[The morphology features of bone marrow in the prefibrotic-early primary myelofibrosis].

OBJECTIVE: To compare the morphologic features of bone marrow (BM) between the prefibrotic-early primary myelofibrosis (PMF) and essential thrombocythaemia (ET). METHOD: Seven cases of prefibrotic-early PMF were selected and analyzed. Based on the diagnostic standard of prefibrotic-early PMF by WHO, BM aspirate smears, trephine biopsy sections and imprints of 156 uncertain ET cases conducted simultaneously were recruited into this study, the BM morphologic features between the prefibrotic-early PMF and ET groups were analyzed. The morphological difference in 22 cases of prefibrotic-early PMF and 27 ET were compared between the JAK2V617F mutation positive and negative groups. RESULTS: Of the 156 uncertain ET cases, it was reclassified 61 prefibrotic-early PMF (34 MF-0, 27 MF-1), 12 PMF and 83 ET. The platelet count and LDH level in MF-1 group were obviously higher than that of ET group (P < 0.05). The blast percentage of BM smear in MF-1 group was also higher than that of ET group (P < 0.05). As to BM section, cases with increased nucleated cells (granulocyte), compact megakaryocytic cluster, megakaryocyte near bone trabecula, cloud-like megakaryocyte, small bare nucleus of megakaryocyte and large ball-like megakaryocyte in MF-0 and MF-1 group were significantly higher than that of ET group (all P < 0.05), cases with megakaryocytic cluster of various size in MF-1 group were significantly higher than that of MF-0 and ET groups (P < 0.05). The JAK2V617F mutation rate in prefibrotic-early PMF and ET groups were 54.5% and 48.1%, respectively. Hb level in JAK2V617F mutation positive group was obviously higher than the negative group (P < 0.05), no special change with megakaryocytic morphology was found between the positive and negative groups. CONCLUSION: Morphology of BM section, especially megakaryocytic morphologic characteristics are the main basis in distinguishing prefibrotic-early PMF from ET. The importance of morphologic index were megakaryocytic cluster with various size, cloud-like megakaryocyte, large ball-like megakaryocyte, increased nucleated cells (granulocyte), small bare nucleus, megakaryocyte near bone trabecula and compact megakaryocytic cluster in order. JAK2V617F mutation provides no specific effect on the megakaryocytic morphology.

[Value of imprint in bone marrow morphological examination].

OBJECTIVE: To explore the role of bone marrow (BM) imprint in the diagnosis of hematological diseases. METHODS: Between January 2002 and June 2008, a total of 3024 cases with BM smears, imprints and sections conducted simultaneously were recruited. There were 1667 males and 1357 females with a median age of 55 years old (range: 7 to 92). The cellularity on imprint and smear was evaluated with the standard cellularity on BM section. With the integrative diagnosis (including all examinations and clinical outcomes) as the standard, the diagnostic accuracy of hematological diseases were compared between BM imprint, smear and section groups. Another 79 cases of lymphoma and 114 cases of plasma cell myeloma (PCM) were selected for a correlation analysis of tumor cell infiltration patterns. RESULTS: BM imprint contained hematopoietic and non-hematopoietic regions and cells retained integrated structure. The cellularity evaluation by imprint was superior to smear overall. In BM imprint group, the diagnostic accuracy for hypersplenism (n = 130), metastatic carcinoma (n = 67), refractory anemia with excess blasts, myeloproliferative neoplasm (n = 174), and PCM (n = 94) were better than smear group (96.9% vs 80.7%, 91.0% vs 76.1%, 92.6% vs 81.5%, 92.5% vs 76.4%, and 97.8% vs 92.6% respectively, all P < 0.05); And the diagnostic accuracy for megaloblastic anemia (n = 69), acute myeloid leukemia (n = 104), refractory cytopenia with unilineage dysplasia (n = 15), refractory cytopenia with multilineage dysplasia (n = 22), and lymphoplasmacytic lymphoma (n = 12) were higher than biopsy section group (100% vs 84.0%, 91.3% vs 74.0%, 86.7% vs 60.0%, 90. 9% vs 72.7%, and 66.6% vs 50.0% respectively, all P < 0.05); And the diagnostic accuracy for myelodysplastic/myeloproliferative neoplasm (n = 26) was higher than smear group (76.3%, P < 0.05) and biopsy section group (78.2%, P < 0.05). Excellent correlations existed between BM imprint and section of the patients with lymphoma or with PCM (r = 0.90, r = 0.78, both P < 0.05). CONCLUSIONS: BM imprint contains the characteristics of both smear and section. BM imprint is superior to smear for an evaluation of cellularity. And it is also better than section for an analysis of cytological changes.

[Improvement of Phi bodies stain and its clinical significance].

The aim of this study was to improve the dyeing method of hydroperoxidase (HPO), to analyze the morphologic features of Phi bodies and to evaluate the clinical application of this method. 128 bone marrow or peripheral blood smears from patients with myeloid and lymphoid malignancies were stained by improved HPO staining. The Phi bodies were observed with detection rate of Phi bodies in different leukemias. 69 acute myeloid leukemia (AML) specimens were chosen randomly, the positive rate and the number of Phi bodies between the improved HPO and POX stain based on the same substrate of 3, 3'diaminobenzidine were compared. The results showed that the shape of bundle-like Phi bodies was variable, long or short. while the nubbly Phi bodies often presented oval and smooth. Club-like Phi bodies were found in M(3). The detection rates of bundle-like Phi bodies in AML M(1)-M(5) were 42.9% (6/14), 83.3% (15/18), 92.0% (23/25), 52.3% (11/21), 33.3% (5/15) respectively, and those of nubbly Phi bodies were 28.6% (4/14), 66.7% (12/18), 11.1% (3/25), 33.3% (7/21), 20.0% (3/15) respectively. The detection rate of bundle-like Phi bodies in M(3) was significantly higher than that in (M(1) + M(2)) or (M(4) + M(5)) groups. The detection rate of nubbly Phi bodies in (M(1) + M(2)) group was higher than that in M(3) group. In conclusion, after improvement of staining method, the HPO stain becomes simple, the detection rate of Phi bodies is higher than that by the previous method, the positive granules are more obvious, and the results become stable. This improved method plays an important role in differentiating AML from ALL, subtyping AML, and evaluating the therapeutic results.

Novel distribution pattern of fibrinolytic components in rabbit tissues extract: a preliminary study.

OBJECTIVE: The purpose of this work was to investigate the distribution pattern of fibrinolytic factors and their inhibitors in rabbit tissues. METHODS: The components of the fibrinolytic system in extracts from a variety of rabbit tissues, including tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasminogen (Plg), plasmin (Pl) and alpha(2) plasmin inhibitor (alpha(2)PI), were determined by colorimetric assay. RESULTS: The tissue extracts in renal, small intestine, lung, brain and spleen demonstrated strong fibrinolytic function, in which high activity of tPA, Plg and Pl was manifested; whereas in skeletal muscle, tongue and stomach, higher activity of PAI-1 and alpha(2)PI showed obviously. Also excellent linear correlations were found between levels of tPA and PAI-1, Pl and alpha(2)PI, Plg and Pl. In related tissues, renal cortex and renal marrow showed distinctly higher activity of tPA and lower activity of PAI-1, with the levels of Plg and Pl in renal cortex being higher than those in renal marrow, where the alpha(2)PI level was higher than that in renal cortex. Similarly, the levels of tPA, Plg and Pl in small intestine were higher than those in large intestine, but with respect to PAI-1 and alpha(2)PI, the matter was reverse. In addition, the fibrinolytic activity in muscle tissue was lower, however, the levels of tPA, Plg, and Pl in cardiac muscle were obviously higher than those in skeletal muscles, and the levels of PAI-1 and alpha(2)PI were significantly lower than those in skeletal muscle. CONCLUSION: Our data demonstrate that a remarkable difference of the fibrinolytic patterns exists in rabbit tissues, which has probable profound significance in understanding the relationship between the function of haemostasis or thrombosis and the physiologic function in tissues.

Clinical study of plasma thrombomodulin detection.

The purpose of this study was to investigate the clinical value of plasma thrombomodulin (PTM) in different diseases or in different severity or complications of diseases, PTM in 979 patients and 60 healthy controls was determined by ELISA method. The results showed that the PTM level in the control group was 20.40 +/- 7.72 microg/L, there was no difference in sex and ages. In chronic primary glomerular disease, the PTM level in chronic renal failure (CRF) group was higher than that in non-CRF group (P < 0.01). PTM level > 70 microg/L was defined as its positive criterion. The sensitivity, specificity and positive predictive value in PTM were 85.7%, 82.4% and 77.8% respectively. The PTM level in septemia group was higher than that in non-septemia group (P < 0.01), the sensitivity, specificity and positive predictive value were 86.6%, 89.5% and 76.5% respectively (> 50 microg/L as its positive criterion). With respect of multiple trauma, the PTM level in multiple organ failare (MOF) group was higher than that in non-MOF group (P < 0.01), while the sensitivity, specificity and positive predictive value were 77.8%, 77.3% and 73.7% respectively (> 40 microg/L as its positive criterion). For systemic lupus erythematosus (SLE), the PTM level in the patients with albuminuria was higher than that in the patients without albuminuria (P < 0.01), and the sensitivity, specificity and positive predictive value were 77.8%, 92.3% and 93.3% respectively (> 35.54 microg/L as its positive criterion). For diabetes, the PTM level in complication group was higher than that in group without complications, the sensitivity, specificity and positive predictive value were 53.4%, 97.1% and 98.6% respectively (> 35.54 microg/L as its positive criterion). The PTM level in microangiopathy group was higher than that in macroangiopathy group (P < 0.01). The sensitivity, specificity and positive predictive value were 71.2%, 97.1% and 97.9% respectively. Acute leukemia (AL) and multiple myeloma (MM) had higher PTM level and PTM level was extremely high when renal failure developed (P < 0.01). As compared the acute stage with the restoration stage in stroke, pre-chemotherapeutics with post-chemotherapeutics in AL and MM, and pre-operation with post-operation in cancer, the PTM level was connected with clinical development. The PTM level in the patients with microangiopathy was higher than that in the patients with macroangiopathy (P < 0.01). The defined PTM level was higher than its normal upper limit as PTM positive criterion in microangiopathy diseases, the sensitivity, specificity and positive predictive value were 77.7%, 71.2% and 75.6% respectively. It is concluded that PTM level is a good criterion in evaluating the microangiopathy, and PTM is also a valuable indicator in prediction or assessment of the severity of diseases, or evaluation of therapeutic effectiveness.

Plasma level of thrombomodulin is an early indication of pancreatic necrosis in patients with acute pancreatitis.

BACKGROUND: The potential to predict pancreatic necrosis within the first 48 h by using plasma soluble thrombomodulin (sTM) in 104 patients with acute pancreatitis (AP) was analyzed in a prospective 5-year investigation performed at a single institution. METHODS: According to Balthazar CT grade, pancreatitis was classified as no necrosis in 72 patients, one-third necrotic in 18 patients, one-half necrotic in 10 patients and more than one-half necrotic in 4 patients. Blood was collected at the first 48 hours after the onset of pain and analyzed for sTM. RESULTS: In the healthy volunteers, plasma levels of TM were 16.49+/-5.24 microg/L. By comparison, the mean plasma levels of TM in each group of pancreatitis patients were as follows: CT grade A group, 34.21+/-10.73 microg/L; CT grade B group, 36.18+/-12.50 microg/L; CT grade C group, 49.39+/-18.38 microg/L; CT grade D group, 114.46+/-39.44 microg/L; CT grade E group, 100.22+/-15.97 microg/L (p<0.01). And for the patients, the Pearson correlation coefficient between the CT grade and TM values was 0.784 (p<0.01). No necrosis group, 39.22+/-13.75 microg/L; one-third necrotic group, 71.44+/-18.02 microg/L; one-half necrotic group, 123.50+/-28.57 microg/L; more than one-half necrotic group, 129.00+/-33.28 microg/L (p<0.01); And for the patients, the Pearson correlation coefficient between the degree of necrosis and TM values was 0.888 (p<0.01). ROC analysis indicated the area under the ROC curve (AUC +/- SE) for sTM was 0.949+/-0.020, clearly supportive of the high accuracy of this index in predicting the necrosis of AP. CONCLUSION: Plasma soluble thrombomodulin (sTM) is a potential marker to predict pancreatic necrosis within the first 48 h, and further investigation in a multicentre study is necessary.

[Application of bone marrow biopsy imprint in evaluating cellularity].

OBJECTIVE: To study the value of bone marrow biopsy imprint in evaluating cellularity. METHODS: The bone marrow tissues were obtained by trephine biopsy from 272 patients, and then put on the slides to make the imprints. The imprints was stained by Wright-Giemsa method, and the bone marrow smears and imprints were examined simultaneously according to the bone marrow cellularity criteria. RESULT: In bone marrow cellularity, four grades (distinct decrease, extreme decrease, distinct increase, and extreme increase) were significantly higher in bone marrow imprints than those in bone marrow smears (P <0.05), but there was no significantly differences between bone marrow imprints and sections (P >0.05). Using bone marrow sections as standard, in cellularily decreasing samples, the consistent rate of bone marrow imprints and smears were both high (84.4% and 97.9%), in the group of the normal and increased cellularity, the consistent rate of the bone marrow imprints (84.4% and 97.7%) was significantly higher than that in smears (60% and 64%, P<0.01). The sensitivity, specificity, Youden index, positive predictive value and positive likelihood rate of bone marrow imprints were all higher than those of the smears. Using the bone marrow sections as gold standard, in 124 cases with decreased cellularity in smears, the positive diagnosis rate for aplastic anemia and dyshaematopoiesis based on bone marrow imprints was 37.1% with a false positive rate of 7.3% which was lower than that of the bone marrow smears (false positive rate of 29.8%, P<0.01). CONCLUSION: To evaluate bone marrow cellularity, bone marrow imprint is better than bone marrow smear. The combination of the two examinations can make the diagnosis more convenient and quicker.

Morphological study on the megakaryocytes with nuclear extrusion and nucleocytoplasmic separation in four cases.

To investigate the morphological changes of megakaryocytes with nuclear extrusion and nucleocytoplasmic separation, the morphological characteristics of megakaryocytes in peripheral blood films, bone marrow smears, and bone marrow biopsies from 4 newly diagnosed patients with primary myelofibrosis (PMF), myelodysplastic syndrome (MDS), myeloblastic leukemia with maturation (M(2)) and erythroleukemia (M(6)) were studied by using light microscope. The results showed that many kinds of dysmegakaryocytes were observed in bone marrow smears of 4 cases, while in case A (PMF) and case D (M(6)) micromegakaryocytes were ripped apart; in case B (MDS) and case C (M(2)) megakaryocytes were accompanied by nuclear extrusion or nucleocytoplasmic separation, and their bodies were large or giant, the part of nucleus separated from their body and little cytoplasm remained as micromegakaryocytes. The nucleocytoplasmic separation could be displayed by immunocytochemistry stain. It is concluded that the phenomenon of nuclear extrusion and nucleocytoplasmic separation in megakaryocytes suggested the process that dispersed multinuclear releasing towards surround or even totally left the cell body during the megakaryocyte maturation. It also showed that the micromegakaryocytes may be the result of nucleocytoplasmic separation or splittings from multi-separated nucleus.

[Detection of soluble Apo-1/Fas in plasma, pleural and ascites fluid of malignant tumor patients and its clinical significance].

OBJECTIVE: To study the changes of soluble Apo-1/Fas levels in plasma, pleural and ascites fluid of malignant tumor patients and to evaluate their clinical significance. METHODS: The soluble Apo-1/Fas levels were measured by enzyme-linked immunosorbent assay (ELISA) in the plasma of 157 malignant tumor patients and 25 normal controls as well as in the pleural and ascite fluids of 129 patients with various diseases. RESULT: The plasma soluble Apo-1/Fas levels in acute and chronic leukemia and multiple myeloma were significantly higher than those in normal controls (P <0.05). The plasma soluble Apo-1/Fas levels in chronic myeloid leukemia and chronic lymphocytic leukemia were significantly higher than those in acute myeloid leukemia and acute lymphocytic leukemia, respectively (P <0.05). After chemotherapy, the plasma soluble Apo-1/Fas levels in complete remission group were distinctly decreased(P <0.05),whereas the levels in no remission and recurrence groups remained high. Compared with normal controls, the plasma soluble Apo-1/Fas levels in solid tumors were significantly increased (P <0.01), and the levels in metastasis cancers were significantly higher than those in non-metastasis cancer (P <0.0 1). Simultaneously the levels in remission cancer patients after operation and radiotherapy were distinctly lower than those before treatment(P <0.01), but were significantly increased in recurrence cancer patients (P <0.01). The soluble Apo-1/Fas levels in pleural and ascites fluid of malignant tumors were significantly higher than those in tuberculous effusions and transudates. CONCLUSION: The soluble Apo-1/Fas levels in plasma, pleural and ascites fluid of malignant tumor patients are markedly increased, which might be associated with the progress of cancers. The changes of soluble Apo-1/Fas levels may be useful for understanding the pathologic process of cancers and to differential diagnosis of various pleural and ascites fluids.

[Detection of thrombomodulin in both plasma and tissue extracts of cancer patients and its clinical significance].

OBJECTIVE: To study the changes of thrombomodulin(TM) in both plasma and tissue extracts of cancer patients for evaluating its clinical significance. METHODS: Plasma TM levels were measured by enzyme-linked immunosorbent assay(ELISA) in plasma of 188 cancer patients and in 24 cancer tissue extracts including their adjacent normal tissue. RESULTS: The plasma TM levels both in cancer patients and in metastasis patients were significantly higher than that in controls [(33.47+/-14.25) microg/L/ (41.68 +/-16.96) micro/L, compared with (20.40+/-7.22) microg/L, P<0.01]. The plasma TM levels in cancer patients after operation decreased obviously [(18.45+/-9.96) microg/L, compared with (28.29+/-11.74) microg/L,P<0.01]. Whereas, the plasma TM levels in patients with recurrence and metastasis after operation increased obviously [(34.50+/-12.57 micro/L]. The plasma TM levels in metastasis of lung cancers, gastric cancers and pancreatic cancers were significantly higher than that in non-metastasis (P<0.05 approximate, equals 0.01) respectively, but no significant differences were found between controls and non-metastasis cancers including gastric cancers, pancreatic cancers, nasopharyngeal cancers, large intestine cancers and laryngeal cancers (P>0.05). The TM levels in cancer tissue extracts were significantly lower than that in their adjacent normal tissue extracts [(647.71+/-317.51)) microg/L,compared with (1455.63+/-772.22) microg/L,P<0.01]. On the contrary, the plasma TM levels in these cancers were higher than that in controls. CONCLUSION: The rise of plasma TM levels in cancer patients is associated with metastasis and diffusion of cancers. The TM levels can be used as an sensitive index for judging progression and metastasis of cancers.

Dynamically monitoring tissue factor and tissue factor pathway inhibitor following secondary brain injury.

OBJECTIVE: To study the altering rule of coagulation function at molecular level in patients with secondary brain injury (SBI). METHODS: Tissue factor (TF) and tissue factor pathway inhibitor (TFPI) were studied in 32 patients 1, 2, 3 and 7 days after craniocerebral injury. Repeated cranial CT scans and platelet counts were made simultaneously. Same measurements were done in 30 normal adults except CT scan. RESULTS: No obvious difference was found in age, sex and platelet count between the injured and the normal groups. TFPI/TF decreased markedly in the first week after injury in patients with SBI, but only decreased on the 7th day in the patients without obvious SBI. For the patients who developed delayed intracranial hematoma (DIH) or hematoma enlargement, TF rose only 1 and 2 days after injury, but TFPI had a tendency to rise again after a fall on the 3rd day. For those patients who developed no DIH, TF rose all the time within the 1st week. CONCLUSIONS: Decrease of TFPI/TF for a long time, especially within 3 days after injury, may be one of the most important reasons for SBI. High expression of TF for a relative short time and increase of TFPI after a fall within 3 days may be one of the important reasons for DIH or hematoma enlargement.

[Detection and clinical significance of plasma urokinase-type plasminogen activator and its soluble receptor in patients with multiple myeloma].

OBJECTIVE: To study the plasma levels of urokinase-type plasminogen activator(u-PA) and its soluble receptor(suPAR )in patients with multiple myeloma(MM) and to evaluate their clinical significance. METHODS: Plasma u-PA and suPAR levels in 34 MM cases were measured with enzyme- linked immunosorbent assay (ELISA). The changes of plasma u-PA and suPAR levels in 6 MM cases were observed in succession before and after chemotherapy. RESULT: The plasma u-PA and suPAR levels of MM patients were significantly higher than those of controls. The plasma u-PA and suPAR levels in the progress period was significantly higher than those in the stable period of MM patients as well as in controls (P<0.01), whereas there were no significant difference between the stable period of MM patients and controls (P>0.05). Among 6 cases,the plasma u-PA and suPAR levels after chemotherapy were significantly lower than those before chemotherapy (P<0.05). MM patients with tumor cells >20% in bone marrow smear had higher levels of plasma u-PA and suPAR than those with tumor cells <19% (P<0.05 P<0.01). The plasma u-PA and suPAR levels were positively correlated with the levels of serum globulin and the percentage of tumor cells in bone marrow,but negatively correlated with the levels of serum albumin. CONCLUSION: Plasma u-PA and suPAR levels can serve as an index for clinical staging and assessing the therapeutic effect in MM patients.