Iliac Crest Bone Marrow Aspirates Research Articles

A Comparative Gene Expression Study of CD3+ T-Cells In Bone Marrow From Patients with Chronic ITP and Healthy Controls by DNA Microarray.

AbstractAbstract 1445 Background:The pathophysiology of ITP (immune thrombocytopenia) is more complex than initially believed. The importance of regulatory T-cells, platelet specific cytotoxic T-cells, suppression of megakaryocyte proliferation and maturation, functional thrombopoietin deficiency and inappropriately low bone marrow production of platelets has been unraveled over the last decade. T-cells are important in all forms of autoimmunity including ITP. However, very little is known about the T-cell activity in organs where platelets and their precursors are targeted by effectors of the immune system. To provide new insights into the pathophysiology of ITP, we performed a DNA microarray analysis on isolated bone marrow derived T-cells from chronic ITP patients and healthy controls. Patients and Method:Bone marrow was collected from 6 chronic ITP-patients (3 males and 3 females, mean age 39.5 years) and 6 healthy controls (3 males and3 females, mean age 33.8 years) through iliac crest bone marrow aspiration. T-cells were isolated by immunomagnetic cell sorting (Miltenyi Biotec, Surrey, UK). RNA was prepared using the Chomczynski method (Chomczynski and Sacchi, 1987), followed by RNeasy minielute clean-up (Qiagen, Hilden, Germany). RNA was amplified from 20 ng of starting total RNA with the Ovation RNA Amplification System V2 (NuGEN Technologies, Inc.), and cDNA was synthesized using the Encore Biotin Module kit (NuGEN Technologies, Inc.). After standard labeling, each sample was hybridized to an Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA). Results:We identified 397 differently regulated genes in the bone marrow derived T-cells between ITP patients and controls by the DNA microarray analysis (P-value <0.05 and fold change ± 1.3). Some of the regulated genes have previously been implicated in ITP, such as integrin alpha 4 (ITGA4 or CD49D or VLA-4), Fas ligand (FASLG) and cytotoxic lymphocyte-associated 4 (CTLA-4). Furthermore, several apoptotic genes were lower expressed in ITP patients, such as Fas apoptotic inhibitory molecule 2 (FAIM2), BCL2-like 11 (BCL2L11), BCL2-like 13 (BCL2L13), peptidyl-tRNA hydrolase 2 (PTRH2) and the enzyme inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB).Integrin alpha 6, (ITGA6) and the adhesion molecule endothelial cell adhesion molecule (ESAM) were higher expressed in ITP patients compared to controls and are important for T-cell adhesion and migration or “homing” of lymphocytes to different lymphoid organs and inflammatory sites. Conclusion:We have identified a number of regulated genes in T-cells from bone marrow that differed between patients with ITP and healthy controls. The importance of several of the identified genes has already been implicated in ITP, proving the usefulness of this strategy. The identification of novel regulated genes may provide new insights into the physiology and pathophysiology of ITP and for autoimmunity in general. Disclosures:No relevant conflicts of interest to declare.

Chemokine Profile of Human Serum from Whole Blood: Migratory Effects of CXCL-10 and CXCL-11 on Human Mesenchymal Stem Cells

Autologous human serum is used in cartilage repair and may exert its effect by the recruitment of mesenchymal stem and progenitor cells (MSC). Aim of our study was to analyze the chemokine profile of human serum and to verify chemotactic activity of selected chemokines on MSC. Human MSC were isolated from iliac crest bone marrow aspirates. Chemotactic activity of human serum made from whole blood and pharma grade serum was tested in 96-well chemotaxis assays and chemokine levels were analyzed using human chemokine antibody membrane arrays. The chemotactic potential of selected chemokines on MSC was tested dose dependently using chemotaxis assays. Human serum derived from whole blood significantly attracted human MSC, while pharma grade serum did not recruit MSC. Human chemokine antibody array analysis showed that the level of chemokines CXCL-3, 5, 7-8, 10-12, 16; CCL- 2, 5, 11, 13, 16-20, 24-25, 27; as well as XCL-1 was elevated (fold change >1.5) in serum derived from whole blood compared to nonrecruiting pharma grade serum. Chemotaxis assays showed that the chemokines IP-10/CXCL-10 and I-TAC/CXCL-11 significantly recruit human MSC. PARC/CCL-18, HCC-4/CCL-16, CTACK/CCL-27, and Lymphotactin/XCL-1 showed no chemotactic effect on MSC. Therefore, human serum derived from whole blood contains chemokines that may contribute to serum-mediated recruitment of human mesenchymal progenitors from bone marrow.

HIV-Infection, Hodgkin Lymphoma, and Pregnancy: A Case Report and Recommendations On Treatment.

Abstract 4638According to the CDC the incidence of HIV-infection in women of child bearing age continues to increase in the era of Highly Active Anti-Retroviral Therapy (HAART). In 1992, women accounted for 14% of all adults and adolescents living with HIV/AIDS, but by the end of 2005 women accounted for 23% of all HIV/AIDS cases [1]. As Hodgkin lymphoma (HL) is the most common non-AIDS defining malignancy, we anticipate that the number of cases of HIV-associated Hodgkin lymphoma (HIV-HL) in pregnant women will increase in the near future. Herein, we describe the case of a pregnant 30-year-old HIV-infected Ethiopian woman with a CD4+ count of 254 cells/μ;L and an HIV viral load of 1200 copies/mL who presented to medical attention with progressive neck adenopathy and fatigue, but no fevers, night sweats, or significant weight loss. An incisional biopsy of a cervical lymph node revealed Reed-Sternberg cells (CD30+, CD15+, CD20-, CD3-) and an absence of sclerosis consistent with Classical Hodgkin Lymphoma, mixed cellularity subtype. A subsequent unilateral posterior iliac crest bone marrow aspirate and biopsy was unremarkable with normal trilineage hematopoiesis. Following a spontaneous miscarriage ten weeks into her pregnancy, a 18F-fluorodeoxy-D-glucose PET and fusion CT scan demonstrated disease above and below the diaphragm, establishing stage IIIA HL. The patient subsequently began HAART consisting of a co-formulation of emtricitabine and tenofovir (Truvada®) and nevirapine, in conjunction with chemotherapy (AVD x 8 cycles). Thirty three months post-completion of chemotherapy, the patient remains disease free without evidence of recurrent HL. Through a literature search, we identified only two additional case reports describing HIV, HL, and pregnancy. One patient received three cycles of chemotherapy, refused further treatment, delivered a HIV-positive girl, and died shortly after from complications of presumed pneumocystis jiroveci pneumonia [2]. The second patient received both active antiretroviral therapy and chemotherapy, delivered a HIV-negative boy, and remained without evidence of HL at nine months follow-up [3].The paucity of reported cases in the medical literature precludes any evidence based recommendations for the care of pregnant patients with HIV-HL. However, we recommend that medical providers use the same precautions to ensure the safety of both the mother and the child as recommended for pregnant HIV-negative patients with HL. Pregnant patients with HL should not be staged with imaging techniques that require significant radiation exposure including plain radiographs, CT, and PET scans. The extent of substantial mediastinal and pulmonary disease can be safely determined with a postero-anterior radiograph of the chest with proper shielding of the abdomen. Abdominal ultrasounds and magnetic resonance imaging may provide adequate information for the management of disease without placing the fetus at risk [4]. We also suggest controlling the underlying HIV infection when initiating HL treatment as using HAART in parallel with chemotherapy has been correlated with a dramatically improved prognosis for HIV-HL patients [5]. In the hopes of developing more specific management guidelines, we encourage other clinicians to publish their experiences with HIV-HL in pregnant patients.1.Centers for Disease Control and Prevention. HIV/AIDS Among Youth–United States, 2008. http://www.cdc.gov/hiv/resources/Factsheets/youth.htm2.Okechukwu CN, Ross J. Hodgkin's Lymphoma in a Pregnant Patient with Acquired Immunodeficiency Syndrome. J Clin Oncol 1998; 10:410-411.3.Kelpfish A, Schattner A, Shtalrid M,et al. Advanced Hodgkin's Disease in a Pregnant, HIV Seropositive Woman: Favorable Mother and Baby Outcome Following Combined Anticancer and Antiretroviral Therapy. Am J Hematol 2000; 63:57-58.4.Connors, Joseph. “Challenging Problems: Coicident Pregnancy, HIV infection, and Older Age.” In: Hematology 2008: American Society of Hematology Education Program Book. Gewirtz AM, Muchmore EA, Burns LJ, editors. Washington, D.C.: American Society of Hematology; 2008. p. 334-39.5.5.Tirelli U, Errante D, Dolcetti R, et al. Hodgkin's Disease and Human Immunodeficiency Virus Infection: Clinicopatholgic and Virologic Features of 114 Patients From the Italian Cooperative Group on AIDS and Tumors. J Clin Oncol 1995; 13:1758-67. Disclosures:No relevant conflicts of interest to declare.

The differentiation of anaemia in rheumatoid arthritis: parameters of iron-deficiency in an Indian rheumatoid arthritis population

Iron deficiency anaemia (IDA) is common in Indian patients with rheumatoid arthritis (RA). We evaluated red blood cell indices, serum iron related and bone marrow iron stores measurements in diagnosing iron deficiency in patients with RA. Fifty consecutive anaemic patients with RA had their complete blood counts, red cell indices, serum iron, serum ferritin and serum total iron binding capacity (TIBC) measured and underwent posterior iliac crest bone marrow aspiration. Fixed smears were stained for iron with Perl's Prussian blue and patients who had no (grade 0) or minimal stainable iron (grade I) were regarded as iron deficient and rest iron replete (grade II-IV) and hence as having anaemia of chronic disease (ACD). To determine diagnostic power of tests used for diagnosing iron deficiency in addition to positive likelihood ratio, sensitivity, specificity and negative predictive values; receiver operating characteristics (ROC) curves were plotted and areas under the receiver-operating curves were compared. Eighteen patients (36%) had IDA and 32 (64%) had ACD. Correlation between the bone marrow iron stores and serum ferritin was poor in the IDA group (r = -0.09, P = 0.57) and significant in the ACD group (r = 0.79, P < 0.0001). Areas under the ROC curves for mean corpuscular haemoglobin (MCV), serum iron, TIBC and mean corpuscular haemoglobin concentration (MCHC) were relatively low (0.52, 0.71, 0.75 and 0.77, respectively) and these tests had low positive likelihood ratios (1.08, 2.13, 4.62 and 1.5, respectively). Both area under ROC curve [0.98, 95% confidence interval (0.94, 0.99)] and negative predictive value (97%) were highest when cut off serum ferritin was <82 microg/l. In patients with RA serum iron, TIBC, MCV and MCHC have limited value in diagnosing iron deficiency. In this study compared to American and European studies a much higher cut off value of serum ferritin had most diagnostic power for detecting iron deficiency. Bone marrow iron stores measurements appears to be the most reliable method for diagnosing IDA however, it needs to be taken in conjunction with other laboratory findings and the clinical scenario.

Percutaneous Autologous Bone Marrow Harvest from the Calcaneus and Proximal Tibia: Surgical Technique

Several studies have advocated the use of autologous bone marrow aspirate (BMA) to augment traditional treatment of nonunions, chronic wounds, tendon rupture, and multiple other disease states (1–3). Bone marrow contains 2 types of stems cells, hematopoietic and mesenchymal. The hematopoietic stem cells differentiate into red and white blood cells, platelets, and macrophages. The mesenchymal stem cells form multiple cell types involved in tissue repair when placed in the appropriate microenvironment. These include osteoblasts, chondrocytes, fibroblasts and myogenic cells (1–3). Traditionally, BMA has been harvested from the iliac crest and occasionally from the sternum (2). Alternative sites include the proximal ends of long bones, which also contain red marrow, although no studies have evaluated the relative concentration of stem cells in these locations in comparison with the iliac crest. For foot and ankle surgeons, both the calcaneus and the proximal tibial metaphysis offer convenient harvest sites with the ability to obtain a significant amount of BMA through a percutaneous approach. The maximum amount of BMA that should be aspirated from a single site should be 2 mL to maximize the number of alkaline phosphatase–positive, colony-forming units, which are indicative of cells that will differentiate into a mature osteoblastic phenotype. Muschler et al found that aspiration of more than 2 mL from a single site resulted in progressive dilution from peripheral blood, which, theoretically, would decrease the bone growth stimulation provided by the BMA (4). Hernigou et al treated tibial atrophic nonunions with iliac crest BMA and found that patients who

Comparative analysis of bone marrow (BM) micrometastasis (M) with sentinel lymph node (SLN) status in early breast cancer (BRCa)

508 Background: While sentinel lymph node (SLN) metastasis (mets) and bone marrow (BM) micrometastases (M) provide prognostic information in breast cancer (BRCa) patients (pts), a definitive relationship between the two has not yet been proven. We hypothesize that the presence of BMM may not infer an increased risk of SLN mets due to differences in their mechanisms of spread. Methods: We conducted a 6-year retrospective study of 251 T1/T2 BRCa pts who underwent bilateral iliac crest bone marrow aspiration, SLN mapping, and either lumpectomy, partial mastectomy, modified radical mastectomy, or total mastectomy by a single surgeon. BM samples were examined for micrometastases by Automated Cellular Imaging System using Chroma Vision Cytokeratin Detection Kit to detect cells expressing CK 8 as defined by CAM 5.2 monoclonal antibody. Pts with BMM underwent repeat BM analysis after completion of adjuvant chemotherapy. Data was collected for SLN, BM, ER/PR and HER2/neu status. Results: There were 251 consecutive pts entered into the study. Of these, 27.5% (69/251) had SLN mets and 9.6% (24/251) had BMM. Of the pts with SLN mets, 11.6% (8/69) had BMM vs. 88.4% (61/69) with no BMM ( Table ). In pts with ER/PR negative (-ve) BRCa, 7.1% (2/28) had BMM vs. 92.9% (26/28) with no BMM and 25% (7/28) had SLN positive (+ve) vs. 75% (21/28) SLN -ve pts ( Table ). Of the pts with HER2/neu +ve BRCa, 13.1% (8/61) had BMM vs. 86.9% (53/61) with no BMM and 26.2% (16/61) were SLN +ve vs. 73.8% (45/61) SLN -ve pts. After completion of adjuvant chemotherapy all pts with BMM (n=24) converted to BM -ve status. Conclusions: No correlation was identified between BMM and SLN status in T1 and T2 BRCa pts. This study suggests that BMM can occur independently of SLN status. Furthermore, (-ve) ER/PR and (+ve) HER2/neu status did not have a direct correlation with either BMM or SLN mets. Pts with BMM status may benefit from adjuvant therapy. Long-term implications of BM (+ve) have to be evaluated by larger multi- center trials. [Table: see text] No significant financial relationships to disclose.

Towards in situ tissue repair: Human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.

Effect of donor characteristics, technique of harvesting and in vitro processing on culturing of human marrow stroma cells for tissue engineered growth of bone

The aim of this study was to assess the effect of donor characteristics and the technique of harvesting and in vitro processing on the efficacy of culturing of human mesenchymal stem cells (hMSCs) for tissue engineered growth of bone. Cultures of hMSCs were derived from iliac crest bone marrow aspirates (21 donors, age 11-76) and from cancellous bone grafting material (32 donors, age 13-84). Age had no significant effect on the ability to isolate and culture hMSCs, although the failure rate was 55.6% in donors beyond the age of 60, while it varied between 14.3% and 22.2% in donors under 60 years of age. Male and female donors had comparable failure rates (27.3% and 28.6%, respectively). Culturing of hMSCs was successful in 90.4% of marrow aspirates from 21 donors and in 62.5% of cancellous bone specimens from 35 donors. This difference was statistically significant (P=0.023). Regression analysis confirmed that at simultaneous testing of the three variables, only the source of cells significantly affected the result (P=0.043). Morphological evaluation of the unfractionated primary population showed a change in cell shape of the adherent cells from a triangular into thin spindle-shaped elongated form, which remains until confluence. When the cultures were exposed to osteoinductive medium, various morphotypes expressing different levels of alkaline phosphatase and secreting different amounts of mineral were evident. Morphology of marrow stroma cells (MSCs) from marrow aspirates was not different from MSCs derived from cancellous bone specimens. Expression of osteogenic markers in MSCs as shown by PCR as well, did not differ between the two sources. It is concluded that marrow aspirates and cancellous bone specimens produce comparable populations of MSCs. However, bone marrow aspirates from donors under the age of 60 years rather than cancellous bone chips are favourable for isolation and expansion of hMSCs for tissue engineered growth of bone.

The impact of bone marrow micrometastases on metastatic disease‐free survival in patients with colorectal carcinoma

The biological relevance of bone marrow micrometastases (BMM) in colorectal cancer remains unknown. Here, we investigate their nature by examining the impact of the presence of BMM on metastatic disease-free survival in a cohort of patients with this disease. Sixty-three consecutive patients undergoing surgery for colorectal cancer of any stage were studied after approval of the study protocol by the local ethics committee and with full individual informed consent. All had bilateral iliac crest bone marrow aspirates prior to operation. Aspirates were then examined for the presence of aberrant cytokeratin-18-positive cells by a blinded observer using both flow cytometric and APAAP immunohistochemical techniques. Mean follow-up after surgery was 4.6 years (range 1.9-6.9) for those without hepatic metastases at diagnosis. Seven of 34 patients with Dukes' stage A or B developed metastatic disease after a mean interval of 4.7 years (range 3.8-6.8). However, only 2 of these patients demonstrated BMM at the time of surgery. Nine of 15 patients with Dukes' C carcinoma at the time of surgery subsequently developed metastases after a mean interval of 4.4 years (range 1.9-6.9). Again, only two of these patients had BMM detectable initially. In only three of the 14 patients known to have metastases at the time of operation (i.e. Dukes''D' disease) were BMM found. The presence of BMM as detected by this methodology was not predictive of tumour recurrence or metastasis. This study does not support the consideration of adjuvant therapy based on the presence of BMM at a single pre-operative time point in patients with colorectal cancer.

CELLULAR PHENOTYPES AFFECTED BY INDUCTION THERAPY WITH CAMPATH-1H VS THYMOGLOBULIN VS ZENAPAX IN KIDNEY ALLOGRAFT RECIPIENTS

P387 Introduction: Several independent studies have shown the safety and efficacy of antibody induction in renal transplantation using several different maintenance immunosuppressive protocols. This describes an 18 month follow-up in a randomized study, comparing the cytoablative effect of Campath-1H (C) 0.3 mg/kg-2 dosages, Thymoglobulin (T) 1mg/kg-5 dosages and Zenapax (Z) 1mg/kg- 5 dosages on peripheral blood and iliac crest bone marrow of kidney allograft recipients. Methods: In C half maintenance dosages of tacrolimus (tacro) and mycophenolate (MMF) but no corticosteroid were given. In T and Z higher maintenance doses of tacro, MMF as well as steroids were given. Depletion of discrete mononuclear cell phenotypes in peripheral blood and iliac crest bone marrow samples taken sequentially post-transplant at 10, 60, and 200 days were compared. The absolute number of mononuclear cells in peripheral blood or iliac crest bone marrow aspirates per mm3 and the CD3, 19, 56/16 (NK cells), 25, 14 (Monocytes) and 34 (Stem cells) phenotypes in flow cytometry were calculated. Results: In peripheral blood, when compared to normal values, in the C group, there was depletion of virtually all lymphocytes, T cells by 100%, B cells by 98%, NK cells by 95%, monocytes by 83% and Stem cells by 40%, with an earlier recovery (in descending order) of stem cells, B cells, monocytes, and NK cells, and later (at >6 months) with T cells. In contrast, T depleted T cells (99%) and NK cells (98%), but B cells, and monocytes were only moderately affected (20% and 45% respectively). Z depleted all CD25 positive cells with early modest depletion of T cells, B cells and NK cells by 50%, 72% and 62% respectively, and with no effect seen on monocytes. In iliac crest marrow, at 10 days C also strongly depleted all lymphocyte, CD3+, CD19+, CD56/16+, and CD14+ counts by 99%, 98%, 96%, and 68% respectively. However, virtually normal numbers of CD34+ cells were present. At 200 days post-transplant, an increased number of B cells (double the normal values) were seen. The early depletion of affected subsets by T in this compartment were 50% that of C with an earlier return to normal ranges in all phenotypes, while with Z there was only a minimal effect. Conclusion: C is the more potent lymphoablative induction agent (followed by T) allowing in most cases for early and total avoidance of corticosteroids and lower doses of maintenance immunosuppressive agents.

Lower Leg Pain in a 17-Year-Old BoyOrthopaedic-Radiology-Pathology Conference

Lower Leg Pain in a 17-Year-Old BoyOrthopaedic-Radiology-Pathology Conference

Percutaneous Ethibloc injection in aneurysmal bone cysts.

To investigate whether the injection of Ethibloc into aneurysmal bone cysts can be an effective treatment modality. Ethibloc is an alcoholic solution of zein (corn protein) which has thrombogenic and fibrogenic properties. Ten patients with aneurysmal bone cysts were treated with CT-guided percutaneous injection of Ethibloc into the cyst cavity. Ethibloc injection was the primary treatment in five patients. Four patients had recurrence following previous curettage and bone grafting and one patient had not responded to injection into the lesion of autologous iliac crest bone marrow aspirate. Three patients needed a second injection. The median follow-up was 27 (6-60) months. Symptoms were relieved in all patients. At imaging, seven patients had resolution of the lesion and three had partial response at the most recent follow-up. Complications consisted of a local transitory inflammatory reaction in two patients and an aseptic abscess in one patient. This relatively simple, minimally invasive procedure makes an operation unnecessary by stopping the expansion of the cyst and inducing endosteal new bone formation. This technique may be used as the primary management of aneurysmal bone cysts excluding spinal lesions.

DETECTION OF CIRCULATING PROSTATE SPECIFIC ANTIGEN EXPRESSING PROSTATIC CELLS IN THE BONE MARROW OF RADICAL PROSTATECTOMY PATIENTS BY SENSITIVE REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION

Purpose The reverse transcriptase polymerase chain reaction (RT-PCR) assay for prostate specific antigen (PSA) expressing cells in the blood circulation has been under intense investigation since 1992. Although it has been suggested that this technology could be used as molecular staging for occult prostatic hematogenous metastases, we have been unable to confirm RT-PCR PSA positivity of peripheral blood to predict stage or recurrence in radical prostatectomy cases. We performed bone marrow RT-PCR PSA assay on a large cohort of radical prostatectomy cases and evaluate the use of this assay in improving prostate cancer staging and detecting early recurrence. Materials and Methods Unilateral anterior iliac crest bone marrow aspirates were performed on 116 patients immediately before radical prostatectomy between February 1995 and September 1997. Radical prostatectomy specimens were processed as whole mounts. A sensitive nested RT-PCR assay with specific primers derived from the PSA sequence was used, which enabled us to detect PSA expressing LNCaP prostate cancer cells at the sensitivity of 1 cancer cell per 10 million lymphocytes (1/10 7). A minimum of 3 RT-PCR PSA reactions were performed on all patients and at least 2 positive tests were required to define positivity. Patients were followed for PSA recurrence (mean followup 14.7 months). Results PSA expressing cells were detected in bone marrow of 51 of 116 patients (44.0%) when at least 2 of 3 RT-PCR PSA assays per patient were positive. A much higher rate of RT-PCR PSA positivity was noted (77/116 patients, 66.3%) when any RT-PCR PSA positivity was considered. In 10 randomly selected cases the RT-PCR product was confirmed as PSA by deoxyribonucleic acid sequencing. Of 51 bone marrow RT-PCR positive cases 25 (49%) had organ confined disease and 26 (51%) had nonorgan confined disease. Similarly, bone marrow RT-PCR PSA was not associated with age, race, grade, pretreatment PSA or prostatic acid phosphatase value, clinical stage or margin status. However, the 2-year disease-free survival was 96.6% in RT-PCR negative patients versus 77.5% in RT-PCR positive patients (p = 0.054), and bone marrow RT-PCR PSA was an independent prognostic factor in multivariate analysis including PSA, Gleason grade and pathological stage. Conclusions Bone marrow RT-PCR PSA positivity in this study did not predict pathological stage, grade or margin positivity as determined from whole mount prostate cancer specimens. Furthermore, no relationship with age, grade or serum markers and bone marrow RT-PCR PSA positivity was noted. However, bone marrow RT-PCR PSA was associated with early disease recurrence. Further studies and longer followup are warranted to define the metastatic potential of the PSA expressing cells in the bone marrow of prostate cancer patients.

Continuing observations on the regulatory effects of donor-specific bone marrow cell infusions and chimerism in kidney transplant recipients.

Continued follow-up of a series of donor bone marrow cell (DBMC)-infused first cadaver renal transplant recipients is described (n=58), now at a 36-month actuarial time point postoperatively. Serial polymerase chain reaction-flow cytometry (PCR-Flow) and cellular immune assays of iliac crest bone marrow aspirates and peripheral blood have begun to be compared with concomitantly transplanted recipients of living-related donor (LRD) kidneys and donor marrow infusions given the same immunosuppressive regimen (n=16). There have also been comparisons (36 months) with 188 controls transplanted concomitantly, i.e., recipients of first cadaver kidney transplants, who did not receive bone marrow. Each group was given equivalent immunosuppressive regimens of OKT3 anti-T cell induction and maintenance tacrolimus, mycophenolate mofetil, and methylprednisolone. Actuarial patient and graft survival have been 96% and 93%, respectively, in the controls and 91% and 91%, respectively, in the DBMC-infused recipients. Trough levels of tacrolimus were significantly lower in the DBMC-infused group. In PCR-Flow measurements, in peripheral blood up to 6 months postoperatively, there were higher levels of chimerism, i.e., in the total number of donor cells, as well as the donor CD3+ and CD34+ subsets in the LRD recipients administered DBMC infusions, compared with cadaver DBMC recipients, supporting the notion of a positive effect of histocompatibility on chimerism levels. In PCR-Flow measurements of recipient iliac crest bone marrow aspirates as in previous studies on peripheral blood, early acute rejection episodes (<1 month) were found to be associated with a later (6-14 months) decrease in donor cell lineage chimerism. However, a trend toward recovery of chimeric levels occurred by 21-28 months in a second iliac crest marrow aspirate 1 year after the first aspirate in the DBMC-infused recipients who experienced such early rejection episodes. This was in contrast to the controls in whom there were sustained low levels of iliac crest bone marrow chimerism at both the earlier and later intervals (i.e., no chimeric recovery), with 17/183 surviving controls progressing into chronic rejection. This has not yet been seen in the DBMC-infused group (0/54). In in vitro observations on cellular immune reactivity at 1 year postoperatively, decreased peripheral blood lymphocyte proliferative reactions were seen in response to phytohemagglutinin and Staph-A mitogens, as well as to cytomegalovirus and Epstein-Barr viral protein antigens in the DBMC-infused group versus the controls. Chronic immunosuppression did not seem to effect a vigorous in vitro inhibitory (regulatory) activity of bone marrow taken from these transplant recipients 2 years postoperatively in mixed lymphocyte culture and cell-mediated lympholysis reactions, using allogeneic responding cells from "normal" laboratory volunteers. Autologous peripheral blood lymphoproliferative responses to phytohemagglutinin and Staph-A mitogens, as well as to cytomegalovirus and Epstein-Barr virus protein antigens, were also regulated by either organ donor (non-immunosuppressed) bone marrow cells or by transplant recipient (immunosuppressed) bone marrow cells. What appeared to be disparate between the DBMC-infused and control groups (both immunosuppressed) was the trend for the (autologous) bone marrow suppressive effect on antiviral lymphoproliferative responses, to be stronger in the DBMC-infused group, who also had significantly (>one order of magnitude) higher levels of chimerism (P=0.01). It is concluded that the establishment of a chimeric state in DBMC-infused recipients, albeit of relatively low magnitude (approximately 1% at 2 years in recipient iliac crest bone marrow), has had a definite regulatory effect on immune responses. These results, therefore, add weight to the "causal" horn of the dilemma as to whether donor cell chimerism is a cause or an effect of

Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.

An assessment of the effects of cadaver donor bone marrow on kidney allograft recipient blood cell chimerism by a novel technique combining PCR and flow cytometry.

A new technique, the PCR-flow assay is described that has allowed for the serial identification and quantitation of discrete mononuclear cell subsets of donor (or recipient) bone marrow derived cells in cadaver kidney transplant recipients infused postoperatively with donor vertebral body bone marrow cells. With fixed permeabilized cells in flow cytometry the amplification power of the polymerase chain reaction (PCR), using fluorescent-labeled primers to identify single copy HLA class II DRbeta1 genes of either donor or recipient origin, is combined with multi-color fluorochrome-labeled CD epitope-specific monoclonal antibodies. The details of the methodology are described; these support the utility of the assay. Initial observations were made on the chimeric makeup of the peripheral blood as well as iliac crest bone marrow between six months and one year posttransplantation in recipients serially followed weekly and then monthly, concomitantly compared with a control group of stable kidney transplant recipients using similar therapeutic protocols, who did not receive cadaver bone marrow. Several findings are of note. In 14 recipients of two bone marrow infusions totalling a mean of 6.29+/-2.18x10(10) cells, donor CD34 positive (+) (immature) cells were fourteen times as numerous in peripheral blood six months postoperatively as in six recipients given half as many bone marrow cells in one infusion (averaging 3.02+/-0.5x10(10)). These donor CD34+ cells unexpectedly averaged 36+/-7% of the total (donor plus recipient) CD34+ subset counted. Moreover, iliac crest bone marrow aspirates contained an average of thirteen times this number of CD34+ cells than in the peripheral blood, supporting the notion of engraftment. Of additional interest, between six months and one year posttransplant although no donor cells could be detected in peripheral blood of the controls there was an identifiable presence of donor CD34+ cells in their iliac crest bone marrow, albeit 10-fold less than the marrow-infused patients. In the clinical follow-up, although there were three unrelated mortalities, there were no additional kidney losses with current serum creatinine concentrations averaging 1.3+/-0.06 mg/dl. In conclusion, the PCR-flow assay presents the possibility of identifying discrete subsets of donor or recipient cells that may have an immunoregulatory function.

Stomatococcus Mucilaginosus Meningitis in a Patient with Acute Lymphoblastic Leukemia

Stomatococcus Mucilaginosus Meningitis in a Patient with Acute Lymphoblastic Leukemia

ACQUIRED HYPOPROTHROMBINAEMIA AND LUPUS ANTICOAGULANT: RESPONSE TO STEROID THERAPY

An iliac crest bone marrow aspiration in a 24-year-old man was followed by severe haemorrhage into the iliopsoas muscle. A lupus anticoagulant and severe hypoprothrombinaemia, as well as clinical and laboratory pointers to suggest the presence of a systemic lupus erythematosus-like syndrome, were demonstrated. Therapy with prednisone was commenced following recurrent severe epistaxis. His prothrombin time, activated partial thromboplastin time and prothrombin activity improved promptly and his bleeding ceased. The lupus anticoagulant is commonly encountered in the laboratory, but acquired hypoprothrombinaemia is extremely rare. The condition is reviewed and its treatment discussed.

Bronchoscopy and Bone Marrow Examinations: An Efficient Strategy to Establish the Diagnosis of Small Cell Carcinoma of the Lung

The utility of various diagnostic procedures, including fiberoptic bronchoscopy and bone marrow aspirate/biopsy for small cell carcinoma, was examined in 150 patients. From a chart review of the first 100 patients, 55 required an invasive procedure such as mediastinoscopy or thoracotomy to establish the tissue diagnosis. In 50 consecutive prospective patients, only four required an invasive procedure for tissue confirmation. Eighteen of the 50 prospective patients had positive iliac crest bone marrow aspirates or biopsies diagnostic for small cell carcinoma. Fiberoptic bronchoscopy was nondiagnostic in 15 prospective patients, but the bone marrow examination was diagnostic in 12 of this subgroup. The combination of fiberoptic bronchoscopy and bone marrow examination in patients with centrally located small cell carcinoma greatly reduced the need for more expensive and invasive diagnostic procedures.

Flow cytometry of DNA content in human bone marrow: a critical reappraisal

Because cytokinetic studies of the human bone marrow aspirate as a prognostic factor and as a monitor of drug perturbation are frequently inconsistent, we investigated reproducibility of DNA distribution measured by flow cytometry of DNA content in patients with morphologically normal bone marrow. In 15 patients, correlation was noted between DNA distributions simultaneously obtained on right and left iliac crest bone marrow aspirates (r = .588), although considerable variation in individuals was encountered. Much better reproducibility (r =.879) was achieved using bilateral core biopsy of bone marrow in these same patients. In 60 samples, comparison of DNA distribution between bone marrow aspirate and simultaneously obtained biopsy revealed higher relative proportions of S and G2 + M phase cells in biopsies (p < 0.001), suggesting peripheral blood contamination of aspirate material. Brisk shaking of biopsy specimens in saline expelled a representative sample in the supernatant that could be subjected to simultaneous cytomorphological and cytokinetic analysis. To improve reproducibility of DNA content determinations in normal human bone marrow, bone marrow biopsy should be utilized.