acute leukaemia (AL) and eight patients with chronic myelocytic leukaemia (CML) were prepared without decalcification for electron microscopy. A new type of transmission electron microscope (LEM 2000) was used for wide-field observation, and the venous sinuses were morphometrically evaluated on micrographs using an automatic image analyser. No differences were found between either AL or CML bone marrows and the controls in the diameter, circumference, or luminal and endothelial area of sinuses. No degenerative changes were observed in the sinus endothelium of either the AL or CML patients, but the adventitial cell cover in sinuses was significantly less in both AL (P less than 0.001) and CML (P less than 0.001) patients than in the controls, indicating a close relationship between the decrease in adventitial cell cover and increase in migrating leukaemia cells. In addition, the sinus endothelium in CML had significantly (P less than 0.03) more pores than in AL patients and three or more leukaemic cells in migration through the same large pore were occasionally found in the CML patients. These findings suggest that leukaemic cell migration in AL differs from that in CML.

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Cases of increased OKT 8-positive T cells were found among patients with multiple myeloma, and the suppressor function of these fractions was studied. The suppressor activity of OKT 8 cells with myeloma was not increased in the Igs synthesis system. Therefore it was suggested that the increase of OKT 8 cells was not correlated to the activity of suppressor T cell function.

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In the clinical course of a patient with progressive facial hemiatrophy associated with ipsilateral body atrophy (total hemiatrophy), signs and symptoms of localized scleroderma were noted. The patient subsequently was found to have Schönlein-Henoch purpura with renal involvement and, later, paroxysmal nocturnal hemoglobinuria. To our knowledge, such an association has not been reported before.

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We investigated the ultrastructural changes in the hematopoietic microenvironment of the bone marrow obtained from 15 untreated patients with chronic myelocytic leukemia (CML) in the chronic phase by transmission and scanning electron microscopy using the cryofracture technique. Examination of the undecalcified bone marrow specimens confirmed extensive hyperplastic granulopoiesis. In the stroma, fat cells were scarce or absent. Macrophages were increased and scattered throughout the marrow. The cytoplasm contained abundant cellular debris and crystals of the Charcot-Leyden type. Slender reticular cells were inconspicuously located between proliferating myeloid cells. A few foci of fibrosis were occasionally observed. The sinus endothelium generally retained its continuity, and no features suggesting complete deterioration of the sinus were evident. However, certain alterations in the sinus wall were noted in the process of leukemia cell migration. Many cells migrated transcellularly into the circulation through transient large openings in the sinus endothelium.

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