The role of maternal serum C-reactive protein and white blood cell count in the prediction of chorioamnionitis in women with premature rupture of membranes.

This study was performed to evaluate the diagnostic performance of maternal serum C-reactive protein, maternal white blood cell (WBC), and neutrophil counts in the detection of histologic chorioamnionitis. One hundred and twenty six pregnant women after at least 28 weeks of gestation with premature rupture of membranes (PROM) were studied. Blood samples for C-reactive protein, WBC and neutrophil counts were taken at delivery. Placental histology was evaluated for histologic chorioamnionitis. Maternal and neonatal complications were observed. Among women with and without histologic chorioamnionitis, the maternal WBC and neutrophil counts were different (P<0.05) but the maternal serum C-reactive protein was not. Cutoff values for C-reactive protein, WBC, and neutrophil counts were 0.5 mg/dL, 15,000 cell/mm3, and 80 per cent, respectively. Sensitivity and specificity were 56 per cent and 58 per cent for C-reactive protein, 60 per cent and 63 per cent for WBC count, and 62 per cent and 54 per cent for neutrophil count, respectively. In conclusion, the maternal serum C-reactive protein, WBC, and neutrophil counts have poor diagnostic performance for histologic chorioamnionitis.

Clinical associations and prognostic significance of serum anti-p53 antibodies in Thai patients with hepatocellular carcinoma.

Mutations of the p53 gene have been reported to be of prognostic significance in hepatocellular carcinoma (HCC). However, the clinical associations and prognostic value of anti-p53 antibodies, known to be products of the host immune response to these mutations, have been controversial. Serum anti-p53 antibodies were measured in 121 Thai patients diagnosed with HCC using a specific enzyme-linked immunosorbent assay (ELISA) kit. The clinical/pathological characteristics of the patients were compared with respect to the presence of serum anti-p53 antibodies. Cox regression analysis was performed to assess factor interaction and association with survival. Anti-p53 antibodies were detected in 13.2% (16 of 121) of our patients. There were no differences between groups with regard to age, sex, viral markers (HBsAg or anti-HCV), severity of liver disease and tumor advancement. The median survival rates for patients positive and negative for anti-p53 antibodies were 4.0 and 3.0 months, respectively (p = 0.443, by log-rank test). Multivariate analysis demonstrated that an advanced Okuda stage, lack of therapy and presence of portal vein thrombosis were independent factors related to the prognosis of the patients. Nonetheless, the presence of anti-p53 antibodies did not constitute a predictive variable associated with a poorer prognosis. Serum assay of anti-p53 antibodies, although rapid and easily performed, may not be suitable as an alternative to molecular detection of mutations in assessing tumor advancement and prognosis of patients with HCC.

Erythropoietin level and hematologic parameters in healthy adults.

Since the major physiological control of erythropoiesis is related to the eryhtropoietin (EPO) level, correlating the EPO level with hematologic parameters in healthy adults, which constitutes an inexpensive and simple routine laboratory report, would be very useful. Two hundred healthy adult blood donors, 100 males and 100 females, aged between 17 and 60 years old were randomly chosen. The EPO reference range was determined by enzyme linked immunosorbent assay (ELISA) using a reagent kit from Research & Development Systems Inc. The hematologic values for reticulocyte and red blood cell parameters were assessed using the Technicon H*3 RTC, an automated blood cell analyzer. The EPO reference range in the studied population was 2.21-20.95 mU/ml. The correlations between the EPO level and hematologic parameters were between -0.302 (p < 0.01) and 0.294 (p < 0.01). The results suggested that there were none or low correlations between the EPO level and hematologic parameters in healthy adults. According to our results, these parameters could not be used to indicate the level of EPO in healthy adults.

P53 expression and polysomies of chromosome 9, 17 in head and neck cancer prognosis.

Sixty-nine cases of head and neck squamous cell carcinoma were examined by immunohistochemistry for p53 and chromosome in situ hybridization for chromosome 9 and 17 to determine the relationship between p53 expression and polysomies of chromosome 9 and 17 with the development of a second primary tumor as well as recurrence of primary tumor of head and neck squamous cell carcinoma. We found early expression of p53 in the normal and premaligant lesions adjacent to tumor which was associated with a gradual increase in the fraction of positive nuclei as well as numbers of cancer. We also found statistically significant increments of polysomies of chromosome 9 and 17 in terms of the polysomy index seen through the histologic changes occurring during multistep tumorigenesis. Our results could not demonstrate statistically significant correlation between p53 expression and PI 9 and 17 in tumorigenesis. Interestingly, however, there was a strong correlation between p53 expression and second primary tumor as well as recurrence of primary tumor. The p53 expressed group had a seven fold increased incidence in developing second primary tumor and a two and a half times increased incidence for recurrence of primary tumor, compared to the non-expressed group. We conclude that p53 expression and polysomies of chromosome 9 and 17 have an important role in multistep tumorigenesis in HNSCC. There was no significant correlation between p53 expression and polysomies of chromosome 9 and 17. However, the expression of p53 was statistically significant for association with second primary tumor and recurrence of primary tumor of head and neck squamous cell carcinoma.

Reference ranges of reticulocytes in adults.

According to the International Committee for Standardization in Haematology (ICSH), we determined the reference values for reticulocytes using an automated blood cell analyzer Technicon H*3 RTC in 200 healthy adult blood donors, aged between 17 and 60 years, 100 of whom were male and 100 female. The parameters included reticulocyte count, and its corpuscular indices; mean reticulocyte corpuscular volume (MCVr), mean reticulocyte corpuscular hemoglobin concentration (CHCMr), mean reticulocyte hemoglobin content (CHr), reticulocyte distribution width (RDWr), reticulocyte hemoglobin distribution width (HDWr) and reticulocyte corpuscular hemoglobin concentration distribution width (CHDWr). The reference ranges were established by setting the reference limits at two standard deviations from the arithmetic reference mean.

Evaluation of the reticulocyte count portion of technicon H*3 blood analyzer: lowering the test expense by reducing the reticulocyte reagent.

Automated reticulocyte counting has become an essential instrument of the hematology laboratory. This automatic technique has lead to diminishing labour tasks and to significant improvements in accuracy and precision compared with the manual microscopic methods. In any event, it adds a considerable expense to the laboratory budget. Here, we report the modified method of applying the new mixture of 1 microL of whole blood with 1 mL of reticulocyte reagent, which we evaluated for its accuracy and precision, instead of using the mixture of 3 microL of whole blood with 3 mL of reticulocyte reagent recommended by the company. We demonstrated the accepted accurate and precise results of percentage and absolute number of retculocyte count, low-stained reticulocyte count and its corpuscular indices; the mean reticulocyte corpuscular volume (MCVr), mean reticulocyte corpuscular hemoglobin concentration (CHCMr), and mean reticulocyte hemoglobin content (CHr). These suggested that, for every red cell assessed, the number, the cell volume, hemoglobin content and concentration are accurately and precisely measured by the modified method while the sub-populations of reticulocyte count and distribution width of reticulocyte indices are variable. In conclusion, our results provided the information that 1) the modified method can be used as a routine test and it provides accurate and precise results; 2) with the modified method, two-thirds of the expense spent for reticulocyte reagent can be saved; 3) it should not be used for research purposes.

Squamous cell carcinoma of head and neck.

Head and neck cancers are a major heath problem and common malignancies in Thailand. Up to 80 per cent of cases are caused by smoking and alcohol consumption. Epithelial mucosa of the aerodigestive tract exposed to carcinogens results in cellular mutations at different areas by a process called field cancerization and causes multistep carcinogenesis. Over 90 per cent of cases are squamous cell carcinoma. Prognostic factors depend on the patients, diseases and treatment. Currently, several molecular pathogenesis have been discovered such as abnormalities of c-myc, c-ras, c-erbB-1, bcl, int-2, hst1 oncogenes, p53 and p16 tumor suppressor genes. Common chromosomal abnormalities are 3p, 9p, 11q, 13q, 17p. Diagnosis requires symptoms and signs, radioimaging, and pathology. Stage I and II can be treated by surgery or radiotherapy. However, stage II requires and combination of surgery and radiotherapy, and studies of chemotherapy and local treatment to increase therapeutic efficacy by several approaches such as combination chemotherapy, new drugs, and biologic therapy.

Effect of cold exposure on platelet concentrates: changes in platelet indices and aggregation states.

Samples from platelet concentrates (filtered/non-filtrated, at the beginning/the end of shelf life) were exposed to 4 degrees C overnight, subsequent to dilution in platelet storage media (PSM) and/or exposure to EDTA to induce shape changes. Paired sampling protocol (+/- EDTA) was used and changes in cellular indices and induced-aggregation states were measured by Technicon H*1. Cold induced changes in platelets as identified by increase in MPV (0.5-0.8 fL for EDTA; 1.5-2.0 fL for citrated samples) with concomitant reverse changes in PDW ranging from 2-13 per cent was observed. Processing, storage and cold exposure also induced disparity between leucocyte peroxidase/basophil counts. This in conjunction with changes in platelet counts and cellular indices upon exposure to EDTA provide a unique new tool for assessing the aggregation states of platelets during processing and storage. Both filtration and dilution in PSM affect platelet storage stability. Platelets which have already undergone shape changes (i.e. exposure to EDTA) responded to a lesser degree to cold exposure. Our findings indicate that platelets's response to cold exposure can be used as a simple, reliable and accurate test for assessment of platelet morphological and function integrities.

Genetic instabilities of chromosome 9, 17 and accumulation of p53 overexpression during multistage tumorigesis in head and neck cancer.

Malignant transformation and tumor progression are currently thought to be the result of the accumulation of genetic alterations in critical genes, the proto-oncogenes and the tumor suppressor genes. Among the tumor suppressor genes, the p53 tumor suppressor gene mutations are the most prevalent. In order to determine genetic instability and p53 expression, we analyzed the genetic changes of chromosome 9 and 17 by non-isotopic in situ hybridization in formalin-fixed, paraffin embedded tissues and calculated for normalized chromosome index (NCI) and polysomy index (PI), and the expression of p53 by using immunohistochemistry (IHC). The means of chromosome 9 and 17 NCI were found to increase gradually as the tissues progressed from normal to squamous cell carcinoma; 1.02 and 1.03, respectively, in normal adjacent tissue (ANL), 1.19 and 1.20 in hyperplasia (HYP), 1.28 and 1.31 in mild dysplasia (MD), 1.38 and 1.43 in moderate dysplasia (ModD), 1.39 and 1.66 in severe dysplasia/carcinoma in situ (SD/CIS), and 1.65 and 1.83 in squamous cell carcinoma (SCC). Moreover, the PI 9 and 17 means also increased as the tissues passed from histologically normal epithelium to HYP to dysplasia (DYP) to cancer. In ANL, PI 9 and 17 means were 0.90 and 1.53 percent, compared to 3.78 and 3.38 percent in HYP, 3.73 and 5.12 percent in MD, 5.66 and 8.47 percent in ModD, 13.56 and 20.99 percent in SD/CIS, and 17.74 and 22.50 percent in SCC. Interestingly, p53 expression also increased continuously, not only in amount but also in the incidence of its expression, as the tissues progressed from normal to cancer, 2.29 percent in ANL, 4.65 percent in HYP, 9.09 per cent in MD, 9.58 per cent in ModD, 29 percent in SD/CIS, and 38.67 per cent in SCC in the amount; and 3 of 33 (9%) in ANL, 6 of 37 (16%) in HYP, 5 of 21 (24%) in MD, 3 of 12 (25%) in ModD, 8 of 18 (44%) in SD/CIS, and 24 of 49 (49%) in SCC in the incidence. Our studies demonstrated that genetic instability and p35 expression occurred very early from ANL to SCC and increased gradually through HYP, DYP, to SCC in head and neck cancer. The genetic instability and the loss of normal p53 function play the potential role in multistep tumorigenesis in head and neck cancer and might be the useful biomarkers in assessing the risk of tumor development.

Leukocyte alkaline phosphatase activity and peripheral changes of granulocyte following administration of granulocyte colony-stimulating factor.

The rhG-CSF had specificity of stimulation proliferation and differentiation of the neutrophil lineage in which there was an increase of younger stages, the earliest was myelocyte, of granulocyte in circulation. The effect of it was demonstrated within 24 hours of administration and reduced immediately after withdrawal. LAP activity in this condition was normal. The pattern of hematologic change in this condition may mimic CML and leukemoid reaction. It differed clearly from CML, since LAP activity in CML was lower than normal, but LAP activity could not define it from leukemoid reaction. The detection of young cells in peripheral blood through automated measurement of nuclear density by TechniconH*1 was observed for the high sensitivity (100%) and low specificity (54.8%). Furthermore, the study of cell kinetic in bone marrow should be evaluated to complete the cell kinetic effect of rhG-CSF and give a better evaluation for the H*1 blast flag.

Genetic instability and the development of recurrence of primary tumor and second primary tumor during head and neck tumorigenesis.

To determine whether the degree of genetic instability is associated with the development of recurrence of primary tumor (RPT) and second primary tumor (SPT), we examined 46 cases of head and neck squamous cell carcinomas (HNSCC) by nonisotopic in situ hybridization using chromosome specific DNA probes for chromosome 9 and 17. Forty-six cases were classified into three groups; group I, 15 cases without developing RPT and SPT; group II, 21 cases with RPT, and group III, 10 cases with SPT. We demonstrated the statistical significant increment of genetic instability in terms of normalized chromosome index (NCI) and polysomy index (PI) of chromosome 9 and 17 from normal adjacent to malignant lesions (ANL), to hyperplasia (HYP), to dysplasia (DYP), to squamous cell carcinomas (SCC). Our results demonstrated the trend of increased chromosome indices as the tissue progressed from ANL to SCC in group II over group I. However, when we compared the genetic instability between group I and the specimens from the patients who developed RPT within 6 months (group III), we found the significant increment of PI of both chromosome 9 (0.84 +/- 0.54 vs 1.25 +/- 0.46, p = 0.10) and 17 (1.02 +/- 0.62 vs 1.89 +/- 0.87, p = 0.06) on ANL in the later group. Our results also demonstrated the higher trend of genetic instability on ANL in group III over group I as shown by the statistical significance of NCI of both chromosome 9 (0.98 +/- 0.12 vs 1.06 +/- 0.02, p = 0.05) and 17 (1.02 +/- 0.09 vs 1.10 +/- 0.05, p = 0.05). These results suggested that the degree of genetic instability might be used as a potential molecular marker for the risk assessment of early RPT and SPT development during head and neck tumorigenesis.

Molecular biology of head and neck tumorigenesis: the role of p53 expression and genetic instability.

Head and neck cancers progress as multistep tumorigenesis through accumulation of genetic instability. The p53 tumor-suppressor gene encodes a cell-cycle checkpoint protein that functions in the G1 phase of the cell cycle. When DNA damage is incurred, p53 transactivates a number of downstream genes whose products, with diverse biologic activities, contribute to the cellular response to DNA damage. One major p53-mediated function in response to DNA damage is to induce the G1 cell-cycle arrest, or delay, which probably allows time for the cell to repair DNA damage prior to S-phase entry. In cell lacking of p53 function, a condition of genetic instability results from checkpoint loss (Fig. 4.). These events occur early from ANL to SCC and increase gradually through multistep tumorigenesis. Due to the potential role of p53 expression and genetic instability, both might be useful biomarkers in assessing the risk of head and neck tumorigenesis.

In situ hybridization: a new tool in molecular medicine.

In situ hybridization (ISH) is a molecular technique that been used over three decades to detect specific nucleic acid sequences of gene expression at the cellular level. It is a morphologic method of localizing specific DNA or RNA sequences in the individual cell. The technique can be applied to cells frozen or fixed tissues or whole organisms and cytologic preparations; various types of probes can be utilized and the reaction can be visualized by autoradiography using isotopic markers or by colorimetric methods using fluorochromes or enzymes. It has been used primarily for localization of DNA sequences and applied recently to the localization of viral DNA sequences which provides insight into the pathology of viral infection and is enabling the diagnosis of viral infection. Analysis of gene expression by in situ hybridization messenger RNA (mRNA) is a crucial step towards understanding gene function and biology at molecular level. Nowadays, ISH is applied in three major categories: 1) infectious disease 2.) cytogenetics and 3.) gene expression. This technique has undoubtedly become a powerful new tool for molecular diagnostic techniques and is increasingly important in several areas of molecular medicine.

Changes in white blood cells and myeloperoxidase activity in rhG-CSF prophylactic patients receiving cytotoxic chemotherapy.

The patterns of white blood cell parameters and mean peroxidase index (MPXI) changes in recombinant human granulocyte colony-stimulating factor (rhG-CSF) prophylactic patients, receiving myelosuppressive chemotherapy, have been studied in 8 cases, using flow cytochemistry blood autoanalyser (Technicon R H*1). No severe neutropenia (absolute neutrophil count, ANC < 0.50 x 10(9)/L) appeared in 6 rhG-CSF primary prophylactic patients, but severe neutropenia was noticed in 2 rhG-CSF secondary prophylactic patients for a period less than 1 week. In most of the cases the significant increase of neutrophil during leukocytosis occurred within 24 hours after starting rhG-CSF prophylaxis, and decreased within 24 hours after the end of rhG-CSF prophylaxis. There was a small degree of lymphocytosis, monocytosis, and basophilia in some cases. From this study, there were no significant changes of MPXI during rhG-CSF prophylaxis, the neutrophils produced during proliferative response to rhG-CSF possessed normal myeloperoxidase (MPO) activity. We concluded that the information obtained from automated blood cell analyser Technicon R H*1 especially MPXI and ANC, could be very useful for monitoring rhG-CSF prophylactic patients receiving myelosuppressive chemotherapy. The advantages of the MPXI over other methods is its simplification when performed as part of a routine complete blood count (CBC) on an automated hematology instrument, and its ability to measure even severe neutropenic samples.