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PubMed1994

Investigation of an organic delivery system for demineralized bone matrix in a delayed-healing cranial defect model.

The osteogenic potential of demineralized bone matrix (DBM) has been demonstrated in multiple animal models and clinical applications. A particulate form of DBM is generally used to fill defects because it is easily packed into a defect site without operative planning or shaping. One potential disadvantage in the use of a particulate is the migration of particles from the defect site. To stem this, glycerol was added to DBM to improve handling properties. A study was undertaken to compare two forms of DBM with glycerol, with DBM particulate and autograft in a bony defect site. The model chosen consisted of bilateral 8-mm trephine defects created in the parietal skull of 40 rabbits. Animals were sacrificed at 2 and 8 weeks. Results demonstrated both cartilage and bone induction with the three forms of DBM, with 75-90% of the linear width of the defect filled with new bone by 2 weeks. This was similar to the autograft (86%) and significantly greater than the unfilled defects (26%) at 2 weeks. The addition of glycerol to the DBM did not affect the inductive capacity, and produced a response similar to that of DBM particulate alone or autogenous bone.

Animals↗
PubMed2003

Biological performance of biomimetic calcium phosphate coating of titanium implants in the dog mandible.

The aim of the present study was to analyze the in vivo effect of biomimetic calcium phosphate coating of titanium implants on periimplant bone formation and bone-/implant contact. Five types of implants were used: 1) Ti6Al4V implants with a polished surface; 2) Ti6Al4V implants with collagen coating; 3) Ti6Al4V implants with a mineralized collagen layer; 4) Ti6Al4V implants with sequential coating of hydroxyapatite (HA) and collagen; and 5) Ti6Al4V implants with HA coating only. All implants had square cross sections with an oblique diameter of 4.6 mm and were inserted press fit into trephine burr holes of 4.6 mm in the mandibles of ten beagle dogs. The implants of five animals each were evaluated after a healing period of 1 month and 3 months, respectively, during which time sequential fluorochrome labeling of bone formation had been performed. Bone formation was evaluated by morphometric measurement of the newly formed bone around the implants and the percentage of implant bone contact. After 1 month, there was a significantly higher percentage of mean bone/implant contact in the HA-coated implants compared to those with polished surface and those with the collagen-coated surface. After 3 months, these differences were not present anymore. Bone apposition was significantly higher next to implants with sequential HA/collagen coating compared to polished surfaces and mineralized collagen layer. It is concluded that biomimetic coating of titanium implants with HA has shown the clearest trend to increase bone-implant contact in the early ingrowth period. The addition of collagen to an HA coating layer may hold some promise when used as sequential HA/collagen coating with mineralized collagen as the surface layer.

Alloys↗
PubMed2005

Osteogenic potential of injectable tissue-engineered bone: a comparison among autogenous bone, bone substitute (Bio-oss), platelet-rich plasma, and tissue-engineered bone with respect to their mechanical properties and histological findings.

Recently, tissue engineering has become available as a regenerative treatment for bone defects. However, the evaluation of its success is limited to histological analysis, and its effects on mechanical hardness remain to be investigated. This study investigated mechanical strength in support of histological findings, specifically for tissue-engineered bone with mesenchymal stem cells (MSCs) and platelet-rich-plasma (PRP). Initially, teeth were extracted, and bone defects on both sides of the mandible were prepared with a trephine bar. The defects were implanted by using the following graft materials: 1) PRP, 2) PRP and dog MSCs (dMSCs), 3) autogenous bone (PCBM), 4) bone substitute (Bio-Oss), and 5) control (defects only). After 2, 4, 8, and 12 weeks of implantation, the defects were histologically assessed to examine their mechanical properties. According to histological observations, the dMSCs/PRP groups had well-formed mature bone compared with the control (defects only), Bio-Oss, and PRP groups. The Vickers hardness test values were 8 (control), 9 (PRP), not detected (Bio-Oss), 11 (PCBM), and 17 (dMSCs/PRP) after 2 weeks. Therefore, tissue-engineered bone can be used for early stage bone regeneration from the viewpoint of histology and mechanical properties.

Animals↗
PubMed1987

The assessment of bone formation and bone resorption in osteoporosis: a comparison between tetracycline-based iliac histomorphometry and whole body 85Sr kinetics.

Bone formation and resorption have been measured in patients with idiopathic osteoporosis by histomorphometry of 7.5-mm trephine biopsies and in the whole body by 85Sr radiotracer methodology and calcium balances. The studies were synchronized and most were preceded by double in vivo tetracycline labeling. Correlations between histological and kinetic bone formation indices were better when better when based on the extent of double tetracycline labels than on measurements of osteoid by visible light microscopy. Correction of the kinetic data for long-term exchange, using 5 months' serial whole body counting of retained 85Sr, improved the fit of the kinetic to the histological data. A statistical analysis of the measurement uncertainties showed that the residual scatter in the best correlations (between exchange-corrected bone formation rates and double-labeled osteoid surface indices) could be attributed to measurement imprecision alone. The exchange-corrected resorption rate correlated fairly well with iliac trabecular resorption surfaces, and using a volume referent rather than a surface referent for the histological index improved the statistical fit when patients with therapeutically accelerated bone turnover were included. A much better correlation was obtained by including osteoid volume acting as an independent predictor of bone resorption in a bivariate regression with a resorption surface index. The residual errors could then be accounted for by known measurement uncertainties. Whereas osteoid taking a double label closely predicted the kinetic rate of bone formation, further analysis suggested that osteoid that took no label or a single label was more closely related to bone resorption, presumably as a secondary result of the coupling of bone formation to bone resorption. The idea that continued bone loss in some patients is associated with defective osteoblastic bone formation is supported by the low rates found in some patients by both techniques. Heuristically these studies validate both in vivo tetracycline labeling for dynamic histomorphometry and corrections for long-term exchange in kinetic studies of bone formation, providing a quantitative framework for the design and analysis of future studies of bone remodeling in the osteoporoses.

Adult↗
PubMed2001

MR-guided biopsy of musculoskeletal lesions in a low-field system.

Thirty magnetic resonance (MR)-guided biopsies were obtained from 20 skeletal and 10 soft-tissue lesions in 31 patients using an open 0.2 T MR system equipped with interventional accessories. The results from aspiration (N = 3), core biopsy (N = 15), and transcortical trephine biopsy (N = 12) were evaluated for accuracy and clinical efficacy. Specimens were successfully obtained from 29 patients. Results were clinically effective in 23 patients, rated definitive in 16, nonconclusive in 9, and unspecific in 2 patients. A false diagnosis due to sampling error occurred in 2 patients, and biopsy sampling was impossible in one case. The best diagnostic yield was achieved from nontranscortical biopsies of osteolytic or soft-tissue masses. Results from transcortical biopsies were less specific due to the predominance of benign lesions. MR fluoroscopy for needle guidance was applied in 13 patients. Complete needle placement inside the magnet could be performed in 16 patients. MR-guided biopsy using an open low-field MR imager is feasible and clinically effective and will become a valuable tool in the management of musculoskeletal lesions. J. Magn. Reson. Imaging 2001;13:761-768.

Adolescent↗
PubMed1995

Herpes simplex virus DNA in normal corneas: persistence without viral shedding from ganglia.

Herpes simplex virus type 1 (HSV-1) DNA has been shown to persist in the cornea not only after inoculation of experimental animals but also in surgical samples from patients with herpes keratitis. The further observation of corneal HSV-1 DNA in subjects without known HSV eye disease prompted the present study of the presence and distribution of HSV-1 in eye bank corneas. Prior to DNA extraction, the corneas were trephined, separating the central and peripheral cornea. With polymerase chain reaction (PCR) for HSV-1 thymidine kinase (TK) and glycoprotein D (gD) gene sequences, we found HSV-1 in 10 of 24 eye bank corneas, from the 4 mm wide corneal rim in 8 eyes and from the 8 mm diameter central cornea in 2 eyes. In 9 subjects, both eyes were assayed, and HSV-1 was detected in 6 subjects. In only one subject was HSV-1 detected in both eyes and in only one subject was HSV-1 detected in the central and peripheral cornea of the same eye. The biological role of HSV-1 DNA corneal sequences is unknown. To investigate this, a rabbit animal model was established by transplantation of corneas containing viral DNA sequences in HSV-1 naive recipients. Followed for 5 months, there was no evidence of sheeding of HSV-1 in the tear film or seroconversion of the recipient rabbits. At the end of this time, HSV-1 DNA was detected in the corneal graft at a similar intensity to the PCR signal from the donor rims.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Mid-infrared laser ablation of the cornea: a comparative study.

The ablation thresholds and patterns of collateral damage in cornea produced by Er:YAG (2.94 microns) and Er:YSGG (2.79 microns) lasers were measured. Two different pulse durations, 200 microseconds (normal spiking mode) and 100 ns (Q-switched mode), were used at both wavelengths. In the normal spiking mode, damage zones of 16 +/- 2 microns and 39 +/- 7 microns and ablation thresholds of 250 +/- 20 mJ/cm2 and 420 +/- 35 mJ/cm2 were measured at 2.94 microns and 2.79 microns, respectively. In the Q-switched mode, damage zones of 4 +/- 2 microns and ablation thresholds of 150 +/- 10 mJ/cm2 were found irrespective of the laser used. The similarity between the results using the Er:YAG and Er:YSGG lasers in the Q-switched mode suggest that either laser can be used with equal effectiveness for corneal trephination.

Animals↗
PubMed1979

Bone marrow biopsy in children: a study of 111 patients.

Closed trephine needle biopsy of the bone marrow has become an established procedure in the evaluation of many malignant and benign diseases in adults; however, its role in pediatric pathology has not yet been defined. In the period from February 1974 to April 1978 we have performed 164 such biopsies in 111 children under 15 years of age. A representative specimen has been obtained in over 80% of cases. This series included, in order of frequency, non-Hodgkin lymphomas, Hodgkin lymphomas, aplastic anemias, rhabdomyosarcomas, neuroblastomas, miscellaneous solid tumors, and single cases of histiocytosis X, malignant histiocytosis, sarcoidosis, malignant histiocytoma, and Castleman lymphoma of the hyaline-vascular type. Histology has been found superior to cytology in the detection of neuroblastoma invasion; the evaluation of the true cellularity in aplastic anemia, and the detection of granulomatous tissue in the only case of sarcoidosis. In other diseases histology and cytology gave similar information, except for the few cases of acute leukemia in partial relapse, which has been better defined in the aspirate smears than in the core specimen. Further evaluation of this technique in other patient series appears advisable.

Adolescent↗
PubMed1989

Bone marrow biopsy in the initial staging of Hodgkin's disease.

Results of bone marrow biopsy were retrospectively evaluated in 120 previously untreated patients with Hodgkin's disease. The incidence of bone marrow involvement was 13%. All patients with marrow invasion had B symptoms and/or clinically advanced disease. When patients with bone marrow involvement were compared to those without there were significant differences in the incidence of B symptoms, the clinical stage, hemoglobin levels, leukocyte counts, platelet counts, and serum levels of lactate dehydrogenase and alkaline phosphatase. None of 59 patients with clinical stage IA and IIA had evidence of marrow invasion. This study demonstrates that trephine bone marrow biopsy is of value in detecting marrow involvement in specific subgroups of untreated patients with Hodgkin's disease, i.e., those patients with constitutional symptoms and/or clinical stage III or IV. However, bone marrow biopsy adds little to the initial staging of patients with clinical stage IA and IIA. Routine use of this procedure in such patients may be unnecessary.

Adolescent↗
PubMed1988

An immunocytochemical study of lymphocyte and macrophage populations in the bone marrow of patients with non-Hodgkin's lymphoma.

Pairs of bone marrow trephine samples from 67 patients with known or suspected non-Hodgkin's lymphoma (NHL) were collected. One sample was processed for morphological examination, the other for frozen section immunohistology, using a panel of monoclonal antibodies (MAB) reacting with lymphocyte and macrophage determinants, detected by the immuno-alkaline-phosphatase (APAAP) method. Forty-one cases showed definite (36) or suspected (5) involvement of the marrow by NHL. Most were examples of lymphocytic or centroblastic/centrocytic NHL. The pattern of immunostaining confirmed the presence of NHL in these cases: the phenotype of the neoplastic cells was broadly consistent with nodal histology, where available. In ten cases, the marrow showed no evidence of involvement by NHL, and in 15, the eventual diagnosis was an abnormality of the myeloid series. These two groups of marrows not involved by NHL both showed a 'reactive' pattern of immunostaining, comprising polyclonal B-cells, T-helper and suppressor/cytotoxic cells and macrophages. We conclude that immunohistological examination of the bone marrow is useful in cases where the specimen shows morphological evidence of NHL, including those that are only 'suspect', but does not detect lymphoma where there is no morphological evidence of involvement.

B-Lymphocytes↗
PubMed1994

Ki-S1 and PCNA expression in erythroid precursors and megakaryocytes--a comparative study on proliferative and endoreduplicative activity in reactive and neoplastic bone marrow lesions.

The monoclonal antibody Ki-S1 reacts with a cell proliferation-associated nuclear antigen which is expressed in the G1 through G2/M phases of the cell cycle and is resistant to formalin fixation. We have studied Ki-S1 and PCNA (PC10) immunostaining of erythroid precursors (proliferative activity) and megakaryocytes (endoreduplicative activity) in bone marrow trephine biopsies in a variety of reactive and neoplastic lesions using double immunohistochemistry to identify both cell lineages. A significant increase in Ki-S1 labelling compared with PCNA positivity was found in all conditions studied. In particular, specimens derived from secondary polycythaemia (SP), polycythaemia vera (P. vera), and primary osteomyelofibrosis (OMF), and from splenic tissue with myeloid metaplasia (MM), revealed a disproportionally high labelling index of erythropoiesis, which was not present in chronic myelogenous leukaemia (CML), AIDS, and autoimmune (idiopathic) thrombocytopenia (ITP). Enhancement of Ki-S1 (PCNA) staining in SP and P. vera is in keeping with the relevant increase in erythroid precursor proliferation, but in OMF and MM there is overexpression of both proliferation markers, possibly due to secondary folic acid deficiency, which is known to cause a block in the S-phase of the cell cycle. A significant correlation was observed between the sizes of megakaryocytes and their nuclei with Ki-S1 (and also PCNA) staining. Ki-S1 (and PCNA) labelling of predominantly smaller elements of this lineage supports a hypothesis that the phases of the cell cycle have different durations in the various steps of polyploidization, with a prolongation of G1/G2 at higher ploidy levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Neoplasm↗
PubMed2006

Multicentric osteosarcoma presenting as retrobulbar mass: a diagnostic enigma.

Osteosarcoma is the most common primary malignant bone tumor of children and adolescents. It often presents as a solitary lesion; multicentric osteosarcoma with synchronous lesions occurring at multiple skeletal sites is very rare. We report a 9-year-old boy with multicentric osteosarcoma who presented with a left retrobulbar non-sclerotic mass. The multiple lesions in bone were mostly non-sclerotic on radiological examination except for a single lesion in the left tibia. Biopsy of the retrobulbar mass showed an unclassifiable poorly differentiated malignant tumor. Marrow aspiration smears showed many large, often segregated, round cells that expressed NB84a. However, trephine biopsy showed the formation of tumoral osteoid by the malignant cells, finally permitting the definitive diagnosis of osteosarcoma to be made. A hypertetraploid clone with complex structural abnormalities was demonstrated by cytogenetic study.

Bone Marrow↗
PubMed2001

Two freezing cycles ensure interface sterilization by cryosurgery during bone tumor resection.

Cryosurgery utilizing an argon-based system allows bone-tumor interface sterilization, while avoiding the risks of conventional cryosurgery. This study was conducted in order to evaluate the number of freezing cycles required for interface sterilization in cases of aggressive human bone tumors. Sixteen tumors were included (six chondrosarcomas, eight metastatic carcinomas, and two giant cell tumors). All occurred within long bones. In all cases a standardized marginal resection was performed. Following thorough curettage, we sampled five different locations within the tumor interface by a cylindrical hollow trephine. The interface viability was assessed using the XTT method. Quantitative histological evaluation was based on the percentage of live cells divided by total lacunae number in five random medium-power fields. One freezing cycle (5 min, -40 degrees C) reduced tumor viability to approximately 5% of prefreezing. However, there were still live specimens. Two or three freezing cycles led to complete interface sterilization. The difference between a single freezing cycle and two freezing cycles was significant (ANOVA, F = 130, P < 0.01). The difference between two freezing cycles and three freezing cycles was not significant (ANOVA, F = 0.14, P < 0.6). The results of the XTT method for the assessment of interface viability correlated well with histological evaluation of the percentage of viable cells (r = 0.89), as well as with cell culture results of frozen vs. prefreezing tumor samples. In conclusion, two freezing cycles are adequate to achieve tumor-bone interface sterilization in aggressive human bone tumors.

Adult↗
PubMed1996

A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization.

The effects of growth factors on re-epithelialization of wounded human and bovine corneas were studied in a simple organ culture system. Excisional trephine and epithelial scrape wounds were created on bovine and human corneo-scleral rings in which the endothelial corneal concavity was then filled with an agar-collagen mixture. Organ culture was undertaken at 37 degrees C in a humidified 5% CO2 incubator with serum-free Medium 199 maintained at the level of the conjunctival epithelium. Rates of reepithelialization in response to addition of exogenous epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor type beta 1 (TGF-beta 1) were assessed by image analysis. Corneal cultures could be maintained for up to 3 weeks without significant stromal oedema or keratocyte deterioration and with little loss of epithelial architecture. Following wounding the cornea reepithelialized in a similar fashion to that observed in vivo i.e. a lag phase followed by migration/proliferation and the reformation of an intact multilayered epithelium. EGF accelerated, basic FGF had no effect on, and TGF-beta 1 inhibited the rate of corneal re-epithelialization. Our organ culture model maintains corneal integrity and provides a practical system in which to study factors that modulate corneal reepithelialization following wounding.

Animals↗
PubMed2000

The role of endogenous growth factors to support corneal endothelial migration after wounding in vitro.

Previous studies have shown that corneal endothelial cells contain mRNA and protein of various growth factors. However, the role of these endogenous growth factors in corneal endothelial wound healing is not fully elucidated. In the present study, we investigated the role of endogenous factors and several growth factor inhibitors on migration of corneal endothelial cells in an in vitro model of wound healing. Bovine corneal endothelial cells (BCEC) were grown to confluency and experiments performed at passage 2 under serum reduced [2% fetal calf serum (FCS)] conditions. A central circular 'wound' (5 mm diameter) was made with an especially designed trephine. In different experiments, cells were incubated over different time periods (1-72 hr) either with the cellular debris produced by the wounding procedure or with previously prepared endothelial cell lysates (protein content 50-500 microg ml(-1)). Additionally, purified bovine polyclonal anti-BFGF antibodies (Ab) (4.5-27 microg ml(-1)), suramin (0.5 m M) or anti-FGF receptor-Ab (1 microg ml(-1)) were added to both experimental approaches, respectively. Migration was quantitated by counting the cells inside the denuded area in four different sections from the wound edge after 5 days. Cellular migration of cells adjacent to the wound was significantly stimulated by factors released during wounding or by endothelial cell lysates at protein concentrations >100 microg ml(-1). This increase in migrating cells was partially inhibited when the anti-bFGF antibody was incubated with the cell debris or the lysates. The addition of suramin at 0.5 m M almost completely blocked the migration activity. Incubation of the anti-FGF-receptor antibody prior to and >5 hr after wounding significantly reduced migration to nearly 50% of the rate in control cultures (P<0.001). In the present study, we demonstrate that intracellular growth factors released from corneal endothelial cells enhance the migration of surviving cells in vitro. The strong inhibitory effect of suramin indicates a major role of heparin-binding growth factors for cellular migration. bFGF and the regulation of bFGF-receptor expression on cells at the wound margin seem to be of crucial importance for the wound healing process.

Analysis of Variance↗
PubMed2001

Intracellular signaling pathway of FGF-2-modulated corneal endothelial cell migration during wound healing in vitro.

After wounding, the corneal endothelium heals primarily by migration of adjacent cells into the denuded wound area. In this study, it has been attempted to identify elements of the intracellular signaling pathway activated through basic Fibroblast Growth Factor (FGF-2)- and Protein Kinase C (PKC)-modulated migration, using specific inhibitors and stimulators of second messengers in a cell culture model. Bovine corneal endothelial cells (BCEC) were grown to confluency and experiments performed with first passage cells under serum-free conditions. A central circular 'wound' was made with a specially designed trephine. In different experiments, cells were incubated with either FGF-2 (10 ng ml(-1)), pertussis toxin (PTX; 1-50 ng ml(-1)), phorbol 12-myristate 13-acetate (PMA; 50 ng ml(-1)), 2,4'-di-bromoacetophenone (DAP; 5 microM), 1-(5-iosquinolinesulphonyl)-2-methyl-piperazine dihydrochloride (H7; 10 microM), indomethacin (5 ng ml(-1)), nordihydroguaiaretic acid (NDGA; 10 ng ml(-1)), 2-(4-morpholinyl)-8-pheny-4H-1-benzopyran-4-one (LY294002; 10 microM) or different combinations of these agents. Unsupplemented cultures served as controls. Migration was quantitated by counting the cells inside the denuded area in one randomly chosen section from the wound edge 72 hr after wounding. Cell toxicity was determined with the trypan blue exclusion test. Results were statistically analysed by Student's t-test. FGF-2 and PMA (a protein kinase C activator) both stimulated migration of endothelial cells at 2.2- and 3.1-fold, respectively. The PLA(2) inhibitor DAP and the PKC inhibitor H7 both significantly reduced PMA-stimulated migration to control levels but had no effect (DAP) or even stimulated (H7) FGF-2-modulated migration. PTX did not affect FGF-2-stimulated migration. The phosphoinositol (3)-kinase inhibitor LY294002 significantly reduced FGF-2-mediated stimulation of endothelial migration similar to the rate of control cultures. LY294002 had no effect when applied together with PMA. The cyclooxygenase inhibitor indomethacin did not influence migration rates of the cells added either alone or in combination with PMA and FGF-2, respectively. The lipoxygenase inhibitor NDGA significantly reduced the number of migrating cells in cultures with no other supplements, or of those supplemented with either PMA or FGF-2. FGF-2-induced endothelial migration in vitro is not dependent on PKC/PLA(2) or pertussis-toxin sensitive G-protein pathways but rather requires activation of a phosphoinositol (3)-kinase-like enzyme and/or arachidonic acid release with subsequent liberation of lipoxygenase products. Independent of FGF-2, PKC is a major intracellular effector of corneal endothelial migration activity after wounding and stimulates migration via the PLA(2)-dependent generation of lipoxygenase metabolites.

Acetophenones↗
PubMed2002

Modifying subsceral fluid pressure in an experimental model of mitomycin-C diffusion.

BACKGROUND: Episcleral application of mitomycin-C (MMC) during glaucoma filtration surgery hinders the post-operative wound healing. Diffusion through the sclera might result in a toxic effect on the ciliary body resulting in reduced aqueous humor production leading to post-operative hypotony. We developed an experimental model to investigate the influence of intraocular pressure on the diffusion of MMC through the sclera and in subscleral compartments. METHODS: Scleral quadrants of 10 human donor eyes were mounted on PMMA tubes filled with saline imitating the intraocular volume. By height variation of a coupled infusion line different intraocular pressures were simulated (0, 8, 23 and 80 mmHg). Additionally the model included a subscleral sponge to mimic the compartment of the ciliary body. The episcleral sides of the scleral quadrants were exposed for 1 min to sponges soaked with 200 microg ml(-1) MMC. An 8-mm-diameter scleral disk was punched out with a trephine and horizontally dissected with a kryotome. The MMC concentrations of scleral layers, epi-and subscleral sponges and the fluid within the tubes were analysed by means of high-performance liquid chromatography. RESULTS: The MMC concentration gradually declined from the episcleral sponge (165 microg ml(-1)) to the superficial (3.3 microg ml(-1)) and deep scleral layers (1.2 microg ml(-1)), and to the subscleral sponge (0.2 microg ml(-1)). We were able to detect very small concentrations of MMC in the fluid within the PMMA tubes (0.01 microg ml(-1)). CONCLUSION: We developed a new experimental in vitro model for investigating transscleral MMC diffusion. The different simulated intraocular pressures had no effect on the concentration gradient through the investigated compartments of our model.

Diffusion↗
PubMed1999

Apoptotic fraction in childhood ALL assessed by DNA in situ labelling is ploidy independent.

BACKGROUND: Apoptotic cell fraction and presence or degree of aneuploidy may both affect treatment outcome in childhood acute lymphoblastic leukaemia (ALL), which is largely defined by drug resistance. Independence of the variables is at present not established. Until the development of in situ labelling of cells committed to the apoptotic pathway, the fraction of cells in apoptosis could not be determined objectively. AIM: To determine the relationship between apoptotic cell fraction and karyotype in childhood ALL using in situ labelling. METHODS: 1.3.1. Study Groups and Samples. Diagnostic, pretreatment bone marrow trephine and aspirate samples of 24 consecutive, unselected cases of childhood ALL were included in the study: Normal karyotype (n = 11, 5M,6F), high hyperdiploid aneuploidy (DNA index > 1.5, n = 7, 1M,6F), complex karyotypic anomalies (n = 6, 5M,1F). 1.3.2. Apoptotic Cell Labelling. In situ labelling of the 3'-OH ends of the apoptosis specific DNA (Klenow) fragment (Frag-EL, CalBiochem, USA). 1.3.3. Quantitation. Apoptotic cell fraction was established using 10 systematically random fields of > 20 nuclei. Results were tabled per group. After calculations of means, differences between groups were assessed using t-test. RESULTS: Apoptotic cell fraction, ranging from < 1 to 95%, did not differ statistically significant between the three study groups. CONCLUSION: Apoptotic cell fraction in childhood leukaemia is independent of ploidy status and euploid karyotypic anomalies.

Apoptosis↗