January 2004

Clinical pathology abstracts editors: Michael Bissell, MD, PdD, MPH, professor and director of clinical services and vice chair, Department of Pathology, Ohio State University Medical Center, Columbus, and Ronald Domen, MD, professor or pathology, medicine, and humanities, Penn State University College of Medicine, Hershey, Pa.

• Preventing pain related to bone marrow aspiration
• Transcobalamin polymorphism and spontaneous abortion
• Stabilization of mRNA in whole blood samples
• Hyperglycosylated human chorionic gonadotropin and Down syndrome
• Homogeneous vs. precipitation assays for HDL in diabetics

Preventing pain related to bone marrow aspiration

Pathologists are often called on to perform bone marrow aspirations in their local hospitals. Although local anesthesia is routinely used in performing the aspiration, little is known about the prevalence and prevention of pain related to this invasive procedure. In the first part of this study, the authors interviewed 132 adult patients scheduled to undergo bone marrow aspiration. A visual analogue scale was used to assess the level of pain experienced by the patients. No pain was a VAS score of 0 mm, minimal pain was a score of one to 30 mm, moderate pain was a score of 31 to 54 mm, and severe pain was a score of 55 mm or higher. In the second part of the study, patients were randomized in a double-blind single-dose trial to receive oral premedication with tramadol or a placebo. Tramadol is a centrally acting analgesic agent that binds to opiate receptors and inhibits norepinephrine and serotonin reuptake in the central nervous system. All patients received local anesthesia with two percent lidocaine. Eighty (60.6 percent) of the patients were male and were a mean age of 58.5 ± 1.4 years. A total of 111 (84.1 percent) patients reported pain during the bone marrow aspiration. The pain intensity averaged 27.2 ± 2.1 mm. Pain was absent or minimal in 85 (64.4 percent) patients, moderate in 26 (19.7 percent), and severe in 21 (15.9 percent). Pain intensity was significantly less when the procedure took less than 10 minutes (P=0.004) or was performed by an experienced physician (P=0.04). Pain during aspiration was significantly less in tramadol recipients. No pain was reported in 18 (36 percent) tramadol recipients versus in seven (14 percent) placebo recipients (P=0.021). Moderate pain also occurred less often in the tramadol group than in the placebo group (20 versus 40 percent; P=0.029). Similar side effects, none serious, were reported between the tramadol and placebo groups. The authors concluded that despite the use of local anesthesia, the level of pain experienced with bone marrow aspiration is not insignificant. Pretreatment of patients with tramadol significantly lowers pain intensity and is well tolerated.

Vanhelleputte P, Nijs K, Delforge M, et al. Pain during bone marrow aspiration: prevalence and prevention. J Pain Symptom Manage. 2003;26:860–866.

Correspondence: Dr. Steven Vanderschueren, Multidisciplinary Pain Center, University Hospital Leuven,

Transcobalamin polymorphism and spontaneous abortion

Cobalamin is an essential co-factor in folate-dependent homocysteine metabolism and is frequently deficient in pregnant women. The products formed are the essential amino acid methionine and tetrahydrofolate. The deficiency state is commonly associated with hyperhomocysteinaemia, a risk factor for neural tube defects and recurrent embryo loss. Insufficient methylation of crucial metabolites and direct toxicity of homocysteine have been suggested as possible mediators of teratogenesis. Two proteins bind vitamin B₁₂ in serum-haptocorrin and transcobalamin. The latter is the critical transporter that delivers vitamin B₁₂ to peripheral tissues. Polyacrylamide gel electrophoresis reveals four common phenotypic isotypes of transcobalamin: M, X, S, and F, which influence vitamin B₁₂ levels. The phenotypic variability of transcobalamin is multifactorial, but the substitution of proline for arginine at codon 259 of the transcobalamin gene is the major determinant of transcobalamin variability in Caucasians and affects transcobalamin levels in plasma. The authors hypothesized that there may be a relation between fetal transcobalamin Pro259Arg genotypes and spontaneous abortion because of the differential effects of the genotypes on transcobalamin levels, vitamin B₁₂ bioavailability, and homocysteine metabolism. The authors analyzed DNA samples from 77 spontaneously aborted human embryos and 115 adult controls for the transcobalamin Pro259Arg polymorphism using the solid-phase minisequencing technique, which directly detects the transcobalamin Pro259Arg polymorphism and is not sensitive to silent polymorphisms. The DNA samples were taken from embryos that had been spontaneously aborted between the sixth and 20th week after conception and adult controls. The 259-Pro allele was significantly less frequent in the spontaneous abortion group than in the control group (42.2 and 57 percent, respectively; P=0.005), while the frequency of 259-Arg was significantly increased. There was a lower prevalence of 259-Pro homozygotes in the spontaneous abortion group compared with the control group (9.1 and 32.2 percent, respectively; P<0.001). The authors concluded that the 259-Pro allele seems to have beneficial influence during embryogenesis, perhaps through increased vitamin B₁₂ intracellular bioavailability.

Zetterberg H, Regland B, Palmér M, et al. The transcobalamin codon 259 polymorphism influences the risk of human spontaneous abortion. Hum Reprod. 2002; 17: 3033-3036.

Reprints: Henrik Zetterberg, Dept. of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, S-413 45 Gotheburg, Sweden; henrik.zetterberg@clinchem.gu.se

Stabilization of mRNA in whole blood samples

Profiling of gene transcription by microarray or quantitative PCR has become an important research tool. Quantitative reverse transcription-PCR (RT-PCR) assays for specific gene transcripts have been developed to indicate the presence or prognosis of disease, predict or monitor response to drug therapy, and track disease kinetics. Analysis of blood samples is most often conducted on leukocytes. Whole blood is collected in various anticoagulants, including salts of citrate, heparin, and EDTA. Though these additives inhibit clotting, they do little to maintain in vivo gene transcript quantities or control other preanalytical variables added by the cell preparation method. Consequently, accurate analysis of in vivo gene expression in whole blood may be complicated by changes in cellular transcript patterns caused by sample collection, handling, storage, or uncontrolled coagulation. Intracellular RNA may be rapidly degraded ex vivo by endogenous nucleases. In addition, unintentional gene expression can be induced by phlebotomy, contact with foreign surfaces, or exposure to the contents of lysed cells, such as hemoglobin. The authors developed the PAXgene blood RNA system (for research use only), which includes a stabilizing additive in a blood collection tube, the PAXgene blood RNA tube (PAXgene), and a companion sample-processing reagent set, the PAXgene blood RNA kit, for purifying intracellular RNA from whole blood. Using agarose gel electrophoresis, Northern blot analysis, and quantitative real-time PCR, the authors compared the stability of total RNA and specific mRNAs in blood collected in PAXgene and EDTA tubes. They collected parallel blood samples from healthy donors in the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for five days at room temperature and for up to 90 days at 4°C and 20°C. Samples were analyzed by Northern blot analysis or RT-PCR. Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors. The authors concluded that storing whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.

Rainen L, Oelmueller U, Jurgensen S, et al. Stabilization of mRNA expression in whole blood samples. Clin Chem. 2002;48: 1883-1890.

Reprints: Lynne Rainen, Becton Dickinson MC 338, 1 Becton Drive, Franklin Lakes, NJ 07417; lynne_rainen@bd.com

Hyperglycosylated human chorionic gonadotropin and Down syndrome

Human chorionic gonadotropin exists in maternal pregnancy serum in many different forms, such as intact hCG, free hCG subunits, nicked forms, and the recently identified hyperglycosylated forms of intact hCG, and presumably free β-hCG, in which there are additional sialyl N-acetyllactosamine antennae. Attempts to improve Down syndrome detection rates have been ongoing in recent years. Cole et al (1997) initially reported increased levels of hyperglycosylated variants of hCG in urine from women carrying a fetus affected by Down syndrome. The authors of the current study examined the value of the hyperglycosylated hCG (HhCG) measurement with a sialic acid-specific lectin immunoassay in the first trimester of pregnancy in 54 cases of pregnancy complicated by Down syndrome and 224 unaffected pregnancies. They compared these levels with those of other potential first trimester serum markers-free β-hCG, pregnancy-associated plasma protein A, and total hCG-and modeled detection rates and false-positive rates of various biochemical markers in conjunction with fetal nuchal translucency and maternal age using a maternal age-standardized population. Maternal serum HhCG in cases of Down syndrome was significantly elevated (median MoM, 1.97), with 24 of 54 cases (44 percent) above the 95th centile for unaffected pregnancies. Maternal serum HhCG levels were not correlated with fetal nuchal translucency but showed significant correlation with total hCG and free β-hCG and with pregnancy-associated plasma protein A (PAPP-A) in the Down syndrome group (r=0.536). Maternal serum HhCG levels in cases with Down syndrome had a significant correlation with gestational age, increasing as gestation increased. When HhCG was combined with fetal nuchal translucency, PAPP-A, and maternal age, at a five percent false-positive rate the modeled detection rate was 83 percent, approximately six percent lower than when free β-hCG was used and some four percent better than when ThCG was used. The authors concluded that maternal serum HhCG is unlikely to add additional value to existing modalities for first trimester Down syndrome screening.

Spencer K, Talbot JA, Abushoufa RA. Maternal serum hyperglycosylated human chorionic gonadotropin (HhCG) in the first trimester of pregnancies affected by Down syndrome, using a sialic acid-specific lectin immunoassay. Prenat Diagn. 2002; 22: 656-662.

Reprints: Kevin Spencer, Endocrine Unit, Clinical Biochemistry Dept., Harold Wood Hospital, Gubbins Lane, Romford, Essex RM3 OBE, United Kingdom; kevinspencer1@cs.com

Homogeneous vs. precipitation assays for HDL in diabetics

Total and low-density lipoprotein cholesterol are directly associated and high-density lipoprotein is inversely associated with clinical atherosclerotic disease. Diabetic patients have an increased risk of atherosclerosis, and diabetic dyslipidemia with elevated LDL cholesterol and low HDL cholesterol is common, especially in type 2 diabetic patients in whom the degree of glycemic control and presence of late complications seem to be associated with these lipid levels. The routine use of calculated LDL cholesterol by the Friedewald formula in patients with type 2 diabetes has been questioned. The authors demonstrated earlier systematic differences in HDL and LDL cholesterol levels determined by the phosphotungstic acid/magnesium chloride precipitation method compared with the ultracentrifugation method in type 1 diabetic patients. They compared a homogeneous assay for direct HDL cholesterol analysis with the phosphotungstic acid/MgCl₂ precipitation method in 55 type 1 diabetic patients, 70 type 2 diabetic patients, and 82 nondiabetic normal control subjects with plasma triglyceride levels of less than 4.6 mmol/L. The cholesterol content of HDL determined by the direct assay was, overall, 0.1 mmol/L higher in all three groups than HDL cholesterol measured after precipitation, but the two methods were closely correlated. Neither HbA_1c, blood glucose, serum albumin, serum bilirubin, nor triglyceride affected the differences of the two HDL cholesterol measurements. The authors concluded that the direct homogeneous assay for HDL cholesterol determination in diabetic patients, unlike the precipitation method, seems not to exhibit a negative bias when compared with the ultracentrifugation method. Furthermore, the direct assay saves time and is not influenced by type of diabetes or degree of metabolic control.

Jensen T, Truong Q, Frandsen M, et al. Comparison of a homogeneous assay with a precipitation method for the measurement of HDL cholesterol in diabetic patients. Diabetes Care. 2002;25:1914-1918.

Reprints: Dr. Tonny Jensen, Clinic of Endocrinology, Hvidovre Hospital, 30 Kettegaard Alle, DK-2650 Hvidovre, Denmark; tonny.jensen.tj@hh.hosp.dk