Table 1. Mean positlvity scoresa of five fecal ... - Clinical Chemistry

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gene amplification and cleavage with Hhal. I Lipid Res 1990:31:545-8. Influence of Delay in Stool Sampling on Fecal Occult. Blood. Test Sensitivity,. Graeme P.
Clinical Chemistry

polyacrylamide using continuous and pulsed electric fields. I Chromatogr 1990:516:33-48. 3. Liu MS. Zang J, Evangelista RA, Rampal 5, Chen FTA. Double-stranded DNA analysis by capillary electrophoresis with laser-induced fluorescence using ethidium bromide as an intercalator. BioTechniques 1995;18:316-23. 4. Pao CC, Kao SM, Hor ii, Chang SY. Lack of mutational alteration in the conserved regions of ZP’ and SPY gene of 46. XY females with gonadal dysgenesis. Hum Reprod 1993:8:224-8. 5. Hixson JE, vernier DI. Restriction isotyping of human apolipoprotein F by gene amplification and cleavage with Hhal. I Lipid Res 1990:31:545-8.

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42, No. 7, 1996

test for each time point

at which the tests were to be subsequently developed. The remainder of each supplemented stool was placed in a standard specimen jar, sealed, and left at room temperature (19-22 #{176}C) to provide samples for delayed testing. Test cards were prepared from the contents of the specimen jars and developed at the same time points as were used with the cards that had been prepared immediately after supplementation. Test cards were developed and read in bright, direct lighting, the card being viewed for a full 60 s after the addition of developer. Any blue color, no matter how transient, was recorded as positive. The intensity of blue color in each window was graded as follows: 0 for no color, I for a trace of blue, 2 for Influence of Delay in Stool Sampling on Fecal Occult Blood an intermediate degree of blue, and 3 for bright blue covering an Test Sensitivity, Graeme P. Young,l* Marc A. Sinatra,’ and D. extensive area [9]. For each test card, a graded score was derived James B. St. John [‘Univ. of Melbourne, Dept. of Med., Royal for the pair of windows by averaging. The person developing the Melbourne Hosp., Melbourne, Victoria 3050, Australia (present test was unaware of the hemoglobin concentration and the address and *address for correspondence: Univ. of Adelaide, nature of storage of the test cards and fecal samples. Dept. of Med., Queen Elizabeth Hosp., Woodville (Adelaide), Hemoccult II SENSA was slightly more sensitive for blood South Australia 5011; fax 61 8 222 6042); 2 Dept. of Gastroenadded to feces in vitro than Hemoccult II. Threshold analytical terol., Royal Melbourne Hosp., Melbourne, Victoria 3050, sensitivities for hemoglobin in each stool sample (g/g feces) Australia were 200, 200, 200, 200, 300 for Hemoccult II SENSA and 200, 200, 300, 300, 500 for Hemoccult II when the test cards were Hemoglobin is not stable in stools. The globin molecule is developed after immediate smearing. steadily degraded and the heme molecule is modified [1-5]. The Table 1 shows the test scores for each of three hemoglobin latter effect is relevant to fecal occult blood tests that are based concentrations, for each occult blood test, when smearing was on the pseudoperoxidase activity of heme. This peroxidase immediate or delayed. In general, the intensity scores for activity depends on the presence of an iron atom within the Hemoccult II SENSA were greater than those for Hemoccult II, porphyrin ring [5-7]. Colonic bacteria can remove the iron atom and both scores increased as the concentration of added hemoduring colonic transit; furthermore, evidence indicates that globin increased. Importantly, from day 2 onwards, for each removal of the iron atom can continue after passage of a stool concentration of hemoglobin, higher scores were obtained for [7]. Therefore, the manufacturers of most guaiac-based fecal cards that had been smeared with feces directly after suppleoccult blood tests recommend that samples be taken from menting than for those in which the smearing was delayed. freshly passed stools and used, immediately, to form thin smears Thus, Hemoccult II SENSA has a higher analytical sensitivity on a test card, as is the case with the Hemoccult#{174}’ (SmithKline Diagnostics, San Jose, CA) tests. However, clinical than Hemoccult H for blood in feces, confirming the results in a previous study where the same tests were compared by hospital studies have not always followed this recommendation [8], and ward staff [9]. This increased analytical sensitivity translates into the same is probably also true in general pathology laboratories. improved clinical sensitivity for colorectal neoplasia [10]. HowTo examine the effect of delaying smearing vs immediate smearing of fecal samples onto test cards for the Hemoccult IT#{174} ever, it is critically important for sensitivity that the samples be stable. and Hemoccult II SENSA#{174} guaiac-based tests, we supplemented fresh stool specimens from five healthy individuals with The present study demonstrates a loss of sensitivity when the smearing of feces onto the test card is delayed. Hemoccult II and whole-blood lysate to give various hemoglobin concentrations. Hemoccult II SENSA give stable results for at least 19 days Samples were taken from the supplemented specimens without delay after preparation, one test card being prepared for each when cards are smeared immediately; however, if the supple-

Table 1. Mean positlvity

scoresa of five fecal samples In relationship to time after supplementing with hemoglobin: ILL.” Days after Hb

Test Hb added,

0 200

400

1000

7

10

16

19

0.2/0.2 0.7/0.6

0.4/0.1 0.8/0.3

0.4/0.0 1.0/0.4

0.4/0.0 0.6/0.2

0.3/0.0 0.7/0.2

0.0/0.0 0.6/0.0

0.3/0.0 0.2/0.0

1.2/1.2

1.2/0.1

1.2/0.2

1.5/0.0

1.4/0.0

1.3/0.0

1.4/0.0

1.7/1.6

1.9/0.6

1.6/0.5

1.6/0.2

1.5/0.2

1.5/0.2

1.2/0.2

2.3/2.5 2.9/2.9

2.2/0.9 3.0/1.6

2.0/0.7 3.0/0.9

2.2/0.4 2.6/0.6

2.3/0.1 2.8/0.4

2.5/0.1 2.7/0.4

2.3/0.2 2.5/0.2

i-’-g/g

Hemoccult II Hemoccult SENSA Hb added,

$

,.Lg/g

Hemoccult II Hemoccult SENSA Hb added,

2

supplementadon

Lg/g

Hemoccult II Hemoccult SENSA

aFrom 0 (negative) to 3 (strongly positive); see text. b cards smeared immediately after fecal supplementation with hemoglobin; L, cards smeared later after supplementation and tested

24 h

after smearing.

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Technical

in moist form, rather than as a dry is delayed, the positivity score falls rapidly. Apparently, the degradation of heme through the removal of the iron atom [5, 7] is more rapid in moist samples than in the thin, relatively dry smears on the sample cards. Thus, erroneous results (false negatives) will tend to occur when smearing of stools onto the sample collection device is delayed. This finding is particularly relevant to two areas. First, pathology laboratories that receive stools in an unaltered moist form are likely to miss pathological amounts of blood because of bacterial degradation of the heme. Second, studies in which occult blood testing is performed on moist samples sent to the laboratory, with a resulting delay in smearing of the test card, may not give reliable estimates of the clinical performance of these tests, e.g., in screening for colorectal neoplasia. We conclude that the test cards for fecal occult blood tests based on the pseudoperoxidase activity of heme should be prepared immediately after defecation. Transport of moist samples to the laboratory should be avoided; that procedure results in reduced sensitivity.

Briefs

P450c21, is encoded by the gene CYP2IB, which is found in the human leukocyte antigen (HLA) class III gene region on chromosome t5p21 .3, together with a nonfunctional pseudogene [1]. HLA genotyping has been used to detect 21-OH deficiency carriers and perform prenatal diagnosis in families with a previously haplotyped affected child, but de novo mutations or intra-HLA recombination has sometimes invalidated the approach [2]. Frequent homozygosity and poor expression of some of these markers in amniocytes have made prenatal diagnosis difficult [3]. Molecular identification of these markers after easy PCR techniques has only been developed for the class II genes. Prenatal diagnosis and carrier detection have also been performed through biochemical evaluation of 17-OH progesterone (17OHP), either in amniotic fluid or after corticotropin (adrenocorticotropic hormone; ACTH) stimulation, respectively. Unfortunately, dexamethasone treatment to the mother, to prevent prenatal virilization, suppresses 17OHP in amniotic fluid [4], and the results for post-ACTH 17OHP carriers overlap those of controls [1, 3, 5]. Direct analysis of the gene is now feasible and offers a more accurate identification of patients and carriers in affected families [6-9]. Moreover, the strong genotype-phenotype relationship found in this disease [7-9] reinforces interest in finding the This study was supported in part by a grant from SmithKline mutations underlying this deficiency. The severity of the disorDiagnostics Inc., San Jose, CA. der is related to the degree of impairment of the enzymatic activity, which depends, in turn, on the severity of the mutation. References In addition, we must consider that patients with mild forms of 1. Rose IS, Young OP. St John DiG, Deacon MC, Blake D, Henderson RW. Effect of ingestion of hemoproteins on fecal excretion of hemes and porphyrins. the deficiency may carry severe mutations [7-9] that will not be Clin Chem 1989:35:2290-6. detected by methods other than direct gene analysis (see below, 2. McDonald CA, Walls RS, Burford Y, Goulston ‘ci. Yuen AC. Immunochemical Fig. IA). detection of faecal occult blood. Aust N Z I Med 1984;14:105-10. Point mutations in the CYP2IB gene are the molecular defect 3. Young GP, St John DJB. Selecting an occult blood test for use as a screening tool for large bowel cancer. Front Gastrointest Res 1991:18:135-56. in 70% of the alleles for severely affected patients and in almost 4. Burton RM, Landreth KS, BarrowsGH, Jarrett DD, Songster CL. Appearance. all of the alleles associated with the late-onset forms of 21-OH properties and origin of altered human hemoglobin in feces. Lab Invest deficiency [5-9]. These point mutations are probably products 1976;35:111-5. of gene conversion events between the pseudogene and the 5. Schwartz S. DahI J, Ellefson M. Ahiquist D. The “HemoQuanV’ test: a specific and quantitative determination of heme (hemoglobin) in feces and other CYP2JB gene, and screening for a limited number of mutations materials. Clin Chem 1983:29:2061-7. allows the characterization of most of the mutated alleles, 6. Poh-Fitzpatrick MB. Increased levels of fecal protoporphyrin and guaiac including de novo mutations [6]. We have recently reported the testing [Letterl. Arch Dermatol 1982:118:538. distribution and frequency of CYP2IB mutations in the 21-OH7. Ahlquist DA, McGill DB. Schwartz S. Taylor WF, Ellefson M, Owen RA. HemoQuant, a new quantitative assay for fecal hemoglobin. Ann Intern Med deficient Spanish population [9]. 1984;1O1:297-3O2. Allele-specific oligonucleotide (ASO) hybridization methods, 8. Ahlquist DA, Wieand HS, Moertel CG, McGill DB, Loprinzi CL, O’Connell Mi. based on isotopic labeling, are used to detect these mutations et al. Accuracy of fecal occult blood for colorectal neoplasia. JAMA 1993: [2, 4, 7-11]. Highly sensitive isotopic methods have the disad269:1262-7. 9. Petty MI, Deacon MC, Alexeyeff MA, St John DiG, Young GP. Comparison of vantage of the short half-life of labeled oligonucleotides and are readability and sensitivity of an established and a new faecal occult blood not feasible in many clinical laboratories, given that special test in the hospital ward environment. Med J Aust 1992:156:420-3. installations are needed. We have designed a protocol for the 10. St John DJB. Young GP, Alexeyeff MA, Deacon MC, Cuthbertson AM. Macrae nonradioactive detection of point mutations in the 21-OH gene. FA, Penfold JCB. Evaluation of new occult blood tests for detection of colorectal neoplasia. Gastroenterology 1993:104:1661-8. This nonisotopic protocol was applied to 15 families with 21-OH-deficient family members-5 with classical (CL) and 10 with late-onset (LO) forms. We also used a protocol involving radiolabeling to study 38 21-OH-deficient families (25 CL and 13 LO, some reported previously [9J). Five families were studied with both methods. Genomic DNA was isolated from peripheral Nonisotopic Detection of Point Mutations in CYP2IB Gene in Steroid 21 -Hydroxylase Deficiency, Begona Ezquieta,’ * Jos#{233} blood leukocytes. Oligonucleotides were synthesized on an M. Varela,’ Carlos Jariego,’ Antonio Oliver,2 and Ricardo Gracia2 Applied Biosystems (Foster City, CA) 391 DNA synthesizer. (Depts. of’ Biochem. and 2 Pediatr. Endocrinol., Hosp. La Paz, PCR amplifications were carried out with a Pharmacia-LKB Paseo Castellana 261, 28046 Madrid, Spain; *author for corre(Uppsala, Sweden) Gene ATAQ thermal controller. The CYP2IB gene was amplified in three overlapping fragments: spondence: fax 34 13582733) exon 1 to exon 3 (fragment a), exon 3 to the noncoding 3’ Classical steroid 21-hydroxylase (21-OH; EC 1.14.99.10) defitermini (b), and exon 2 to exon 6 (c) as described previously [9]. ciency is the enzymatic defect underlying 90 -95% of congenital Exons 3 and 6 are the regions selected for specific amplification adrenal hyperplasias. The affected enzyme, cytochrome of the gene [7-11]. The denatured PCR-amplified fragments mented

fecal sample

is stored

smear on a test card, and sampling