Influence of delay in stool sampling on fecal occult blood test sensitivity.

Table 1. Mean

positlvity

scoresa of

five fecal samples In relationship to time after supplementing with hemoglobin: ILL.”

Days after Hbsupplementadon

Test 0 2 $ 7 10 16 19

Hb added, 200

,.Lg/g

Hemoccult II 0.2/0.2 0.4/0.1 0.4/0.0 0.4/0.0 0.3/0.0 0.0/0.0 0.3/0.0

Hemoccult SENSA 0.7/0.6 0.8/0.3 1.0/0.4 0.6/0.2 0.7/0.2 0.6/0.0 0.2/0.0

Hb added, 400

i-’-g/g

Hemoccult II 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

Hemoccult SENSA 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

Hb added, 1000 Lg/g

Hemoccult II 2.3/2.5 2.2/0.9 2.0/0.7 2.2/0.4 2.3/0.1 2.5/0.1 2.3/0.2

Hemoccult SENSA 2.9/2.9 3.0/1.6 3.0/0.9 2.6/0.6 2.8/0.4 2.7/0.4 2.5/0.2

aFrom 0 (negative) to 3 (strongly positive); see text.

b cards smeared immediately after fecal supplementation with hemoglobin; L, cards smeared later after supplementation andtested^{24 h}after smearing.

Clinical Chemistry 42, No. 7, 1996 1107

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.

Influence

of Delay in Stool Sampling on Fecal Occult Blood Test Sensitivity, Graeme P. Young,l* Marc A. Sinatra,’ and D.

James B. St. John [‘Univ. of Melbourne, Dept. of Med., Royal

Melbourne

Hosp., Melbourne, Victoria 3050, Australia (present address and *address for correspondence: Univ. of Adelaide, Dept. of Med., Queen Elizabeth Hosp., Woodville (Adelaide),

South Australia 5011;

fax 61 8 222 6042); 2 Dept. of Gastroen-

terol., Royal Melbourne

Hosp., Melbourne, Victoria 3050, Australia

Hemoglobin is not stable

in stools. The globin molecule is

steadily degraded and the

heme molecule is modified [1-5]. The latter effect is relevant to fecal occult blood tests that are based on the pseudoperoxidase activity of heme. This peroxidase

activity depends on

the presence of an iron atom within the porphyrin ring [5-7]. Colonic bacteria can remove the iron atom during colonic transit; furthermore, evidence indicates that removal of the iron atom can continue after passage of a stool

[7].

Therefore, the manufacturers of most guaiac-based fecal occult blood tests recommend that samples be taken from freshly passed stools and used, immediately, to form thin smears on a test card, as is the case with the Hemoccult#{174}’

(SmithKline Diagnostics, San Jose, CA) tests. However, clinical studies have not always followed this recommendation [8], and

the

same is probably also true in general pathology laboratories.

To examine the effect of delaying smearing vs immediate smearing of fecal samples onto test cards for the Hemoccult IT#{174}

and Hemoccult II SENSA#{174}guaiac-based tests, we supple- mented fresh stool specimens from five healthy individuals with whole-blood lysate to give various hemoglobin concentrations.

Samples were taken from the supplemented specimens without delay after preparation, one test card being prepared for each

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 re- corded as positive. The intensity of blue color in each window was graded asfollows: 0 for no color, I for a trace of blue, 2 for an intermediate degree of blue, and 3 for bright blue covering an extensive area [9]. For each test card, a graded score was derived for the pair of windows by averaging. The person developing the test was unaware of the hemoglobin concentration and the nature of storage of the

test

cards and fecal samples.

Hemoccult II SENSA was slightly more sensitive for blood added to feces in vitro than Hemoccult II. Threshold analytical sensitivities for hemoglobin in each stool sample (g/g feces) were 200, 200, 200, 200, 300 for Hemoccult II SENSA and 200, 200, 300, 300, 500 for Hemoccult II when the test cards were developed after immediate smearing.

Table 1 shows the test scores for each of three hemoglobin concentrations, for each occult blood test, when smearing was immediate or delayed. In general, the intensity scores for Hemoccult IISENSA were greater than those for Hemoccult II, and both scores increased as the concentration of added hemo- globin increased. Importantly, from day 2 onwards, for each concentration of hemoglobin, higher scores were obtained for cards that had been smeared with feces directly after supple- menting than for those in which the smearing was delayed.

Thus, Hemoccult II SENSA has a higher analytical sensitivity than Hemoccult H for blood in feces, confirming the results in a previous study where the same tests were compared

by

hospital ward

staff [9].

This increased analytical sensitivity translates into improved clinical sensitivity for colorectal neoplasia [10]. How- ever, it is critically important for sensitivity that the samples be stable.

The present study demonstrates a loss of sensitivity when the smearing of feces onto the test card is delayed. Hemoccult II and Hemoccult II SENSA give stable results for at least 19 days when cards are smeared immediately; however,

if the supple-

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1108 Technical Briefs

mented fecal sample is stored in moist form, rather than as a dry

smear

on a test card, and sampling 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 isdelayed.

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 isperformed 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.

This study was supported in part by a grant from SmithKline Diagnostics Inc., San Jose,

CA.

References

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.

Clin Chem 1989:35:2290-6.

2. McDonald CA, Walls RS, Burford Y, Goulston ‘ci. Yuen AC. Immunochemical detection of faecal occult blood. Aust N Z I Med 1984;14:105-10.

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.

4. Burton RM, Landreth KS,BarrowsGH, Jarrett DD, Songster CL. Appearance.

properties and origin of altered human hemoglobin in feces. Lab Invest 1976;35:111-5.

5. Schwartz S. DahI J, Ellefson M. Ahiquist D. The “HemoQuanV’ test: a specific and quantitative determination of heme (hemoglobin) in feces and other materials. Clin Chem 1983:29:2061-7.

6. Poh-Fitzpatrick MB. Increased levels of fecal protoporphyrin and guaiac testing [Letterl. Arch Dermatol 1982:118:538.

7. Ahlquist DA, McGill DB. SchwartzS. Taylor WF, Ellefson M, Owen RA.

HemoQuant, a new quantitative assay for fecal hemoglobin. Ann Intern Med 1984;1O1:297-3O2.

8. Ahlquist DA, Wieand HS, Moertel CG, McGill DB, Loprinzi CL, O’Connell Mi.

et al. Accuracy of fecal occult blood for colorectal neoplasia. JAMA 1993:

269:1262-7.

9. Petty MI, Deacon MC, Alexeyeff MA, St John DiG, Young GP. Comparison of readability and sensitivity of an established and a new faecal occult blood test in the hospital ward environment. Med J Aust 1992:156:420-3.

10. St John DJB. Young GP, Alexeyeff MA, Deacon MC, Cuthbertson AM. Macrae FA, Penfold JCB. Evaluation of new occult blood tests for detection of colorectal neoplasia. Gastroenterology 1993:104:1661-8.

Nonisotopic Detection of Point Mutations in CYP2IB Gene in Steroid 21 -Hydroxylase Deficiency, Begona Ezquieta,’ ^*Jos#{233}

M. Varela,’ Carlos Jariego,’ Antonio Oliver,2 and Ricardo Gracia2 (Depts. of’ Biochem. and 2 Pediatr. Endocrinol., Hosp. La Paz, PaseoCastellana 261, 28046 Madrid, Spain; *author for corre- spondence: fax 34 13582733)

Classical steroid 21-hydroxylase (21-OH; EC 1.14.99.10) defi- ciency is

the

enzymatic defect underlying 90 -95% of congenital adrenal hyperplasias. The affected enzyme, cytochrome

P450c21, isencoded by the gene CYP2IB, which is found in the human leukocyte antigen

(HLA)

class

III

gene region on chromosome t5p21 .3, together with anonfunctional pseudogene [1]. HLA genotyping hasbeen 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 ap- proach [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 per-

formed through biochemical evaluation

of 17-OH progesterone

(17OHP),

either in amniotic fluid or after corticotropin (adre- nocorticotropic 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 amore accurate identification of patients and carriers in affected fami- lies [6-9]. Moreover, the strong genotype-phenotype relation- ship found in this disease [7-9] reinforces interest in finding the mutations underlying this deficiency. The severity of the disor- der is related to the degree of impairment of the enzymatic activity, which depends, in turn, on the severity of the mutation.

In addition, we must consider that patients with mild forms of the deficiency may carry severe mutations

[7-9]

that will not be detected by methods other than direct gene analysis (seebelow, Fig. IA).

Point mutations in the CYP2IB gene are the molecular defect in 70% of the alleles for severely affected patients and in almost all of the alleles associated with the late-onset forms of 21-OH deficiency [5-9]. These point mutations are probably products of gene conversion events between the pseudogene and the CYP2JB gene, and screening for a limited number of mutations allows the characterization of most of the mutated alleles, including de novo mutations [6]. We have recently reported the distribution and frequency of CYP2IB mutations in the 21-OH- deficient Spanish population [9].

Allele-specific oligonucleotide (ASO) hybridization methods, based on isotopic labeling, are used to detect these mutations [2, 4, 7-11]. Highly sensitive isotopic methods have the disad- vantage of the short half-life of labeled oligonucleotides and are not feasible in many clinical laboratories, given that special installations are needed. We have designed a protocol for the nonradioactive detection of point mutations in the 21-OH gene.

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 blood leukocytes. Oligonucleotides were synthesized on an Applied Biosystems (Foster City, CA) 391 DNA synthesizer.

PCR amplifications were carried out with a Pharmacia-LKB (Uppsala, Sweden) Gene ATAQ thermal controller. The CYP2IB gene was amplified in three overlapping fragments:

exon 1 to exon 3 (fragment a), exon 3 to the noncoding 3’

termini (b), and exon 2 to exon 6(c) as described previously [9].

Exons 3 and 6 are the regions selected for specific amplification of the gene [7-11]. The denatured PCR-amplified fragments

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