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Fig. 1. Subtyping strains of hepatitis C virus. Hepatitis C virus ANA of different subtypes (provided by Innogenetics) was amplified with the Roche Amplicor HCV kit. Denatured amplicons (50 L) were hybridized to membrane-bound probes at 46’C as outlined in the Info LIPa HCV kit manual. The accompanying scheme for subtyping Is shown on the right. Subtype la refers to the original HCV US isolate (HCV-1) and Isolates HCV-H and HC-J1. Subtype lb comprises isolates HCV-J, HCV-BK, HCV-T, HCV-JK1, HC-J4, HCV-TA.HCV-JT, and HCV-China. HCV type 2a is represented by isolate HC-J6, type 2b by isolate HC-J8. Subtype 3a includes isolates E-bl, Ta, BR33, BR36, and HDIO; subtype 3b, Tb and HCV-TR. Isolates similar to Z5 and Z6 and EG-16 and -33 can be detected as type 4, and isolates similar to SAl, 3, 7, and 11 can be classified as type 5.

In summary, the direct detection of viral RNA was reliable for ascertaining infection with HCV. Using the combination of two HCV assay systems permitted quick and economical subtyping of the viral genome. References 1. Choo QU, Kuo G, Weiner Ad, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a bloodborne non-A, non-B viral hepatitis genome. Science 1989;244:359-62. 2. Choo QL, Richman KH, Han JH, Berger K, Lee C, Dong C, et al. Genetic organization and diversity of the hepatitis C virus. Proc Nati Acad Sci USA 1991;88:2451-5. 3. Takamizawa A, Mon C, Fuke I, Manabe S, Murakami S, Fujita J, et al. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol 1991;65: 1105-13. 4. Kate N, Hijikata M, Ootsuyania Y, Nakagawa M, Ohkoshi 5, Sugimura T, Shimotohno K Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc NatI Acad Sci USA 1990;87:9524-8. 5. Okamoto H, Kurai K, Okada S-I, Yamamoto K, Uizuka H, Tanaka T, et al. Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes. Virology 1992;188:331-41. 6. Cha TA, Beau E, Irvine B, Kolberg J, Chien D, Kuo G, Urdea MS. At least five related, but distinct, hepatitis C viral genotypes exist. Proc Nati Acad Sci USA 1992;89:7144-8. 7. Brechot C. Polymerase chain reaction for the diagnosis of hepatitis B and C viral hepatitis. J Hepatol 1993;17(Suppl 3): S35-S41. 8. Yoshioka K, Kakumu S, Wakita T, Ishikawa T, Itoh Y, Takayanagi M, et al. Detection of hepatitis C virus by polymerase chain reaction and response to interferon-a therapy: relationship to genotypes of hepatitis C virus. Hepatology 1992;16:293-9. 9. Cha TA, Kolberg J, Irvine B, Stempien M, Beall E, Yano M, et al. Use of a signature nucleotide sequence of hepatitis C virus for detection of viral RNA in human serum and plasma. J Clin Microbiol 1991;29:2528-34. 10. Bukh J, Purcell RH, Miller RH. Sequence analysis of the 5’ noncoding region of hepatitis C virus. Proc Natl Acad Sci USA 1992;89:4942-6.

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Rapid Ultrafiltration Method for Detecting Myoglobinuria, Daniel Robitaille, Fran#{231}ois Rousseau,’ Wiener Audouin, and Jean-Claude Forest (Service de Biochim., Hopital Saint-Fran#{231}ois-d’Assise,10, rue de l’Espinay, Qu#{233}bec, P0, Canada GiL 3L5; corresponding author: fax 418-525-4481) Myoglobin is the 02-binding protein of striated (cardiac and skeletal) muscle. Unlike hemoglobin (Hb), myoglobin exists only as a monomer (molecular mass --16 kDa). Myoglobinuria may be secondary to: (a) important crush injuries, severe exercise, seizures, muscular ischemia; (b) diminished energy production (hypokalemia, hypophosphatemia), (c) toxic substances (alcohol overdose, phencyclidine, carbon monoxide), and (d) infections (e.g., Legionnaires disease, influenza). Thus, myoglobinuria testing is often requested in emergency situations. In the past, myoglobin has been separated from Hb by molecular sieve chromatography (1) or centrifugation through a microconcentrator membrane (2) and identified spectrophotometrically. Latex agglutination can also be used, but this method is expensive and requires an experienced technologist to perform the analysis and to interpret the results reproducibly. Here, we propose a simple and quick method based on ultrafiltration of the urine sample with a 30-kDa cutoff filter and enzymatic (dipstick) detection of myoglobun to determine the presence of myoglobinuria. Both Hb and myoglobin give a positive peroxidase reaction on Hb dipsticks, but only myoglobin is present in the filtrate, the Nb (64 kDa) having been retained by the membrane. The procedure is as follows: 1) Centrifuge the urine specimen (5 mlii, 1500g). 2) Test the supernate with a peroxidase-sensitive dipstick (e.g., Chemstrip bA; Boehringer Mannheim Canada, Laval, P0, Canada) used for hemoglobinuria, and read the results [we used a Urichem 1000 (Boehringer Mannheim) analyzer, which gives these ranges of results:

0, erythrocytes (ery) 1071L; +, 25 X 1061L; + +, 50 X 1061L; +++, 150 X iO6fL; ++++, 250 x bO6fL]. 3) If the Nb reaction in the unfiltered urine indicates i07 eryfL, report “absence of myoglobin in significant quantity”; if the reaction indicates 25 x 106 eryfL [+], proceed to step 4. 4) Add 1 mL of urine to the sample reservoir of a Centricon30TM concentrator (Amicon, Beverly, MA; 30kDa cutoff) and centrifuge (10 min, 4000g). 5) Perform the peroxidase test (Hb dipstick) again on the filtrate and read the results. 6) If the Hb reaction is negative, report “myoglobin not detectable”; if the reaction indicates 10 ery/L, report “presence of myoglobin.” We compared the performance of this test with that of a latex agglutination kit (Rapitex-Myoglobin; Behring, Montreal, Canada), modified for urine analysis. We analyzed urine specimens to which various concentrations of purified equine myoglobin (Sigma, St. Louis, MO) had been added and samples from 10 patients having various degrees of rhabdomyolysis. In the absence of hematuria/hemoglobinuria, a dipstick result of 25 X 106 eryfL [+] before ultrafiltration corresponded to a #{235}oncentration of equine myoglobin of --300 pg/L. The Rapitex method gave a definite positive result with a urine specimen containing 100 p.g/L myoglobin. A myoglobin-negative urine sample with lysed red blood cells added to the level of visible hematuria (reddish color) gave a 250 X 10 ery/L{++++] reading on direct analysis with the dipstick but a negative result for the ultrafiltrate. All 10 patients with positive Rapitex results also tested positive with the dipstick/ultraffltration method. Filtrates from all urine specimens to which purified equine myoglobin was added before filtration gave dipstick results one [+] sign less than they did before ultrafiltration. This suggests that a small percentage of myoglobin is retained by the membrane. Multiple analyses made on the same specimens (even after frozen storage for 3 months) gave identical results. We find the proposed method easier to perform, cheaper, less time-consuming and less labor-intensive than the latex agglutination methods. Complete analysis takes about 20 mm. The analytical sensitivity is slightly better with the Rapitex than with the method described here but the clinical importance of this difference is not apparent. Finally, this method appears to be reproducible and is easier to interpret than the latex agglutination methods. References 1. Theil GB. Separation and identification of myoglobin and hemoglobin. Am J Clin Pathol 1968;49:190-5. 2. Kelner MJ, Alexander NW. Rapid separation and identification of myoglobin and hemoglobin in urine by centrifugation through a microconcentrator membrane. Clin Chem 1985;31: 112-4.

PCR Analysis of Hair Root Specimens to Detect Tay-. Sachs Disease Camers in Ashkenazi Jews, Ruby Brillante,’ Victoria Yang,2 Anne Proos,2 and Leslie Burnett2’3 (‘School of Med., Repatriation General Hosp., Concord NSW 2139, Australia; 2 Dept. of Chin. Chem., Inst. of Clin. Pathol. and Med. Res., Westmead Hosp., Westmead NSW 2145, Australia; author for correspondence: fax +61-2-633-7947)

Tay-Sachs Disease (TSD) is an autosomal recessive lysosomal disorder caused by deficiency of the enzyme f3-N-acetylhexosaminidase A (HEXA), usually due to mutations in the HEXA gene (1). The deficiency is expressed with high frequency in the Ashkenazi Jewish population. Population-based testing programs based on enzyme assays of serum or leukocytes have been introduced worldwide to detect asymptomatic heterozygote carriers of the defective gene within the Ashkenazi Jewish community. More recently, direct DNA testing has become available, and three mutations-TATC 1278 in exon 11, iWS12(G-C) in intron 12, and 1IVS9(G-*A) in exon 7-have been found to account for between 92% and 98% of all HEXA mutant alleles in the Ashkenazi Jewish population (2). Techniques have recently become available for diagnosis of genetic diseases by analyzing DNA in hair roots (3-5) and buccal swab specimens (5). We have developed a method of extracting DNA from single hair roots and identi!ring the HEXA alleles present. Hairs with visible roots were collected by the subjects, who plucked them directly from their scalp, sealed in a clean envelope, and sent to our laboratory through the normal postal service. We cut the hairs -5 mm from the root end with sterile scissors and digested the roots in 70 L of lysis buffer (6) containing 50 g of Proteinase-K (Amresco, Solon, OH). For comparison, we extracted DNA from whole blood by use of salting-out methods (6). The quantities of DNA were estimated by means of DNA Quick Strip (Kodak, New Haven, CT). Amplification of DNA by PCR was conducted in 20 L containing 1 L of genomic DNA, 10 pmol of each ohigonucleotide primer, 200 mol/L dNTPs, 4 mmoIfL MgC12, and 1 U of Taq DNA polymerase (Promega Corp., Annandale, NSW, Australia) in a Corbett Research Capillary Thermal Sequencer (Corbett Research, Mortlake, NSW, Australia) at an annealing temperature of 55#{176}C for 40 cycles. Afterwards a 4-L aliquot of amplified products was digested with restriction enzyme (except exon lb PCR products) and analyzed in a 10% nondenaturing polyacrylamide gel. For all subjects, serum and leukocyte HEXA activity was measured by standard techniques (7). As Fig. 1 shows, there was complete agreement between the results obained by HEXA enzyme analysis, DNA analysis from blood, and DNA from hair roots for the three most common mutations found in Ashkenazi Jews and for the normal alleles. DNA quantitation confirmed that the yield of genomic DNA from single hair roots was lower than that from six hair roots and much lower than that from blood (results not shown). The DNA from hair roots of one subject (of the 50 subjects we tested) could not be successfully amplified, presumably because this individual used anti-dandruff shampoo (8, and results not shown). Use of alternative specimens for DNA analysis (such as hair roots, buccal mouthwash, or buccal swabs) instead of blood has advantages for collection, transport, storage, and overall cost (5), and may also decrease subjects’ disinclination to participate in screening (9). The extreme ease of transport of hair roots contrasts with the rigorously standardized conditions required for collection and transport of blood samples for analysis of the thermolabile HEXA enzyme (5, 10). This property is particularly important for providing access to testing to communities that CLINICAL CHEMISTRY, Vol. 41,

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