Placental pathologic conditions in anticardiolipin antibody positive women whose infants had congenital heart defects.

Anticardiolipin antibodies (ACLA) are present in 10% of women with recurrent pregnancy loss. Other associations with ACLA are arterial and venous thrombosis, cerebral infarction, pulmonary hypertension, preterm delivery, and fetal growth retardation. A previous prospective study of infants of mothers with positive ACLA identified an increased incidence of congenital heart disease in this population. As a follow-up, the placentas of the initial 40 ACLA-positive patients were studied to determine whether there was an increased incidence of infarct or thrombosis compared with that in control subjects matched for maternal age and gestational age within the same 2-year period. The age of ACLA-positive mothers was 30 +/- 5 years versus 29 +/- 5 years in the ACLA-negative mothers. Gestational age was 37 +/- 2 weeks in both groups; placental weight was 553 +/- 169 gm in the ACLA-positive group versus 593 +/- 117 gm in the ACLA-negative group. The birth weight was 2972 +/- 709 gm in infants of ACLA-positive mothers and 2920 +/- 674 gm in infants of ACLA-negative mothers. There was no statistically significant difference between the two groups in gestational age, maternal disease, placental histologic findings, placental weight, type of delivery, or type of ACLA. Twenty-seven ACLA-positive women were receiving prednisone. Chi square analysis showed the ACLA-positive mothers to have more spontaneous abortions (p = 0.02) and to have more children with congenital heart disease (5 ventricular septal defects and 2 atrial septal defects) (p = 0.006). In summary, infants born with congenital heart defects in women positive for ACLA did not have significant placental pathologic conditions when compared with control infants.

Novel methods of DNA analysis.

The need for improved ways to analyze nucleic acids for the tools of molecular biology to become more prevalent in the routine clinical laboratory has led to the creation of unique methods for DNA analysis. Automation of sequencing techniques can provide a more rapid turnaround for results and can be more cost effective for the diagnosis of genetic disease and cancer than conventional techniques. Miniaturization of sequencing on DNA chips should be even more effective than our current means and make screening for these diseases easier. Improved amplification techniques will increase the sensitivity of infectious disease detection, and new methods to label and detect DNA probes will help alleviate the need to use radionuclides. This article centers on the nature of some recent advances in DNA analysis and on their future application in the clinical laboratory.

Clinical measurement of serum amiodarone and desethylamiodarone by using solid-phase extraction followed by HPLC with a high-carbon reversed-phase column.

We describe a rapid, simple HPLC method routinely used in our clinical laboratory for determining amiodarone and its metabolite desethylamiodarone. These compounds are released from serum proteins by pretreatment with an acidic solution and then extracted onto a C2 reversed-phase clean-up column. After elution from the extraction column, the compounds are separated and quantified by HPLC with a C18 reversed-phase column and spectrophotometric detection. The standard curves for the drug and metabolite are linear up to 20.0 mg/L, with a lower limit of detection of 0.16 mg/L. The CVs for intra-assay precision were 5.0% at 0.58 mg/L and 2.9% at 5.96 mg/L; for inter-assay precision, they were 9.6% at 0.52 mg/L and 6.1% at 2.09 mg/L. Lipemia, hemoglobin, and bilirubin up to 300 mg/L do not interfere with this assay. None of > 550 cardiac patients' samples tested contained a compound that interferes with this assay.

Measurement of antimicrobial agents in cerebrospinal fluid using the Abbott TDx analyzer.

Occasionally, requests are made by our physicians for the measurement of gentamicin, tobramycin, or vancomycin in cerebrospinal fluid (CSF) specimens during the course of treating patients for bacterial meningitis. We evaluated CSF as a specimen type for the measurement of amikacin, gentamicin, tobramycin, and vancomycin on the Abbott TDx analyzer. Coefficients of variation for CSF spiked with these antimicrobial agents ranged from 0.8% to 6.5% for intra-assay values and from 2.1% to 2.3% for inter-assay values. Serum and CSF specimens were spiked at various levels with equal amounts of the antibiotics. Correlation coefficients for serum vs. CSF for these agents were 0.999. Recoveries ranged from 86% to 134%. Sensitivity for these assays is about fourfold better for CSF than for serum. CSF appears to be an acceptable specimen type for the measurement of these antibiotics using the Abbott TDx analyzer.

A comparison of two common clinical methods with high-pressure liquid chromatography for the measurement of creatinine concentrations in neonates.

The accurate measurement of low serum creatinine levels is necessary for estimating clinically useful creatinine clearances in the pediatric population. This study compares two routine clinical methods: the kinetic Jaffé with the newer Kodak enzymatic method against our reference method, high-pressure liquid chromatography, for the measurement of serum and urine creatinine levels in neonates. One hundred and twenty-five serum and 59 urine creatinines and 56 absolute creatinine clearances were measured in neonates ranging from 23 to 46 weeks (mean 32 weeks) post-conceptional age and weighing 480-4398 g (mean 1650 g). Urine creatinine levels, and serum creatinine levels greater than 0.8 mg/dl were equivalent for both clinical methods. However, the enzymatic method was much more accurate (P less than 0.001) than the kinetic Jaffé method for serum creatinine measurements of less than or equal to 0.8 mg/dl. We conclude that the enzymatic methodology is a better clinical choice for the accurate measurement of serum creatinine levels when using these values for the determination of neonatal renal function.

Clinical evaluation of five therapeutic drugs using dry film multilayer technology on the OPUS immunoassay system.

Five therapeutic drug assays, carbamazepine, phenobarbital, phenytoin, theophylline, and valproic acid, were evaluated using an automated random access system for performing thin dry film multilayer competitive immunoassays, the OPUS analyzer. All reagents for the therapeutic drug assays are contained in a coated multilayer film chip encased within a plastic bar-coded test module and require no external or supplementary reagents. A serum or plasma sample is applied to the test module by the instrument and the fluorescence intensity from the module is measured after 6 minutes. We found the OPUS assays acceptable for clinical use. Within-run coefficient of variations were 2.3-6.7%, between-run, 2.9-7.6%. These methods correlated well with the Abbott TDx, having correlation coefficients of 0.92-0.97. Because of the instrument design and the stability of the reagents, weekly calibration is not needed and samples can be run immediately upon receipt in a random access fashion or can be batched together.

Rhabdomyolysis in a case of free-base cocaine ("crack") overdose.

This is the case of a 27-year-old black man who was admitted to Loyola University Medical Center after a one-time experience of smoking free-base ("crack") cocaine. Clinical manifestations of the resulting cocaine intoxication were rhabdomyolysis, acute renal failure, and transient liver failure. This patient came to our attention because of the striking alterations in his blood-chemistry values, which indicated acute tissue damage, and his remarkable recovery within 96 h. We discuss the dramatic changes in the laboratory findings and the clinical course of this patient.

Construction of cosmid genomic libraries for the normal and myopathic Syrian hamsters.

Cosmid genomic libraries from both normal and myopathic Syrian hamsters have been constructed. MboI was used to generate 35- to 50-kilobase DNA fragments which were isolated from a 5-25% NaCl gradient. The 35- to 50-kilobase DNA fragments were ligated to the cosmid vector pCV108 and packaged into Escherichia coli DK1. Approximately 3 X 10(5) - 4 X 10(5) clones were obtained per microgram of ligated DNA. Thirteen clones have been isolated from 2 X 10(5) colonies using a cardiac myosin heavy chain clone as a probe. Restriction maps of two of these clones are presented here.

Two different forms of beta myosin heavy chain are expressed in human striated muscle.

We have found evidence for two beta-like myosin heavy chains in humans, one cardiac and one skeletal. The cDNA sequences of the cardiac beta myosin heavy chain cDNA clone pHMC3 and the skeletal beta-like myosin heavy chain cDNA clone pSMHCZ, were compared to each other. It was found that the 3' untranslated regions as well as 482 nucleotides specifying the carboxyl coding region, were 100% homologous. Further examination revealed that the skeletal clone pSMHCZ diverges from the human cardiac beta myosin heavy chain cDNA clone pHMC3 at the 5' end. We present evidence in this report which indicates that the cardiac beta myosin heavy chain mRNA is expressed in skeletal muscle tissues. The human cardiac beta myosin heavy chain cDNA clone, pHMC3, which codes for a portion of the light meromyosin section of the myosin heavy chain, was used as a probe for S1 nuclease mapping studies with RNA derived from cardiac tissue, smooth muscle and skeletal muscle tissues consisting of fast-twitch, slow-twitch and mixed fast- and slow-twitch muscle fibres. Two probes were used to examine the expression of the mRNA. One probe (406 nucleotides) constitutes the 3' untranslated region and a portion of the coding region of the beta cardiac myosin heavy chain cDNA clone, which is 100% homologous to pSMHCZ, the skeletal cDNA clone. The other constitutes the majority of the coding region (1017 nucleotides) of the cardiac clone pHMC3 in which the first 216 nucleotides from the labelled end are 100% homologous to the skeletal clone pSMHCZ. In the soleus muscle, which is rich in slow-twitch type I muscle fibres, the expression of the cardiac beta myosin heavy chain mRNA was very prominent. In gastrocnemius muscle, a mixed fibre muscle, the expression of this mRNA was detected to a lesser degree than that for the soleus muscle. In vastus lateralis and vastus medialis, which consist of predominantly type II, fast-twitch fibres, there were trace amounts of the cardiac beta myosin heavy chain mRNA. When expression of this mRNA was tested in smooth muscle tissue none could be detected.

Construction of a human ventricular cDNA library and characterization of a beta myosin heavy chain cDNA clone.

We have constructed and characterized for the first time a complementary DNA (cDNA) clone, pHMC3, which codes for a cardiac myosin heavy chain mRNA from human heart. This clone contains a 1.7 kb DNA segment and specifies 543 amino acids of the carboxyl portion of the myosin heavy chain. The DNA sequence and encoded amino acid sequence were compared to the hamster alpha (pVHC1) and beta (pVHC2/pVHC3) cardiac myosin heavy chain cDNA and amino acid sequences and the rat cardiac myosin heavy chain sequences as well. The myosin heavy chain mRNAs are highly conserved and this is reflected in our cDNA clone. The pHMC3 clone is 87.9% homologous to the hamster alpha cDNA and 92.2% homologous to the hamster beta cDNA clones. The 3' untranslated region of pHMC3 is 64.1% homologous to the hamster beta clone while the hamster alpha myosin heavy chain shows only 25% homology to pHMC3 and exhibits extensive diversity. Similar results were obtained when pHMC3 was compared to the rat cardiac myosin heavy chain cDNA sequences. The comparisons showed that pHMC3 is a beta cardiac myosin heavy chain cDNA clone.

Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequences, the carboxyl-terminal peptide and the 3'-untranslated region are highly divergent and specific for this cDNA clone. There appears to be an amino acid deletion in the 3' end of the hamster alpha myosin heavy chain as compared to the rat alpha myosin heavy chain. S1 nuclease mapping experiments have shown that the mRNA represented by this cDNA clone is scarcely expressed in neonatal development, but its expression increases with age and reaches maximal levels in adult life. This cDNA clone provides a useful tool to follow the myosin heavy chain mRNA changes during development and during the genesis of a cardiomyopathy, an autosomal recessive defect carried by the Syrian hamster.