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2009 by the American College of Gastroenterology The American Journal ofGASTROENTEROLOGY

INTRODUCTIONAutoimmune gastritis is an autoimmune disease with a prev-alence o ~2% in the general population (1,2), which mayincrease up to three- to fve-old in patients with other autoim-mune disorders, such as type I diabetes or autoimmune thyroiddisease (35). Te pathological lesion o autoimmune gastritisis atrophic body gastritis (ABG), which is characterized by thedisappearance o the oxyntic glands leading to loss o produc-tion o hydrochloric acid and intrinsic actor (6). Hypochlo-rhydria causes loss o eedback on gastrin production, thushypergastrinemia together with low Pepsinogen I levels are

well-established biochemical markers o ABG (7,8), which is achronic, oen silent pathological lesion. It may maniest itselclinically as iron defciency anemia (9,10) or in a later stage othe disease as pernicious anemia (PA), defned as cobalaminmalabsorption caused by an inadequate secretion o gastricintrinsic actor (11). Recent experimental and clinical data sug-gest that Helicobacter pylori inection may be a trigger o gastricautoimmunity, as well as subsequent autoimmune gastritis andPA (1214), but this issue awaits a defnite clarifcation.

wo circulating autoantibodies, autoantibodies to the gastricparietal cells (PCAs) directed to the gastric H + /K + APase

Reassessment of Intrinsic Factor and Parietal Cell

Autoantibodies in Atrophic Gastritis With Respect toCobalamin Deficiency

Edith Lahner, MD1, Gary L. Norman, PhD2, Carola Severi, MD3, Susan Encabo, MD2, Zakera Shums, MS2, Lucy Vannella, MD1,Gianranco Delle Fave, MD1 and Bruno Annibale, MD1

OBJECTIVES: Atrophic body gastritis (ABG) is an autoimmune condition eventually manifesting itself as

pernicious anemia (PA). Parietal cell autoantibodies (PCAs) and intrinsic factor autoantibodies

(IFAs) are considered characteristics of these conditions. Recent studies on IFA and PCA

frequency with respect to cobalamin deficiency in biopsy-proven ABG patients are lacking.

We addressed this issue using new enzyme-linked immunosorbent assay (ELISA)-based assays.METHODS: Sera from 165 patients with histologically diagnosed ABG and 113 controls were tested for IFA

and PCA using ELISA. A total of 81 ABG patients had cobalamin deficiency and macrocytic

anemia (Group 1-PA), 36 had cobalamin deficiency without macrocytic anemia (Group 2), and

48 had normal cobalamin levels (Group 3).

RESULTS: IFAs were detected in 44/165 ABG patients (27% sensitivity) and in 0/113 controls (100%

specificity). PCAs were detected in 134 ABG patients (81% sensitivity) and in 11 controls (90%

specificity). In Group 1, IFAs showed 37% sensitivity and 100% specificity, whereas PCAs showed

81% sensitivity and 90% specificity. Combining IFA and PCA testing increased the sensitivity to

61% in all ABG patients and to 73% in Group 1, while maintaining 100% specificity.

CONCLUSIONS: IFAs are 100% specific for biopsy-proven ABG and occurred in 27% of patients. PCAs occurred

in 81% of ABG patients and in 10% of controls. Combining IFA and PCA testing significantlyincreases their diagnostic performance for ABG and PA, yielding a 73% sensitivity for PA. The

non-invasive combined PCA and IFA assessment may be useful in selecting patients at risk for

autoimmune gastritis to be confirmed by gastroscopichistologic examination.

Am J Gastroenterol2009; 104:20712079; doi:10.1038/ajg.2009.231; published online 2 June 2009

1Department of Digestive and Liver Disease, 2nd Medical School, University Sapienza , Rome, Italy; 2INOVA Diagnostics, San Diego, California, USA; 3Department

of Clinical Sciences, University Sapienza, Rome, Italy. Correspondence: Bruno Annibale, MD, Department of Digestive and Liver Disease; University Sapienza,

Ospedale SantAndrea, Via di Grottarossa 1035, 00189 Roma, Italy. E-mail: [email protected] 11 December 2008; accepted 13 March 2009

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(6,11,21); (b) the presence o ECL (enterochroma n-like) cellhyperplasia (3,6); (c) the presence o cobalamin defciency withor without concomitant macrocytic anemia, or the presence oiron defciency anemia (3,11,24); and (d) concomitant thyroidand/or extrathyroid autoimmune disease (35).

According to these criteria, 165 patients with ABG (61%emale, median age 54 years; 95% CI (95% confdence inter-val): 5057) were included. Table 1 gives details regarding theclinical, hematological, and histological eatures o the includedpatients.

Pattern of anemia

With respect to anemia and cobalamin defciency, ABGpatients were classifed into the ollowing three groups:

Table 1. Study population: patients with ABG and controls

ABG patients(n= 165)

Controls(n= 113)

Female gender, n (%) 109 (66.1) 83 (73.4)

Age, years, median (95% CI) 54 (5057) 47 (3954)

Fasting gastrin, pg/ml, median (95% CI) 500 (445536) 55 (1572)

Pepsinogen I, ng/ml, median (95% CI) 10 (510) 78 (54126)

Gastric mucosa histology

Corpus-restricted atrophic gastritis

with spared antrum, n (%)122 (73.9)a

Antral and corporal atrophic

gastritis, n (%)43 (26.1)b

Presence of ECL cell hyperplasia,

n (%)165 (100)

H. pylori-positive chronicsuperficial antral gastritis

44 (38.9)

Normal stomach 69 (61.1)

Pattern of anemia

Macrocytic anemia and cobalamin

deficiency (Group 1)

81 (49.1)

Iron deficiency anemia or

macrocytosis with normal

hemoglobin concentration and

cobalamin deficiency (Group 2)

36 (21.8)

Iron deficiency anemia and normal

cobalamin levels (Group 3)

39 (23.6) 21 (18.6)

Normal hemoglobin concentration and

normal cobalamin levels (Group 3)

9 (5.5) 92 (81.4)

Autoimmune thyroid disease 66 (40.0)

Extrathyroid autoimmune disease 20 (12.1)

Any associated autoimmune disease 79 (47.9) 113

ABG, atrophic body gastritis; CI, confidence interval; ECL, enterochromaffin-like;

H. pylori, Helicobacter pylori.a23/122 (18.8%) with active H. pyloriinfection. b11/43 (25.6%) with active

H. pyloriinfection.

and autoantibodies to intrinsic actor (IFAs), are commonlydescribed in patients with autoimmune gastritis and PA. Ear-lier studies report that PCAs are associated with ABG regard-less o PA and thereore, are generally considered as a markero autoimmune gastritis (1521). IFAs are described to occur

rarely in autoimmune gastritis not associated with cobalamindefciency and are thus, viewed as a less sensitive, but a morespecifc diagnostic marker o PA (1722). In clinical practice,with the vanishing availability o Schillings test, increasingreliance is placed on the detection o IFAs or the diagnosis oPA (21). However, the real diagnostic utility o these circulat-ing autoantibodies remains largely unknown, especially usingupdated enzyme-linked immunosorbent assay (ELISA)-basedassays. Moreover, many o the earlier studies assessing the prev-alence o PCAs and IFAs in autoimmune gastritis patients werecompleted long beore routine gastroscopy, and a histologicalconsensus classifcation o gastritis was introduced (23). o ourknowledge, recent studies assessing the requency o IFAs and

PCAs in patients with biopsy-proven ABG are lacking. Tere-ore, the aim o this study was to reassess the occurrence oIFAs and PCAs in well-characterized patients with ABG and todetermine their diagnostic utility with respect to the pattern oanemia and cobalamin defciency.

METHODSPatients and study design

A gold standard or the diagnosis o autoimmune gastritis isstill lacking (3,5). A hallmark or the diagnosis o this conditionis the presence o PCAs and/or IFAs (21). As the assessment oPCAs and IFAs was the goal o this study, their presence could

not be used as selection criteria or the study population. Tus,as an alternative, well-characterized patients with ABG at highrisk o autoimmune gastritis were selected or inclusion in ourstudy by combining the well-accepted eatures or this condi-tion (36,11).

Patients were selected rom the cohort o those with ABGdiagnosed in our Gastroenterology Department (an academic,tertiary-care medical institution). ABG has been diagnosed atour institution since 1992, as the result o a screening programor the early detection o this disorder in patients who presentwith long-standing dyspepsia or who are reerred rom theHematological Department or anemia or the EndocrinologyDepartment or autoimmune thyroid disease (4).

In this study, ABG patients were included when they met

the ollowing inclusion criteria: (i) complete clinical question-naire at the time o diagnosis o ABG comprising anagraphical,lie style, and amily history items; (ii) complete hematologi-cal data at the time o diagnosis o ABG, including completeblood cell count and assessment o serum erritin and cobala-min; (iii) availability o a serum sample drawn at the time oABG diagnosis and preserved at 20C or the assessment oIFA and PCA autoantibodies; (iv) the presence o at least twoo the ollowing eatures indicative o autoimmune gastritis: (a)the presence o corporal mucosa atrophy with a spared antrum

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Intrinsic Factor and PCAs in Atrophic Gastritis

Group 1: the presence o macrocytic anemia (hemoglobinconcentration < 14 g/dl or males and < 12 g/dl or emales,MCV (mean corpuscolar volume) 100 ) associated with lowlevels o serum cobalamin (normal values 190950 pg/ml) andresponse to vitamin B

12treatment (probable overt PA).

Group 2: concomitant presence o low levels o serumcobalamin and iron defciency anemia (hemoglobinconcentration < 14 g/dl or males and < 12 g/dl or emales,level o erritin < 20g/dl) and/or low levels o cobalamin andmacrocytosis (MCV > 100 ) with normal hemoglobin con-centration together with the response to vitamin B

12treatment

(probable latent PA).Group 3: normal serum levels o cobalamin and normal

hemoglobin concentration.Other causes o cobalamin defciency, such as veganism,

pancreatic insu ciency, bacterial overgrowth, ileal diseaseor surgery, drugs, such as proton pump inhibitors and/or H

2

antagonists, as well as other causes o macrocytic anemia, such

as olate defciency, drugs, hemolytic anemia, myelodysplasia,and chronic liver, and pulmonary disease (25) were excludedon a clinical basis by specifc questions during the clinical inter-view, hematological records, and a physical examination per-ormed at the time o diagnosis o atrophic gastritis.

Diagnosis of ABG

Te presence o ABG was defned on the basis o histologi-cal confrmation o gastric corporal mucosal atrophy, astinghypergastrinema, and low Pepsinogen I levels (9). All patientsunderwent gastroscopy with standardized biopsy samplingrom the antrum (n = 3) and body (n = 3) mucosa or conven-tional histopathological examination (9). Te degree o gas-

tritis was assessed according to the updated Sydney System(26). Atrophy o the gastric body mucosa was defned as ocalor complete oxyntic gland loss and/or their replacement bymetaplastic pyloric or intestinal glands. Atrophy o the gastricantral mucosa was defned as ocal or complete disappearanceo antral glands and/or their replacement by intestinal meta-plastic epithelium (9).

Te ECL cell prolierative pattern was evaluated in non-intestinalized areas o the gastric body mucosa and was assessedas described elsewhere (27). All patients underwent serologicalstudies. Fasting gastrin levels were evaluated by a specifc radi-oimmunoassay (RIA) and Pepsinogen I levels were measuredusing a commercial RIA kit (Pepsik, Sorin, Saluggia, Italy) (9).H. pylori immunoglobulin G antibodies were determined using

a commercial ELISA kit (GAP test IgG, Biorad, Milan, Italy).ABG patients were defned as having active H. pylori inectionwhen both histology and ELISA serology were positive (14).

Controls

o investigate the diagnostic accuracy o IFAs and PCAs inABG patients, these autoantibodies were also assessed in anon-ABG control group. Subjects were recruited rom patientswith dyspepsia and/or iron defciency anemia presenting toour Gastroenterology Department or gastroscopy with antral

and corporal biopsies. Te inclusion criteria or controls were(i) the presence o normal fndings at the histological evalu-ation o antral (n = 3) and corporal (n = 3) biopsies, or thepresence o antral superfcial H. pylori gastritis with normalcorporal mucosa; (ii) complete hematological data, including

complete blood cell count, assessment o serum erritin, andserum cobalamin; (iii) normal serum cobalamin levels; (iv)the absence o thyroid or extrathyroid autoimmune disease;and (v) consensus to participate in the study and to donate avenous blood sample.

According to these criteria, 113 controls (emale 73.4%,median age 47 years; 95% CI: 3954) were included. Detailsregarding clinical, hematological, and histological eatures aregiven in Table 1.

None o the patients or controls included in the study wereon treatment with anti-secretory drugs (proton pump inhibi-tors and/or H

2antagonists) and/or cobalamin. All patients and

controls gave written inormed consent to the study, which was

approved by the local ethics committee.

Assessment of IFAs and PCAs

IFAs and PCAs were measured on stored rozen ( 20C) serao patients and controls using ELISA (Quanta Lite IntrinsicFactor Antibody and Quanta Lite Gastric Parietal Cell Anti-body ELISAs, INOVA Diagnostics, San Diego, CA). Bothassays are FDA 510(k)-cleared and were perormed accordingto the manuacturers recommendations. Te IFA ELISA uses arecombinant intrinsic actor antigen, whereas the PCA ELISAuses Na + /K + APase purifed rom porcine stomach.

For IFAs and PCAs, a titer o 25 Units was considered posi-tive, a titer o < 20 Units was considered negative, and a titer

between 20 and 25 Units was considered equivocal.IFA and PCA ELISA were perormed at the laboratorieso INOVA Diagnostics. Te researchers who perormed theELISA measurements (G.L.N., S.E., Z.S.) were blinded to theclinical data and to the diagnoses o patients and controls.

Statistical evaluation

Data were expressed as median (range or 95% CI), and/ornumber/total (percentage, %), and dierences betweenpatients and controls, as well as subgroups o patients (pat-tern o anemia and cobalamin defciency) were analyzed usingFishers exact test, Students t-test, or MannWhitney test,as appropriate. wo-tailed P values < 0.05 were consideredstatistically signifcant.

Te diagnostic perormance o IFA and PCA testing inpatients with respect to controls was expressed in terms osensitivity, specifcity, positive predictive value (PPV), andnegative predictive value (NPV) expressed in percentage. Forthe calculations regarding the diagnostic perormance o IFAand PCA testing, equivocal IFA and PCA ELISA titers wereconsidered as negative to make results dichotomic.

Descriptive statistics were determined using a dedicatedsoware package (MedCalc Soware, Mariakerke, Belgium,version 9.5).

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RESULTSIFA and PCA testing in ABG patients and controls

As shown in Table 2, out o the 165 patients with ABG, 44(26.7%) were positive or IFAs and 134 (81.2%) were posi-tive or PCAs. All 113 controls (100%) were negative or IFAs

whereas 102 (90.3%) were negative (or equivocal) or PCAs.Figure 1a illustrates the requency and magnitude o IFAsdetected in both patients with ABG and controls. Te IFA resultso ABG patients (median values o 6.4 Units, range 2.2137.9Units) were signifcantly higher than that measured in con-trols (median titer 3.0 Units, range 1.913.2 Units, P< 0.0001),whose values in turn were clearly in the negative range.

PCAs were detected in more than 81% o the 165 ABGpatients. Figure 1b shows that the profle o PCA values or theABG patients is airly evenly distributed over the range o 3.4153.8 Units (median titer 65.2 Units). In contrast to the 100%specifcity o IFA testing, 11 individuals (9.7%) showed valuesranging rom 28 to 119.8 Units (median 53.3 Units).

IFAs, PCAs, and pattern of anemia

Overall, o the 165 ABG patients, 117 (70.9%) had cobalamindefciency, 81 (49.1%) had an associated macrocytic anemia

(Group 1), whereas the other 36 (21.8%) had iron defciencyanemia (n = 31) or a macrocytosis with normal hemoglobinconcentration (n = 5) (Group 2). Te remaining 48 (29.1%)ABG patients had normal cobalamin levels (Group 3), 39 hadiron defciency anemia, whereas the remaining 9 patients had

normal hematological data.Positivity to IFA was observed in 30 (37%) patients o Group 1,

7 (19.4%) o Group 2, and in 7 (14.6%) o Group 3. Positivity toPCA was observed in 66 (81.5%) patients o Group 1, 30 (83.3%)o Group 2, and in 38 (79.2%) patients o Group 3. As shown inFigure 2, a decreasing trend o positivity to IFA was observedrom Group 1 to Group 3 (P= 0.008), although the percentagedistribution o PCA positivity was not dierent in the threegroups classifed according to the pattern o anemia (Groups 1and 2 vs. Group 3: P= 1.00 and P= 0.82, respectively).

Figure 3 illustrates the IFA and PCA titers o ABG patientswith respect to the pattern o anemia and cobalamin defciency.Patients with macrocytic anemia and low cobalamin levels

(Group 1) had the highest median level o IFAs. Patients withnormal cobalamin and hemoglobin levels (Group 3) had thelowest median IFA values, whereas those with low cobalaminlevels without macrocytic anemia (Group 2) had intermediate

Figure 1. Titers of intrinsic factor and parietal cell autoantibodies in patients with atrophic body gastritis and controls. (a) Enzyme-linked immunosorbent

assay (ELISA) titers of intrinsic factor (left graph) and (b) parietal cell autoantibodies (right graph) in patients with atrophic body gastritis with respect to

controls. *Patients vs. controls: P< 0.0001 by MannWhitney test. #Patients vs. controls: P< 0.0001 by MannWhitney test. = Autoantibody titer of

each subject.

160

140

120

100

80

60

40

20

0

Patients with atrophic body gastritis Controls

160

140

120

100

80

60

40

20

0

Patients with atrophic body gastritis

#

Units

Units

Controls

a b

Table 2. Occurrence of IFAs and PCAs in the entire study population and in age- and gender-matched patients with ABG and controls

Entire study population Age- and gender-matched

ABG patients (n= 165) Controls (n= 113) ABG patients (n= 79) Controls (n= 79)

IFA-positive, n (%) 44 (26.7) 0 20 (25.3) 0

IFA-negative, n (%) 116 (70.3) 113 (100) 57 (72.1) 79 (100)

IFA-equivocal, n(%) 5 (3.0) 0 2 (2.5) 0

PCA-positive, n (%) 134 (81.2) 11 (9.7) 64 (81.0) 4 (5.1)

PCA-negative, n (%) 24 (14.5) 99 (87.6) 12 (15.2) 73 (92.4)

PCA-equivocal, n(%) 7 (4.3) 3 (2.7) 3 (3.8) 2 (2.5)

ABG, atrophic body gastritis; IFAs, intrinsic factor autoantibodies; PCAs, parietal cell autoantibodies.

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and PCA autoantibodies was observed. Positivity to IFA wasshown in 31 (25.4%) patients with corpus-restricted atrophicgastritis and in 13 (30.2%) patients with antral and corporal

atrophic gastritis (P= 0.68). Positivity to PCA was detected in101 (82.8%) patients with corpus-restricted atrophic gastritisand in 33 (76.7%) o patients with antral and corporal atrophicgastritis (P= 0.52).

Diagnostic performance of IFA and PCA testing

Table 4 shows the diagnostic perormance o IFA and PCAtesting. As none o the control subjects were positive or IFA,IFA yielded a specifcity and a PPV o 100% in all patientswith ABG, regardless o the pattern o anemia and cobalamin

levels o IFA. PCA values showed a reversed trend with the mostseverely aected ABG patients (Group 1) showing the lowestlevel o PCAs and those with milder hematological abnormali-

ties showing higher levels o PCAs.

IFA and PCA testing and pattern of gastric atrophy

Overall, o the 165 ABG patients, 122 (73.9%) had corpus-restricted atrophic gastrits and 43 (26.1%) had concomitantantral and corporal atrophic gastritis. As shown in Table 3,these two groups o ABG patients were similar regarding gen-der, gastrin levels, the presence o ECL cell hyperplasia, patterno anemia, associated autoimmune diseases, and the pres-ence o active H. pylori inection. A similar positivity to IFA

Figure 2. Positivity to intrinsic factor (left graph) and parietal cell autoantibodies (right graph) expressed in percentage of patients with atrophic body gastritis

with respect to the pattern of anemia and cobalamin deficiency. Group 1 (n= 81): the presence of macrocytic anemia associated with low cobalamin levels;

Group 2 (n= 36): concomitant presence of low cobalamin levels and iron deficiency anemia and/or low cobalamin levels and macrocytosis with normal

hemoglobin concentration; Group 3 (n= 48): the presence of normal cobalamin levels and normal hemoglobin concentration. = positive enzyme-linked

immunosorbent assay (ELISA) values; = negative ELISA values; = equivocal ELISA values. *Indicates Group 3 vs. Group 1, P< 0.01 by Fishers exact test.

100

90

80

70

60

50

40

30

20

10

0

%

1 2

Groups Groups

3

100

90

80

70

60

50

40

30

20

10

0

%

1 2 3

Figure 3. Titers of intrinsic factor (left graph) and parietal cell autoantibodies (right graph) in patients with atrophic body gastritis with respect to the pat-

tern of anemia and cobalamin deficiency. Group 1 (n= 81): the presence of macrocytic anemia associated with low cobalamin levels; Group 2 (n= 36):

concomitant presence of low cobalamin levels and iron deficiency anemia and/or low cobalamin levels and macrocytosis with normal hemoglobin concen-

tration; Group 3 (n= 48): the presence of normal cobalamin levels and normal hemoglobin concentration. *Indicates Group 3 vs. Group 1, P= 0.001(by

MannWhitney test). #Indicates Group 2 vs. Group 1, P= 0.02 (by MannWhitney test). Indicates Group 3 vs. Group 1, P= 0.05 (by MannWhitney test).

160

140

120

100

80

60

40

20

0

Units

1 2

Groups Groups

3

160

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80

60

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0

Units

1 2 3

#

#

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defciency. Te sensitivity and NPV were inuenced by thepattern o anemia and the presence or absence o cobalamindefciency. Overall, in the whole population o ABG patients,IFA testing yielded a sensitivity and a NPV o 26.7 and 48.3 %,respectively, but these fgures increased to 37 and 68.9%,respectively, when only ABG patients o Group 1 were consid-ered. Te sensitivity decreased to 14.6% and NPV increased to~80% when these parameters were calculated or ABG patientso Group 3. In contrast, the diagnostic perormance o PCA

testing was not inuenced signifcantly by the pattern o ane-mia and cobalamin defciency because sensitivity, specifcity,PPV, and NPV were similar in the three groups o patientsconsidered.

O the 165 patients with ABG, the presence o both IFAs andPCAs was observed in 37 (22.4%) patients, whereas 24 (14.5%)were negative to both autoantibodies. O the 81 patients oGroup 1, 24 (29.6%) had both autoantibodies positive and 48(59.3%) both negative. In Group 2, 6 o the 36 patients (16.7%)were positive and 5 (13.9%) were negative or both IFAs

and PCAs. In the 48 patients without cobalamin defciency(Group 3), positivity and negativity to both autoantibodieswere observed in 7 (14.6%) and 10 (20.8%) patients, respec-tively. As shown in Table 4, when the test results or both IFAand PCA autoantibodies were combined, the sensitivity and

the NPV dramatically increased to 60.6% sensitivity and 80.9%NPV or all ABG patients and to 72.7% sensitivity and 91.9%NPV or those in Group 1, while maintaining 100% specifcityand 100% PPV.

DISCUSSIONTis study reassessed the occurrence o IFAs and PCAs inpatients with biopsy-proven ABG using updated ELISA assays.raditionally, PCAs have been detected on mouse kidneystomach tissue section using indirect immunouourescenceassays. Although these tests are generally highly sensitiveand specifc, interpretation o the immunouorescent pat-

terns is operator-dependent and labor-intensive. New ELISAassays, such as those used in this study, measure antibodies tothe specifc target o PCA (purifed gastric H + /K + APase)and thus, oer an objective detection o PCA. Until recently,measurement o IFA had been taken using RIA procedures. Inaddition to the issue o using and disposing radioactive waste,the RIA method can give alse positive results i the patienthas high levels o circulating vitamin B

1221. In our study, IFAs

were measured by an ELISA assay using recombinant intrinsicactor antigen or detecting IFA. Te assay provides accurateresults even i the patient has high levels o vitamin B

12and will

measure both type I and type II IFAs (RIA will only measuretype I antibodies) (21).

In our study, IFAs were detected in 27% o cases, but in noneo the control subjects. PCAs were ound in the majority o ABGpatients (81.3%), but also in 9.7% o controls. Immunouores-cent testing on mouse kidneystomachliver tissue slides othe 11 PCA ELISA positive sera confrmed a PCA pattern oreactivity in 9 o the 11 sera. Te two remaining sera showedsmooth muscle and anti-nuclear antibody staining.

Te measurement o circulating autoantibodies to intrinsicactor and parietal cells are commonly used in clinical practiceas markers or autoimmune gastritis and PA (11,21). However,as a gold standard or the diagnosis o autoimmune gastritis isstill lacking, the diagnostic utility o these autoantibodies orABG is unclear. o assess the utility o IFA and PCA testingin a clinical setting, which reected typical clinical practice,

patients at high risk o autoimmune gastritis were selected orinclusion in our study. Determination o a high risk o autoim-mune gastritis was made by combining well-accepted eaturesor this diagnosis; (i) all patients had the pathological lesion obody mucosa atrophy, ~ o them with a spared antrum, and allpatients showed ECL cell hyperplasia (6,11,21); (ii) > 50% o thepatients had concomitant thyroid and/or extrathyroid autoim-mune disease, conditions, which are requently associated withautoimmune gastritis (35); and, (iii) a large proportion opatients (71%) had an associated cobalamin defciency and hal

Table 3. Characteristics of patients with corpus-restricted

atrophic gastritis compared with patients with concomitant

antral and corporal atrophic gastritis and occurrence of IFAs

and PCAs

Corpus-restrictedatrophic gastritis

(n= 122)

Antral andcorporal atrophic

gastritis (n= 43)

Female gender, n (%) 82 (67.2) 27 (62.8)

Age, years, median (95% CI) 53.5 (4758) 57 (5162)

Fasting gastrin, pg/ml, median

(95% CI)

500 (438554) 500 (310541)

Pepsinogen I, ng/ml, median

(95% CI)

9 (512) 10 (513)

Presence of ECL cell

hyperplasia, n (%)

122 (100) 43 (100)

Pattern of anemia, n (%)

Group 1 60 (49.2) 21 (48.8)

Group 2 28 (22.9) 8 (18.6)

Group 3 34 (27.9) 14 (32.6)

Autoimmune thyroid disease 48 (39.3) 18 (41.9)

Extrathyroid autoimmune

disease

15 (12.3) 5 (11.6)

Active H. pyloriinfection, n (%) 23 (18.8) 11 (25.6)

IFA positive, n (%) 31 (25.4) 13 (30.2)

PCA positive, n (%) 101 (82.8) 33 (76.7)

CI, confidence interval; ECL, enterochromaffin-like. H. pylori, Helicobacter pylori;

IFAs, intrinsic factor autoantibodies; PCAs, parietal cell autoantibodies.

Group 1: n= 81, presence of macrocytic anemia associated with low cobalamin

levels; Group 2: n= 36, concomitant presence of low cobalamin levels and iron

deficiency anemia and/or low cobalamin levels and macrocytosis with normalhemoglobin concentration; Group 3: n= 48, presence of normal cobalamin levels

and normal hemoglobin concentration.

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cobalamin defciency. Tus, our data are in agreement with thehypothesis that autoimmune gastritis may be associated with

variable hematologic presentations, and that PA is probably theresult o a long-standing end-stage autoimmune process, whichmay be preceded by iron defciency anemia or clinically silentcobalamin defciency, as reported recently (24).

Te requency o IFAs, observed in our study, is lower than inearlier studies, which reported IFA in 4060% o patients withPA (19,20), increasing to 6080% with an increasing durationo disease (22). Tis apparent dierence may result rom dier-ences in the study populations. In our study, all patients had abiopsy-proven atrophy o the body mucosa and ECL cell hyper-plasia. Furthermore, in addition to patients with overt PA, westudied a wide spectrum o ABG patients at risk o autoim-mune gastritis, including those with iron-defciency anemia,with normal cobalamin levels and even without anemia, as well

as patients with concomitant antral atrophy. Dierences alsolikely result rom the assays used in earlier studies.

In our study, IFAs were observed in seven ABG patientswho had neither cobalamin defciency nor anemia. Te sevenpatients were ollowed-up or a median o 5 years. Over thistime, one patient developed PA, whereas the others showed nocobalamin defciency or anemia. Considering the specifcity oIFA or PA, the diagnostic interpretation o the ABG patientswith IFA, but not with cobalamin defciency or anemia posi-tivity suggests several possibilities. One explanation is that as

o them concomitant macrocytic anemia, thus with the likelypresence o overt or latent PA (5,9,22,24). In this clinical setting,

the IFA assay yielded a specifcity and a PPV o 100%. Tesefgures mean that having a positive IFA titer indicates atrophicdamage in the gastric body mucosa. Tis result is novel andclinically signifcant because IFAs are generally considered spe-cifc markers or the presence o PA and not or ABG withoutPA (11,21). Tereore, serious consideration should be given tothe inclusion o IFA measurement in the clinical evaluation opatients with suspected ABG. Our fnding urther suggests thatwhen positive IFA values are detected, irrespective o the pat-tern o anemia, gastroscopy with antral and body mucosa biop-sies should be carried out to confrm the presence o ABG.

Te occurrence o IFA was related to the pattern o anemiaand cobalamin defciency. When ABG patients with associatedmacrocytic anemia and cobalamin defciency (overt PA, Group

1) were considered, positivity to IFA was 37% (Figure 2). In con-trast, the presence o IFA decreased to 19% in individuals withcobalamin defciency and without macrocytic anemia (latentPA, Group 2) and to 15% in those with normal hemoglobinand cobalamin levels (Group 3). Tese fndings suggest thatthe occurrence o IFA is primarily related to the presence o PAwhen it maniests itsel explicitly. Tis observation is supportedby the results regarding the ELISA IFA titers, which showed adecreasing trend in the three groups, being signifcantly higherin ABG patients with concomitant macrocytic anemia and

Table 4. Diagnostic performance of intrinsic factor and parietal cell autoantibodies in patients with ABG classified according to

pattern of anemia and cobalamin deficiency

Sensitivity (%) Specificity (%) PPV (%) NPV (%)

Intrinsic factor autoantibodies

Group 1 37.0 100 100 68.9

Group 2 19.4 100 100 79.6

Group 3 14.6 100 100 73.4

All ABG patients 26.7 100 100 48.3

Parietal cell autoantibodies

Group 1 81.5 90.3 85.7 87.2

Group 2 83.3 90.3 73.1 94.4

Group 3 79.2 90.3 77.5 91.1

All ABG patients 81.2 90.3 92.4 76.7

Combined testing for intrinsic factor and parietal cell autoantibodies

Group 1 72.7 100 100 91.9

Group 2 54.5 100 100 95.3

Group 3 41.2 100 100 91.1

All ABG patients 60.6 100 100 80.9

ABG, atrophic body gastritis; NPV, negative predictive value; PPV, positive predictive value.

Group 1: n= 81, presence of macrocytic anemia associated with low cobalamin levels; Group 2: n= 36, concomitant presence of low cobalamin levels and iron deficiency

anemia and/or low cobalamin levels and macrocytosis with normal hemoglobin concentration; Group 3: n= 48, presence of normal cobalamin levels and normal hemo-

globin concentration.

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PA is known to develop over a long period, it is possible thatthe presence o IFAs in these patients presages the developmento clinically recognizable PA. Second, a positivity to IFA maybe indicative o polyglandular syndrome (21); indeed, in threeo these seven patients, an associated thyroid and extrathyroid

autoimmune disease was observed, whereas one had only anassociated thyroid autoimmune disease, thus making possiblethe diagnosis o polyglandular syndrome.

With regard to the PCA assay, in our clinical setting o ABGpatients, a specifcity o 90% was obtained, with 9.7% o con-trol subjects testing PCA-positive. Tis result is similar to otherobservations that PCAs may be ound in 25% o the generalpopulation (21) and in patients with H. pylori gastritis withoutany atrophic damage o gastric mucosa, involvement o the cor-poral mucosa, or even without any pathological alteration inthe gastric mucosa (28,29). Tus, our study confrms the resultso earlier reports (1520) that the presence o PCAs indicatesautoimmune damage in the gastric mucosa, but is not 100%

specifc or this condition.PCAs have been suggested to be present in all patients with

autoimmune gastritis (21). In our study, PCAs were detected in81% o ABG patients, with 4% giving an equivocal ELISA titer.Tis apparent contrast may result rom the loss o PCA mass andantigenic drive during the progression o autoimmune gastritis(20). Tis idea is supported by the signifcantly lower PCA titerobserved in ABG patients with concomitant macrocytic anemiaand cobalamin defciency (likely overt PA and longer durationo disease) compared with the PCA titers obtained in the ABGpatients without macrocytic anemia and/or cobalamin defciency.

In contrast to IFA, the patterns o anemia and cobalamin def-ciency had no inuence on the occurrence o PCAs, which was

similar (~80%) in patients o Groups 1, 2, and 3. Although thisresult diers rom earlier fndings reporting a similar occur-rence o IFAs (55%) and PCAs (54%) in patients with PA (22),the observed dierences may be explained in part by dierentgenetic origins o the investigated patients, clinical dierencesin the patient groups, as well as by the dierent assays used orPCA and IFA detection.

In this study, 26% o the investigated ABG patients did nothave a corpus-restricted atrophic gastritis with a spared antrum,the most classical histological presentation o autoimmune gas-tritis, but had concomitant antral atrophic gastritis, a histologi-cal eature more oen related to H. pylori inection. Althoughstill under debate, there is evidence that H. pylori inection itselmay be implicated in gastric autoimmunity and subsequent

autoimmune gastritis (1214). Our results show that neitherthe occurrence o IFAs and PCAs, nor the presence o macro-cytic anemia and cobalamin defciency, ECL cell hyperplasia,associated autoimmune diseases, nor the presence o activeH. pylori inection were related to the pattern o gastric atrophy(Table 3). aken together, these fndings indicate that the histo-logical criterium o the spared antrum is not able to character-ize a particular subset o ABG patients, thus suggesting that theinvolvement o the gastric antrum in the atrophic damage doesnot necessarily exclude the presence o gastric autoimmunity.

A key fnding o our study is the demonstration that thediagnostic perormance o these updated ELISA assays in theclinical setting o ABG patients at high risk o autoimmune gas-tritis may be increased notably when the test results o IFAs andPCAs are combined. For the total cohort o 165 ABG patients,

the sensitivity increased dramatically rom 27 to 61% and theNPV increased rom 48 to 81%. When only ABG patients withmacrocytic anemia and cobalamin defciency were considered,these fgures urther improved to 73% sensitivity and 92% NPV,respectively. Te PPV was 100% or the total cohort o ABGpatients and the subgroup o ABG with macrocytic anemia andcobalamin defciency. Tis fnding is critically important orclinical practice, because as a result o the vanishing availabilityo Schillings test, increasing reliance is being placed on theseautoantibodies or the diagnosis o PA. Tus, the results o ourstudy suggest that in an appropriate clinical setting, in whichpatients are well-characterized rom a clinical and hematologi-cal point o view, the non-invasive combined serological testing

or PCAs and IFAs may help in identiying patients in whoman invasive gastroscopichistologic procedure should be per-ormed or a diagnosis o defnite autoimmune gastritis.

Some limitations should be considered in interpreting thefnding o our study. As methylmalonic acid and homocysteinewere not assessed, ailure to identiy patients with silent cobala-min defciency would result in a misclassifcation o thesepatients. Second, ABG patients with cobalamin defciencyresulting rom hypochlorhydria-induced malabsorption maynot have IFAs or PCAs, as the cobalamin defciency is unrelatedto autoimmune processes (11,30).

In conclusion, the fndings o this study show that IFAs, tradi-tionally assessed only or the diagnosis o PA, are 100% specifc

or biopsy-proven ABG, occurring in about one-third o thesepatients, whereas PCAs are highly indicative, but not exclusiveor this condition. Te combined testing or IFA and PCA signi-icantly increases their diagnostic perormance, in particular orABG patients with concomitant macrocytic anemia and cobala-min defciency, yielding a 73% sensitivity and a 92% NPV. Tus,the non-invasive combined assessment o PCA and IFA may beuseul in selecting patients at high risk o autoimmune gastritisto be confrmed by gastroscopichistologic examination.

CONFLICT OF INTEREST

Guarantor o the article: Bruno Annibale, MD.Specifc author contributions: Literature review, studydesign, data collection, data input and analyses, and writing

this paper: Edith Lahner; study design, monitoring ELISAmeasurements, contribution to discussion and interpreta-tion o the study results, and revision o this paper: Gary L.Norman; supply o part o the sera and related data o controlsubjects and contribution to data collection: Carola Severi;ELISA measurements, contribution to data collection andinput: Susan Encabo; ELISA measurements, contribution todata collection and input and revision o this paper: ZakeraShums; contribution to data collection and input: LucyVannella; revision o this paper: Gianranco Delle Fave;

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6. Dixon MF. Te components o gastritis. Histology and pathogenesis. In:Graham DY, Genta RM, Dixon MF (eds). Gastritis. Lippincott Williams &Williams: Philadelphia, 1999, pp. 5166.

7. Kekki M, Samlo IM, Varis K et al. Serum pepsinogen I and serum gastrinin the screening o severe atrophic corpus gastritis. Scand J Gastroenterol1991;186 (Suppl): 10916.

8. Vnnen H, Vauhkone M, Helske et al. Non-endoscopic diagnosis oatrophic gastritis with a blood test. Correlation between gastric histologyand serum levels o gastrin-17 and pepsinogen I: a multicentre study. Eur JGastroenterol Hepatol 2003;15:88591.

9. Marignani M, Delle Fave G, Mecarocci S et al. High prevalence o corpus-predominant atrophic gastritis in patients with unexplained microcyticand macrocytic anemia: a prospective screening study. Am J Gastroenterol1999;94:76672.

10. De Block CE, Van Campenhout CM, De Leeuw IH et al. Soluble transer-ring receptor level: a new marker o iron defciency anemia, a commonmaniestation o gastric autoimmunity in type 1 diabetes. Diabetes Care2000;23:13848.

11. oh BH, Alderuccio F. Pernicious anemia. Autoimmunity 2004;37:35761.12. Bergman JP, Vandenbroucke-Grauls CM, Appelmelk BJ et al. Te story

so ar: Helicobacter pylori and gastric autoimmunity. Int Rev Immunol2005;24:129.

13. DElios MM, Appelmelk BJ, Amedei A et al. Gastric autoimmunity: therole oHelicobacter pylori and molecular mimicry. rends Mol Med

2004;10:31623.14. Annibale B, Lahner E, Santucci A et al. CagA and VacA are immunoblot

markers o past Helicobacter pylori inection in atrophic body gastritis.Helicobacter 2007;12:2330.

15. Irvine WJ, Davies SH, eitelbaumj S et al. Te clinical and pathologicalsignifcance o gastric parietal cell antibody. Ann NY Acad Sci1965;124:65791.

16. Fisher JM, Mackay IR, aylor KB et al. An immunological study o catego-ries o gastritis. Lancet 1967;i:17680.

17. aylor KB, Roitt IM, Doniach D et al. Autoimmune phenomena in perni-cious anemia: gastric autoantibodies. Br Med J 1962;2:134752.

18. Samlo IM, Kleinman MS, urner MD et al. Blocking and binding anti-bodies to intrinsic actor and parietal cell antibody in pernicious anemia.Gastroenterol 1968;55:57583.

19. Ungar B, Whittingham S, Francis CM. Pernicious anaemia: incidence andsignifcance o circulating antibodies to intrinsic actor and parietal cells.Aus Ann Med 1967;16:2269.

20. Davidson RJL, Atrah HI, Sewell HF. Longitudinal study o circulating

antibodies in pernicious anemia. J Clin Pathol 1989;42:10925.21. oh BH, Alderuccio F. Parietal cell and intrinsic actor autoantibodies.

In: Shoeneld Y, Gershwin ME, Meroni PL (eds). Autoantibodies, 2nd edn,Elsevier: Amsterdam, 2007, pp. 47986.

22. Carmel R. Reassessment o the relative prevalence o antibodies to gastricparietal cell and to intrinsic actor in patients with pernicious anaemia:inuence o patient age and race. Clin Exp Immunol 1992;89:747.

23. Price AB. Te Sydney system: histological division. J Gastroenterol Hepatol1991;6:20922.

24. Hershko C, Ronson A, Souroujon M et al. Variable hematologic presenta-tion o autoimmune gastritis: age-related progression rom iron defciencyto cobalamin depletion. Blood 2006;107:16739.

25. Babior BM. Erythrocyte disorders: anemias related to disturbance o DNAsynthesis (megaloblastic anemias). In: Williams JW, Beutler E, Erslev AJ,Lichtman MA (eds). Hematology, 4th edn, McGraw-Hill: New York, 1998,pp. 45381.

26. Dixon MF, Genta RM, Yardley JH et al. Classifcation and grading o gastri-tis: the Update Sydney System-International Workshop on the Histopathol-ogy o gastritis, Houston 1994. Am J Surg Pathol 1996;20:116181.

27. Bordi C, Annibale B, Azzoni C et al. Endocrine cell growths in atrophicbody gastritis. Critical evaluation o a histological classifcation. J Pathol1997;182:33946.

28. Negrini R, Savio A, Poiesi C et al. Antigenic mimicry between Helicobacterpilori and gastric mucosa in the pathogenesis o body atrophic gastritis.Gastroenterology 1996;111:65565.

29. Kohlstadt IC, Antunez de Mayolo EA, Ramirez-Icaza C. Parietal cellantibodies among Peruvians with gastric pathologic changes.Gastrointestinal Physiology Working Group. Scand J Gastroenterol1993;28:9737.

30. Carmel R. Cobalamin, the stomach, and aging. Am J Clin Nutr1997;66:7509.

study design, ull access to all the data in the study, and fnalresponsibility or the decision to submit or publication:Bruno Annibale. All authors have seen and approved the fnalversion o this paper.Financial support: Gary L. Norman, Susan Encabo, and

Zakera Shums are employees o INOVA Diagnostics. Noother authors received any fnancial support rom INOVADiagnostics. Te direct involvement o INOVA Diagnostics inthe assessment o the ELISA assays had no inuence on studydesign, conduct, or reporting.Potential competing interests: None.

ACKNOWLEDGMENTS

We are indebted to Proessor Cesare Bordi rom the Depart-ment o Pathology, University o Parma, who perormedthe histological evaluation o gastric biopsies, or his highlyqualifed contribution. We are grateul to Mrs AmeliaPasquali or her skilled laboratory assistance. Tis study

was unded by Grants o the Italian Ministry or Universityand Research PRIN 2005 and by the University Sapienza 8.1.111.13611/2007.

Study Highlights

WHAT IS CURRENT KNOWLEDGE

3Gastric parietal cell autoantibodies (PCAs) aregenerally considered as markers of autoimmune gastritisand intrinsic factor autoantibodies (IFAs) as markers of

pernicious anemia.

3The real diagnostic and practical utility of thesecirculating autoantibodies, however, remains largely

unknown.

WHAT IS NEW HERE

3IFAs are 100% specific for atrophic body gastritis (notonly for pernicious anemia), showing that the IFA titer

indicates atrophic damage in the gastric body mucosa.

3PCAs occurred in 81% of atrophic body gastritis patientsand in 10% of controls, showing that the presence ofPCAs indicates atrophic damage in the gastric body

mucosa, but are not specific for this condition.

3Combining IFA and PCA testing significantly increasesdiagnostic performance for atrophic gastritis patients

with suspected pernicious anemia.

REFERENCES1. Jacobson DL, Gange SJ, Rose NR et al. Epidemiology and estimated

population burden o selected autoimmune diseases in the United States.Clin Immunol Immunopathol 1997;84:22343.

2. Carmel R. Prevalence o undiagnosed pernicious anemia in the elderly.Arch Intern Med 1996;156:1097100.

3. De Block CEM, Leeuw IH, Van Gaal LF. Autoimmune gastritis in type1 diabetes: a clinically oriented review. J Clin Endocrinol Metabol2008;93:36371.

4. Lahner E, Centanni M, Agnello G et al. Occurrence and risk actors orautoimmune thyroid disease in patients with atrophic body gastritis. Am JMed 2008;121:13641.

5. oh BH, Gleeson PA, Whittingham S et al. Autoimmune gastritis and per-nicious anemia. In: Rose NR, Mackay IR (eds). Te Autoimmune Diseases,3rd edn. Academic Press: San Diego, 1998, pp. 45976.

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