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Bone marrow aspiration and trephine biopsy. An approach to a thorough study.

Recent advances in the treatment of hematologic malignancies have been paralleled by renewed interest on the part of pathologists and hematologists in methods of obtaining and preparing bone marrow for diagnostic studies. A thorough bone marrow morphologic study involves examination of peripheral blood smears, direct, particle, and buffy coat bone marrow smears, trephine biopsy imprints, particle and trephine biopsy sections, and marrow volumetric data. The information obtained from the study of these various specimens is complementary. Frequently it is a combination of clues gathered from examination of several different preparations that leads to a correct diagnosis. Utilization of biopsy material by the methods described provides complete, accurate and reproducible information and minimizes the necessity for repeating a biopsy for morphologic diagnosis or ancillary studies.

Anemia, Aplastic↗

Trephination for acute pain management.

Surgical trephination can and does provide immediate relief of pain, surgical drainage of the infection and related fluids, and in most cases does not require supplementary administration of antibiotics and only minimal amounts of analgesics. This paper outlines the diagnosis and technique of surgical trephination.

Drainage↗

Promising results using the Hanna Corneal Trephine System in penetrating keratoplasty.

PURPOSE: To evaluate the outcome of penetrating keratoplasties using the Hanna Corneal Trephine System. METHODS: Fifty-five patients out of 70, who had penetrating keratoplasty performed by a single surgeon (KHO), from 1995 to 2001, were reviewed in 2002, at Department of Ophthalmology, Hospital of Hilleroed, Denmark. The reviews were performed by a single observer (NJ). All donor corneas were trephined from the epithelial side. Visual acuity, refraction, corneal astigmatism, and graft survival were main outcome measures. RESULTS: Seventy-two percent of the patients achieved a visual acuity > or =0.5. In major subgroups, that is, Fuchs endothelial dystrophy, secondary endothelial dystrophy, and keratoconus, a best corrected visual acuity of > or =0.5 was achieved in 57, 67, and 100%, respectively. The mean (+/-SD) postoperative spherical refraction was +0.84 D (+/-3.72), whereas mean keratometric and refractive astigmatism were -3.24 D (+/-1.51) and -3.06 D (+/-1.95), respectively. The overall graft survival rate was 96.7% after 2 years and 81.2% after 5 years. At least 90% of all patients were content with the result and course of the operation. CONCLUSIONS: The best corrected visual acuity was better, and the degree of astigmatism was less compared to previously reported studies. We suggest that these findings can be partly explained by the operative technique, that is, cutting the donor and the recipient cornea from the epithelial side by the use of an artificial chamber.

Aged↗

Experiences with a new corneal suction trephine.

The importance of exact trephining for successful keratoplasty is well known. We employ a new suction trephine, thus obtaining exact and sharp cutting margines which are examined by scanning electron microscopy (SEM). The first observed clinical results in 25 cases are good.

Cornea↗

"Orientation teeth" in non-mechanical laser corneal trephination for penetrating keratoplasty: 2.94 microm Er:YAG v 193 nm ArF excimer laser.

BACKGROUND/AIMS: "Orientation teeth" at the donor trephination margin and correspondent "notches" at the host margin facilitate graft orientation and avoid "horizontal torsion" induced by asymmetric suture placement. In this study the quality and reproducibility of these structures created by non-mechanical laser corneal trephination were compared using two laser emissions. METHODS: The procedure was performed in 20 enucleated pigs' eyes using open metal masks with eight "orientation teeth/notches" (0.3 x 0.15 mm, base x height), an automated globe rotation device, and either a 193 nm ArF excimer laser or a Q switched 2.94 microm Er:YAG laser. "Teeth/notches" were analysed by planimetry and scanning electron microscopy (SEM). RESULTS: Mean size was 0.30 (0.027) x 0. 16 (0.017) mm for "teeth" and 0.30 (0.035) x 0.15 (0.021) mm for "notches" (excimer), and 0.31 (0.022) x 0.16 (0.015) mm and 0.30 (0.031) x 0.14 (0.021) mm respectively (Er:YAG). Overall, variability of notches was higher than that of teeth. By SEM, comparable cut regularity and sustained ablation profile were observed with both lasers. However, the corneal surface at the cut edge appeared slightly elevated (</=35 microm) in the Er:YAG group. CONCLUSION: Orientation teeth/notches resembling those obtained with the excimer laser can be created using the Q switched Er:YAG laser, with potential advantages of lower costs, convenient equipment size, and solid state safety.

Animals↗

Use of APAAP technique on paraffin wax embedded bone marrow trephines.

The immunoalkaline phosphatase (APAAP) technique was applied to the labelling of decalcified sections of formalin fixed, paraffin wax embedded bone marrow trephine biopsy specimens. A panel of monoclonal antibodies reactive with haemopoietic and epithelial antigens, which survive routine formalin fixation, was assessed on 72 cases of haematological malignancy (including acute and chronic leukaemias and lymphomas) showing bone marrow infiltration. The APAAP method showed clear distinct labelling of antigen positive cells without loss of antigens due to decalcification. Both normal or reactive single cells present in the sample and neoplastic cell populations could be identified morphologically and their antigenic phenotype and cellular origin, whether lymphoid or myeloid, established. The application of the APAAP method to routinely prepared paraffin wax embedded trephines has many advantages over the assessment of specially prepared cryostat sections of bone marrow.

Alkaline Phosphatase↗

Peripheral T cell lymphoma: value of bone marrow trephine immunophenotyping.

Bone marrow infiltrates taken from 11 patients with peripheral T cell lymphoma were immunophenotyped as T cell lymphoma using monoclonal antibodies on frozen bone marrow trephine biopsy specimens. In nine these were taken at diagnosis and in two after failure of treatment to eradicate lymphoma in the marrow. Patterns of infiltration were as follows: diffuse (n = 4), interstitial (n = 1), nodular (n = 1), focal (n = 5). All cases were CD3 positive and 10 were CD2 positive; five lacked expression of either CD5 or CD7, or both markers. In nine the determination of T cell phenotype depended on analysis of the frozen bone marrow trephine biopsy specimen as there was no other biopsy tissue available for study. In the other two cases there was agreement between the immunophenotypes seen in lymph node and bone marrow infiltrates.

Adult↗

Haemopoietic regrowth after chemotherapy for acute leukaemia: an immunohistochemical study of bone marrow trephine biopsy specimens.

AIM: To analyse haemopoietic regrowth and residual disease in bone marrow trephine biopsy specimens after treatment for acute leukaemia, using immunohistochemical staining. METHODS: Biopsy specimens before and after treatment were studied from patients diagnosed as having acute myeloid or lymphoblastic leukaemia. Specimens after treatment encompassed periods from two to 56 weeks from the start of treatment. Routine haematoxylin and eosin and Giemsa stained sections were evaluated in association with immunostained preparations. A panel of antibodies was used, which reacts with epitopes showing restricted expression dependent on the lineage or maturation stage of cells. Results were evaluated in the light of clinical, peripheral blood, and marrow aspirate findings. RESULTS: The speed and sequence of regrowth of haemopoietic cells were more variable than expected. Immunostaining highlighted features of dysplasia after treatment and in some cases assisted detection of residual or relapsed leukaemia. Peripheral blood and aspirate cell counts reflected accurately the amount of regrowth, but not the dysplasia, seen in biopsy samples. Delayed regrowth was associated with complex individual factors. CONCLUSIONS: Morphological and immunohistochemical study of trephine biopsy specimens from patients treated for acute leukaemia provides information complementary to that obtained from peripheral blood and aspirated marrow. Variation in the timing and sequence of regrowth is highlighted. Immunostaining can aid in the detection of relapse or minimal residual leukaemia. The clinical relevance of dysplastic changes in biopsy specimens after treatment is uncertain, but such changes may persist for long periods.

Acute Disease↗

Late relapse in IgD myeloma: value of immunohistochemistry in trephine biopsy specimens.

IgD myeloma is a rare disorder with a very poor prognosis. An unusual case is reported where prolonged remission was obtained. Immunohistochemical staining of the bone marrow trephine biopsy specimen proved invaluable in documenting early relapse. It is suggested that immunohistochemistry should be adopted more widely in view of the disparities evident in aspirates and trephines.

Biopsy↗

Immunohistochemistry in apparently normal bone marrow trephine specimens from patients with nodal follicular lymphoma.

AIM: To establish the role of immunohistochemistry (using a limited panel of antibodies) in detecting minimal involvement by follicular lymphoma in routinely processed bone marrow trephine specimens, which show no obvious morphological (light microscopic) evidence of lymphoma; to determine whether bcl-2 immunostaining in bone marrow distinguishes between benign and malignant infiltrates in a patient with nodal follicular lymphoma. METHODS: Twenty seven consecutively selected paraffin wax embedded, formalin fixed bone marrow trephine specimens were stained with the following antibodies: anti-bcl-2, anti-CD79a, anti-CD3, and kappa and lambda light chains, using the Streptavidin biotin complex technique. RESULTS: Five of the 27 cases, which showed no evidence of involvement by follicular lymphoma on routine stains, showed monotypic B cells on immunohistochemistry. Two of the cases were diffuse, while the remaining three showed mini-aggregates around bony trabeculae. In all five cases the lymphomatous infiltrates were strongly bcl-2 positive. Reactive B lymphoid nodules did not show the same degree of bcl-2 positivity, and negative cells could be discerned within the reactive nodules. CONCLUSIONS: There is merit in studying so-called negative bone marrows immunohistochemically in order to detect minimal involvement by follicular lymphoma. A limited panel of antibodies including anti-bcl-2, anti-CD79a and anti-CD3 is usually adequate to accomplish this. Strongly bcl-2 positive lymphoid aggregates in the bone marrow of patients with nodal follicular lymphoma are indicative of lymphoma.

Biopsy↗

Adequacy of bone marrow trephine biopsy specimens in children.

AIMS: To evaluate success in obtaining adequate bone marrow trephine biopsy cores from children. METHODS: Sections of trephine biopsy cores submitted by 25 centres from children with neuroblastoma over a five year period were reviewed centrally. In cores containing no tumour adequacy was defined as 0.5 cm of well preserved bone marrow after processing. Occasional smaller cores containing obvious tumour were also considered adequate. RESULTS: Of 822 biopsy specimens, 139 (17%) were inadequate. In 13 centres submitting at least 20 cores failure rates ranged from 2.6 to 50%. There was no improvement over the five years of the study. There was no practically important correlation between the numbers of cores submitted and success in obtaining adequate specimens. Although a lower rate of inadequate biopsy specimens was found when haematologists rather than paediatricians (13 v 29%) were the predominant operators this should not be overinterpreted, not least because of the potentially confounding association between haematologist operators and larger numbers of biopsy specimens, and because the arbitrary subdivision of centres according to operator specialty was crude. The skill of individual operators could not be assessed. CONCLUSIONS: Many operators do not obtain adequate bone marrow biopsy specimens from children. Improvement is necessary because this is an invasive investigation, often performed under general anaesthesia. Reporting pathologists are well placed to influence practice by pointing out inadequacies in the specimen and suggesting retraining or even a change in operator. Improvement would almost certainly occur if this investigation was restricted to locally recognised successful operators, whatever their specialty. Most centres should review their practice and devise strategies to improve their ability to obtain adequate cores.

Biopsy, Needle↗

Deterioration in performance in obtaining bone marrow trephine biopsy cores from children. European Neuroblastoma Study Group.

AIM: To complete an audit of bone marrow trephine biopsy adequacy in children MATERIAL: 605 specimens from children with neuroblastoma submitted by 25 centres were reviewed centrally. This reassessment ran between January 1995 and August 1998. RESULTS: 25% of specimens (95% confidence interval (CI) 21% to 29%) were inadequate compared with 17% (95% CI 14% to 20%) in a previous study. Variation between individual centres' performance remains high (5-54% of specimens inadequate). Had five centres performed as well as previously, the inadequate biopsy rate would have been unchanged from that found in the previous study. There was no important improvement in any centre's performance. Earlier suggestions about change in practice have had no discernible impact on centres' ability to obtain adequate bone marrow trephine biopsies from children. CONCLUSIONS: The responsibility for improving the rate of adequate biopsies lies with individual centres. Reporting pathologists might help by making even more positive attempts to influence operators within their own centres.

Biopsy↗

Trained nurses can obtain satisfactory bone marrow aspirates and trephine biopsies.

AIMS: To assess the feasibility of training nurse practitioners to perform bone marrow aspiration and trephine biopsy, and to compare the quality of these samples with those obtained by medical staff. METHODS: A retrospective audit was undertaken of nurse practitioner and medical staff performance in bone marrow procedures in a busy haematology day unit. RESULTS: Nurse practitioners fared favourably in comparison with medical staff in performing bone marrow trephine biopsies, with mean biopsy lengths of 11 mm and 10.7 mm respectively. However, only 78% of the smears obtained by the nurses were judged technically satisfactory, compared with 91% prepared by doctors. This discrepancy was thought to be due largely to the quality of slide spreading. CONCLUSIONS: With motivated staff and a structured educational and training programme it is possible for nurse practitioners to perform the techniques of bone marrow aspiration and biopsy, and obtain specimens of satisfactory quality, thus improving efficiency of the haematology day unit and increasing quality of patient care.

Biopsy, Needle↗

Thoracic spine: percutaneous trephine biopsy.

Forty-one percutaneous trephine biopsies of thoracic vertebral bodies or disks from T-3 to T-12 were performed under fluoroscopic guidance without complications. Biopsy technique was improved by a new set of trephine needles using a procedure similar to vascular catheterization and specific prone-oblique positioning of the patient. On a 35 degrees prone-oblique view, the heads (dorsal extremities) of the ribs determine the direction of puncture. Both pleura and spinal canal are easily recognized and avoided. Of 40 cases, there were 31 true-positive results, six contributory results, and three false-negative results. Percutaneous biopsy of the thoracic spine with this technique is a safe, rapid, and reliable procedure.

Biopsy↗

Colonoscopy-assisted 'trephine' sigmoid colostomy.

Fecal diversion is often required to treat complex traumatic, malignant or inflammatory anorectal conditions. In such circumstances, the formation of a proximal, 'trephine' sigmoid colostomy would avoid the need for, and the associated morbidity of, a formal laparotomy. We describe a technique which combines intraoperative colonoscopy with a diverting, 'trephine' sigmoid colostomy, thereby helping the surgeon to identify the correct loop of bowel, to avoid inadvertent maturing of the wrong end of the divided colon, and to exclude intracolonic lesions.

Anastomosis, Surgical↗

Tyramide signal amplification: an enhanced method for immunohistochemistry on methyl-methacrylate-embedded bone marrow trephine sections.

The detection of cellular antigens in bone marrow sections depends on the method of embedding, the nature of antigen and antibody, antigen retrieval techniques and the sensitivity of the immunohistochemical method. This study evaluated a fluorescyl-tyramide-enhanced immunostaining method on methyl-methacrylate-embedded bone marrow trephine biopsies. Compared with the standard immunostaining technique, fluorescyl-tyramide-enhanced immunostaining showed a superior signal intensity on methyl- methacrylate-embedded bone marrow trephines. Thus, the fluorescyl-tyramide-based immunostaining method, while retaining specificity, is highly sensitive and provides optimal staining results for a broad panel of antibodies commonly used for the routine diagnosis and classification of hematological disorders.

Antibodies, Monoclonal↗

Percutaneous trephine lung biopsy: evolving role.

The Steele trephine was employed to perform 134 transthoracic biopsies in 120 patients. Resultant cores of tissue and "lung juice" provided a diagnosis in 89 percent (107) of the patients. Complications occurred in 41 percent (49) of the patients, the incidence being greatest in those with diffuse parenchymal disease. Most complications were of a minor nature. Although the precise role of trephine lung biopsy in the physician's diagnostic armamentarium remains to be defined, there is no doubt that it is a useful and safe technique in the elucidation of intrathoracic disease.

Adult↗

In situ hybridization of immunoglobulin light chain mRNA on bone marrow trephines using biotinylated probes and the APAAP method.

A non-radioactive in situ hybridization method has been established for the detection of mRNA for the constant region of light chain immunoglobulin genes in routinely processed bone marrow trephines. The method utilizes a cocktail of biotinylated synthetic oligonucleotide probes to kappa or lambda mRNA. The method entails dewaxing of the paraffin-embedded sections, proteinase K treatment and overnight hybridization with the biotinylated probe. Detection of probe hybridization was performed by 2 immunocytochemical detection methods, utilizing either streptavidin-alkaline phosphatase or monoclonal anti-biotin followed by the alkaline phosphatase: anti-alkaline phosphatase (APAAP) labelling system. The new fuchsin/naphthol phosphate substrate yielded the strongest signal with specific localization of the hybridization signal to positive cells. Morphological preservation was excellent enabling both polyclonal and monoclonal plasma cells to be detected in bone marrow trephines. We conclude that this in situ hybridization method is no more difficult than standard immunohistochemical techniques and can be used in routine diagnostic laboratories.

Biopsy↗