Thrombocytopenia as a Prognostic Marker in Patients with Acute Encephalitis at a Tertiary Care Centre in Northern India.

Encephalitis is challenging to manage given the diversity of clinical and epidemiologic features. Various predictors of outcome have been studied so far including thrombocytopenia, cerebral edema, hypoglycaemia, development of status epilepticus and need for endotracheal intubation. Thrombocytopenia represents one of the potentially modifiable risk factors for poor prognosis in encephalitis. A better understanding of the epidemiology of this devastating disease and identification of predictors of outcome and management of reversible factors will pave the way for better management of the disease. MATERIAL: A total of 98 Hospitalised patients of Acute Encephalitis were enrolled in the study. Diagnoses were confirmed by CSF and Neuroimaging studies. Platelet count <150,000/cumm was considered as thrombocytopenia. Mild, moderate and severe thrombocytopenia was categorized at platelet count 100,000-150,000, 50,000-100,000 and <50,000/ cumm, respectively. Outcome at discharge was assessed using the Modified Ranking Score, categorized into 3 groups - good (0-2), fair (3-4), and poor (5-6). Chi-square, ANOVA and Independent samples 't'-tests were used to compare the data. OBSERVATION: Mean age of patients was 34.06±18.76 (range 14-85) years. Majority of patients were males (54.1). Mean GCS at admission was 9.41±1.90. Acute viral encephalitis(unclassified) (n=33; 33.7%), Scrub typhus (n=24; 24.5%) and Japanese encephalitis virus (n=12; 12.2%) were the most common underlying etiologies. A total of 74 (75.5%) patients had thrombocytopenia. Mild, moderate and severe thrombocytopenia was seen in 34 (34.7%), 30 (30.6%) and 10 (10.2%) cases. Thrombocytopenia was significantly higher in Dengue and Scrub as compared to other etiologies. Thrombocytopenia and its severity showed a significant association with lower GCS and higher mRS scores indicating a poor outcome. CONCLUSION: Thrombocytopenia is associated with a poor clinical status and adverse outcomes in patients with encephalitis of all causes.

A randomized study of very low-dose factor VIII prophylaxis in severe haemophilia - A success story from a resource limited country.

INTRODUCTION: Current factor prophylaxis strategy practised in developed countries is not feasible in resource constraint developing countries like India. AIM: The aim of this study was to investigate the efficacy and safety of very low-dose factor prophylaxis in India. METHODS: Children of 1-10 years of age with severe haemophilia were randomized to Prophylaxis group and Episodic (On demand) group. Children in prophylaxis group received very low-dose factor VIII (FVIII) concentrate, i.e. 10 units kg(-1) body weights on 2 days a week. Episodic group received factor concentrate in standard recommended doses. The study period was 11.5 months. RESULTS: In total 21 children were enrolled in this study, 11 assigned to prophylaxis and 10 to episodic group. Children on prophylaxis had 11 joint bleeds in comparison to 57 joint bleeds in episodic group. Mean number of haemarthrosis per patient per month were 0.08 (0.08 ± 0.13) in prophylaxis group compared to 0.48 (0.48 ± 0.34) in episodic group (P < 0.05). Total FVIII consumption was 87.51 and 56.32 units kg(-1) month(-1) in prophylaxis and episodic group respectively (P = ns). Overall median hospital emergency visits were 1 day in prophylaxis group and 9 days in episodic group (P ≤ 0.05). Median days of absenteeism from school were 25 days in episodic group and 3 days in prophylaxis group (P < 0.05). No significant complications were noted in prophylaxis group and compliance was 98%. CONCLUSION: To conclude, low-dose FVIII prophylaxis is cost effective, efficacious and a safe method of preventing joint bleeds and consequent joint damages.

A protein anomaly in erythrocyte membranes of patients with Duchenne muscular dystrophy.

Raman spectroscopic comparisons of erythrocyte membranes from 20 patients with Duchenne muscular dystrophy and 8 age-matched controls indicate a prominent and consistent protein anomaly in the patient samples. This was apparent in the following: (a) CH-stretching signals from control membranes reveal a thermotropic transition at 15.6 degrees C, attributable to a protein/lipid phase that is lacking in dystrophic membranes. (b) CH-stretching signals from control membranes also show a protein transition at 39 degrees C [pH 7.4] that is shifted to 45 degrees in dystrophic membranes. (c) A reduction in pH to 5.7 shifts this transition from 39 degrees C to 7 degrees C in normal membranes and from 45 degrees C to 24 degrees C in dystrophic membranes. (d) The Amide I/Amide III regions indicate a significant proportion of beta-structured peptide in dystrophic but not normal membranes. (e) Analysis of tyrosine signals indicates greater polar exposure of tyrosine hydroxyl groups in dystrophic vs normal membranes. All of the differences between dystrophic and normal membranes are highly significant (P less than 0.001).

Separation and quantification of lanthanides in synthetic standards by capillary electrophoresis: a new experimental evidence of the systematic "odd-even" pattern observed in sensitivities and detection limits.

The systematic zigzag pattern of sensitivities and detection limits (LODs) of lanthanides, previously observed in mass spectrometric and chromatographic measurements, was once more investigated through the indirect photometric detection with capillary electrophoresis. Well-designed chemometric experiments were performed for the electrophoretic separation and detection of lanthanides using standards with similar concentrations. A fused silica capillary 355 mm x 75 microm was used. Complete separation for all 14 lanthanides was achieved in approximately 3 min at a capillary temperature of 15 degrees C. Indirect photometric detection at 214 nm using a voltage of +25 kV and a hydrostatic injection (100 mm for 20 s) were used. The background electrolyte used consisted of an optimum mixture of 0.004 M HIBA (as complexing agent) and 0.010 M UVCat-1 (as a UV-absorbing co-ion) with a pH 4.4. A good reproducibility in migration times (<2.7% RSD), peak areas (<3.8% RSD) and peak heights (2.7% RSD) were systematically obtained. Calibration curves based on both peak areas and peak heights (from seven replicates) were prepared using weighted least-squares regressions, which were employed for the correct estimation of individual sensitivities and LODs. For a better estimation of LODs, the lowest concentration standard was injected 30 times. A new experimental evidence of the systematic "odd-even" pattern was again observed in the lanthanide sensitivities (and therefore in LODs). The calculated sensitivities were greater for lanthanides with an odd-atomic number than for their corresponding neighboring element with an even-atomic number (i.e., (57)La-(58)Ce, (59)Pr-(60)Nd; (63)Eu-(64)Gd, etc.). Concerning the LODs, a systematic zigzag pattern was observed where the odd atomic number elements have lower LODs than the even atomic number neighbor elements (i.e., (57)La-(58)Ce; (59)Pr-(60)Nd; (63)Eu-(64)Gd, etc.). The possible origin of this "odd-even" effect is briefly discussed. Accuracy errors were less than 5% for lanthanide concentrations of three synthetic standard solutions, which were considered as "unknown" samples.

Mutation Analysis in Chronic Myeloid Leukemia Patient in Chronic Phase on Imatinib Having Delayed Achievement of Milestones or Loss of Response.

Tyrosine kinase inhibitors (TKI's) are currently the drug of choice for management of chronic myeloid leukemia. Imatinib is the most commonly used first line TKI in India. Mutations leading to resistance to imatinib are the most common cause for imatinib failure. We studied pattern of kinase domain mutations in 40 patients of CML who either lost their response or did not achieve it in defined timepoints. Loss of molecular response was the most common indication for asking mutation analysis. Sixteen patients were found to have detectable mutations. M351T was the most common tyrosine kinase mutation followed by Y253H and H396R. Two patients had 2 mutations simultaneously. M351T is the most common mutation in our patient population.

Brain stem auditory evoked responses and visual evoked responses in children with tubercular meningitis.

Thirty two patients between 6 months and 14 years of age with tubercular meningitis were evaluated for brain stem auditory evoked response (BAER) and Visual evoked responses (VER), within 7 days of admission. Absolute latencies and interpeak latencies were compared with values obtained from normal children. BAER abnormality was found in 56.25% and VER in 28%children, respectively. BAER abnormality correlated with Glasgow Coma Scale at admission and discharge, stage of meningitis, raised intracranial pressure, seizure activity, and poor outcome. VER abnormality correlated with abnormal fundus findings only.

Differences between the structural dynamics of plasma membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40 as revealed by laser Raman spectroscopy.

The Raman spectra of highly purified plasma membranes from SV40-transformed GD248 lymphocytes have been compared with the spectra of the membranes of normal cells over the spectral region 100 cm-1 to 3010 cm-1. Striking differences between the two membrane categories were observed in the thermal response of the CH-stretching and acoustical regions. Analysis of CH-stretching shows that the membranes of normal cells exhibit a thermal transition centered at 7 degrees and approximately 5 degrees wide. The membranes of GD248 cells, in contrast, show a lipid transition centered at -5 degrees and 12-18 degrees wide. Analysis of the acoustical region yields equivalent results. The membrane proteins of normal membranes undergo a large thermotropic transition, starting at 39 degrees (sample temperatures), whereas this transition begins at 23 degrees with GD248 plasma membranes. The results suggest the possibility that SV40-specific membrane proteins may modify the collective thermotropic behavior of both normal membrane proteins and membrane lipids.

Haemostatic Profile in Patients of Myeloproliferative Neoplasms-A Tertiary Care Centre Experience.

INTRODUCTION: Patients of MPN commonly present with abnormalities in laboratory coagulation tests that are consistent with hypercoagulable state. Some individuals with MPN exhibit a pattern of exclusive bleeding or thrombotic events; many others have both bleeding and thrombosis during the course of the disease. AIM: This study was undertaken to assess the haemostatic defects and platelet functions in patients of MPN. MATERIALS AND METHODS: One year prospective study was conducted at a tertiary care centre in North India in Department of Pathology in collaboration with Department of Clinical Haematology. All recently diagnosed cases of MPN along with 30 age and sex matched controls were included. Patients on antiplatelet drugs, antimyeloproliferative treatment, vitamin K agonists or antagonists, OCPs, Platelet count <1,00,000/µl, high grade fever, liver disease, pregnancy were excluded from this study. All the patients underwent screening investigations like CBC, peripheral smear evaluation, BT, PT, aPTT, Protein C and S measurement (clot based assay) and aggregation studies with ADP (5µM) (Optical Aggregometry with AGGRO/LINK 8 software and CHRONOLOG 700 aggregometer). RESULTS: In present study, 50 cases were included. There was an occult prothrombotic state, suggested by significantly (p<0.001) reduced levels of Protein C and Protein S, but no patient presented with frank thrombosis while 8 out of 50 patients had haemorrhagic manifestations ranging from subdural haematoma to pin point petechial haemorrhages. Patients of CML-CP, ET, PV, PMF, MPN-NOS showed significantly reduced maximal aggregation with ADP (5µM) when compared to control (p<0.001). MPV also showed a statistically significant increase in these patients. CONCLUSION: Thrombohaemorrhagic complications significantly affect the morbidity and mortality of MPN patients. This can be assessed by the use of platelet aggregation studies, Protein C and S activities and other coagulation studies. Timely diagnosis of these prothrombotic/haemorrhagic states can decrease the morbidity in these patients.

Low levels of the pesticide, delta-hexachlorocyclohexane, lyses human erythrocytes and alters the organization of membrane lipids and proteins as revealed by Raman spectroscopy.

We studied the nature of the interaction of delta-hexachlorocyclohexane (delta-HCCH), a pesticide having a stereoisomeric structure similar to inositol, with red blood cells. Cell survival data, measured as percent of hemoglobin released by delta-HCCH, show that the cell lysis increases with post exposure time. delta-HCCH at 55-60 micrograms/ml causes about 70% cell lysis after 24 h of exposure. The nature of interaction of delta-HCCH with membrane components was evaluated by studying the thermotropic transitions and protein structure of ghosts using Raman spectroscopy. Control ghosts show transitions with onset/completion temperatures 30 degrees C/38 degrees C (high temperature transition) and 3 degrees C/10 degrees C (middle temperature transition) when monitored by the I2935/I2850 ratio. The interaction of delta-HCCH drastically broadens the high temperature transition and shifts it to the temperature range of 10-29 degrees C. The plots of (I2880-90/I2850) vs. temperature show two transitions for control ghosts, one extending from -10 degrees C to 3 degrees C (lower temperature transition) and the other from about 7 degrees C to about 15 degrees C (middle temperature transition). Ghosts lysed with delta-HCCH shows only a single and a very broad transition in the range of about -3 degrees C to about 15 degrees C. These changes in the thermal transition properties suggest that delta-HCCH alters lipid and lipid-protein phases of erythrocyte membranes. The comparison of Raman spectra in the amide I and III regions of erythrocyte ghosts and purified band 3 with several amidated compounds reveals that cytoskeleton proteins contain highly amidated residues (probably glutamine and asparagine). The interaction of delta-HCCH with erythrocytes drastically alters the environment of these amidated residues indicating the involvement of cytoskeleton proteins. We conclude that the interaction of delta-HCCH with red blood cells disrupt membrane structure and change the environment of cytoskeleton proteins that could cause cell lysis.

Raman and resonance-Raman scattering by erythrocyte ghosts.

1. We present the laser-Raman spectra of human erythrocyte ghosts, isolated by standard conditions and compare these with the spectra of lecithin liposomes and fat-free serum albumin. 2. The hydrocarbon stretching modes of membrane lipids are temperature sensitive and may serve as a index of hydrocarbon chain motion. 3. The Amide I and Amide III bands of ghosts in H-2O and 2-H-2O, indicate a mixture of alpha-helical and unordered conformation, but do not allow a quantitative estimate of secondary structure. 4. Strong, scattering bands at 1530 and 1165 cm-1 are attributable to conjugated double bond systems, probably of membrane-associated carotenoids. Their high intensity is due to resonance enhancement.

Evidence for constrained lipid mobility in the erythrocyte ghost. A spin label study.

Our data show that the ESR spectrum of 5-nitroxide stearate bound to erythrocyte membranes varies with the amount of label bound and suggest that, at high binding, a significant proportion of label molecules lie within approximately equal to 15-10--8 cm; this gives rise to spin-exchange (ambient temperature) and dipole-dipole interactions. We find that these spectral manifestations due to label clustering can be abolished by reduction of pH and the conjoint action of lysolecithin and trypsin, although both perturbations increase 5-nitroxide stearate binding. Both perturbations are known to mobilize intramembranous particles by modifying or extracting some membrane proteins. We accordingly suggest that the lipids and proteins of erythrocyte membranes exist in a relatively fixed mosaic, and that the mobility of both components is restricted by some membrane-associated protein framework.

Raman spectra of some saturated, unsaturated and deuterated C18 fatty acids in the HCH-deformation and CH-stretching regions.

We have analyzed the Raman spectra of stearic acid and two deuterated derivatives thereof, 2,2-dideutero stearic acid and 18,18,18-trideutero stearic acid, as well as of four unsaturated, C18 acids, (cis,-9; cis, cis-9,12, cis, cis, cis-9,12,15; and trans, trans-9,12) in their solid and liquid states. The different spectra of stearic acid and the two deuterated derivatives exhibit distinctive HCH-deformation and CH-stretching features for CH2 residues adjacent to CO2H or CH3 residues. Unsaturated hydrocarbon chains reveal additional features attributed to CH2 residues adjacent to one or two HC=CH groups. The spectra of the various fatty acids in their solic or liquid states show that both the HCH-deformation and CH-stretching regions are very sensitive to chain architecture, the CH-stretching region most dramatically so. Evaluation of the spectral features in the two regions indicates that the detailed structure of the CH-stretching region depends strongly upon interaction, enhanced by Fermiresonance, between CH-stretching fundamentals and HCH-deformation overtones. The data further suggest that the differences between HCH-deformation, in the solid and liquid states, and the resulting altered interactions between CH-stretching fundamentals and HCH-deformation overtones, produce the large modifications of the CH-stretching region that accompany solid-liquid transitions. The results thus provide a basis for the changes in the CH-stretching region, that accompany order-disorder transitions in model and biological membranes.

Carotenoids as a Raman-active probes of erythrocyte membrane structure.

1. Erythrocyte ghosts exhibit resonance-enhanced Raman bands at 1530 cm(-1) and 1165 cm(-1) attributable to v(-C=C-) and v(=C-C=), respectively, of the conjugated polyene chains in carotenoids. In lipid extract of ghosts, these resonance-enhanced bands lie at 1527 and 1158 cm(-1). The spectra indicate the presence of membrane-bound beta-carotene. 2. The resonance-enhanced Raman spectrum of beta-carotene in lecithin liposomes is identical to that obtained with hexane or chloroform solutions. 3. Increasing proportions of cholesterol in cholesterol-lecithin liposomes up to a cholesterol: phospholipid molar ratio of 0.8-0.9 drastically decreases the intensity of both resonance-enhanced bands. 4. In ghosts the carotenoid bands respond to membrane perturbations. Trypsinization, lysolecithin treatment and reduction of pH increase the intensities of the 1530 and 1165 cm(-1) bands. In contrast, a decrease in the intensity of both bands follows equilibration of ghosts for 15 min at approx. 50 degrees C or addition of (0.1%) sodium dodecyl sulfate. 5. We suggest that perturbants known to change lipid-protein interactions in erythrocyte membranes modify the microenvironment and/or configuration of the membrane-bound carotenoid.

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes.

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).

Multiple thermotropic state transitions in erythrocyte membranes. A laser-Raman study of the CH-stretching and acoustical regions.

1. We have examined the Raman scattering from erythrocyte ghosts at 2700 cm-1 -3000 cm-1 (CH-stretching region), that at low frequencies due to acoustical vibrations, as well as that due to v (-C=C-) and v (=C-C=) of beta-carotene, in the temperature range of -30 degrees C to +30 degrees C. For comparison we have also evaluated the Rama spectra of liposomes composed of lecithins+/-cholesterol. 2. Plots vs. temperature of the intensities (I) of the approx. 2880 cm-1 and 2932 cm-1 bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the (I2880/I2850) and (I2932/I2850) ratios, revealed discontinuities in both erythrocyte membranes and liposomes. 3. In erythrocyte membranes, plots of (I approximately 2880/I2850) and (I approximately 2932/I 2850) reveal several discontinuities. Those reported by (I approximately 2880/I 2850) occur at +17 degrees C and approx. -4 degrees C. The ratio (I approximately 2932/I 2850) reveals a transition between -20 degrees C and +10 degrees C, 1/2 maximal at -8 degrees C. The ratio (I 1527/I 1158), representing the relative intensity of v (-C=C-) vs. v (=C-C=) of membrane-bound beta-carotene, changes discontinuously with the 17 degrees C transition, but is stable over the rest of the temperature change studied. 4. The data indicate that a major membrane-state transition takes place below 0 degrees C but that an additional transition occurs at approx. 17 degrees C.

Organic pesticides modify lipid-lipid and lipid-protein domains in model membranes. A laser Raman study.

The effect of hexachlorocyclohexane (all isomers) on the thermal transition properties of phospholipid liposomes was determined by Raman spectroscopy. Raman spectra of liposomes with and without the presence of hexachlorocyclohexanes were recorded in the C-H stretching region which shows three major bands around 2850, 2880 and 2930 cm-1. Thermal transition properties were estimated from plots of I2880/I2850 and or I2930/I2850 vs. temperature, where I represents the intensity of the respective band. Our data on phospholipid liposomes reveal that delta- and gamma-hexachlorocyclohexanes drastically reduce and broaden the main thermal transitions of phospholipids at toxic level concentrations. These effects are more pronounced in liposomes containing 18 or more carbon atom long acyl chains. Alpha- and beta-isomers at similar concentrations show a minimum effect on the thermal transition properties of phospholipids. Raman analysis of phospholipid liposomes containing melittin, interestingly, reveal that the delta-isomer unlike the gamma-isomer strongly alters the transition properties of boundary lipids. These data suggest that the effect of hexachlorocyclohexanes on the thermal transition properties of membranes is stereo specific and that the delta-isomer preferably disrupts the lipid-protein domains. Results are explained on the basis of the dynamic flexibility owing to the equatorial and axial chlorine atoms of various hexachlorocyclohexane isomers.

The structure and thermotropism of thymocyte plasma membranes as revealed by laser-raman spectroscopy.

1. Plasma membranes from rabbit thymocytes have been analyzed by laser-Raman spectroscopy over the 800-3000 cm-1 region and the spectra compared with those of endoplasmic reticulum, as well as relevant liposome systems. 2. Evaluation of the Amide I and Amide III regions indicates that thymocyte plasma membranes, but not endoplasmic reticulum, contain appreciable beta-structure peptide. This conclusion is supported by infrared spectroscopy. 3. Evaluation of the 2890 cm-1: 2850 cm-1 intensity ratio of plasma membranes as a function of temperature, using an integration technique, demonstrates a thermotropic lipid transition centered near 23 degrees C. This transition is less sharp than one observed with egg lecithin in this temperature range. 4. The significance of the thermotropic transition is evaluated in view of the lack of thermotropic lipid-protein segregation detectable by freeze-fracture electron microscopy (Wunderlich, F., Wallach, D.F.H., Speth, V. and Fischer, H. (1973) Biochim. Biophys. Acta 373, 34-43).