Hemostatic/fibrinolytic protein changes in C3H/HeN mice infected with Rickettsia conorii--a model for Rocky Mountain spotted fever.

Changes in plasma hemostatic and fibrinolytic proteins were determined during courses of a murine model of fatal and non-fatal Rocky Mountain spotted fever. C3H/HeN mice were infected with Rickettsia conorii and coagulation and histopathologic studies were performed at prescribed periods of time. A significant decrease in plasma factor VIII activity and rise in plasma factor V procoagulant activity correlated with a fatal infection. Factor VII levels were unchanged; factor XI levels dropped early in the course in the lethally infected animals, but returned to normal. Factor XII, high molecular weight kininogen, and prekallikrein levels were unchanged by the sublethal infection. Prekallikrein levels fell during the lethal infection. Antithrombin concentrations were decreased significantly in all animals, but plasma plasminogen levels did not change in either group of animals. Nonocclusive thrombi were microscopically observed rarely and only in animals surviving a sublethal infection. A fall in tissue plasminogen activator activity and a rise in plasminogen activator inhibitor activity highly correlated with a lethal outcome. Lethal infection with R. conorii is associated with primary endothelial cell injury resulting in decreased tissue plasminogen activator and increased plasminogen activator inhibitor.

T-gamma gene rearrangement and CMV mononucleosis.

A clonal T-gamma rearrangement was found in peripheral blood and bone marrow in a 57-year-old female who presented with 6-week history of fevers, night sweats, and weight loss. Splenomegaly, hemolytic anemia, atypical lymphocytosis, a marrow lymphoid aggregate, and elevated LDH had suggested lymphoproliferative disease. However, IgM serology for cytomegalovirus (CMV) was positive. With observation alone, her clinical features improved over 4 weeks with normalization of the blood count and disappearance of CMV viremia and the aberrant T-gamma clone. Acute CMV infection may mimic lymphoproliferative disease. T-gamma gene rearrangement may be part of the immune response to CMV infection and is not specific to lymphoid neoplasia.

Griscelli syndrome: rare neonatal syndrome of recurrent hemophagocytosis.

Griscelli syndrome (GS) is a rare inherited disease characterized by immunodeficiency and partial albinism. The microscopic findings of the skin and hair are highly suggestive of the disease. The GS locus colocalizes on chromosome 15q21 with the myosin-Va gene (MYO5a), and mutations have been identified in few patients. We describe a 2-month-old Hispanic girl with severe pancytopenia secondary to hemophagocytosis. Even though a mutation at the Griscelli locus had not been identified, her clinical features and outcome were typical of GS. The purpose of this article is to alert physicians to the association between GS and hemophagocytosis. We suggest that GS should be considered in infants with hemophagocytosis because the features of partial albinism can be subtle. The relevant literature is summarized.

CD13-positive anaplastic large cell lymphoma of T-cell origin--a diagnostic and histogenetic problem.

The expression of myelomonocytic-associated antigens in anaplastic large cell lymphomas (ALCLs), particularly those presenting in extranodal sites, can make their distinction from extramedullary myeloid cell tumors (EMCTs) or histiocytic tumors problematic. Yet, this distinction is clinically significant because of its therapeutic and prognostic implications. Herein, we describe a case of extranodal anaplastic lymphoma kinase-positive CD30-positive ALCL of T-cell origin in a 12-year-old boy, which was initially called an EMCT because of the expression of CD13 and HLA-DR detected by flow cytometry and the absence of other T-cell-related surface markers. However, the detection of cytoplasmic CD3 by flow cytometry prompted further studies. The tumor was composed of large cells with abundant slightly eosinophilic vacuolated cytoplasm and ovoid or reniform nuclei with a few small nucleoli. Using immunohistochemistry, the tumor was positive for CD45, CD30, CD45RO, and CD43 with a strong cytoplasmic and nuclear anaplastic lymphoma kinase stain. The tumor cells showed a T-cell clonal genotype. Electron microscopy revealed no ultrastructural features of myelomonocytic or histiocytic origin. The patient responded well to the chemotherapy and was in complete remission for 10 months at the time of submission of this manuscript. Review of the literature showed inconsistencies regarding the diagnosis, nomenclature, and, therefore, treatment and prognosis of these tumors. In addition, the CD13 expression in ALCL raises some histogenetic questions and may indicate origin from a pluripotent stem cell, misprogramming during malignant transformation, or a microenvironmental effect on lymphoid cell expression of surface antigens. Therefore, ALCL should be considered in the differential diagnosis of EMCTs or histiocytic tumors, particularly when surface marker lineage assignment is ambiguous.

P53 overexpression in bone marrow biopsies in refractory anemia and aplastic anemia: impact of antibody selection.

There is a growing interest in studying cell cycle and apoptosis in the myelodysplastic syndromes (MDS). p53 has been a major target of several studies. We examined the impact of antibody selection on p53 overexpression in bone marrow (BM) biopsies of 28 patients with refractory anemia (RA) in addition to 10 cases of aplastic anemia (AA) using three antibodies DO-7, PAb 1801, and PAb 240. DO-7 was positive in 68%, PAb 1801 in 18% and PAb240 in 4% of RA patients. All three antibodies were negative in AA. We conclude that antibody selection is an important variable in studying p53 in MDS regardless of the method of fixation or decalcification of BM trephine biopsies.

Stability of prothrombin time and activated partial thromboplastin time tests under different storage conditions.

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are common laboratory tests that are useful in the diagnosis of coagulation disorders and monitoring anticoagulant therapy. Recent expansions in the outreach laboratory services at our institution prompted us to investigate the shipping limitations for some tests, including PT and aPTT. Although we followed NCCLS guidelines for the collection of blood specimens, we observed falsely elevated PT and aPTT values due to the different storage conditions. The objective of this study is to determine the effect of conditions and duration of storage on PT and aPTT tests using plasma and whole blood samples, respectively. For this study, 36 plasma samples with normal and prolonged PT and aPTT were exposed to different storage conditions. Blood was centrifuged immediately and plasma was stored at room temperature (RT), refrigerated at 4 degrees C, or frozen at -20 degrees C. The samples were analyzed at 0 h and repeated at 6, 12 and 24 h under various conditions. Although statistically significant differences were observed for plasma samples for normal PT tests after 12 h at refrigerated and frozen storage conditions, the differences would not change the clinical interpretation of the results. On the other hand, samples stored refrigerated or at RT showed significant differences for aPTT at 24 h. These differences would change clinical interpretation, especially for samples with normal or near normal aPTT times. Interestingly, aPTT was significantly higher for samples stored frozen when compared to refrigerated and RT conditions at 6 h. Similar patterns were also observed on ten whole blood samples with normal PT and aPTT values. In conclusion, either plasma or whole blood samples can be accepted for PT testing up to 24 h and for aPTT testing up to 12 h only, when transported either at RT or at 4 degrees C.

Fanconi anemia: myelodysplasia as a predictor of outcome.

The adverse potential of the development of myelodysplastic syndrome (MDS) in Fanconi anemia (FA) was examined in a retrospective study of 41 FA patients who had bone marrow morphology and chromosomes reviewed by a single group. Thirty-three patients had adequate cytogenetic studies, and 16 (48%) had one or more abnormal studies: nine initially, and seven more on follow-up. Cytogenetic clonal variation was frequent, including disappearance of clones, clonal evolution, and appearance of new clones. The estimated five-year survival with a cytogenetic clone is 0.40, compared to 0.94 without a clone. Morphologic myelodysplasia (MDS), independent of a cytogenetic clone, was found in 13/41 patients (32%). The estimated five-year survival with MDS is 0.09, versus 0.92 without MDS. Leukemia developed in three patients whose initial cytogenetic clones prior to leukemia were t(1;18), t(5;22) and monosomy 7; the one with t(1;18) also had MDS. Our results focus on marrow morphology, and suggest that morphologic MDS may be more important than classical cytogenetics in prediction of an adverse outcome.

Bilateral breast MALT lymphoma: a case report and review of the literature.

Breast lymphoma is a rare disease. Both primary and secondary breast involvement have been reported. Most primary breast lymphomas are high-grade malignant neoplasms, mainly large cell and Burkitt type. Low-grade lymphomas of the breast, particularly mucosa-associated lymphoid tissue (MALT) lymphomas, have been exceedingly rare. In this report we present a patient with bilateral breast involvement by MALT lymphoma. Our patient developed localized MALT lymphoma in both breasts in a sequential fashion. She was treated with bilateral lumpectomy, followed by radiation therapy to both breasts. The patient is alive and well more than 1 year after therapy with no recurrence. We believe this is the first such case described in detail in the literature.

Double esterase staining of the bone marrow contributes to lineage identification in a case of minimally differentiated acute myeloid leukaemia (AML M0).

Minimally differentiated acute myeloid leukaemia (AML-M0) may pose difficulty in diagnosis since morphological criteria alone are not reliable. Other studies such as electron microscopy, immunocytochemistry and surface marker analysis are needed. The role of cytochemistry has been limited to the negative staining of blasts for Sudan Black B and myeloperoxidase. An abnormal morphology and cytochemistry of bone marrow maturing cells may indicate the myeloid nature of acute leukaemia. In this case of AML-M0, increased numbers of maturing and mature bone marrow granulocytic cells staining simultaneously for both specific and non-specific esterase (double esterase) are described. In acute leukaemia, this abnormal cytochemical finding seems to be specific for myeloid leukaemia and may be used as supplementary evidence of myeloid differentiation of morphologically undifferentiated blasts in cases of AML-M0.

Hemostatic changes in Rocky Mountain spotted fever and Mediterranean spotted fever.

Rocky Mountain spotted fever and Mediterranean spotted fever are rickettsial infections primarily of endothelial cells that normally have a potent anticoagulant function. As a result of endothelial cell infection and injury, the hemostatic system is perturbed and shows changes that vary widely from a minor reduction in the platelet count (frequently) to severe coagulopathies, such as deep venous thrombosis and disseminated intravascular coagulation (rarely). Changes favoring a hypercoagulable state include endothelial injury and release of procoagulant components, activation of the coagulation cascade with thrombin generation, platelet activation, increased antifibrinolytic factors, consumption of natural anticoagulants, and possibly high levels of coagulation-promoting cytokines. Yet, most studies have been performed on endothelial cell cultures that provide nonphysiologic, reductionistic, experimental conditions. The lack of flow, platelets, and WBCs makes these experiments far from simulating the response of endothelial cells in the human body. Coagulopathies and thrombotic events should be considered as potential complications of severe Rocky Mountain spotted fever and Mediterranean spotted fever.

Double esterase staining and other neutrophilic granule abnormalities in 237 patients with the myelodysplastic syndrome studied by the cancer and leukemia group B.

We investigated double (specific and nonspecific) esterase (DE) staining in marrow cells of 237 patients with the myelodysplastic syndromes (MDS). Additional abnormalities of neutrophilic granules were examined cytochemically and immunocytochemically for myeloperoxidase activity and antigen elastase, lactoferrin and CD15 granule-membrane glycoproteins. Abnormal DE staining (>/=3% of all nucleated marrow cells) was present in 27% of patients with no difference among different MDS subtypes. However, the prevalence of high abnormal DE staining (>/=10%) was significantly lower in refractory anemia with excess blasts in transformation (1%) compared to other MDS subtypes (12-15%; p = 0.004). The prevalence of other granule abnormalities was not statistically different in the DE normal and DE abnormal groups. Abnormal DE staining is relatively common among all MDS subtypes. High DE staining may identify a subgroup of patients with a lower grade MDS.

Surface marker abnormalities in myelodysplastic syndromes.

BACKGROUND AND OBJECTIVE: The myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with a variety of abnormalities of mature and maturing cells, including surface antigen abnormalities. Granulocytes and monocytes function as members of the immune system. Surface antigens serve as biological sensors allowing various cells to interact with different stimuli. Abnormalities of surface antigens may be associated with defective cell function and may indicate a more severe or more advanced stage of the disease. INFORMATION SOURCES: The author has a great interest in bone marrow changes in MDS and has several previous publications in this field. In addition, relevant articles published since 1966 were retrieved using Medline of English literature and were included. STATE OF THE ART AND PERSPECTIVES: Several surface antigens in MDS have shown abnormal expression either in the intensity of fluorescence or the percentage of positive cells. These abnormalities include increased, decreased or lineage-aberrant expression. Abnormalities of several surface markers have prognostic significance. MDS patients with a low percentage of bone marrow cells expressing CD11b had a higher risk of evolution to acute myeloid leukemia and shorter survival compared to patients with more than 53% of marrow cells expressing CD11b (29 weeks versus 160 weeks). On the other hand, an increased percentage of bone marrow cells expressing early or immature markers, such as CD 13, CD33, CD34 and HLA-DR, has been associated with a worse outcome and with progression to a higher risk MDS or to acute myeloid leukemia. However, there are numerous discrepancies and inconsistencies in the literature when reviewing surface marker changes in MDS. These discrepancies may be related, at least in part, to the presence of an intracellular storage compartment of numerous surface antigens in the granulocytes and monocytes. Because of these storage pools, the techniques of preparing more mature granulocytes and monocytes, such as density gradient separation, and the interpretation of results must be carefully evaluated. Furthermore, various methods have been used to express abnormal results including percentage of positive or negative cells, fluorescent intensity (FI) of individual patients or a group of patients using a mean fluorescent channel (256 or 1024 channel mode), and finally the expression of FI as molecules of equivalent soluble fluorochromes or antibody binding capacities. Several mechanisms may be involved in the abnormal expression of surface antigens in MDS including defective granulopoiesis, defective intracellular storage pool, abnormal membrane of cytoplasmic granules, and the effect of high levels of marrow cytokines such as tumor necrosis factor alpha and transforming growth factor-beta. Standardization of the methods of preparing and studying mature and maturing granulocytes and monocytes in MDS has to be achieved in order to produce comparable results, thus allowing surface marker studies to be utilized as diagnostic and prognostic tools in MDS.

Significance of p53 overexpression in bone marrow biopsies from patients with bone marrow failure: aplastic anemia, hypocellular refractory anemia, and hypercellular refractory anemia.

Among patients with bone marrow failure, differentiating acquired aplastic anemia (AA) from hypocellular refractory anemia (hypo RA) can be a difficult and challenging task. Morphological, cytochemical, immunocytochemical, and cytogenetic studies may provide tools for discriminating between both entities. In addition, differences in the pattern of proliferation and apoptosis of bone marrow cells in AA and in the myelodysplastic syndrome have been reported. Because of the correlation between p53 and apoptosis, we examined the overexpression of p53 on bone marrow biopsies in RA and AA. Our study included 14 patients with hypo RA, 14 patients with hypercellular (hyper) RA, ten patients with classic acquired AA, and 37 hematologically normal individuals. p53 was overexpressed in eight (57%) hypo RA patients and 11 (79%) hyper RA patients. All normal individuals and patients with AA showed no overexpression of p53 in their marrow. These results were statistically significant:p < 0.01 (AA vs hypo RA), p<0.001 (AA vs hyper RA), while the difference between hypo RA and hyper RA was not statistically significant. We conclude that p53 overexpression in bone marrow biopsies is a valuable tool for studying bone marrow failure and may provide additional information to help differentiate hypo RA from acquired AA.

Deficiency of neutrophilic granule membrane glycoproteins in the myelodysplastic syndromes: a common deficiency in 216 patients studied by the Cancer and Leukemia Group B.

Previous studies on neutrophils in patients with the myelodysplastic syndromes (MDS) have indicated deficiencies in the contents of primary and secondary granules. However, the granule membrane remains virtually unstudied despite its essential role in the dynamic function of the cytoplasmic granules. In this study, we examined the membrane glycoproteins of primary and secondary granules of peripheral blood and/or bone marrow neutrophils using the monoclonal antibody H36/71 to CD15 glycoproteins. In addition, myeloperoxidase activity and antigen, elastase and lactoferrin were also studied using cytochemical and immunocytochemical stains. A total of 216 patients were included. Deficiencies of granule membrane glycoproteins were the most common, detected in 49%, followed by myeloperoxidase activity (17%), elastase (16%), myeloperoxidase antigen (9%), and lactoferrin (8%). Multiple deficiencies always included granule membrane deficiency. We conclude that granule membrane defects are common in MDS, may provide a common mechanism for multiple granule deficiencies, and may prove to be an additional abnormality associated with granulocyte dysfunction.

True histiocytic lymphoma: is it an entity?

Monocytes and macrophages are closely related in origin, structure and function. True histiocytic lymphoma is a neoplasm of phagocytic histiocytes and is treated as non-Hodgkin's lymphoma. Tissue involvement with myeloid leukemia, including monocytic leukemia, is called extramedullary myeloid cell tumor or granulocytic sarcoma and treated with antileukemic chemotherapy. Differentiating true histiocytic lymphoma from tissue involvement with monocytic leukemia is a rather impossible task using available morphologic, cytochemical, immunocytochemical and molecular methods. The two entities need to be unified probably more appropriately as extramedullary myeloid cell tumor of monocytic origin, and should therefore be treated as such.

Peripheral blood picture in primary hypocellular refractory anemia and idiopathic acquired aplastic anemia: an additional tool for differential diagnosis.

BACKGROUND AND OBJECTIVE: Hypoplastic myelodysplastic syndromes (MDS) are being reported with increasing frequency. Aplastic anemia (AA) needs to be differentiated from hypoplastic MDS particularly primary hypoplastic refractory anemia (PHRA) because of the impact on management and prognosis. This distinction may be morphologically difficult even with careful marrow examination which may provide insufficient material due to extreme hypocellularity. The value of peripheral blood (PB) parameters in making the distinction between AA and PHRA is not well studied. In this work, we attempt to examine peripheral blood findings as an additional tool for differentiating PHRA from acquired idiopathic AA. METHODS: PB findings in ten cases of PHRA, which are selected based on the following: less than 30% cellularity, multilineage dysplasia and/or clonal cytogenetic abnormality, are compared to ten cases of classic AA. The PB is examined for automated parameters, differential white cell count, morphologic changes in red cells, white cells, platelets, and the presence of circulating blasts, megakaryocytic fragments and micromegakaryocytes. RESULTS: AA patients tend to have lower platelet and monocytic counts and higher lymphocytic percentages. The following morphologic findings are seen only in PHRA but not in AA: hypochromic red cells, left shift, circulating blasts, hypersegmentation with long filaments, hypogranular, ring, and pelgeroid neutrophils, Dohle bodies, circulating micromegakaryocytes and megakaryocytic fragments. INTERPRETATION AND CONCLUSIONS: We conclude that careful examination of peripheral blood may provide sufficient information to allow for the distinction between PHRA and AA early in the course of the disease. Similarly, patients with classic AA who subsequently develop unusual blood findings during routine follow up should be suspected of having a clonal evolution which needs to be confirmed by marrow examination and cytogenetic analysis.

Hodgkin's disease coexisting with myelodysplastic syndrome prior to therapy: a case report and a review of the association of Hodgkin's disease with stem cell disorders.

The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are well-documented complications secondary to chemotherapy and radiation therapy for Hodgkin's disease (HD). The coexistence of primary MDS with HD prior to therapy is an extremely rare event which has been reported only once in the English literature. This is the second case of such combination. Both patients with MDS developed AML only 7 months after diagnosis and both died shortly after the initiation of treatment. Since these cases raise the possibility of a stem cell association with HD, we reviewed the literature for other stem cell disorders with similar association with HD prior to aggressive therapy. Four cases of stem cell disorders other than MDS were reported. These included two cases of aplastic anemia, one case of myeloid metaplasia with myelofibrosis, and one of polycythemia vera. Two of the four patients died, one of AML and the other of thrombocytopenia-related cerebral hemorrhage. The association between HD and stem cell disorders, although rare, may need to be investigated further.