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A 62-year-old Virginia man in good health was found on routine testing to have a decreased platelet count of 124k/μL (reference interval: 150–450k/μL). There was no evidence of bruising or bleeding, and there was no family history of thrombocytopenia. His weight was stable, and he had no complaints of pain and no gastrointestinal, musculoskeletal, or hematological symptoms. He denied fatigue, fevers, chills, and night sweats. Past medical history included hypothyroidism and chronic obstructive pulmonary disease. Physical examination was unremarkable. Other laboratory test results included hemoglobin of 13.8 g/dL (reference interval: 14.0–18.0 g/dL) and mean red-cell volume of 98.1 fL (reference interval: 83.0–95.0 fL). Plasma potassium was 4.1 mmol/L (reference interval: 3.5–4.5 mmol/L), calcium 8.8 mg/dL (reference interval: 8.8–10.5 mg/dL), albumin 4.23 g/dL (reference interval: 3.4–5.0 g/dL), and alkaline phosphatase 61 U/L (reference interval: 40–150 U/L). Review of a peripheral blood smear found no obvious evidence of pseudothrombocytopenia. Serum ferritin was 53 ng/mL (reference interval: 20–275 ng/mL) and iron saturation was 18% (reference interval: 16%–48%). Serum protein electrophoresis (Fig. 1A) revealed 2 restricted bands in the γ region, identified by immunofixation (IFE)3 electrophoresis (Fig. 1B) as IgG κ proteins. The combined concentration of these 2 bands was

Although the majority of all clinical testing is performed in a central laboratory environment within the hospital or reference laboratory, there is a growing trend to provide laboratory results in near-patient or point-of-care testing environments. In addition, there is growing evidence of testing being performed outside of a healthcare facility, with sample collection and/or testing being performed by the patient in an at-home setting. For waived, low-complexity testing, this approach may be appropriate; however, direct-to-consumer (DTC)3 marketing has also made highly complex testing available to the consumer via self-collection of a buccal (cheek) swab and routine mailing to specialized laboratories for genetic testing and risk assessment. We asked 5 professionals representing academic clinical laboratories, medical genetics subspecialties, and DTC laboratories to comment on this growing trend. Lew Bender4 : One of the goals of personalized healthcare is to prevent disease through proactive understanding of our health risks, setting goals to become healthier, and ultimately giving consumers the tools they need to make more informed decisions to manage their health. DTC testing enables individuals to hone in on a particular condition they’re interested in, learn more about it from their test kit and online support, and take a simple cheek swab in the privacy of their own home. There are significant advantages to this type of testing, provided DTC genetic tests have analytic and clinical validity, are clinically useful, and at the same time provide actionable, personalized guidance that consumers can then follow to minimize their genetic risks. Lawrence M. Silverman5 : Accessibility of esoteric testing is a mixed blessing. On one hand, the consumer is able to choose which laboratory and what tests are being performed, which is often difficult when third-party payers control the choice of laboratories. However, removing healthcare professionals from the process makes quality …

The tumor suppressor genes BRCA1 and BRCA2, which confer increased susceptibility to breast and (or) ovarian cancer, were recently identified. Mutation analysis of BRCA1 has demonstrated significant allelic heterogeneity; however, some distinct mutations have been detected in unrelated individuals. The most notable is the 185delAG mutation, which occurs at an estimated frequency of ∼1% in individuals of Ashkenazi Jewish descent [1]. Although consensus has not been reached regarding clinical testing for mutations in BRCA1, a tiered strategy may be appropriate, in which direct testing for the more common mutations is one component. Specific alleles can be detected by using PCR-mediated site-directed mutagenesis (PSM), which alters the PCR products derived from either the wild-type or mutant allele to create or destroy a restriction endonuclease recognition site. Recognition sites are introduced by a base substitution in one of the primers. The alleles are then resolved by electrophoresis of the digested PCR products. We have applied this technique to the detection of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and R1443X. Another mutation, 1294del40, can be resolved from the wild-type allele by high-resolution gel electrophoresis alone. The PSM technique is sensitive, does not require radioactivity, and is specific for individual mutations.

Amplification of specific gene target sequences has become a routine molecular procedure in a variety of laboratories. When coupled with either a direct or indirect method of detecting amplified product, in situ amplification offers an extremely powerful investigative tool. We describe a protocol for a localized in situ amplification (LISA) reaction that includes tissue-culture cloning rings and allows for the amplification of gene target sequences in specific regions of paraffin-embedded tissue sections. Digoxigenin-11-dUTP was added to the amplification reaction and thus incorporated into the amplified products, providing a mechanism by which direct nonisotopic detection could be performed. To demonstrate the approach, LISA was performed on known positive Pneumocystis carinii rat lung tissues, with primers specific for the P. carinii rRNA gene sequence.

In this issue, Castaldo et al. (1) describe the use of denaturing gradient gel electrophoresis to scan the CFTR gene in cystic fibrosis (CF) patients from Southern Italy who were negative for the common CF mutations. As expected, rare mutations were found, five of which were useful in population screening. These findings are particularly important for laboratories in Italy and in areas with families of Italian descent. Advanced molecular techniques provide a double-edged sword because they often detect sequence changes whose deleterious natures are by no means established. In the CFTR gene, such variants may cause classic CF, elicit atypical phenotypes with similarities to CF, or have no consequence to health. Among recent letters in the New England Journal of Medicine on mild phenotypic variants of CF (2)(3)(4), one suggested that “the diagnosis of cystic fibrosis is too nebulous to preserve in clinical practice and that perhaps, as is the case with Cushing’s disease and syndrome, we need to have both a cystic fibrosis ‘disease’. . . and a cystic fibrosis ‘syndrome’ (which would include the pancreatic manifestations, congenital bilateral absence of the vas deferens, or lesser pulmonary manifestations)” (3). As improved mutation detection technologies enter the clinical laboratory and identify rare CFTR mutations, laboratorians and clinicians must be cognizant of the changing nature of genotype-phenotype associations. The severity of the pancreatic and hepatic features of CF is highly variable. The gastrointestinal presentations endured by CF patients take many forms (5). The majority of patients (85–90%) experience pancreatic exocrine insufficiency. Those who retain pancreatic exocrine function (10–15%) typically carry at least one of the CFTR gene mutations that have been associated with residual function. Other CF patients have impaired endocrine insulin production associated with diabetes (10–15% of patients) or liver dysfunction (5), although no specific CFTR …

Detection of carriers of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), in the deletion cases, involves calculating gene dosage from Southern blots. We show that the analysis of dosage can also be made from the polymerase chain reaction (PCR) with use of allele-specific oligonucleotides (ASOs). The deletion-prone exons are amplified, transferred to a membrane, and hybridized with ASOs complementary to the exons; the autoradiographic bands are then quantified with a densitometer. After determining the quantitative conditions of the amplification reaction, we were able to identify deletions in a DMD/BMD carrier female. The determination of carrier status via PCR removes several of the technical limitations of Southern analysis and is also cost- and labor-effective.

The cystic fibrosis (CF) gene has been recently cloned, and a deletion of 3 basepairs (bp) of DNA was found on most of the CF chromosomes. This deletion leads to the synthesis of a protein that lacks a phenylalanine residue at position 508. Using two polymerase chain reaction protocols to study the frequency of this mutation in a series of 192 CF patients, we found the mutation on 72% of affected chromosomes. We then used this value to calculate the predictive value of a negative test result in a population-based screening program for CF carrier status. Haplotype analysis with the polymorphic markers XV.2c and KM-19 on 239 CF chromosomes revealed that 90.7% of CF chromosomes with the deletion had a single haplotype. This haplotype was also associated with 60.4% of CF chromosomes with unknown mutations. These values can be used to calculate the probability of whether an individual from the general population is a carrier of any CF mutation.

Gene mutations responsible for the majority of Duchenne/Becker muscular dystrophy (DMD/BMD) and cystic fibrosis (CF) chromosomes have been identified. We describe a DNA-based strategy, rather than the traditional biochemical assays, for screening newborns. DNA sequences spanning the CF mutation and several DMD/BMD deletion-prone exons are amplified simultaneously via a multiplex polymerase chain reaction. The gel is visually inspected for DMD/BMD deletions and then blotted and hybridized with allele-specific oligonucleotides to determine the presence or absence of the CF mutation. We determined that blood spots provide sufficient DNA for the molecular analysis, so the procedure can be used in screening programs of newborns.

We compared relative increases in creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) after reperfusion in myocardial infarction for four popular methods: electrophoresis, immunoinhibition, the "Magic Lite" (Ciba-Corning) system, and the Stratus (Dade). In a method comparison study, we confirmed that all four methods correlated (r greater than 0.95). Electrophoresis demonstrated the greatest scatter about the regression line, immunoinhibition the least. For CK-MB quantities near each method's "positive cutoff" indicating myocardial infarction, results by all methods agreed in 95% of samples. To characterize relative increases in CK-MB, we computer-fitted data obtained from each method for serial specimens collected from six acute myocardial infarction patients during myocardial reperfusion. Although for each individual patient the four methods appeared to exhibit parallelism, the methods differed significantly in terms describing their appearance rate, peak-time & fall-off, and time-to-peak activity. Consistent with these data, we found that the relative CK-MB increases at various times after reperfusion, compared with baseline concentrations, are method-dependent. Therefore, when using CK-MB for indicating coronary patency, one must develop specific limits for each method utilized.

By use of cDNA probes, molecular deletions were identified in 66.6% of 42 patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). Owing to this high deletion rate, a new strategy for detecting DMD/BMD carriers is feasible in which the polymerase chain reaction is used as an initial screen for detecting the deletions occurring in specific deletion-prone exons. Because the deletions do not occur randomly, specific cDNA probes are utilized first with Southern blot analysis. Identification of a deletion permits direct analysis for DMD carrier status and removes the inherent limitations of the conventional restriction fragment length polymorphism technique. Carrier status is determined by scanning the autoradiographs with a densitometric spectrophotometer or by detection of a junction fragment.

A case of adenocarcinoma of the pancreas and mild lung disease in a 39-year-old man homozygous for the delta F508 cystic fibrosis mutation is presented. Cystic fibrosis is the most common lethal genetic disease in Caucasians, and is most commonly associated with severe obstructive lung disease. To our knowledge, this is only the fifth case of adenocarcinoma of the pancreas in a CF patient to be reported and the first case for which molecular data are available. The rare incidence of this type of malignancy in the general population suggests a possible association of CF with this malignant disease.

The polymerase chain reaction (PCR) has been applied in a novel manner to detect the multiple mutations causing cystic fibrosis (CF). PCR-mediated site-directed mutagenesis (PSM) has been applied to create allele-specific restriction enzyme cutting sites for three of the more common mutations. Two other mutations after cutting sites on their own. We discuss the implications for the expedient detection of five different CF-causing mutations.

The tumor suppressor genes BRCA1 and BRCA2, which confer increased susceptibility to breast and (or) ovarian cancer, were recently identified. Mutation analysis of BRCA1 has demonstrated significant allelic heterogeneity; however, some distinct mutations have been detected in unrelated individuals. The most notable is the 185delAG mutation, which occurs at an estimated frequency of approximately 1% in individuals of Ashkenazi Jewish descent [1]. Although consensus has not been reached regarding clinical testing for mutations in BRCA1, a tiered strategy may be appropriate, in which direct testing for the more common mutations is one component. Specific alleles can be detected by using PCR-mediated site-directed mutagenesis (PSM), which alters the PCR products derived from either the wild-type or mutant allele to create or destroy a restriction endonuclease recognition site. Recognition sites are introduced by a base substitution in one of the primers. The alleles are then resolved by electrophoresis of the digested PCR products. We have applied this technique to the detection of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and R1443X. Another mutation, 1294de140, can be resolved from the wild-type allele by high-resolution gel electrophoresis alone. The PSM technique is sensitive, does not require radioactivity, and is specific for individual mutations.

A case of adenocarcinoma of the pancreas and mild lung disease in a 39-year-old man homozygous for the delta F508 cystic fibrosis mutation is presented. Cystic fibrosis is the most common lethal genetic disease in Caucasians, and is most commonly associated with severe obstructive lung disease. To our knowledge, this is only the fifth case of adenocarcinoma of the pancreas in a CF patient to be reported and the first case for which molecular data are available. The rare incidence of this type of malignancy in the general population suggests a possible association of CF with this malignant disease.

Amplification of specific gene target sequences has become a routine molecular procedure in a variety of laboratories. When coupled with either a direct or indirect method of detecting amplified product, in situ amplification offers an extremely powerful investigative tool. We describe a protocol for a localized in situ amplification (LISA) reaction that includes tissue-culture cloning rings and allows for the amplification of gene target sequences in specific regions of paraffin-embedded tissue sections. Digoxigenin-11-dUTP was added to the amplification reaction and thus incorporated into the amplified products, providing a mechanism by which direct nonisotopic detection could be performed. To demonstrate the approach, LISA was performed on known positive Pneumocystis carinii rat lung tissues, with primers specific for the P. carinii rRNA gene sequence.

Although this collection of laboratory techniques is directed primarily to research scientists, it has a wealth of information for clinicians and laboratorians who are involved with cystic fibrosis. As such, it delivers diverse protocols that cover topics ranging from the basic pathophysiology of cystic fibrosis to animal models and gene therapy. Therein lie the strengths of this volume. Basic researchers will probably be familiar with techniques related to their specialty, but other methods covered in this book should be of great interest. For clinicians and laboratorians, familiarity with these techniques …

The polymerase chain reaction (PCR) has been applied in a novel manner to detect the multiple mutations causing cystic fibrosis (CF). PCR-mediated site-directed mutagenesis (PSM) has been applied to create allele-specific restriction enzyme cutting sites for three of the more common mutations. Two other mutations after cutting sites on their own. We discuss the implications for the expedient detection of five different CF-causing mutations.

Gene mutations responsible for the majority of Duchenne/Becker muscular dystrophy (DMD/BMD) and cystic fibrosis (CF) chromosomes have been identified. We describe a DNA-based strategy, rather than the traditional biochemical assays, for screening newborns. DNA sequences spanning the CF mutation and several DMD/BMD deletion-prone exons are amplified simultaneously via a multiplex polymerase chain reaction. The gel is visually inspected for DMD/BMD deletions and then blotted and hybridized with allele-specific oligonucleotides to determine the presence or absence of the CF mutation. We determined that blood spots provide sufficient DNA for the molecular analysis, so the procedure can be used in screening programs of newborns.

The cystic fibrosis (CF) gene has been recently cloned, and a deletion of 3 basepairs (bp) of DNA was found on most of the CF chromosomes. This deletion leads to the synthesis of a protein that lacks a phenylalanine residue at position 508. Using two polymerase chain reaction protocols to study the frequency of this mutation in a series of 192 CF patients, we found the mutation on 72% of affected chromosomes. We then used this value to calculate the predictive value of a negative test result in a population-based screening program for CF carrier status. Haplotype analysis with the polymorphic markers XV.2c and KM-19 on 239 CF chromosomes revealed that 90.7% of CF chromosomes with the deletion had a single haplotype. This haplotype was also associated with 60.4% of CF chromosomes with unknown mutations. These values can be used to calculate the probability of whether an individual from the general population is a carrier of any CF mutation.

By use of cDNA probes, molecular deletions were identified in 66.6% of 42 patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). Owing to this high deletion rate, a new strategy for detecting DMD/BMD carriers is feasible in which the polymerase chain reaction is used as an initial screen for detecting the deletions occurring in specific deletion-prone exons. Because the deletions do not occur randomly, specific cDNA probes are utilized first with Southern blot analysis. Identification of a deletion permits direct analysis for DMD carrier status and removes the inherent limitations of the conventional restriction fragment length polymorphism technique. Carrier status is determined by scanning the autoradiographs with a densitometric spectrophotometer or by detection of a junction fragment.

The authors state that the purposes of this book are to provide a “brief survey of the field” and to “provide an intermediate-level challenge” to the reader. They try to accomplish this by offering 750 multiple-choice, matching, and true/false questions. Each question has an explanation of the correct answer and an appropriate reference. For the most part, I found the questions to be challenging at an “intermediate level”, thus accomplishing one of the purposes; however, …

It used to be simple. Physicians observed patients, asked questions, examined them, and established a defined clinical phenotype. Sometimes a laboratory analyte, with a sufficient and significant correlation with the observed clinical phenotype, could be measured to provide a basis for hypothesis-driven approaches to disease pathogenesis. Two classic examples of diseases are cystic fibrosis (CF), which is associated with increased sweat chloride concentrations, and diabetes mellitus, which is associated with increased glucose. The road to gene discovery has been considered tractable for CF because of its inheritance pattern, whereas diabetes has been considered a polygenic disorder quite intractable to such approaches. …

Characteristics of CK-MB, the MB1 and MB2 isoforms, and the MB2/MB1 ratio are described in six acute myocardial infarction (AMI) patients in whom the infarct-related artery was identified and, after intervention, normal coronary flow was re-established. After myocardial reperfusion, washout of CK-MB and the MB2 isoform occurred in parallel, with CK-MB peaking between 5.75 and 10.0 h, and MB2 peaking between 4.50 and 8.00 h. In five of the six patients, MB1 peaked between 8.75 and 15.5 h; the MB2/MB1 ratio demonstrated the earliest peak from 0.75 to 2.25 h. When we compared this study group to an additional 10 AMI patients who had achieved myocardial reperfusion earlier, we found a significant difference (P less than 0.005) for all tests, except MB1 isoform activity, as early as 50 min after reperfusion. This same comparison, by logistic-regression analysis, showed that the MB2/MB1 ratio discriminated between the groups 50 min after reperfusion (P less than 0.05); MB2 showed near-significance at 100 min (P less than 0.057); and CK-MB achieved significance after 200 min (P less than 0.05). CK-MB, the MB2 isoform, and especially the MB2/MB1 ratio show potential for the early, noninvasive detection of myocardial reperfusion.

Immunoturbidimetric assays for determination of serum apolipoproteins CII and CIII (apo CII and CIII) were developed for use on the Cobas-Bio centrifugal analyzer with commercially available antisera and reference sera. Within-run and between-run imprecision (CVs) for apo CII was respectively 3.3 and 6.2%, and for apo CIII 2.75 and 5.2%. Addition to sera of as much as 50 g of hemoglobin or 0.15 g of bilirubin per liter did not interfere with determinations of apolipoproteins. Concentrations of apo CII and CIII were measured in sera from 40 normolipidemic and hyperlipidemic subjects by these immunoturbidimetric methods (x) and by radial immunodiffusion assays (y). Apo CII and CIII gave slopes of 0.93 and 0.86, y-intercepts of 7.4 and 9.5 mg/L, and correlation coefficients of 0.96 and 0.94, respectively. Reference intervals for the two apolipoproteins were established with sera from 100 normolipidemic subjects. The distribution of apo CII and CIII among the various lipoprotein fractions for normal subjects differed significantly (p less than 0.005) from that for hypertriglyceridemic subjects.

We report specific findings in the imipramine/serotonin animal model that are consistent with sarcolemmal membrane alterations. Among these findings are cytoplasmic enzyme release, diminished uptake of alpha-aminoisobutyrate (an amino acid analog), decreased oxygen consumption in isolated rat diaphragm, and ribosuria. Furthermore, we describe for the first time the release of the MB isoenzyme of creatine kinase from a source other than cardiac tissue; that is, isolated diaphragms from imipramine/serotonin-treated animals release increased amounts of MB isoenzyme as compared to diaphragms from control animals. We believe the similarities between this animal model and the human disease (Duchenne muscular dystrophy) support a genetically determined generalized membrane abnormality in the pathogenesis of this form of muscular dystrophy.

Albumin and other serum-derived proteins were removed from several types of body fluids by affinity chromatography, to facilitate detection of trace or non-serum-derived proteins in two-dimensional electrophoresis patterns. Albumin was removed by the dye Cibacron Blue F3G-A coupled to Sepharose. Two-dimensional patterns of albumin-depleted serum lack the large albumin spot, and several families of spots become visible that ordinarily are partly or totally hidden by it. However, other proteins also bind to Cibacron Blue. Most serum proteins, including albumin, were effectively removed by anti-human serum antibodies coupled to Sepharose. Two-dimensional patterns of serum-depleted cerebrospinal fluid exhibit five clusters of probable nervous-system protein families not detected in serum. One additional family, probably antigenically related to transferrin, was removed by the affinity step. Two-dimensional patterns of serum-depleted prostatic fluid exhibit five major non-serum families, two of which may be creatine kinase B subunits and prostatic acid phosphatase. Two-dimensional patterns of serum-depleted malignant effusions exhibit one or more of three proteins that possibly are tumor products. Pattern matching suggests the presence of one non-serum-derived protein family common to cerebrospinal fluid, prostatic fluid, and malignant effusions. Prostatic fluid and malignant effusions have in common as many as three non-serum families of proteins.

We describe a group of glycoproteins that are synthesized and released by human breast tumors maintained in organ culture and similar glycoproteins released by a human breast carcinoma cell line (BT-20). The electrophoretic mobility of these glycoproteins on cellulose acetate is consistent with increased glycoprotein-staining material present in the alpha2- to beta-globulin region of serum glycoprotein electropherograms from patients with breast cancer. Moreover, after mastectomy, this glycoprotein material in serum decreases to concentrations seen in a control population of patients with benign breast lesions. Patients with proven metastatic breast cancer have patterns reflecting their clinical status: those who respond to treatment have glycoprotein electropherograms similar to the group of patients with benign breast lesions, while those who do not have increased amounts of alpha2- beta-glycoprotein. We believe serum glycoprotein measurements in breast-cancer patients reflect the presence of glycoproteins that are released by the malignant cells and enter the circulation.

Data are presented demonstrating that adenylate kinase (AK; EC 2.7.4.3) is an oncodevelopmental enzyme in the prostate of Copenhagen rats. We selected the Dunning tumor (dorsal rat prostate) as a model system because it most nearly approximates the human pathology. Four sublines of the tumor (R3327-H, R3327-AT, MAT Lu, and MAT LyLu) were studied. The tumor sublines were maintained as solid tumors in syngeneic rats and as monolayers in tissue culture. AK activity appeared in conjunction with malignant transformation of the dorsal prostate. We also determined the normal developmental enzyme pattern: AK was present in prostates of newborns, but was undetectable in prostates of adults. However, AK increased after castration. Therefore, we propose AK as a potential oncofetal tumor marker in prostatic cancer.

Approximately 85% of patients with multiple sclerosis (MS) can be diagnosed by using magnetic resonance imaging and laboratory tests such as determination of the cerebrospinal fluid (CSF) IgG Index and electrophoresis to detect oligoclonal banding. However, these tests results are abnormal in MS patients whether they are in clinical remission or acute exacerbation. Because apolipoprotein E (apo E) is synthesized in the central and peripheral nervous system, particularly during remyelination, we propose that apo E might be a reliable marker of the remyelination that accompanies clinical remission in MS patients. We studied 33 patients with MS, 22 in remission and 11 in exacerbation, and 26 controls of comparable ages. The apo E Index, calculated from the concentrations of apo E and albumin in CSF and serum, allowed us to discriminate between MS patients in remission and MS patients in exacerbation (P less than 0.001); the IgG Index failed to show similar differences. However, combining the apo E and IgG indices gave maximum discrimination between controls, MS patients in remission, and those in exacerbation. This study suggests that apo E measurements should be included in the laboratory evaluation of MS patients.

Albumin and immunoglobulin G (IgG) were determined in cerebrospinal fluid (CSF) and serum, and the CSF/serum albumin Index (CSF X 10(3)/serum albumin concentration ratio) and IgG Index [(CSF/serum IgG)/(CSF/serum albumin)] were calculated. Data for these indices and oligoclonal banding are described in 23 cases of multiple sclerosis (MS), 19 of systemic lupus erythematosus (SLE), eight of sarcoidosis, 48 cases of miscellaneous disease, and 25 control patients with nonspecific complaints. Of the MS, SLE, and sarcoidosis patient groups, 8.5%, 26%, and 12.5% showed an abnormally high CSF/serum albumin Index; 87%, 16%, and 0% an increased IgG Index; and 87.5%, 42% and 0% showed positive oligoclonal banding. IgG Index and oligoclonal banding results for MS patients differed significantly from the sarcoidosis (p less than .001) and SLE (p less than .05) groups. When the CSF/serum albumin Index is considered also, the control and sarcoidosis patient results differ significantly from the MS group (p less than .001 and p less than .01). A strong correlation between the IgG Index and oligoclonal banding is implicated.

Oligoclonal banding patterns, when present in cerebrospinal fluid (CSF) electrophoresed on agarose gels, are important in the diagnosis of multiple sclerosis and other demyelinating diseases. Typically, an 80-fold concentration of 2.5 mL of CSF has been needed before the oligoclonal banding pattern could be detected with Coomassie Brilliant Blue stain. This excessive volume requirement often is a problem, particularly in children. We describe here an improved silver-stain procedure with which we routinely can detect the oligoclonal banding pattern in 1 to 3 microL of unconcentrated CSF. The procedure is inexpensive and takes less than 45 min. Our method is quick, safe, sensitive, and easily performed in the clinical laboratory.

We describe use of a microconcentrator membrane with a 30 000-Da cutoff for treatment of cerebrospinal fluid (CSF) specimens before detection of oligoclonal bands by electrophoresis on agarose. After centrifugation at 2000 X g for 25 min, 0.5-, 1.0-, and 2.0-mL aliquots of CSF were concentrated 15-, 25-, and 40-fold. Analytical recovery of immunoglobulins G and A from the microconcentrators was about 90% (CV 5-8%). We found good correlation between results by this method and by a silver-stain procedure in a study comparing oligoclonal banding in CSF from multiple sclerosis and control patients. After 40-fold concentration of 2 mL of CSF, 2 mg of immunoglobulin G per liter can be detected, because the analytical sensitivity of the electrophoresis is 80 mg/L.

Characteristics of CK-MB, the MB1 and MB2 isoforms, and the MB2/MB1 ratio are described in six acute myocardial infarction (AMI) patients in whom the infarct-related artery was identified and, after intervention, normal coronary flow was re-established. After myocardial reperfusion, washout of CK-MB and the MB2 isoform occurred in parallel, with CK-MB peaking between 5.75 and 10.0 h, and MB2 peaking between 4.50 and 8.00 h. In five of the six patients, MB1 peaked between 8.75 and 15.5 h; the MB2/MB1 ratio demonstrated the earliest peak from 0.75 to 2.25 h. When we compared this study group to an additional 10 AMI patients who had achieved myocardial reperfusion earlier, we found a significant difference (P less than 0.005) for all tests, except MB1 isoform activity, as early as 50 min after reperfusion. This same comparison, by logistic-regression analysis, showed that the MB2/MB1 ratio discriminated between the groups 50 min after reperfusion (P less than 0.05); MB2 showed near-significance at 100 min (P less than 0.057); and CK-MB achieved significance after 200 min (P less than 0.05). CK-MB, the MB2 isoform, and especially the MB2/MB1 ratio show potential for the early, noninvasive detection of myocardial reperfusion.

Approximately 85% of patients with multiple sclerosis (MS) can be diagnosed by using magnetic resonance imaging and laboratory tests such as determination of the cerebrospinal fluid (CSF) IgG Index and electrophoresis to detect oligoclonal banding. However, these tests results are abnormal in MS patients whether they are in clinical remission or acute exacerbation. Because apolipoprotein E (apo E) is synthesized in the central and peripheral nervous system, particularly during remyelination, we propose that apo E might be a reliable marker of the remyelination that accompanies clinical remission in MS patients. We studied 33 patients with MS, 22 in remission and 11 in exacerbation, and 26 controls of comparable ages. The apo E Index, calculated from the concentrations of apo E and albumin in CSF and serum, allowed us to discriminate between MS patients in remission and MS patients in exacerbation (P less than 0.001); the IgG Index failed to show similar differences. However, combining the apo E and IgG indices gave maximum discrimination between controls, MS patients in remission, and those in exacerbation. This study suggests that apo E measurements should be included in the laboratory evaluation of MS patients.

Albumin and other serum-derived proteins were removed from several types of body fluids by affinity chromatography, to facilitate detection of trace or non-serum-derived proteins in two-dimensional electrophoresis patterns. Albumin was removed by the dye Cibacron Blue F3G-A coupled to Sepharose. Two-dimensional patterns of albumin-depleted serum lack the large albumin spot, and several families of spots become visible that ordinarily are partly or totally hidden by it. However, other proteins also bind to Cibacron Blue. Most serum proteins, including albumin, were effectively removed by anti-human serum antibodies coupled to Sepharose. Two-dimensional patterns of serum-depleted cerebrospinal fluid exhibit five clusters of probable nervous-system protein families not detected in serum. One additional family, probably antigenically related to transferrin, was removed by the affinity step. Two-dimensional patterns of serum-depleted prostatic fluid exhibit five major non-serum families, two of which may be creatine kinase B subunits and prostatic acid phosphatase. Two-dimensional patterns of serum-depleted malignant effusions exhibit one or more of three proteins that possibly are tumor products. Pattern matching suggests the presence of one non-serum-derived protein family common to cerebrospinal fluid, prostatic fluid, and malignant effusions. Prostatic fluid and malignant effusions have in common as many as three non-serum families of proteins.

Data are presented demonstrating that adenylate kinase (AK; EC 2.7.4.3) is an oncodevelopmental enzyme in the prostate of Copenhagen rats. We selected the Dunning tumor (dorsal rat prostate) as a model system because it most nearly approximates the human pathology. Four sublines of the tumor (R3327-H, R3327-AT, MAT Lu, and MAT LyLu) were studied. The tumor sublines were maintained as solid tumors in syngeneic rats and as monolayers in tissue culture. AK activity appeared in conjunction with malignant transformation of the dorsal prostate. We also determined the normal developmental enzyme pattern: AK was present in prostates of newborns, but was undetectable in prostates of adults. However, AK increased after castration. Therefore, we propose AK as a potential oncofetal tumor marker in prostatic cancer.

Creatine kinase isoenzyme BB (CK-BB) is found in the serum of patients with various types of cancer. Using both radioimmunoassay and agarose electrophoresis, we found abnormal amounts of this isoenzyme in the serum of 15 of 17 patients with untreated prostatic carcinoma. Among patients with other types of adenocarcinomas and metastatic disease, serum CK-BB was increased in most of those who were unresponsive to therapy. In benign or malignant prostate tissue and in malignant pleural effusions, cytoplasmic CK-BB as determined by immunoperoxidase staining was found in epithelial cells. Prostatic fluid had high concentrations of CK-BB, as did malignant, but not benign, pleural fluid supernates. We conclude that glandular epithelial cells contain CK-BB, which is released in benign and malignant states and may appear in higher concentrations in the circulation in malignant states. These conclusions are consistent with predictions we have made from a model experimental system concerning characteristics of possible tumor markers. The observations indicate a role for CK-BB as a tumor marker, particularly for adenocarcinoma of the prostate.

Immunoturbidimetric assays for determination of serum apolipoproteins CII and CIII (apo CII and CIII) were developed for use on the Cobas-Bio centrifugal analyzer with commercially available antisera and reference sera. Within-run and between-run imprecision (CVs) for apo CII was respectively 3.3 and 6.2%, and for apo CIII 2.75 and 5.2%. Addition to sera of as much as 50 g of hemoglobin or 0.15 g of bilirubin per liter did not interfere with determinations of apolipoproteins. Concentrations of apo CII and CIII were measured in sera from 40 normolipidemic and hyperlipidemic subjects by these immunoturbidimetric methods (x) and by radial immunodiffusion assays (y). Apo CII and CIII gave slopes of 0.93 and 0.86, y-intercepts of 7.4 and 9.5 mg/L, and correlation coefficients of 0.96 and 0.94, respectively. Reference intervals for the two apolipoproteins were established with sera from 100 normolipidemic subjects. The distribution of apo CII and CIII among the various lipoprotein fractions for normal subjects differed significantly (p less than 0.005) from that for hypertriglyceridemic subjects.