Helicobacter Pylori J Clin Pathol: First Published As 10.1136/Jcp.50.6.461 on 1 June 1997

_J Clin Pathol 1997;50:461-464 461

HpSS: a new silver staining method for

Helicobacter pylori J Clin Pathol: first published as 10.1136/jcp.50.6.461 on 1 June 1997. Downloaded from

Claudio Doglioni, Michela Turrin, Ettore Macri, Concetta Chiarelli, Bastianello Germana, Mattia Barbareschi

Abstract used but are of limited sensitivity (Giemsa) or Aims-To verify whether the proposed lengthy and capricious (Warthin-Starry). More new silver staining method compares recently, Genta et all proposed a panoptic stain, favourably with other well established which achieves the simultaneous sensitive and methods in the detection of Helicobacter clear staining ofHpylori and the morphological pylori in gastric biopsies. evaluation of tissue features. However, the first Methods-One hundred and forty pairs of step in this modified Steiner's stain relies on a antral and fundic biopsies, routinely for- toxic and radioactive compound, uranyl ni- malin fixed and paraffin wax embedded, trate, which is not easily available in many from 70 consecutive unselected patients European countries for safety reasons, thus were stained with haematoxylin and eosin, hampering its widespread use. Immunostain- modified Giemsa, and the proposed H ing with anti-H pylori antiserum is a highly pylori silver stain (HpSS). Hpylori immu- sensitive and certainly more specific method nodetection was performed in the same for identification of H pylori.2 It is currently material with a polyclonal antiserum considered the gold standard morphological against Hpylori. method when an appropriate antigen retrieval Results-Hpylori was detected in 89 biop- protocol is applied; it is especially useful when sies from 48 patients with haematoxylin dealing with coccoid forms but is a costly tech- and eosin; in a further five biopsies (one nique and is not available in all laboratories. antral and four fundic) with Giemsa stain, We propose a simple new silver staining thereby identifying one more H pylori method that clearly delineates H pylori and, infected patient. The new silver staining when coupled with the routine haematoxylin method was positive in all the cases and eosin staining, allows the simultaneous detected by these two methods and de- assessment of morphology. We compared the

tected three extra infected patients (five sensitivity of this new method with haematoxy- http://jcp.bmj.com/ more positive biopsies). Immunohisto- lin and eosin, Giemsa, and H pylori immuno- chemistry detected one more positive case staining in a series of gastric biopsies. (two positive biopsies) not identified by any of the other methods. Conclusions-The HpSS method pro- Material and methods posed is highly sensitive in detecting H Routinely formalin fixed and paraffin embedded of and pylori; it is simple and it compares well pairs antral corpofundic biopsies on September 28, 2021 by guest. Protected copyright. with other methods used routinely for (n = 140) from 70 consecutive, unselected evaluating gastric biopsies for Hpylori. patients were evaluated. Histological slides (7 Clin Pathol 1997;50:461-464) were stained with haematoxylin and eosin, modified Giemsa, the new H pylori silver stain Keywords: Helicobacter pylori; AgNOR; histochemistry (HpSS), and were immunostained with an Department of anti-Hpylori antiserum. Pathology, City In addition, the above methods were applied Hospital, Belluno, Italy The discovery of the role of Helicobacter pylori to archival gastric biopsies of two patients with C Doglioni has revolutionised our understanding, diagno- Helicobacter heilmanni (formerly Gastrospirillum M Turrin hominis) infection. E Macri sis, and treatment of gastroduodenal diseases. C Chiarelli Identification of H pylori in gastric biopsies remains, at present, the most diffuse and HELICOBACTER PYLORI SILVER STAINING METHOD Endoscopy Unit accessible method to diagnose H pylori infec- The HpSS method is summarised in table 1. B Germana tion. Deparaffinised and rehydrated sections were In most cases, H pylori can be recognised rinsed in distilled water and immersed in a Department of Pathology, Santa using the haematoxylin and eosin stain. How- freshly prepared 1% AgNO3 solution. They Chiara Hospital, ever, specialised stains are necessary when bac- were transferred to a microwave oven and irra- Trento, Italy teria are rare or when histological sections diated at 500 W and at 160 W for 3.3 and 2 M Barbareschi show chronic active inflammation but no minutes, respectively; the solution was not bacteria are identified; in particular, special allowed to boil. Slides were rinsed with hot dis- Correspondence to: are in left to and then the Dr Doglioni, Servizio efforts to identify H pylori mandatory tilled water, briefly cool, Anatomia Patologica, control biopsies after antibiotic treatment and silver stain for nuclear organiser regions Ospedale Civile, 32100 in the follow up of patients with gastric (AgNOR) solution was dropped on to the Belluno, Italy. lymphoma. Several staining methods for H slides and allowed to react for 30 minutes in the Accepted for publication pylori have been proposed: a modified Giemsa dark. The AgNOR solution was prepared by 8 April 1997 stain and the Warthin-Starry stain are widely mixing one part of solution A (2 g of gelatin in 462 Doglioni, Turrin, Macri, Chiarelli, Germanai, Barbareschi

Table 1 Helicobacter pylori silver staining (HpSS) method equivocal with haematoxylin and eosin and Giemsa staining, Hpylori peculiar morphology Solutions was demonstrated distinctly by HpSS at x40 Silver staining solution A freshly prepared 1% AgNO, solution AgNOR solution One part solution A (2 g of gelatin in a 1% solution of magnification (fig 1B). Coccoid forms were J Clin Pathol: first published as 10.1136/jcp.50.6.461 on 1 June 1997. Downloaded from formic acid) and two parts solution B (50% AgNO3 also visualised with HpSS in one case, although solution) their definite identification as Hpylori required Blue toning solution 30 mM FeClI, 11 mM potassium hexacyanoferrate (III), 33 mM oxalic acid. Allow to stand overnight the specific immunostaining. and use the supernatant With HpSS, as well as with immunohistochemistry, Hpylori were visualised not only on Procedure Optional pretreatment* Schiff's reagent or Cleland's reagent (1% the surface of the foveolar epithelium but also dithiothreitol) for 10 minutes deep inside the gastric pits and in some cases in Silver staining solution* Irradiated at 500 W and at 160 W for 3.3 and the oxintic glands of the corpus. The new silver 2 minutes, respectively in a microwave oven; the solution must not boil stain also intensely stained H heilmanni; the AgNOR solution* Dropped on to the slides and allowed to react for polar flagella were also highlighted (fig 2A). H 30 minutes in the dark pylori antiserum also strongly labelled this bac- 5% sodium thiosulphate* Two minutes Blue toning solution 20-30 seconds terium (fig 2B). Wash in tap water When coupled with haematoxylin and eosin Routine haematoxylin and eosin stain staining, HpSS enabled a complete assessment *Wash in distilled water after these steps. ofthe morphology to be carried out because no significant background staining was present a 1% solution of formic acid) with two parts of after the blue toning step. solution B (50% AgNO, solution). The Pretreatment with AgNO, was necessary to AgNOR reaction time must be adjusted in each stain the bacteria; when the AgNOR solution laboratory as it may vary depending on the was used alone it highlighted only the nucleolar temperature and the length of fixation of the organiser regions. As in the AgNOR technique, specimen. After rinsing in distilled water, slides an initial pretreatment with Schiffs reagent4 or were immersed in 5% sodium thiosulphate for Cleland's reagent (1% dithiothreitol)' for 10 two minutes and then cleared with blue toning minutes improved silver staining of bacteria solution3 (30 mM FeClI, 11 mM potassium with a cleaner background, but it was not con- hexacyanoferrate (III), 33 mM oxalic acid) for sidered a necessary routine step. 20-30 seconds (blue toning solution remains stable for several months at room tempera- Discussion ture). After washing in tap water, the slides We describe a new simple silver stain that per- were stained with routine haematoxylin and mits an easier identification of H pylori in eosin. histological sections, allowing a simultaneous and complete assessment of the morphological IMMUNOHISTOCHEMISTRY with Hpylori gastritis. HpSS features associated http://jcp.bmj.com/ Rehydrated sections were treated with 2 x 5 allowed the detection of Hpylori in 74% of the minute cycles ofmicrowave heating (700 W) in patients in our series, with a 98% sensitivity citrate buffer, pH 6 and incubated with anti-H compared to immunohistochemistry, which pylori antiserum (1/200 Dako, Glostrup, Den- can be considered the reference method for H mark). Immunoreaction was performed with pylori identification in gastric biopsies.2 HpSS an automated immunostainer (Ventana 320/ proved to be much more sensitive than haema- ES; Tucson, Arizona, USA) developed with toxylin and eosin staining and even more sensi- on September 28, 2021 by guest. Protected copyright. diaminobenzidine and counterstained with tive than the commonly used Giemsa stain. haematoxylin. The high percentage of H pylori infected Histological sections were examined inde- patients in our study reflects the high infection pendently by two pathologists. Each method of rate of the population under study, in which an Hpylori staining was assessed separately, with- elevated incidence of gastric lymphoma and out knowledge of previous results, with a x40 carcinoma has been reported.6 HpSS is simple objective and x10 ocular lens. and does not require special precautions. When followed by haematoxylin and eosin staining it Results enables both the sensitive detection of Hpylori H pylori-like organisms (curved bacilli) were and an adequate evaluation of pathological identified on haematoxylin and eosin stained processes associated with H pylori infection to slides in 89 biopsies (47 antral and 42 fundic be carried out on one slide. The recently (63%)) from 48 patients (68%) and with described Genta's stain also couples a silver modified Giemsa in 94 biopsies (48 antral and stain with a routine stain with the aim of: 46 fundic (67%)) from 49 patients (70%). "allowing the simultaneous evaluation on a HpSS permitted H pylori identification in all single slide of bacteriologic and morphologic cases of infection detected by the previous two aspects of H pylori associated gastritis".' This methods and in a further five biopsies (two stain produces intense and easy to interpret antral and three fundic (70%)), identifying results, but the initial step requires a solution of three more positive patients (74%). Two more uranyl nitrate that is not easily available in sev- positive biopsies (72%) and one infected eral European countries because of radioactive patient (75%) were disclosed by H pylori restrictions. For this reason, Genta's stain has immunostaining. The HpSS method stained not gained popularity in Europe. These bacteria brown-black (fig 1A) and they could problems could be overcome by the use of be detected easily, in most cases, at low magni- HpSS, in which all reagents are widely available fication; in some cases, which were negative or and simple to prepare. Staining results are easy New silver staining methodfor Helicobacter pylori 463

to interpret, well delineating the curved, method was demonstrated in this series. How- seagull-shaped morphology of H pylori and ever, Hpylori immunohistochemistry, as well as giving a sharp contrast between bacteria and another potentially highly sensitive and specific

the epithelial surface or luminal debris, that morphological method-in situ hybridisation J Clin Pathol: first published as 10.1136/jcp.50.6.461 on 1 June 1997. Downloaded from frequently causes interpretative problems with for H pylorn-are not widely used, and most Giemsa or Warthin-Starrry methods. Immuno- histopathological laboratories rely on histo- histochemical detection of H pylori, with an chemical methods for H pylori detection on a adequate pretreatment protocol, is at present routine basis. This flew histochemical method the most sensitive and specific among the could be used in conjunction with haematoxy- methods that permit a direct identification of lin and eosin staining in the evaluation of gas- the bacterium2; the high sensitivity of this tric biopsies when the number of bacteria are

Figure 1 Helicobacter .illeL..C . . . pylorn associated gastritis: A bacteria are stained I r.I brown-black by HpSS; .r * 'Va>< original magnification tsv#,.V-. 0 (A) x400, (B) xlOOO YtrI< *.. r - Fr* %¾ Ar

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I 0 .t '"' w _ l, -J. - -A Figure 2 Gastric mucosa 'p ,'. - infected by Helicobactor heilmanni. (A) HpSS S stain, note the polar I on September 28, 2021 by guest. Protected copyright. flagella; (B) the same t S immunostained with %. anti-Hpylori antiserum (original magnification r - xlOOO)

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JI 464 Doglioni, Turrin, Macri, Chiarelli, Germana, Barbareschi

scanty, in particular, after treatment with biopsies will permit a diagnosis of H pylori proton pump inhibitors or unsuccessful eradi- associated diseases, avoiding the risk ofmissing cation therapy. Successful H pylori eradication or delaying the diagnosis of gastric malignancy:

therapy is followed by the rapid and permanent histological evaluation ofgastric biopsies with a J Clin Pathol: first published as 10.1136/jcp.50.6.461 on 1 June 1997. Downloaded from disappearance of neutrophils from the gastric careful search for Hpylori will remain a signifi- mucosa8; the persistence of neutrophil infiltra- cant part of the pathologist's routine work. tion, albeit focal, should alert the pathologist to the need for a careful search for the bacterium. This work was supported in part by the Regione del Veneto, In cases such as this, a sensitive method like Giunta Regionale, Ricerca Sanitaria Finalizzata, Venezia, Italia. HpSS would be especially useful. The biochemical basis of the HpSS reaction 1 Genta RM, Robin GO, Graham DY. Simultaneous visualiza- are obscure. It is known that carboxyl and sul- tion of Helicobacter pylori and gastric morphology: a new stain. Hum Pathol 1994;25:221-6. phur containing groups are ofgreat importance 2 Ashton-Key M, Diss TC, Isaacson PG. Detection of in the AgNOR reaction and probably act as Helicobacter pylori in gastric biopsy and resection specimens. 7Clin Pathol 1996;49:107-11. reactive sites for an initial silver deposition that 3 Berson G. Silver staining of proteins in polyacrylamide gels. is followed by nucleation offurther silver salts.9 Increased sensitivity by a blue toning. Anal Biochem 1983; It the 134:230-4. could be hypothesised that initial AgNO, 4 Thiebaut F, Rigaut JP, Reith A. Improvement in the specifi- pretreatment confers a NOR-like reactivity to a city of the silver staining technique for AgNOR-associated acidic proteins in paraffin sections. Stain Technol 1984; particular H pylori protein. This reactivity is 59:181-5. shared by other bacteria, as demonstrated by a 5 Lindner LE. Improvements in the silver-staining technique similar reaction with H heilmanni and by other for nucleolar organizer regions (AgNOR). J Histochem Cytochem 1993;41:439-45. non-H pylori bacteria sometimes observed in 6 Doglioni C, De Boni M, Moschini A, Isaacson PG. High the gastric lumina; it remains to be investigated incidence ofgastric lymphoma in northeastern Italy. Lancet 1992;339:834-5. whether this reaction is restricted to a limited 7 Karttunen TJ, Genta RM, Yoffe B, Hachem CY, Graham number of bacteria species or is a common DY, El-Zaatari FAK. Detection of Helicobacter pylori in paraffin-embedded gastric biopsy specimens by in situ property of many bacteria. hybridization. Am J Clin Pathol 1996;106:305-11. Other, non-invasive methods are now avail- 8 Solcia E, Villani L, Fiocca R, Luinetti 0, Boldorini R, Trespi E, et al. Effects of eradication of H. pylori on gastritis in able for the diagnosis ofHpylori infection, such duodenal ulcer patients. Scand J Gastroenterol 1994; as the urea breath test and serology; their use 29(suppl 201):28-34. 9 Smith PJ, Skilbeck N, Harrison A, Crocker J. The effect of a has been advocated as the methods of choice in series of fixatives on the AgNOR technique. J Pathol 1988; evaluating patients with dyspeptic symptoms, 155:109-12. 10 Patel P, Khulusi S, Mendall MA, Lloyd R, Jazrawi R, Max- avoiding the need for endoscopy.'0 However, well JD, et al. Prospective screening of dispeptic patients by only the practice of endoscopy with gastric Helicobacter pylori serology. Lancet 1995;346:1315-18. http://jcp.bmj.com/ on September 28, 2021 by guest. Protected copyright.