Technical methods

donors screened, similar to the proportions of IgAdeficient blood donors reported in other Britishstudies.24 None of the 239 sera agglutinated un-coated latex, but 228 agglutinated IgA coated latexdirectly in the absence of added anti-IgA (includingseven of the 18 confirmed IgA deficient sera). Withethical committee approval, follow up serum sampleswere obtained from the 18 IgA deficient blood donorssix months after the initial samples had been taken: 14of these still showed IgA deficiency (IgA < 0-5 g/l) bylaser nephelometry while four had returned to normalserum IgA values. One IgA deficient donor had mildbut clinically important recurrent infections (sinusitis,otitis media), two had mild allergies, and two hadpossible autoimmune disorders. No other importantclinical abnormalities were elicited at interview.

These results show that the manual indirect latexagglutination test can provide a simple and satis-factory preliminary screen for IgA deficient blood do-nors, reducing the requirement for quantitative IgAtesting by more sophisticated and expensive methods.It is more rapid than the indirect passive hae-magglutination test described by Hunt et al4 whichuses a similar preincubation step; it is free of inter-ference from any anti-red cell antibodies; and IgAcoated latex has been found to be stable for at leasttwo months, unlike IgA coated red cells which mustbe prepared about every week. The relatively simpleammonium-sulphate precipitation for enrichment ofIgA myeloma immunoglobulin for coating renderscoated latex highly sensitive to agglutination by anti-IgA and enables accurate adjustment of cut off to de-sired values to be made. With this degree of sensitivitythe test does not distinguish between IgA deficientdonors and donors with autologous anti-IgA.The lat-ter group would tend not to be distinguishable fromdonors with anti-red cell antibodies in passive hae-magglutination techniques. The importance offinding donors with normal serum IgA values andanti-IgA antibodies is not clear and may merit furtherstudies.

JM Gall was the recipient of a Scottish Home andHealth Department Medical Student Vacation Re-search Grant, 1983.

References

'Yap PL, Pryde EA, McClelland DBL. IgA content of frozen-thawed-washed red blood cells and blood products measured byradioimmunoassay. Transfusion 982;22:36-8.

2Holt PDJ, Tandy NP, Anstee DJ. Screening of blood donors forIgA deficiency; a study of the donor population of south-westEngland. J Clin Pathol 1977;8:1007-10.

3Frommel D, Moullec J, Lambin P, Fine JM. Selective serum IgAdeficiency: frequency among 15,200 French blood donors. VoxSang 1973;25:513-8.

'Hunt AF, Allen DL, Aries DL, Strange JJ. A protocol for sensitive

459large-scale screening of blood donors for IgA deficiency. VoxSang 1985;48:84-8.

5Whicher JT, Perry DE, Hobbs JR. An evaluation of the Hylandlaser nephelometer PDQ system for the measurement of immu-noglobulins. Ann Clin Biochem 1978;15:77-85.

Requests for reprints to Dr GR Barclay, Regional BloodTransfusion Centre, Royal Infirmary, Edinburgh EH3 9HB,Scotland.

PLP fixation for combinedroutine histology andimmunocytochemistry ofliver biopsies

F BRENES, S HARRIS, MOA PAZ,* LM PETROVIC,PJ SCHEUER Academic Departments ofHistopathology and Medicine,* Royal Free Hospitaland School of Medicine, London

The development of monoclonal antibodies has facil-itated both histopathological diagnosis and researchby allowing the presence of various cytoplasmic andcell surface antigens to be shown on lymphoid andother cells.' 2Showing the presence in liver biopsy specimens of

surface markers for lymphocytes, accessory immunecells, and epithelial cells by using monoclonal anti-bodies has largely been limited to frozen tissue3-8;reliable and reproducible results have not beenobtained in paraffin embedded tissue. This createsseveral problems. Frozen sections are inferior in mor-phological detail to paraffin sections and are less easilystored. Dividing the biopsy specimen into two forfrozen and paraffin sectioning increases the possibilityof sampling error, and frozen sectioning introduces arisk of infection for laboratory staff, especially whenmaterial from patients with viral hepatitis is handled.9

Recently Collings et all0 reported the successful useof several monoclonal antibodies using paraffinembedding after fixation of tissues in periodate-lysine-paraformaldehyde (PLP)." In this study we appliedthis technique to liver biopsy specimens to assess itssuitability for routine diagnostic purposes.

Material and methods

The study was carried out in two parts. The first partwas designed principally to test "routine" stainingmethods after PLP fixation and paraffin embedding;

Accepted for publication 11 December 1985

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46020 liver biopsy specimens (16 percutaneous needle andfour wedge biopsies), together with two needle post-mortem examination specimens were divided into twoparts and fixed in 10% neutral buffered formalin andPLP, respectively, before processing, paraffinembedding, and section cutting at 3 pm. Sixteen of thespecimens were from patients with hepatitis B virusrelated chronic liver disease: one showed alcoholiccirrhosis and one was histologically normal. Fourwere from patients with thalassaemia major. Foursurgically removed spleens from these patients, as wellas surgically removed tonsils were used as positivecontrol material for immunocytochemical stainingwith the monoclonal antibodies given in Table 1. Bothsets of sections were stained with haematoxylin andeosin, chromotrope aniline blue, diastase-periodicacid Schiff, Perls Prussian blue for iron, Shikata'sorcein, rhodanine for copper, and by Gordon andSweets' method for reticulin. Spleens and tonsils wereonly stained with haematoxylin and eosin and forreticulin. Staining methods used were those given byScheuer'2 with minor modifications.The second smaller study was designed to compare

the results of PLP fixation and paraffin embeddingwith those obtained in frozen sections. Each of fivespecimens was divided into three parts immediatelyafter it had been obtained from the patient: one wassnap frozen with isopentane in liquid nitrogen whilethe other two parts were fixed in PLP and 10% neutralbuffered formalin, respectively, before embedding.Frozen sections 6-7 gm thick were cut on a cryostatfrom the snap frozen material. Frozen and paraffinsections were stained with haematoxylin and eosinand with the same panel of antibodies used in the firstexperiment (Table 1). Final diagnoses in the fivepatients were fatty liver, fibrosis due to alcoholism,cirrhosis of uncertain cause, mild chronic active hepa-titis, and lymphomatous infiltration.

FIXATION, EMBEDDING, AND STORAGE

PLP fixative was freshly prepared for each specimenfrom two stock solutions:

Technical methods

1 Lysine-phosphate buffer 0-2M lysine-hydrochloricacid was adjusted to pH 7f4 with 0. iMdisodium hydrogen orthophosphate, then dilutedwith 0 IM phosphate buffer (pH 7.4) to a final concen-tration of 0 IM lysine.2 Paraformaldehyde solution an 8% w/v solution ofparaformaldehyde was prepared by continuous stir-ring while heating at 60°C.

Stock solutions were renewed after 10 days. Justbefore use the two stock solutions were mixed, usingthree parts of the lysine-phosphate buffer to one partof paraformaldehyde. Sodium metaperiodate wasthen added to a final concentration of 0-05M.The tissues were fixed in PLP at 4°C for three hours,

four hours, overnight, or for three days, with roughlyequal numbers of specimens in each group. They werethen dehydrated through graded alcohols and clearedin xylene at 4°C, using 30 minute steps. Once clearedthey were infiltrated with three changes of paraffinwax at 56°C and embedded. The paraffin blocks werestored at 4°C until use.

Formalin fixed tissues were processed routinely atroom temperature and embedded at 56°C.

IMMUNOCYTOCHEMICAL STAININGTable I lists the monoclonal antibodies used in thestudy: RFB4, RFB6, RFT2, and RFDR1 were kindlyprovided by Dr LW Poulter, department of immu-nology, Royal Free Hospital School of Medicine.

Sections fixed in PLP were air dried overnight at4°C, while sections fixed in formalin were dried at560 C. Sections were dewaxed in xylene (two changes)for five minutes at room temperature and taken toabsolute ethanol. Endogenous peroxidase wasblocked with 1% hydrogen peroxide in methanol for20 minutes at room temperature; after this sectionswere washed for five minutes in tap water. They werenext treated with 0X025% protease (type XIV, Sigma)prepared in Tris buffered saline (TBS, 0-05M, pH 7 6)for nine minutes at 37°C. The reaction was stopped bywashing in cold tap water for five minutes. The sec-tions were taken to TBS (three changes oftwo minutes

Table 1 Monoclonal antibodies used in this study

Antibody Specificity Source Dilutions

Frozen Paraffin

Dako-LC Leucocyte common antigen Dakopatts 1/50 1/1 and 1/10Pan B Pan B cell Miles 1/300 1/300RFB4 B cells Immunology, Royal Free Hospital 1/5 1/1 and 1/5RFB6 B cells, follicular dendritic cells Immunology, Royal Free Hospital 1/10 1/1 and 1/10RFT2 T blasts Immunology, Royal Free Hospital 1/2 1/1 and 1/2OKT4 T cells (helper/inducer) Ortho Pharmaceuticals 1/5 1/2 and 1/5HB2 T cells (helper/inducer) ATCC 1/1 1/1Dako-T8 T cells (suppressor/cytotoxic) Dakopatts 1/1 1/1Dako-HLA-DR HLA-DR antigens Dakopatts 1/10 1/50RFDR1 HLA-DR antigens Immunology, Royal Free Hospital 1/10 1/5Dako-DRCl Dendritic reticulum cells Dakopatts 1/10 1/10 and 1/50

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Technical methodseach), incubated with normal swine serum (diluted1/20 in TBS) for 15 minutes, drained, and incubatedwith appropriately diluted monoclonal antibody for90 minutes. Working dilutions (Table 1) were deter-mined using spleen and tonsils and varied accordingto the age of the antibody. After a further threewashes in TBS sections were incubated for 30 minuteswith peroxidase conjugated polyclonal rabbit anti-body to mouse immunoglobulin (Dakopatts) for 30minutes, washed three times in TBS, incubated for 30minutes with peroxidase conjugated swine antirabbitserum (Dakopatts), and again washed three times.Peroxidase activity was developed with 0-01%diaminobenzidine (Sigma). Sections were lightlycounterstained with Carazzi's haematoxylin. Nega-tive controls were obtained by substituting the pri-mary antiserum with normal swine serum or TBS.The frozen sections used in the second experiment

were air dried for 60 minutes, fixed in acetone for 10minutes, and stained by a similar method to theabove, but with 60 minutes of incubation in primaryantisera.

Results

Routinely stained sections were equally suitable fordiagnosis after fixing in formalin or PLP; there were

no detectable morphological changes with differentfixation times. In sections stained wth haematoxylinand eosin cell membranes were somewhat betterdefined in tissue fixed in PLP than that fixed in for-malin. Reticulin-diastase periodic acid Schiff, andchromotrope aniline blue sections showed no

detectable differences between PLP and formalin,whereas iron stained with greater intensity by Perls'smethod in tissue fixed in PLP. In orcein preparationselastic fibres stained well after PLP, but hepatitis Bsurface antigen (HBsAg) was very pale and copperassociated protein negative. Due to the presence of theoxidising agent sodium metaperiodate in the PLPfixative, it was thought that there might be over-oxidation of the tissue; oxidation time with potassiumpermanganate in the orcein method was thereforereduced from 15 to five minutes. The results of orcein

461

staining were then similar using both fixatives, withgood staining of HBsAg and copper associated pro-tein.

Immunostaining gave positive results with all anti-bodies in frozen sections of positive control tissues.Different PLP fixation times did not substantiallyaffect subsequent staining with monoclonal anti-bodies, with the exception of Dako-DRCl which waspositive in spleen after three hours and overnightfixation but negative after fixation for three days.Other antibodies gave only slightly weaker resultsafter three days' fixation. Of the 11 monoclonal anti-bodies tested, 10 gave positive results after PLPfixation and embedding, whereas all were negative informalin fixed material (Table 2). Only Dako-T8 gave

negative results in spleen and tonsil, fixed in PLP, as

well as in liver tissue; RFB6 and Dako-DRCI were

positive in spleen and tonsil but negative in the liverbiopsy specimens tested. All the other antibodies gavepositive results in spleen, tonsil, and a variable pro-portion of the liver biopsy specimens. Where the rele-vant cells were present in liver biopsies the strength ofthe reaction was somewhat weaker in PLP sectionsthan in frozen sections. The quality of the PLP sec-

tions, however, was greatly superior with respect totissue detail (Figure).

Discussion

Many surface markers for human T and B lympho-cytes, accessory cells of the immune system, and epi-thelial cells are denatured by routine fixatives used inhistopathology laboratories, including formalin, for-malin sublimate (B5), and glutaraldehyde. 13 - 15Attempts to apply monoclonal antibodies to fixed tis-sues have been restricted to a small range of anti-bodies. 5 16 PLP has the advantage over routinefixatives that it does not interact with proteins butstabilises the carbohydrate moieties, so that antigenicsites are preserved." Subsequent paraffin embeddingdoes not prevent the demonstration of surface anti-gens by means ofmonoclonal antibodies, as shown byCollings et all' using human tonsil and skin. Weconfirmed their results using liver biopsy specimens

Table 2 Results of immunocytochemical staining

Tissue Sections Antibodies

Dako-LC Pan B RFB4 RFB6 RFT2 OKT4 HB2 Dako- Dako- RFDRI Dako-T8 HLA-DR DRCI

Frozen + + + - + + + + + +Liver Formalin

PLP + + + - + + + - + + -

r Frozen + + + + + + + + + + +Spleen, tonsil Formalin

L PLP + + + + + + + - + + +

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Technical methods

b'̂ X 4 g 4 g~4 :

P41> b t ' Rt * " w * A, .

Ak f ,

LI

XaA.;-wtf

Figure Part of liver biopsy specimen from patient with fatty change andfibrosis attributed to alcoholism. Ja) frozensection; ib) paraffin section after PLP; note better tissue definition in Fig. lb. Both show sinusoidal cells positive withmonoclonal antibody Dako-HLA-DR. Formalin fixed material from the same specimen gave a negative result. x 400.

and have further shown that the method is suitable forroutine staining methods with only minormodification in the case of the orcein stain.PLP fixation followed by paraffin embedding is thus

suitable for handling liver biopsy specimens taken fordiagnostic purposes and permits more extensiveinvestigation of the material. Several of the mono-clonal antibodies tested have previously been shownto work after PLP fixation and paraffin embedding.10In addition, we tested Dako-T8, Dako-LC, OrthoOKT4, and Miles pan B; the first of these was negativein all tissues fixed in PLP, including positive controls,while the other three were positive in liver biopsyspecimens. RFB6 and Dako-DRCl, which recognisefollicle centre B cells and dendritic reticulum cells,respectively, were negative in all liver biopsies testedbut positive in control material fixed in PLP andembedded in paraffin. These negative results withRFB and Dako-DRCl, as well as the failure to staincells in all liver biopsies with each antibody, we attri-bute tentatively to absence of the appropriate cellsfrom the liver tissue. This requires confirmation.The PLP processing technique used in the study is

very similar to that routinely used in histopathologylaboratories and overcomes problems of handling,preservation, and storage inherent in frozen sectiontechniques. The immunocytochemical method used isa simple three layer technique, which is convenientwith respect to time and use of reagents. Safe handlingof fixed tissues is particularly important when liver

biopsies are taken from patients with various forms ofhepatitis. Such biopsies provide important researchmaterial for the study of lymphocytes and accessorycells and their part in the pathogenesis of liver disease.

Fixation times were not critical, and it was possibleto obtain good results even with overnight fixation.One potential disadvantage of PLP, however, is thatvery long fixation (over a weekend or during trans-portation of a specimen) is not advisable because ofprobable loss of antigenicity, which might interferewith results obtained with some antibodies. Thefixative has to be prepared fresh for each specimen,and specimens are dehydrated and cleared manuallyat 4°C rather than in a tissue processor. The methodis therefore more time consuming for laboratory staffthan formalin fixation. Nevertheless, PLP fixation fol-lowed by paraffin embedding should prove helpful forspecimens in which morphological diagnosis needs tobe combined with immunocytochemical identificationof surface markers.

We thank Miss L Chidell for technical help and Mr FMoll for help with the Figure. Dr LW Poulter gavevaluable advice and provided antisera. Dr F Breneswas supported by the British Council and CONICIT(Scientific and Technological Research Council),Costa Rica. Dr LM Petrovic was supported by theBritish Council, and Dr MOA Paz by CAPES (Post-graduate Education Federal Agency), Ministry ofEducation and Culture, Brazil.

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Technical methodsReferences

Sikora K. Monoclonal antibodies in oncology. J Clin Pathol1982;35:369-75.

Stein H, Heryet A, Gatter KC, Mason DY. Freeze-driedparaffin-embedded human tissue for antigen labelling withmonoclonal antibodies. Lancet 1984;ii:71-3.

3Barbatis C, Woods J, Morton JA, Fleming KA, McMichael A,McGee JOD. Immunohistochemical analysis of HLA(A,B,C)antigens in liver disease using a monoclonal antibody. Gut1981;22:985-91.

'Eggink HF, Houthoff HJ, Huitema S, Gips CH, Poppema S. Cellu-lar and humoral immune reactions in chronic active liver disease.I. Lymphocyte subsets in liver biopsies of patients with untreatedidiopathic autoimmune hepatitis, chronic active hepatitis B andprimary biliary cirrhosis. Clin Exp Immunol 1982;50:17-24.

'Husby G, Blomhoff JP, Elgjo K, Williams RC Jr. Immuno-histochemical characterization of hepatic tissue lymphocyte sub-populations in liver disease. Scand J Gastroenterol1982;17:855-60.

'Montano L, Aranguibel F, Boffill M, Goodall AH, Janossy G,Thomas HC. An analysis of the composition of theinflammatory infiltrate in autoimmune and hepatitis B virus-induced chronic liver disease. Hepatology 1983;3:292-6.

'Govindarajan S, Uchida T, Peters RL. Identification ofT lympho-cytes and subsets in liver biopsy cores of acute viral hepatitis.Liver 1983;3:13-18.

kBallardini G, Bianchi FB, Doniach D, Mirakian R, Pisi E, BotazzoGF. Aberrant expression of HLA-DR antigens on bileduct epi-thelium in primary biliary cirrhosis: relevance to pathogenesis.Lancet 1984;ii: 1009-12.

463

'Maynard JE. Nosocomial viral hepatitis. Am J Med1981;70:439-44.

'Collings LA, Poulter LW, Janossy G. The demonstration of cellsurface antigens on T cells, B cells and accessory cells inparaffin-embedded human tissues. J Immunol Methods1984;75:227-40.

"McLean IW, Nakane PK. Periodate-lysine-paraformaldehydefixative: a new fixative for immunoelectron microscopy. J Histo-chem Cytochem 1979;27:1131-9.

2 Scheuer PJ. Liver biopsy interpretation. 3rd ed. London: BalliereTindall, 1980.

' Borowitz MJ, Croker BP, Burchette J. Immunocytochemicaldetection of lymphocyte surface antigens in fixed tissue sections.J Histochem Cytochem 1982;30:171-3.

"Hancock WW, Becker GJ, Atkins RC. A comparison of fixativesand immunohistochemical techniques for use with monoclonalantibodies to cell surface antigens. Am J Clin Pathol1982;78:825-3 1.

Hsu S, Zhang H, Jaffe ES. Utility of monoclonal antibodiesdirected against B and T lymphocytes and monocytes inparaffin-embedded sections. Am J Clin Pathol 1983;80:415-20.

6Epenetos AA, Bobrow LG, Adams TE, Collins LM, Isaacson PG,Bodmer NF. A monoclonal antibody that detects HLA-D regionantigen in routinely fixed wax embedded sections of normal andneoplastic lymphoid tissues. J Clin Pathol 1985;38:12-7.

Requests for reprints to: Professor PJ Scheuer, Departmentof Histopathology, Royal Free Hospital, Pond Street, Lon-don NW3 2QG, England.

Letters to the EditorPseudomonas maltophilia

The article by Wheat, Winstanley, andSpencer' discusses discrepant results forsusceptibility testing of Pseudomonas mal-tophilia with aminoglycosides and poly-myosin.

In 1981, in the May 30 edition of theMedical Journal of Australia, Guinness andLevey described a widespread epidemic ofinfection caused by Pseudomonas cepacia.2In this article it was noted that Ps cepaciaappeared to be fully sensitive to kanamycinby disc testing, although quantitative testingshowed this result to be false. A variationwas also noted with carbenicillin.

It was suggested that a false zone of sensi-tivity with-kanamycin may have been a com-bination of factors. Included among the sug-gested causes were the slower growingnature of the organism compared withenteric Gram negative bacilli. Anotherimportant factor could well be that Pscepacia would produce alkaline by-productsof metabolism, which would diffuse into thetest medium and strongly enhance the activ-ity of kanamycin.

With respect, I feel that the main point tobe made is that disc sensitivity testing hasbeen standardised using particular media,taking into account the commonly encoun-tered pathogens. When more unusual patho-gens with special growth requirements arefound the disc method may not be thedefinitive method of choice, and a moreconservative approach to the problem maybe required.

JM LEVEYScience III Microbiology,

Repatriation General Hospital,Concord, New South Wales 2139,

Australia

References

'Wheat PF, Winstanley TG, Spencer RC. Effectof temperature on antimicrobial sus-ceptibilities of Pseudomonas maltophilia. JClin Pathol 1985; 9:1055-8.

2Levy JM, Guinness MDG. Hospital microbialenvironment. Need for continual surveillance.Med JAust 1981;1:590-2.

Dr Spencer and colleagues reply as follows:

With respect, the main point of our paperwas not that discrepant results occurredbetween disc sensitivity and minimuminhibitory concentrations, but that thedifference in results occurred due todifference in temperature. In our studystrains of Ps maltophila had identical sus-ceptibility results whether they were mea-sured by disc or minimum inhibitory con-centrations at the same temperature. DrLevey discusses the problems encounteredwith Ps cepacia, an organism that we havenot investigated, although we were unable toshow this temperature phonomenon withPs aeruginosa. Although it may be true thatdisc susceptibility testing has been standard-ised with common organisms and certainmedia, we have never suggested that the disctechnique is the method of choice, especiallywhen dealing with unusual pathogens.

RC SPENCERTG WINSTANLEY

PF WHEATDepartment of Microbiology,Royal Hallamshire Hospital,

Glossop Road,Sheffield, S1O 2JF, England

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biopsies.immunocytochemistry of liverhistology and PLP fixation for combined routine

ScheuerF Brenes, S Harris, M O Paz, L M Petrovic and P J

doi: 10.1136/jcp.39.4.4591986 39: 459-463 J Clin Pathol 

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