Immunoreactivity of some epitopes in longtime inappropriately stored paraffin-embedded tissues

Technical Note

Immunoreactivity of some epitopes inlongtime inappropriately stored paraffin-embedded tissues

M. A. Seidu1,2, A. R. Adams1, R. K. Gyasi2, Y. Tettey2, D. O. Nkansah1,E. K. Wiredu1,2

1School of Allied Health Sciences, University of Ghana, 2Department of Pathology, University of Ghana MedicalSchool, Accra, Ghana

One advantage associated with paraffin-embedded tissues is their availability for further studies andreview. Where block filing facilities are not available, used blocks are often dumped in neglected rooms.This may affect their appropriateness for follow-up studies such as immunohistochemistry. The goal of thisstudy was to perform immunohistochemical procedures on poorly stored paraffin-embedded blocks after asimple cleaning and re-embedding procedure. Thirty paraffin-embedded Onchocerca nodules, poorlystored for over 15 years, were used. The blocks were soaked overnight in tap water, rinsed in distilledwater, air-dried, and re-embedded in fresh paraffin wax. Immunoperoxidase demonstrations of six majorenzymes (LDH, SDHB, PDK2, G6PD, ME1, and A/BHD4) were performed on their sections and examinedby light microscopy. Fifty-one 0.6% Female worm nodules in the amount of 51.6% had detectable SDHB,56.6% had detectable levels of PDK2, 58.6% had A/BHD4, 61.1% had G6PD, 63.3% had detectable levelsof ME1, and 64.5% had detectable levels of LDH. At a 99% confidence level, §30% of the nodules have allsix enzymes detectable by immunohistochemistry and §35% have four detectable enzymes (LDH, PDK, A/BHD, and ME). Poorly stored archival formalin-fixed, paraffin-embedded Onchocerca nodules over aprolonged period still retain enough antigenicity for immunohistochemical demonstration of the enzymesLDH, SDHB, PDK2, G6PD, ME1, and A/BHD4 and perhaps other antigens.

Keywords: Antibodies, Enzymes, Epitopes, Immunoreactivity, Nodules

IntroductionOne of the numerous advantages associated with

paraffin embedding of tissues is their availability for

review of a previous study or an entirely new

investigation based on new and improved techniques.

This may depend on how appropriately the used

paraffin-embedded blocks were stored. In well-

resourced laboratories, funds are available for the

purchase of filing cabinets that provide adequate

conditions for a large variety of follow-up investiga-

tions. There are indications that these filing systems

can maintain the antigenicity of tissues embedded in

paraffin blocks for up to 65 years.1–3 However, what

is considered normal storage conditions may be

relative to specific environments. It is easy to under-

stand that in very high temperature environments,

some waxes may soften as temperatures rise closer to

their melting points. There are reports that exposure

of up to 5-mm cut tissue sections on glass slides to

long periods of high room temperatures and humid

conditions can result in significant protein degrada-

tion and loss of immunoreactivity.4–7 This suggests

that adverse conditions could affect the immuno-

reactivity of paraffin-embedded tissues. In some

laboratories where block filing facilities are limited,

the used paraffin-embedded tissue blocks are often

stored in neglected rooms exposed to extreme

weather and environmental conditions.

Onchocerca volvulus is a strict human parasite

responsible for the disease called onchocerciasis (river

blindness). When O. volvulus infects its hosts, it is

detected in forms representing two stages of their life

cycle. The larvae (microfilariae) are found mainly

in the dermis of the skin and the adult worms

(macrofilariae) are often found in subcutaneouslyCorrespondence to: MA Seidu, School of Allied Health Sciences,University of Ghana, Accra, Ghana. Email: [email protected]

� National Society for Histotechnology 2013DOI 10.1179/2046023613Y.0000000024 Journal of Histotechnology 2013 VOL. 36 NO. 2 59

placed fibrous nodules. In view of the peculiar habitat

of the O. volvulus adult worms, histology appears to

be the most ideal method for their study including

immunohistochemical procedures.

As a strictly human parasite, the availability of O.

volvulus material is often restricted by ethical concerns.

To overcome the ethical concerns, some researchers

obtain biological information about O. volvulus from

parasites that infect animals.8–10 Animal sources of

parasite material have less stringent ethical concerns

than human sources although ethics are not comple-

tely disregarded. The failure of drugs developed

against those parasites to affect O. volvulus11–17

indicates that there is a need to obtain direct biological

information from O. volvulus itself. However, the

limited availability of O. volvulus material has led to an

over-dependence on cryo-preserved nodules and used

paraffin-processed archival nodules. Based on the

theory that extreme humidity and temperature can

degrade protein in cut sections on glass slides and

antigenicity, it was hypothesized that paraffin-pro-

cessed O. volvulus nodules with cut surfaces, unsealed

and exposed to similar conditions for long periods, will

lose their immunoreactivity

Immunohistochemical detection of lactate dehydro-

genase (LDH), succinate dehydrogenase B (SDHB),

pyruvate dehydrogenase kinase 2 (PDK2), glucose-6-

phosphate dehydrogenase (G6PD), malic enzyme

1 (ME1), and alpha/beta hydrolase 4 (A/BHD4)

was performed on formalin-fixed, paraffin-embedded

archival O. volvulus nodules. These primary antibodies

were part of reagents obtained for a study on the

metabolism of O. volvulus. The nodules had been

stored for up to 15 years under uncontrolled tropical

conditions at a World Health Organization (WHO)

sponsored Onchocerciasis Chemotherapy Research

Centre (OCRC). The immunohistochemical procedure

was carried out after a simple cleaning and re-

embedding of the moldy paraffin-embedded nodules

without the use of detergents or chemicals.

Materials and MethodsTest materialsParaffin-embedded O. volvulus nodules which had

been inappropriately stored were obtained from the

storeroom of a National/WHO co-sponsored OCRC

in Hohoe Hospital of the Volta Region of Ghana.

The nodules were collected by OCRC in early 1995 as

part of an untreated control group for baseline results

in a study of the chemotherapy of onchocerciasis.18

All the nodules had been fixed in 10% phosphate-

buffered formalin, dehydrated through ascending

grades of ethanol to absolute ethanol, and cleared

in xylene before they were embedded in the paraffin

wax. Cut sections were placed in an open cardboard

box in a neglected room at room temperatures

between 20 and 40uC (average yearly extremes of

temperatures as noted by metrological services of

Ghana for the Volta Region) until they were retrieved

in March 2010 (Fig. 1A).

Treatment of retrieved paraffin-embedded nodule

blocks

The moldy and dusty paraffin-embedded nodules

were soaked in tap water overnight, washed thor-

oughly in five changes of tap water, and rinsed with

distilled water. They were air-dried in the laboratory

and transferred into embedding molds on a hot plate

at 62uC to melt the wax and liberate the nodules. The

liberated nodules were transferred into new cassettes

labeled KP1–KP30 and their lids closed, after which

they were transferred into fresh molten paraffin wax

for 2 hours (Leica histowax, mp 57–58uC; Leica

Microsystems GmbH, Wetzlar, Germany). They were

transferred into two further changes of fresh molten

paraffin wax for 2 hours each to ensure complete

infiltration, after which they were re-embedded in

fresh paraffin wax (Fig. 1B). Four micrometer thick

sections were cut with a rotary microtome (Leica RM

2125 RT; Leica Microsystems GmbH) and stained

with hematoxylin and eosin. The stained sections

were examined by light microscopy to rule out

extensive necrosis of nodular tissues.

Six other sections of 4 mm thickness each were cut

from each nodule and mounted on albumin-coated

slides and another six were cut at random as negative

controls. The test samples were put into six groups,

where each group containing one section from each

nodule was labeled for each of the six enzymes (LDH,

SDH, PDK2, G6PD, ME, and A/BHD4). The six

negative control sections were labeled as negative

controls for each enzyme group. All the sections were

dried in a hot air oven at 50uC overnight before

immunohistochemistry was conducted.

Primary antibodies

Primary antibodies included mouse anti-lactate

dehydrogenase (Sigma L7016), rabbit anti-pyruvate

dehydrogenase kinase 2 (Sigma HPA008287), rabbit

anti-malic enzyme 1 (Sigma HPA006493), rabbit anti-

succinate dehydrogenase B (Sigma HPA2868), rab-

bit anti-glucose-6-phosphate dehydrogenase (Sigma

A9521), and rabbit anti-alpha/beta hydrolase 4

(Sigma HPA000600).

Buffers and solutions

The pH values for the citrate buffer and Tris-buffered

saline (TBS) were 6.0 (Sigma C2488) and 7.4. Working

Seidu et al. Immunoreactivity of some epitopes in longtime inappropriately stored paraffin embedded tissues

60 Journal of Histotechnology 2013 VOL. 36 NO. 2

solution contained one part Tris-buffered saline 610

concentrate (Sigma T5912) and nine parts distilled

water. Other supplies included a dimethylaminoben-

zidine tetrahydrochloride solution (Sigma D3939),

streptavidin–horseradish peroxidase polymer (Sigma

S2438), and a peroxidase-block solution (0.03% H2O2

in 95% methanol, i.e. 1 liter contains 3 ml of 30% H2O2

(Sigma 95321) and 997 ml of 95% methanol).

All Sigma products were obtained from Sigma-

Aldrich Chemie GmbH, Taufkirchen, Germany.

MethodThe antibodies were diluted as listed in Table 1, and a

minimum of 200 ml was dispensed per section to ensure

that sections were well covered. A humidified staining

rack was constructed as previously described.19 All the

sections were de-paraffinized in three changes of xylene

for 3 minutes each, followed by three changes in

methanol for another 3 minutes each. The samples

then transferred into a freshly prepared peroxidase

block solution for 5 minutes to block endogenous

peroxidase and then transferred into distilled water.

Antigens were retrieved in citrate buffer (pH 6.0) at

120uC for 5 minutes in a pressure cooker.

Immunohistochemistry procedure

A batch of slides consisting of one slide each from

KP1–KP30 and a negative control was placed on the

Table 1 Preparation of working solutions of antibodies

Table no.of slides

Antibody

Antibodyvol. (ml) TBS vol. (ml)Conc. (mg/ml) Dilution

31 LDH (2.0) 1 : 500 13 650031 ME (0.05) 1 : 75 84 630031 SDH (0.09) 1 : 250 25 625031 PDK (0.03) 1 : 25 250 625021 G6PD (8.6) 1 : 4 1000 4000*31 A/BHD (0.08) 1 : 25 250 625031 STREPT.-HRP (1.0) 1 : 500 13 6500

Note: *G6PD primary antibody could only yield 4000 ml due to low dilution factor.

Figure 1 Photographs of (A) inappropriately stored paraffin-embedded nodules and (B) paraffin wax re-embedded nodules.


Journal of Histotechnology 2013 VOL. 36 NO. 2 61

humidified staining rack and reagents were applied in

the following sequence: TBS (5 minutes62), primary

antibodies (30 minutes), streptavidin-peroxidase poly-

mer (30 minutes), and freshly prepared working DAB

solution (10 minutes). This procedure was used for all

six batches in which the primary antibodies were

different for each batch; and TBS was maintained for

the negative slides during primary incubation to avoid

drying. The slides were rinsed and flooded with two

changes of TBS for 5 minutes each after each

application, except after DAB when tap water was

used. The sections were stained with Mayer’s hema-

toxylin for 1 minute and blued in tap water for

2 minutes, after which they were dehydrated in

ethanol, cleared in xylene, and mounted in DPX with

appropriate cover slips.

Examination of slides

Sections were examined using an Olympus light

microscope (Olympus CX31, model CX31RBSF).

Negative staining showed no DAB reaction and

scored 0, while positive staining appeared as golden-

brown to dark brown reaction products. The

presence of golden-brown to dark brown DAB

reaction products in worm tissue sections was noted

as a positive reaction and the reaction product

intensities were scored 1z for a mild reaction and

2z for a strong reaction (either score was regarded as

positive in the summary of results). The examination

was carried out jointly by a biomedical scientist and a

pathologist, and both agreed on a score before it was

recorded. Micrographs were captured using an

Olympus digital camera (model DP20–50) mounted

on an Olympus microscope (model BX51TF). The

micrographs were captured on a computer, arranged

using Microsoft Office Power Point, and converted to

a TIFF image (Fig. 2).

ResultsA total of 33 worms were detected in the sections

stained for LDH and SDH, 31 worms detected in

sections stained for PDK and ME, and 30 worms in

sections stained for A/BHD. Only 20 worms were

detected in sections stained for G6PD, due to the

limited antibody solution volume. Two male worms

were detected in the sections stained for G6PD,

LDH, and SDH while only one male worm was

detected in each batch of sections stained for PDK,

Figure 2 Micrographs of immunohistochemical staining reactions G6PD (A/a), LDH (B/b), PDK (C/c), ME (D/d), A/BHD (E/e),

and SDH (F/f) (uppercase letters5positive control; lowercase letters5negative control; all 6200 magnification).



ME, and A/BHD, all of which were negative for all

the enzymes.

The summary of the results and proportional

representations are in Table 2, while the tests of

significance results of the presence of all the enzymes

in O. volvulus nodules are in Table 3.

DiscussionThis study was aimed at determining the usefulness

of long period, inappropriately stored paraffin-

embedded O. volvulus nodules for immunohistochem-

ical studies. A demonstration of utility would enable

the use of archival nodules for immunohistochemical

studies when fresh materials are difficult to obtain

from human sources if successful. Results showed

that under the conditions from which these nodules

were retrieved and following the process of re-

embedding, between 52 and 65% of worms in the

nodules still had demonstrable enzymes. At 99%

confidence level, a significant number of worms

(§30%) in nodules stored under the adverse condi-

tions were predicted to be positive for all the six

enzymes (P value: 0.002, 0.001, 0.001, 0.001, 0.005,

and 0.001). It is also predicted that at the same level

of confidence, §35% of worms in nodules will be

found to have four (LDH, PDK, A/BHD, and ME)

demonstrable enzymes (P value: 0.002, 0.006, 0.003,

and 0.007). These results are irrespective of any other

factors that could have contributed to loss of

immunoreactivity other than poor storage. It was

not possible to perform immunohistochemistry on

these nodules 15 years ago, which would have

provided initial results for comparison. However,

after such a long period of inappropriate storage,

there is an appreciable level of maintenance of

immunoreactivity of these epitopes as shown by the

results.

In this study, it is not clear whether inappropriate

storage of tissue blocks under humid and wide range

of temperature conditions for long periods did induce

loss of antigenicity and immunoreactivity. However,

it is clear that there is a significant level of detectable

enzymes in inappropriately stored paraffin-embedded

nodules to merit their use for immunohistochemical

investigations.

Paraffin embedding of formalin-fixed nodules is a

remarkably resilient method of specimen and antigen

preservation, and therefore, can withstand the adverse

effect of extended periods of wide range of room

temperatures (20–40uC) and humid conditions without

significant protein degradation or loss of antigenicity,

hence the successful demonstration of these enzymes in

significant proportions.

ConclusionPoorly stored archival formalin-fixed, paraffin-

embedded Onchocerca nodules over a prolonged

period still retain enough antigenicity for immuno-

histochemical demonstration of the enzymes LDH,

SDHB, PDK2, G6PD, ME1, and A/BHD4 and

perhaps other antigens.

AcknowledgementsThe reagents were purchased through the collabora-

tive funding of the Department of Pathology of the

University of Ghana Medical School and University

of Ghana School of Allied Health Sciences through

their local research funds; the authors thank the

Table 3 Tests of proportional significance of enzymes in O. volvulus (30, 35, and 40%) at 95% significance level

No. of female worms No. with Enzyme present % of female with enzyme 0.30 0.35 0.40

G-6-PD 18 11 61.1 0.002** 0.010* 0.035*LDH 31 20 64.5 0.001** 0.002** 0.022*PDK 30 17 56.6 0.001** 0.006** 0.029*A/BHD 29 17 58.6 0.001** 0.003** 0.018*SDH 31 16 51.6 0.005** 0.031* 0.106ME 30 19 63.3 0.001** 0.007** 0.005**

Note: *Significant at 5%; **Significant at 1%.

Table 2 Summary of results

G6PD LDH PDK ME1 A/BHD4 SDHB

Total no. of worms 20* 33 31 31 30 33No. of male worms 2 2 1 1 1 2No. of female worms 18 31 30 30 29 31Enzyme-positive female worms 11 20 17 19 17 16Enzyme-positive male worms 0 0 0 0 0 0% of female worms with enzyme 61.1 64.5 56.6 63.3 58.6 51.6

Note: *G6PD primary antibody could only cover 21 slides due to low dilution factor.


Journal of Histotechnology 2013 VOL. 36 NO. 2 63

heads of these institutions for their support. The

authors also grateful to the late Dr. K. Awadzi, the

former director of Onchocerciasis Chemotherapy

Research Centre at Hohoe hospital in Ghana and

his staff for the Onchocerca volvulus nodules. Finally,

the authors thank Mr. D. Nana Adjei of the

University of Ghana School of Allied Health

Sciences for his assistance in the statistical analysis.

References1 Manne U, Myers RB, Srivastava S, Grizzle WE. Re: Loss of

tumor marker-immunostaining intensity on stored paraffinslides of breast (letter). J Natl Cancer Inst. 1997;89:585–6.

2 Ibrahim SO, Johannessen AC, Vasstrand EN, Lillehaug JR,Nilsen R. Immunohistochemical detection of p53 in archivalformalin-fixed tissues of lip and intraoral squamous cellcarcinomas from Norway. APMIS. 1997;105:757–64.

3 Camp RL, Charette LA, Rimm DL. Validation of tissue microarraytechnology in breast carcinoma. Lab Invest. 2000;80:1943–9.

4 Jacobs TW, Prioleau JE, Stillman IE, Schnitt SJ. Loss of tumormarker-immunostaining intensity on stored paraffin slides ofbreast cancer. J Natl Cancer Inst. 1996;88:1054–9.

5 Wester K, Wahlund E, Sundstrom C, Ranefall P, Bengtsson E,Russell PJ, et al. Paraffin section storage and immunohisto-chemistry: effects of time, temperature, fixation, and retrievalprotocol with emphasis on p53 protein and MIB1 antigen. ApplImmunohistochem Mol Morphol. 2000;8:61–70.

6 Fergenbaum JH, Garcia-Closas M, Hewitt SM, Lissowska J,Sakoda LC, Sherman ME. Loss of antigenicity in storedsections of breast cancer tissue microarrays. Cancer EpidemiolBiomarkers Prev. 2004;13:667–72.

7 Ran X, Joon-Yong C, Kris Y, Reginald LW, Natalie G,Nathan N, et al. Factors influencing the degradation of archivalformalin-fixed paraffin-embedded tissue sections. J HistochemCytochem. 2011;59:4:356–65.

8 Omar MS, Raoof AM. Onchocerca fasciata: enzyme histo-chemistry and tissue distribution of various dehydrogenases inadult female worm. Parasitology. 1996;82(1):32–7.

9 Ansari AA, Yusufi AN, Siddiqi M. Lipid composition of thefilarial parasite Setaria cervi. J Parasitol. 1973;59:939–40.

10 Barrett J, Mendis AH, Butterworth PE. Carbohydrate meta-bolism in Brugia pahangi (Nematoda: Filaridea). Int JParasitol. 1986;16:465–9.

11 Awadzi K. The chemotherapy of Onchocerciasis II.Quantitation of the clinical reaction to metrifonate. Ann TropMed Parasit. 1980;74:189–97.

12 Awadzi K, Haddock DRW, Gilles HM. The chemotherapy ofonchocerciasis I. Initial open evaluation of metrifonate. AnnTrop Med Parasit. 1980;74:53–61.

13 Awadzi K, Gilles HM. The chemotherapy of OnchocerciasisIII. A comparative study of Diethylcarbamazin (DEC) andmetrifonate. Ann Trop Med Parasit. 1980;74:199–210.

14 Awadzi K, Gilles HM. The chemotherapy of OnchocerciasisIV. Further trials with metrifonate. Ann Trop Med Parasit.1980;74:355–62.

15 Awadzi K, Schultz-Key H, Howells RE, Haddock DRW, GillesHM. The chemotherapy of Onchocerciasis VIII: levamisole andits combination with the benzimidazoles. Ann Trop MedParasit. 1982;76:23–4.

16 Awadzi K, Schultz-Key H, Edwards G, Breckenridge A, OrmeM, Gilles HM. The chemotherapy of Onchocerciasis XIV.Studies with mebendazole-citrate. Ann Trop Med Parasit.1990;41:383–6.

17 Awadzi K, Hero M, Opoku NO, Addy ET, Buttner DW, GillesHM. The chemotherapy of Onchocerciasis XV. Studies withalbendazole. Ann Trop Med Parasit. 1991;42:356–60.

18 Awadzi K, Hero M, Opoku NO, Addy ET, Buttner DW,Ginger CD. The chemotherapy of Onchocerciasis XVIII.Aspects of treatment with suramin. Ann Trop Med Parasitol.1995;46:19–20.

19 Seidu MA, Adams AR, Gyasi RK, Tettey Y, Wiredu EK. Shorttime incubation at low temperature for retrieval of someantigens from formalin-fixed and paraffin-embedded tissues. JHistotechnol. 2012;35:74–9.



Immunoreactivity of some epitopes in longtime inappropriately stored paraffin-embedded tissues

Documents

Transcript of Immunoreactivity of some epitopes in longtime inappropriately stored paraffin-embedded tissues