CLIN. CHEM. 24/5, 756-761 (1978)

756 CLINICALCHEMISTRY,Vol. 24, No. 5, 1978

Evaluationof Fecatest,a New GuaiacTest for OccultBloodin

Feces

Herman Adlercreutz, Kristlan Liewendahi, and Pertti Virkola

A practicable test (Fecatest; Finnpipette Ky) for occultblood in feces, based on the guaiac test, has been evalu-ated, principally by use of specimens from hospitalizedpatients. All steps of the test, including sampling, transport,and sample analysis, are done in a simple plastic case.Occult blood in feces was investigated in about 8000 tests,and the results showed Fecatest to be less sensitive thanthe benzidine (Wagner) test and the Hematest (Ames Co.),but more sensitive than the Hemoccult (Smith Kline Di-agnostics) and guaiac extraction (Weber) tests. A meanblood loss (measured with 51Cr-labeled erythrocytes) ofless than 2.5 ml/24 h in three separate 24-h fecal col-lections from colitis ulcerosa patients in various stages oftreatment gave no positive Fecatest results, but a meanexcretion of 2.5-3.0 ml or more blood per 24 h gave oneor more positive tests if each collection was homogenized.Single samples containing more than 4.8 ml blood per 24h gave positive results if homogenized. Thus the detectionlimit (sensitivity) of Fecatest is 2.5-5.0 ml of blood per 24h for homogenized feces from patients bleeding from thelower gastrointestinal tract. Hemoccult did not give positivetest results for homogenized specimens from similar pa-tients which contain less than 10 ml of blood/24 h andHematest gives frequent false-positive results. The Fe-catest case reduces the risk of contamination of laboratorypersonnel.

AddItIonal Keyphrases: intermethod comparison labo-ratory safety - assessment of gastrointestinal bleeding -

colitis ulcerosa

Current tests for occult blood in feces all involve ex-posure (or at least a risk of exposure) of the sampleduring transport or in the laboratory. This may lead tocontamination of the surroundings with contagiousmaterial. Of the commercially available test kits, someare too sensitive and others too insensitive and theamount of feces used in the test is not standardized.

Despite the fact that there are so many variables in-volved in testing fecal occult blood it seems that someoptimization and standardization of such tests wouldbe of value. Current tests are not very practicable andespecially not hygienic.

We have evaluated Fecatest, a recently developed

Department of Clinical Chemistry, University of Helsinki, MeilahtiHospital, SF-00290 Helsinki 29, Finland.

Received Sept. 30, 1977; accepted Feb. 10, 1978.

method for occult blood in feces. This test seems to usto meet many of the criteria of a good test.

Materials and MethodsCollection of Feces

In most experiments, samples from the routine lab-oratory were used. These samples were from patientson an ordinary hospital diet. In all experiments in whichthe sensitivity of the tests were inter-compared, smallsamples of feces sent from the wards in plastic vials werehomogenized and used. In the studies done to test Fe-catest under routine conditions, the feces samples werenot homogenized. In experiments in which comparisonswere made with the excretion of 51Cr in feces, the whole24-h feces sample was homogenized.

Tests

Fecatest (Finnpipette Ky; Helsinki, Finland): Thistest is carried out in a compact flat plastic case with twolids; one is opened to introduce the sample and the otherfor sample analysis (Figures 1 and 2). A guaiac-im-pregnated paper divides the two compartments withinthe case. The sample lid exerts some pressure on thesample, facilitating its absorption onto the paper. Theamount of feces in contact with the paper is constant.Very dry samples of feces should preferably be kept forabout 2 h in the case before the laboratory lid is opened;samples of normal consistency may be tested within 10mm. To do so, one places 1-2 drops of peroxide reagent(3% peroxide solution) (Figure 2) on the paper and readsthe color after 1 mm. A blue color indicates a positiveresult. Gray or black colors are regarded as negative. Atleast one spot of clearly blue or greenish-blue color mustbe seen before the test is judged as positive. When thetest has been performed, the test-side lid is closed andthe case may be destroyed by burning. The control“feces” samples, which can be used for test standard-ization during production and as laboratory controls,consist of Sephadex G 25 (Pharmacia Ltd., Uppsala,Sweden) and blood hemolysate containing variousknown concentrations of hemoglobin.

Hematest (Ames Co.,Division of Miles Laboratories,Ltd., Stoke Poges, Slough, Bucks., England) was usedaccording to the instructions provided.

Fig. 1. The Fecatest caseRight below: sample lid opened; feces sample Is placed In the central com-partment. Left middle: sample lid closed, case ready for transportatIon. Topcenter: laboratory lId opened (no feces sample)

CLINICAL CHEMISTRY. Vol. 24, No. 5, 1978 757

Hemoccult (Smith Kline Diagnostics, Sunnyvale,Calif. 94086) was also used according to the instructionsprovided. However, as some batches of the peroxidereagent were found to be devoid of peroxide, the rep-resentative in Finland was subsequently called on tocheck that the purchased test packages were fresh.During the study period the peroxide reagent and thecolor of the guaiac-impregnated paper were changed (in1975-1976?) without notice by the producer. In ourexperience, this has decreased the test sensitivity. Mostof our experiments were done with use of the originalreagents bought during the years 1972-1975. However,for the experiments in which patients were studied withthe 51Cr-method, new reagent packs (1976) were used.These were carefully controlled for reagent freshness.

Weber test (guaiac extraction test) (1, 2): About 5 gof feces is homogenized with 1 ml of glacial acetic acid,and 4-5 ml of diethyl ether is added and mixed with thehomogenate. After about 20 s the ether is poured intoa test tube, 8-10 drops of a guaiac solution (40 g/liter)in 94% ethanol, and 3-4 drops of 3% hydrogen peroxidein distilled water are added. If the solution becomes blueor reddish-blue the test is positive.

Wagner test (benzidine test) (3): The benzidine so-lution is freshly prepared by dissolving 0.25 g of benzi-dine in a solution containing 2.5 ml of an equivolumemixture of glacial acetic acid and water, and 2.5 ml of3% hydrogen peroxide. A thin layer of feces is placed ona glass and 2-3 drops of the benzidine solution are

Fig. 2. Fecatest case opened from the laboratory side, perfor-mance of test Illustrated

added. If a deep green or a blue color develops withina minute, the test is positive.

Measurement of fecal blood loss with 51Cr-labelederythrocytes: Gastrointestinal-tract bleeding was as-sessed by labeling the patient’s erythrocytes with 100Ci of sodium [51Cr]chromate in vitro (4, .5). After in-travenous injection of the labeled erythrocytes, stoolswere collected into plastic containers fitted with air-tight caps to avoid offensive odors and a central hollowto increase counting efficiency. The stools were firsthomogenized without adding water and samples takenfor Hematest, Fecatest, and Hemoccult. Water was thenadded, the feces rehomogenized, and the final volumeadjusted to 1 liter. The inconvenience of counting ali-quota of feces was thus avoided. Venous blood samples,5 ml, were taken at the start of each daily stool collec-tion, to allow for delay of intestinal passage, and dilutedin plastic containers to the same volume as the fecalsamples. Blood and fecal specimens were counted in alarge-volume scintillation detector. Daily blood loss (ml)= total counts in feces/counts in blood per milliliter.The sensitivity of the 51Cr-labeled erythrocyte methodallowed detection of 1 ml of fecal blood loss per day.

This method was used in 15 colitis ulcerosa outpa-tients in various stages of treatment, some of whom weresuffering from chronic, low-degree intestinal bleedingof varying intensity. Starting not earlier than two days

758 CLINICALCHEMISTRY,Vol. 24, No. 5, 1978

TIable 1.nvestig

Results of Fecal Occult Bloodations with Different Tests onHomogenized Feces

Test

Fecatest Hemoccult Weber No. samples

Test result

Wagner H.mat.st

0 0 0 0 0 12+ 0 0 0 0 5+ + 0 0 0 8+ + + 0 0 5+ + + + 0 0

+ + + + +

64

0+

+

+

+

+

0 0 0 129

0 0 0 40+ 0 0 17

+ + 0 13

+ 0 + 6

+ + +

251

0 0 0 273+ 0 0 71+ + 0 49+ + + --

455

Table 2. Results of In Vitro Fecal Occult BloodTests in Seven ClinIcal Laboratories a

Labo- Totalratory H.mat.st - H.matest + Hematest - Hematest + no.

code no. F.cat.st - Focat.st - F.cat.st + Fecatest + tests

1 1341 21 29 62 1453

2 9 71 1 81 162

3 240 53 17 68 3784 126 33 2 47 2085 60 8 6 26 1006 47 7 15 10 79

7 62 1 14 6 83Totalno. 1885 194 84

3%30012%

2463100%Percent 77% 8%

The same feces samples were analyzed with Fecatest and Hematest duringdaily routine work.

after theinjectionoflabelederythrocytes, three one-dayspecimens of feces were collected over the next ninedays.

Experimental Trials

Experiment 1. On 64 samples fivetests(Wagner,Hematest, Fecatest, Hemoccult, and Weber), on 251samples four tests (Hematest, Fecatest, Hemoccult, andWeber), and on 455 samples three tests (Hematest,Fecatest, and Weber) were compared. These compari-sons of test sensitivity were done with special care,doubtful analyses were repeated with knowledge of theresults obtained with the other tests.

Experiment 2. Seven clinical laboratories in Finland

did 2463 routine tests for occult blood in feceswithboththe Hematest and Fecatest.

Experiment 3. In the experiments in which fecalblood loss was measured with 51Cr-labeled erythrocytesin 15 colitis ulcerosa patients, the occult blood was alsoanalyzed by Hematest, Fecatest, and Hemoccult, withuse of homogenized feces. These patients were chosen,because we wanted to investigate whether the sensitivityof the test is optimal for detection of colorectal bleeding.Every patient was studied by collecting a 24-h fecessample three times within a nine-day period and themean blood loss on these days as measured by the5tCr-technique was compared to the number of positivetests obtainedfrom each type of reagent kit. Results ofthe radioactivity experiments were first calculated afterthe in vitro tests had been done, by investigators whodidnotknow theresultsofthesetests.

Results

The results of the experiments comparing three tofive different tests for occult blood in feces are showninTable 1 (experiment1).From theseresultsitcan beseen that the Wagner test (benzidine) is the most sen-sitive, followed by Hematest (orthotolidine), Fecatest,Hemoccult, and Weber (guaiac extraction test). He-moccult is so insensitive that it is sometimes negativewhen the Weber test is positive. Despite the fact thatFecatest and Hemoccult are based on the same princi-ple, Fecatest, as judged from the results of this study,is clearly more sensitive; however, Fecatest is somewhatless sensitive than the Hematest.

The results of the large study carried out in sevendifferent routine clinical laboratories (experiment 2) areshown in Table 2. Of the 2463 tests done with use ofboth Hematest and Fecatest, 1885 (77%) were negativefor both tests. In 8% of the tests the Hematest waspositive and Fecatest negative and in 3% of the tests theHematest was negative and the Fecatest positive. Bothwere positive in 12% of the tests. There was a consid-erable variation in results between the different labo-ratories and in our own laboratory we noted that dif-ferent technicians obtained different frequencies ofpositive Hematests.

The results of the measurements of fecal blood losswith 51Cr-marked erythrocytes and the correspondingresults of the three tests for occult blood are shown inTable 3. Hemoccult was not positive in any of thesetests. In our experience, every patient will need to haveat least three Fecatests if one is to see one or two thatare positive if the mean blood loss is in the range 2.5-5.5ml, which is regarded as a pathological loss of blood (6,7). In the four samples that contained more than 4.8ml/24 h, Fecatest (and Hematest also) were positive.Thus it seems that the sensitivity of the Fecatest isoptimal. Hematest was positive for many of the sampleswith normal fecal excretion of blood.

DiscussionThere are several commercial in vitro tests available

for the detection of occult blood in feces. The most

Table 3. Results of in Vitro Fecal Occult BloodTests on Homogenized Feces Compared to In

Vivo 51Cr Blood-loss MeasurementsNo. of posltlv#{149}results

Patientno.

Mean blood loss/24 h In 3 d.tns., ml

Hema-test

1 3.8

6 3.88 4.0

3 5.55 2.52 2.8

15 2.4

9 2.24 1.8

7 <1.010 <1.0

11 <1.0

12 <1.013 <1.0

14 <1.0

Blood loss >4.8 mU24 h in singledetermination

5.6

9.0

4.97.9

1

36

8

(positive+; negative,0)

+ + 0+ + 0+ + 0+ + 0

commonly used aretheHematest (orthotolidine) andHemoccult (guaiac).The Hemoccult slideistransportedlooseina cardboardenvelope,which permitsfecestoescape to the surroundings and possibly cause con-taminationduringtransportationand inthelaboratory.SimilarcontaminationispossiblewithHematest orwiththe other formerlyused testssuch as the benzidine(Wagner) (3) and guaiacextractiontests(Weber) (1, 2)

and theirvariousmodifications. These require a sepa-rate vial for transportation and the sample is exposedin the laboratory. Fecatest clearly differs from the otheravailable tests in being convenient and hygienic; the riskof contamination of the laboratory personnel is reducedbecause the sample is sealed behind the reagent paper.Because of its practicability it is well accepted both bythestaffinthewards and inthelaboratory.Many testscan be done quicklyand fecalodor isclearlylessthanin those testsin which fecessamples have to be ma-nipulated by the technician.

With regard to sensitivity it is generally agreed thatfor adult subjectson an unrestricted diet both thebenzidinetestand theHematest aretoosensitive(see,e.g.,8, 9).For eithertestnoteven a specialdietfreeofred meat and greenvegetablesissufficient to excludefalselypositiveresults(10). They are thereforeun-suitable for screening purposes. Both orthotolidine andbenzidine are carcinogenic, but this problem was over-come in the case of benzidine by using a derivative, te-tramethylbenzidine, in a test called Hemo-Fec (Med-

CLINICALCHEMISTRY,Vol. 24, No. 5, 1978 750

Kjemi A/S,H$n, Norway);thisispackaged inthesameway astheHemoccult test.However, two reagentsareneeded and thepackage must firstbe reopened on thesidecontainingthesample and then on theotherside

InS to read the color.Hematest has been found to showH.moc- great variabilityor has been regardedas too sensitive

F.catest Otilt in several investigations (8, 10-13).

2 2 0 According to theHemoccult product information this2 2 0 testdetectsfecalblood “neartheupper limitofnormal

1 0 (about2 ml ofbloodperday or 100 g feces).”Many in-1 0 vestigatorshave expressedthe opinionthat the He-1 moccultslidetestgivessatisfactoryreproducibilityand0 0 is suitable for screening purposes (11, 12, 14-17).

o o 0 However, inScandinaviait is regarded as being too in-0 0 0 sensitive(see,e.g.,18). Itshouldbe kept inmind that0 0 0 most ofthoseHemoccult studieswere made beforethe

0 0 producermade a change thatdecreasedthesensitivity2 0 0 ofthetest.Our resultsareinagreement with thoseof0 0 0 a recentinvestigation (19), which showed thatwith a0 0 0 blood lossof20.0-29.9ml/day more than 80% of He-2 0 0 moccult testswere positivewhereas a blood lossof

0 0 5.0-9.9and 2.1-4.9mi/daygavea positiveresultsinonly40 and 22% ofcases,respectively.Fecatestisbasedon thesame principleasHemoccult,

butdifferencesinthepreparation of the paper and themore effectiveabsorptionof fecalmaterialinto thepapermake it more sensitive.The upper limitfornor-mal blood loss in feces, measured with 51Cr-labelederythrocytes, is about 2 ml/24 h (6, 7). When the meanvalueforbloodlossduringthreeseparatedayswas lessthan 2.5ml theFecatestwas notpositiveinany ofthe27 samples studied. However, in one single sample witha lossof1.7ml ofbloodper24 h both a positiveHema-testand a positiveFecatest were obtained. This couldnot be regardedas a false-positiveresult,because forothersamples from thissubject the 51Cr studiesdem-onstrated an increased blood loss. It is interesting tonotethatforallthefoursinglesamples with a blood lossof4.8ml ormore per24 h both Hematest and Fecatestwerepositive.However,theHemoccult,carried outwith“new” kits,was negativefortheseclearlypathologicalstools.InsixspecimensHemoccult was negativewhentheveryinsensitiveguaiac extractiontest (positiveonlywhen fecescontains about 5% blood)inadditiontotheHematest and Fecatestwere positive(Table1).The resultsinTable 3 suggest that in subjects with

fecalblood lossfrom the lower gastrointestinal tractslightlyexceedingthe normal upper limit(2.5-5.0mlblood/day) three different feces samples must be ana-lyzed in order to detect a pathological loss of blood, ifFecatest is used. If one of the samples gives a positiveFecatest, it is suggestedthatthepatientshouldbe puton a meat-freediet,devoid of vegetables containingperoxidase or peroxidase activators, for three to fivedays before a new series of tests is made. From theforegoing it can be concluded that Fecatest has optimalsensitivity (sensitivity limit 2.5-5.0 ml blood per 24 h)according to present knowledge of normal and patho-logical blood loss in feces.

In the large clinical studies 3% of the Hematests were

760 CLINICAL CHEMISTRY, Vol. 24, No. 5, 1978

negative when the Fecatest was positive. Some part ofthese results arise from false-negative Hematests dueto faultytechnicalperformance of the testbut somefalse-positive Fecatests cannot be excluded, either. Inotherexperimentswe haveobservedsome false-positiveresultscausedby ingestionofbanana or red meat. Inaddition,ingestionoflargeamounts ofvitaminC maydecrease the sensitivity of the test. If a 3 g/ liter solutionof ascorbic acid is added to the guaiac solution used topreparethetestpaper,thesensitivityassubsequentlytestedwith hemoglobin solutionsisreducedby abouthalf,butstillthetestismore sensitivethanHemoccult.A concentration of 3g ofvitaminC perkilogramoffeceswould mean an intakeof several grams of vitamin C perday,as fecalexcretionofthevitaminisverylow (20),probably lessthan 3%. Despiteslightlydifferentex-perimental conditions it would seem that Hemoccult ismore sensitive to vitamin C (21) than is Fecatest: a so-lution of lysed blood in water (1:100) gave a negative testwith Hemoccult if1.3g ofvitaminC perliterwas alsopresentinthesample (21).

Itisadvisable tostorethesampleinthecaseforabout2 h beforetesting,because very dry samples need sometimetoabsorbintothepaper.Ifthefecessampleisfreshwhen appliedtotheguaiac-impregnated paper, storageforaslongas72 h doesnotcausesignificantalterationsintheresultsoftheFecatest.More prolonged storageof the samples results in a significantdecrease in thenumber of positiveresults,as was alsoobserved forHemoccult (22). Except forthehighestconcentrations,allHemoccult testswere negativeortracewithineightdays (22). However, only 18% of Fecatestsoriginallypositivewere negative after 10 days of storage. ForHemoccult ithas been suggestedthatwatershouldbeadded tothefecalsample sideforoldsamplesand thisrehydration reduces the number of false-negative re-sults due to storage of the specimen (23). However, theinstructions for Hemoccult were recently changed andit is now suggested that the sample side be reopened inthe laboratory and some reagent added to the specimenbeforetheothersideofthepackageisopened.Itisnotstatedhow thisinfluencesthesensitivityofthetest.

Many previous studies have demonstrated that He-moccult and similar procedures for detecting fecal oc-cult blood based on guaiac methods give the most re-producible results (8,9, 11, 12). In our hands Hemoccultand Fecatest gave considerably better reproducibilitythan Hematest. The reproducibility of Fecatest (95%correct values) is of the same order as that of Hemoccult(97% correct values) (11) with two skilled technicianscarrying out the test independently. The correspondingfigureforHematest was only74%.

Larger clinical studies are needed before it can bedefinitelyestablishedwhether thistest,which in our

opinion has optimal theoretical sensitivity, will improvethe diagnostic usefulness of such tests foroccultblood.In the literature there seems to be some disagreementastowhich sensitivityisoptimal(see,e.g.,11). We thinkthatbleedingof 2.5-5.0ml from the colonshould bedisclosed in at least one of three tests carried out on

homogenized fecesifa method isto be regardedashaving optimal sensitivity. Larger amounts of blood inthe feces are needed to give a positive result when theblood derives from the upper gastrointestinal tract.Because, in practice, feces cannot be homogenized, theproportion of false-negative results increases. But if thetest is made more sensitive the number of false-positiveresults is so great, as with Hematest, that its clinicalvalue is greatly diminished. Because of the many vari-ables involved in testing feces for the presence of occultblood (see the excellent review in ref. 24) it must beaccepted that no test can ever be completely free offalse-positive or false-negative results. However, it isobvious that the guaiac tests have many advantages, theprincipal ones being that the reagents are not carcino-genic and the tests are more reproducible.

This work was supported by a grant from the Finnish Ministry ofIndustry and Trade. We are especially grateful for the excellenttechnical assistance of Miss Leena K. Tuomi during many years ofthis work. The following persons gave us much help during testing ofFecatest in other laboratories: Drs. Erkki Leskinen, Teddy Weber,Esko Salo, Knut-Olof Schauman, Matti Puukka, Pentti Karvonen,and chief laboratory nurse Helena Melkas, department laboratorynurses Helena Kekki, Helena Vertanen, Pirjo Makinen, Leena Malila,Rauni Ik#{225}heimonen,Marita Raman, specialist nurse Aino Kekki, andlaboratory nurse Irja Oikarinen. We thank them all very much fortheir kind cooperation.

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