J. clin. Path. (1966), 19, 257

Ribonucleic acid content of Burkitt tumour cellsD. H. WRIGHT AND J. C. McALPINE

From the Department ofPathology, Makerere University College Medical School,Kampala, Uganda

SYNOPSIS Methyl-green-pyronin-Y staining was performed on 57 biopsies of Burkitt's tumour and62 biopsies from other types of malignant lymphoma. The specificity of the pyronin staining forribonucleic acid (RNA) was controlled by staining duplicate slides previously digested in ribo-nuclease. Burkitt's tumour cells have a higher cytoplasmic content of RNA than the cells of mostother malignant lymphomas. The results of acridine orange staining of a smaller number of biopsiessupport these findings.

Intense basophilia of certain cells of the haemopoieticsystem following staining with Romanowsky dyeshas been recognized for many years (White, 1947;Davidson, Leslie, and White, 1948; Rheingold andWislocki, 1948; Harris and Harris, 1949). Both ultra-violet microspectrophotometry and selective aboli-tion of the basophilia with rubonuclease haveindicated that this basophilic substance is ribonucleicacid (Brachet, 1940). Imprint preparations from casesof Burkitt's tumour stained with Romanowsky dyesshow intense cytoplasmic basophilia of the tumourcells (Wright, 1963a and b), a feature that differenti-ates them from most other malignant lymphomas. Itwas decided therefore to stain biopsy sections fromcases of Burkitt's tumour for nucleic acids using amethyl green-pyronin Y method and to compare theresults with those obtained on biopsy sections fromother types of malignant lymphoma. Imprintpreparations from a small number of biopsies ofBurkitt's tumour and other malignant lymphomashave also been studied by the acridine orange methodfor demonstrating nucleic acids.

MATERIALS AND METHODS

Paraffin sections of formalin-fixed tissue and methyl-alcohol-fixed imprints of biopsies from cases of Burkitt'stumour were stained by the methyl green-pyronin Ymethod of Kurnick (1955). The specificity of the pyroninstaining for RNA was assessed by staining duplicate slideswhich had been digested in ribonuclease, 1-0 mg. per ml.,for 60 minutes at 37°C. The comparative study of thepyroninophilia of different types of malignant lymphomawas made by staining all the malignant lymphomabiopsies for 1962 and 1963 on which duplicate sectionswere readily available. Approximately half these biopsiescame from Mulago Hospital, Kampala, and half from'up country' Government and mission hospitals inUganda and the surrounding territories.Received for publication 14 October 1965.

The pyroninophilia of the predominant cell type ineach type of malignant lymphoma was assessed visuallyand graded from 0 to + + +, the + + + gradingcorresponding to the degree of pyroninophilia shown byplasma cells. The grading was made independently byeach of us and the results averaged.

Imprints of Burkitt's tumour and of other lymphomaswere fixed for five minutes in Carnoy's fixative andstained in acridine orange. A fluorochrome concentrationof 0-01 % in Mcllvaine's citric acid disodium phosphatebuffer at pH 4 0 was found to give the best differentiationbetween RNA and DNA. The stained preparations weremounted in buffer solution and examined by blue lightfluorescence using a BG 12 filter and an OG 1 selectiveeyepiece filter.

RESULTS

Occasional biopsies showed a diffuse mauve stainingof the nucleus and the cytoplasm. This was presum-ably due to faulty fixation and these cases werediscarded from the study.The intensity of pyroninophilia in 57 cases of

Burkitt's tumour and 62 cases of other types ofmalignant lymphoma are shown in Fig. 1.Although there is considerable overlap, Burkitttumour cells show a greater degree of pyronino-philia than the cells of lymphocytic lymphomas andhistiocytic lymphomas. Half the cases of stem-celllymphoma showed the same degree of pyronino-philia as Burkitt's tumour.

In keeping with the cytological uniformity ofBurkitt's tumour the cytoplasmic pyroninophiliawasuniform throughout all the cells in the biopsy(Fig. 2). In contrast, most cases of poorly differenti-ated lymphocytic lymphoma and most histiocyticlymphomas showed a range of cells with varyingintensities of cytoplasmic pyroninophilia. Acridineorange staining was in conformity with these

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D. H. Wright and J. C. McAlpine

50 Stem Cell (14 cases)40-

30-

20-

10-

Histiocytic Lymphomas (20coses)50S40-

30-

20-

0w

Lymphocytic Lymphomas (28 cases)40-

w 30-

20-

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rsults Burkitt's Lymphoma (57 cases)40-

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0 0-*-9- + -t--4++ ++ ++--I++I-Intensity of Pyroninophilia

FIG. 1. Intensity of pyroninophilia of Burkitt 's tumourcells compared with the cells ofother malignant lymphomas.

results. Burkitt's tumour cells stained with acridineorange showed an intense, uniform, flame-red cyto-plasmic fluorescence and yellow-green nuclearfluorescence.The large clear or vacuolated histiocytes that are

such a characteristic feature of Burkitt's tumourcontained, in addition to whole lymphoid cells, manyfragments of pyroninophilic material.

DISCUSSION

The results obtained with methyl green-pyronin Ystaining confirm that Burkitt tumour cells have ahigher cytoplasmic content of ribonucleic acid thanthe cells of most lymphocytic lymphomas and mosthistiocytic lymphomas. There is considerable overlap

FIG. 2. Section of Burkitt's tumour stained with methylgreen -pyronin Y ( x 468)... - -------s-MZ

FIG. 3. Section of Burkitt's tumour stained with methylgreen - pyronin Y ( x 1,500).

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Ribonucleic acid content of Burkitt tumour cells

FIG. 4. Section of malignant lymphoma poorly differenti-ated lymphocytic type (lymphosarcoma) stained withmethyl green -pyronin Y (x 468).

with cases of stem-cell lymphoma but these areusually easily distinguishable from Burkitt's tumourusing other cytological criteria. Intense cytoplasmicbasophilia has been a constant feature of over 50Giemsa-stained imprint preparations of freshbiopsies of Burkitt's tumour studied by one of us(D.H.W.). It is probable, therefore, that theoccasional low intensity of pyroninophilia obtainedin sections of Burkitt's tumour was due to poorfixation of the biopsy rather than to a real deficiencyof the ribonucleic acid content of the cells.A high cytoplasmic content of ribonucleic acid is a

feature of protein-secreting cells, as well as ofhaemopoietic blast cells and many embryonic andtumour cells. Protein-secreting cells have a welldeveloped endoplasmic reticulum fringed with

ribonucleoprotein granules (Palade, 1955) whereasblast cells do not have a well-developed endoplasmicreticulum and the ribonucleoprotein granules arescattered freely throughout the cytoplasm (Bernhardand Granboulan, 1960). Electron microscope studieshave shown that the cytoplasm of Burkitt tumourcells is rich in ribosomes which occasionally show atendency towards clumping. The endoplasmicreticulum is poorly developed and most of theribosomes are free within the cytoplasm (O'Conor,personal communication). Electrophoresis of theserum from cases of Burkitt's tumour has shown nospecific protein abnormalities of the type seen inmultiple myelomatosis (Martin, personal communi-cation). The high cytoplasmic level of ribonucleicacid in Burkitt tumour cells may be a reflection ofthe rapid multiplication of these cells and thecorrespondingly high rate of protein synthesis.The abundant cytoplasmic ribonucleic acid in

Burkitt tumour cells gives an amphophilic stainingquality to the cytoplasm of these cells in sections offormalin-fixed material stained with haemotoxylinand eosin. This staining quality, similar to that seenin plasma cells, is best demonstrated in tissue fixedin buffered neutral formalin and is usually absentwhen acid fixatives have been used. It is one of thehistological features used in this department for thediagnosis of Burkitt's tumour. Its intensity and uni-formity throughout the biopsy helps to differentiateBurkitt's tumour from other malignant lymphomasand also from retinoblastomas and neuroblasto-mas which may be confused with Burkitt's tumourclinically.

REFERENCES

Bernhard, W., and Granboulan, N. (1960). In Ciba FoundationSymposium on Cellular Aspects of Immunity, ed. by G. E. W.Wolstenholme and M. O'Connor, p. 92. Churchill, London.

Brachet, J. (1940). C. R. Soc. Biol. (Paris), 133, 88.Davidson, J. N., Leslie, I., and White, J. C. (1948). J. clin. Path., 60, 1.Harris, T. N., and Harris, S. (1949). J. exp. Med., 90, 169.Kumick, N. B. (1955). Stain Technol., 30, 213.Martin, N. H. (1964). Personal communication.O'Concr, G. T. (1964). Personal communication.Palade, G. E. (1955). J. biophys. biochem. Cytol., 1, 59.Rheingold, J. J., and Wislocki, G. B. (1948). Blood, 3, 641.White, J. C. (1947). J. clin. Path., 59, 223.Wright, D. H. (1963a). Brit. J. Cancer, 17, 50.

(1963b). In International Review of Experimental Pathology,edited by G. W. Richter, and M. A. Epstein, vol. 2, p. 97.Academic Press, New York.

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