C reactive protein in the diagnosis of acute appendicitis.

OBJECTIVE: To find out if the C reactive protein concentration is of any value in the diagnosis of acute appendicitis, either alone or in combination with other laboratory tests. DESIGN: Open study. SETTING: Drechtsteden Hospital, Dordrecht, and Spaarne Hospital, Heemstede, The Netherlands. SUBJECTS: 209 consecutive patients admitted with suspected appendicitis. MAIN OUTCOME MEASURES: Correlation of C reactive protein concentration with age, sex, body temperature, duration of abdominal pain, anorexia, nausea, vomiting, white cell count, neutrophil count, erythrocyte sedimentation rate, and histological appearance of the appendix. RESULTS: 125 patients of the 209 patients had their appendixes removed, and of these 101 had histologically confirmed appendicitis: A C reactive protein concentration of > or = 6 mg/1 alone had a sensitivity of 87% and a specificity of 50%. When the selected variables were subjected to multivariate analysis the most important, in decreasing order, were white blood cell count, female sex, and C reactive protein concentration. Combining the variables was of no additional value. CONCLUSION: measurement of the C reactive protein concentration can increase the accuracy in the diagnosis of acute appendicitis.

Multivariate statistical modeling: alternative approach to test evaluation, applied to counting reticulocytes by flow cytometry.

We present a statistical path analysis model for the evaluation of two tests in the absence of a "gold standard" method. This model is applied to the evaluation of flow-cytometric and visual reticulocyte counting by using as the comparison method a combination of three hematological measurements: hemoglobin concentration (HGB), mean cellular volume (MCV), and erythrocyte density width (EDW). We assumed that, in general, a higher reticulocyte count is associated with a lower HGB value and with greater values for MCV and EDW. Applying this assumption and the statistical model, we demonstrated that flow cytometry was superior to visual reticulocyte counting in the low-value range studied. The path analysis model is potentially applicable in other cases where two tests are to be compared, and when no gold standard is available.

Bhattacharya: a new application for quality control.

The Bhattacharya method and the 'average of normals' method for internal quality control were compared. Both are based on unselected patient test results. The Bhattacharya method is mostly used in clinical chemistry for calculation of reference intervals but could be modified for quality control procedures. Using the data of a coagulation test and stimulating systematic errors, it was concluded that the Bhattacharya method is more sensitive to expected shifts and is more flexible than the 'average of normals' method.

Central nervous system involvement in early and late syphilis: the problem of asymptomatic neurosyphilis.

Patients with syphilitic infections are at risk of development of symptomatic neurosyphilis. Adequate treatment with 2.4-7.2 x 10(6) units benzyl penicillin-G intramuscularly within 1 year after infection will rule out this risk. However, more than 1 year after infection this treatment is not fully reliable. In asymptomatic CNS involvement (asymptomatic neurosyphilis) only intravenous penicillin treatment is considered to be adequate in the prevention of neurosyphilis. In this study we redefined criteria for this condition by comparing serum and cerebrospinal fluid (CSF) samples of symptomatic neurosyphilitic patients with those of latent syphilitic patients without CNS involvement. Diagnostic criteria of the World Health Organization and of Centers of Disease Control for asymptomatic neurosyphilis (positive CSF Venereal Disease Research Laboratory (VDRL) test, combined with raised CSF cell count and/or protein content) were studied and compared with some newer parameters such as signs of intrathecal treponemal antibody production (Treponema pallidum haemagglutination assay and intrathecal Treponema pallidum assay index), immunoglobulin G (IgG) and M (IgM) index. The results of this study in 203 syphilitic patients revealed that either a positive CSF-VDRL or combination of a raised IgG and/or IgM index with an elevated CSF cell count both are useful criteria for "ruling-in" asymptomatic neurosyphilis.

Rapid determination of the hypoxanthine increase in ischemic exercise tests.

After ischemic exercise tests, performed to detect glycogenoses or myoadenylate deaminase (EC 3.5.4.6) deficiency, the increases in serum lactate and ammonia usually are measured. Determination of hypoxanthine instead of ammonia can also be used to show myoadenylate deaminase deficiency, but HPLC of hypoxanthine is time-consuming. As a substitute, we developed an indirect enzymatic equilibrium method for hypoxanthine based on coupling the chromogenic system 3,5-dichloro-2-hydroxy-benzenesulfonic acid/4-aminophenazone with formation of hydrogen peroxide by xanthine oxidase (EC 1.1.3.22). The pH optimum is at 7.8 and the absorbance maximum at 510 nm. The calibration curve is linear from 0 to 100 mumol/L and the detection limit is 0.9 mumol/L. Analytical variability (CV) was 1.5% to 3.6% within-run, 4.5% to 8.5% between-run. The assay can be performed with a standard spectrophotometer or a centrifugal analyzer. The coefficient of correlation was 0.68 between hypoxanthine and ammonia increases in plasma from controls who performed the exercise test.

A cytopreparatory method for cerebrospinal fluid in which the cell yield is high and the fluid is saved for chemical analysis.

A method for the concentration of cells from cerebrospinal fluid is described. An adaptation of a commercial cytochamber, consisting of a holder that fixes a disposable chamber directly on a microscope slide, was used. The cells were spun down in a conventional swing-out centrifuge, which was provided with a bucket for the cytochamber system. After removing most of the supernatant with a pipette, the remaining fluid was absorbed by means of a suction device consisting of a disposable pipette tip covered with a piece of Leukopor and filled with Sephadex G10 beads. The method gives a high recovery of cells (90%), together with a good preservation of cell morphology, and leaves about 80% of the fluid available for analysis of the soluble components.

Cerebrospinal fluid IgG and IgM indexes as indicators of active neurosyphilis.

Serological and non-serological tests were performed in matched samples of cerebrospinal fluid and serum from 236 syphilitic patients. An increased IgG or IgM index, or both, was found about 70 times more often in symptomatic neurosyphilis than in latent syphilis without involvement of the central nervous system. An increased Ig index, together with a cell count greater than 5/microL, was only found in symptomatic neurosyphilis. Although the numbers of data are small, we conclude that the IgG and IgM indexes are valuable tests in the diagnosis of syphilitic involvement of the central nervous system.

In vitro inhibition of the classical pathway of human complement by polymyxin B.

Polymyxin B was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC1-9 formation from EAC1-3 and C5-9 though slightly less efficiently. It did not inhibit C3/C5 convertase of the alternative pathway or its formation. The inhibition of the classical pathway was reversible since hemolytic activity was almost completely restored after dialysis.

Differential diagnosis of genetic disease by DNA restriction fragment length polymorphisms.

DNA restriction fragment length polymorphisms (RFLPs) are used for diagnosis of genetic disease in families known to be affected by specific disorders, but RFLPs can be also useful for the differential diagnosis of hereditary disease. An RFLP pattern represents the inheritance of chromosomal markers in a family, and such a pattern may be compatible with the inheritance of a certain disorder in that family. Probabilities to find such a pattern if the disorder were present and if it were absent can be combined with the prior probabilities of disease considered in the differential diagnosis on the basis of previous clinical and laboratory data. Bayes' theorem is used to calculate the posterior probabilities of the diseases in question. This approach is illustrated in a family suffering from either spinal muscular atrophy (an autosomal recessive disease) or Becker muscular dystrophy (an X-chromosomal disorder). Probabilities to exclude a certain disorder can be calculated in advance, as some RFLP patterns are not compatible with the presence of that disorder.

Intra-blood-brain barrier synthesis of human immunodeficiency virus antigen and antibody in humans and chimpanzees.

The presence of human immunodeficiency virus (HIV) antigens in cerebrospinal fluid (CSF) was associated with progressive encephalopathy in adult and pediatric patients with acquired immunodeficiency syndrome (AIDS). HIV antigen was detected in CSF from 6 of 7 AIDS patients with progressive encephalopathy. By contrast, HIV antigen, whether free or complexed, was detected in CSF from only 1 of 18 HIV antibody seropositive patients without progressive encephalopathy and from 0 of 8 experimentally infected chimpanzees without clinical signs. Intra-blood-brain barrier synthesis of HIV-specific antibody was demonstrated in the majority of patients with AIDS (9/12) or at risk for AIDS (8/13) as well as in the experimentally infected chimpanzees, indicating HIV-specific B-cell reactivity in the brain without apparent neurological signs. In 6 of 11 patients with HIV infection, antibodies synthesized in the central nervous system were directed against HIV envelope proteins. Active viral expression appears to be necessary for both the immunodeficiency and progressive encephalopathy associated with HIV infection.

Immunoglobulin D in cerebrospinal fluid.

Immunoglobulin D (IgD) was measured by radioimmunoassay in paired cerebrospinal fluid and serum samples from patients with various neurological diseases. The IgD index was calculated for every patient and compared with the IgG index. An increased IgD index was found in 18 out of 41 patients suffering from multiple sclerosis and in 52 out of all 122 patients investigated. An increased IgD index did not always coincide with an increased IgG index. An increased IgD index suggests an abnormal intrathecal synthesis of this immunoglobulin within the central nervous system. We conclude that the determination of IgD in the cerebrospinal fluid yields additional information on immunological reactions within the central nervous system.

Reinvestigations into the formation and assay of C3bBbP complexes.

C3bBbP complex formation was studied by an enzyme linked immunosorbent assay (ELISA). Microtitre plates were coated with anti-P to trap the complexes and peroxidase labelled anti-C3 was used to detect them with the help of substrates of peroxidase. Incubation of normal serum pool (NSP) at 37 degrees C in the presence of high concentrations (greater than or equal to 0.5 mmol/l) of Mg2+, usually used in alternative pathway (AP) assay systems, caused the generation of C3bBbP complexes. This generation was not observed when NSP was incubated in the presence of low Mg2+ concentration (less than or equal to 0.2 mmol/l) or EDTA. The concentration of Mg2+ required for maximum complex formation was 2.0 mmol/l under the experimental conditions. Complexes could not be generated in B-depleted serum. Incubation of NSP with endotoxin or CoVF in the presence of 0.2 mmol/l Mg2+ caused the generation of the complexes. The generation was influenced by ionic strength in the incubation mixture. Endotoxin and Mg2+-dependent generation of complexes could not be detected when peroxidase-labelled anti-B was used instead of peroxidase-labelled anti-C3. Serum incubated with 0.2 mmol/l Mg2+ or EDTA apparently detected in vivo formed complexes whereas that incubated with 0.2 mmol/l Mg2+ and endotoxin reflected the complex forming capacity of the serum. The serum of a patient with Raynaud's phenomenon having 45% of normal AP activity did not show increased amounts of preformed complexes but had the ability to generate the complexes to a level of about 45% of that attainable by NSP. These observations suggest that the ELISA used here has the potential of detecting activation as well as the integrity of the AP under carefully controlled conditions.

Effect of early and late syphilis on central nervous system: cerebrospinal fluid changes and neurological deficit.

Neurological examination and investigation of the cerebrospinal fluid (CSF) was performed on 24 patients with early and 180 patients with late syphilis. In 21 (12%) patients with late syphilis positive CSF treponemal test results and neurological deficits suggestive of symptomatic neurosyphilis were found. Concomitantly all but three patients with neurosyphilis showed one or more of the following abnormal CSF variables: CSF concentration of albumin X 10(3)/serum concentration (albumin ratio) greater than or equal to 7.9; mononuclear cells greater than 5 microliters: ratio of CSF to serum IgG concentrations/ratio of CSF to serum albumin concentrations (IgG index) greater than or equal to 0.7 or of IgM/albumin (IgM index) greater than or equal to 0.1; or oligoclonal CSF immunoglobulins. In 20 (95%) patients with neurosyphilis evidence of the production of treponemal antibodies within the central nervous system (CNS) was shown. Ten (48%) patients with neurosyphilis had been treated previously for late syphilis. These observations emphasise the need to screen for neurosyphilis in patients with late syphilis. Intrathecal production of treponemal antibodies was detected in six (25%) patients with early and 44 (28%) with late syphilis who did not show any neurological deficit. Intrathecal production of treponemal antibodies indicating that the CNS was affected led us to suspect asymptomatic neurosyphilis in these patients. Seventeen (11%) patients with late syphilis but no neurosyphilis and only one (4%) with early syphilis showed additional abnormal CSF variables. Surprisingly, six out of 22 patients with treated early and 20 out of 68 patients with treated late syphilis showed evidence of treponema antibody production within the CNS. We do not know whether these findings indicate that the CNS was affected because of inadequate treatment or merely reflect persistent synthesis of treponemal antibodies associated with cured infection. In one (4%) patient with early and in 21 (13%) with late syphilis but no neurosyphilis abnormal CSF variables in the absence of positive CSF treponemal test results were observed, which excluded syphilitic inflammation of the CNS.

Rate of synthesis of IgG within the blood-brain barrier and the IgG index compared in the diagnosis of multiple sclerosis.

Receiver-operating characteristic curves of the IgG synthesis rate and the IgG index show that the IgG index has better discriminating properties with respect to multiple sclerosis. This, added to the fact that the precision of determining the index is much higher than that of the synthesis rate, makes the IgG index the parameter of choice in the diagnosis of multiple sclerosis.

In vitro inhibition of the classical pathway of human complement by a natural microbial product, colistin sulphate.

Colistin sulphate was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC14 formation from EA and C2-deficient serum, EAC1-7 formation from EAC1-3, C5, C6 and C7 and the interaction of EAC1-7 with C8 and C9, though less efficiently. It did not inhibit formation of C3/C5 convertase of the alternative pathway. The inhibition of the classical pathway was reversible since hemolytic activity was completely restored after dialysis.

Intrathecal synthesis of antibodies to HTLV-III in patients without AIDS or AIDS related complex.

De novo synthesis in the central nervous system of IgG antibodies to human T cell lymphotropic virus type III (HTLV-III) (lymphadenopathy associated virus) was shown in seven of 10 seropositive men who had syphilis but not the acquired immune deficiency syndrome (AIDS) or AIDS related complex. None of these men showed neurological symptoms when the serum and cerebrospinal fluid were collected. Pleocytosis was present in all 10. Of the seven men who showed evidence of intrathecal synthesis of antibodies, five had increased total concentrations of IgG and four had oligoclonal IgG bands in their cerebrospinal fluid. Oligoclonal bands were also present in one man who did not have any antibodies. Longitudinal study of one man showed that seroconversion preceded intrathecal synthesis of antibody specific to HTLV-III. The appearance of antibody in the cerebrospinal fluid was accompanied by a transient rise in mononuclear cell count and the appearance of oligoclonal bands. The presence of clones of B cells specific to HTLV-III in the central nervous system of these patients without persisting neurological symptoms suggests that HTLV-III enters the central nervous system in the early stages of infection.

Simple method for preparing the cellular intermediate EAC14, and its use for estimation of the second component of complement.

Sensitized erythrocytes carrying the first and fourth components of complement (EAC14) were prepared by incubating optimally sensitized sheep erythrocytes with normal serum appropriately diluted in Mg2+-free diethylbarbiturate buffer containing Ca2+. EAC14 cells so prepared were found to be suitable for use in estimating the second component of complement (C2) in human serum, and the method is described here.