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  IN THIS Article
 ::  Abstract
 ::  Introduction
 ::  Material and Methods
 ::  Results
 ::  Discussion
 ::  Acknowledgements
 ::  References
 ::  Article Figures

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ARTICLE
Year : 1978  |  Volume : 24  |  Issue : 4  |  Page : 226-230

Cytochemical studies in leukaemias


Department of Haematology, K.E.M. Hospital, Bombay 400 012, India

Correspondence Address:
Neelam R Batra
Department of Haematology, K.E.M. Hospital, Bombay 400 012
India
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PMID: 84870

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 :: Abstract 

One hundred cases of acute leukaemia were studied, by Romanowsky stains and by cytochemical stains such as Sudan Black B, Periodic Acid Schiff, Alkaline phosphatase and Peroxidase stains. Cases difficult to diagnose by Romanowsky stained smears were easily classified by these supplementary stains. Their importance as supplements to the routine Romanowsky staining in the diagnosis of leukaemia is emphasized and the division of acute lymphoblastic leukaemia based on these patterns is suggested.



How to cite this article:
Batra NR, Deshpande C K, Mehta B C, Advani S H. Cytochemical studies in leukaemias. J Postgrad Med 1978;24:226-30

How to cite this URL:
Batra NR, Deshpande C K, Mehta B C, Advani S H. Cytochemical studies in leukaemias. J Postgrad Med [serial online] 1978 [cited 2014 Oct 23];24:226-30. Available from: http://www.jpgmonline.com/text.asp?1978/24/4/226/42652



 :: Introduction Top


In chronic leukaemia, the identification of cell type, seldom poses a problem because of abundance of easily identifiable mature cells. However in acute leukaemias, the cell identification often poses a problem because there is a marked similarity between earlier precursors of different cell series. In these cases the cytochemical stains are of great help in recognising the type of precursor cells. As there is asynchronism between nuclear and cytoplasmic maturation, the presence of such cytochemically stainable structures, in earlier precursors, indicate specific cellular differentiation which the precursor cells are undergoing, thus making the identification easy. A number of workers have applied cytochemistry to the classification of leukaemias. [1],[2],[4],[5],[8] We present below our experience with 100 cases of leukaemia studied by various cytochemical stains.


 :: Material and Methods Top


A total of 100 cases of leukaemia were studied, consisting of 70 cases of acute leukaemia and 30 cases of chronic leukaemia. They were taken up for study from the OPDs and from the wards of K.E.M. Hospital, Bombay and from Tata Cancer Hospital, Bombay. Bone marrow and peripheral blood smears were prepared and the patients were followed up during the treatment.

The stains used were Romanowsky stains (Wright's stain and Leishman's stain) Alkaline phosphatase stain (a modified azo-dye coupling technique as described by Kaplow [6],[7] ) . Sudan Black B stain, [9] peroxidase stain, and Periodic acid Schiff stain

Fixatives used

The bone marrow and peripheral smears were fixed in different fixatives for different stains. The fixatives used were as follows:

  1. For alkaline phosphatase reaction: A, mixture of formalin and methanol was used (90%) , the fixative being kept in freezing unit of the refrigerator to chill the solution before fixing for 30 seconds
  2. Periodic acid Schif reaction: Air dried smears were fixed in methanol fo] 10 minutes and washed with tap water before staining.
  3. Sudan Black B staining: Air dried smears were fixed for 5 minutes in formalin vapours and washed in running water.
  4. Peroxidase staining: For peroxidase staining fresh smears were used without fixation.


Staining methods

  1. Sudan Black B staining method as recommended by Sheehan and Storey [9] was used.
  2. Periodic acid Schiff reaction using periodic acid for oxidation and Barger, anc De Lamaters' Schiff reagent was used.
  3. Peroxidase staining was used using benzidine and hydrogen peroxide and later on counter-staining by Wright' , stain was done.
  4. Alkaline phosphatase method used was a slight modification of method uses by Kaplow. [6],[7]


Substrate used in this reaction way alpha naphthyl phosphate, 20 mgm, and fast blue R, 20 mgm, dissolved in 20 ml of the propanediol working buffer with pH around 9.75. Mayer's haematoxylin was used as a counter-stain.

Assessment and Scoring of smears

A detailed study as to the cellular mor­phology of the blast cells was done. Details of cell size, characteristics of cytoplasm and nuclear characteristics were studied in detail in the bone-marrow smears and peripheral blood smears.

Scoring of smears stained with sudan black B, peroxidase, periodic acid schiff, and alkaline phosphatase, were done according to the semiquantitative method of Hayhoe et al. [5] In case of sudan Black B stains and peroxidase stains, the posi­tivity was seen as black granules or localised to heavy diffuse staining of cytoplasm of the blast cells. In case of PAS staining, the positivity was studied in the blast cells in the peripheral blood and bone marrow smears and magenta pink coloured granules or pink coloured diffuse staining of cytoplasm or pink coloured blocks were looked for in the cytoplasm of blast cells. In case of alka­line phosphatase stain, a positive reaction was seen as a faint tinge of brownish black colour, or granules of the same colour, in the cytoplasm of mature polymorphonuc­lear cells in the peripheral blood smears. Hundred consecutive polymorphs were counted, and scored according to the method of Hayhoe et al. [5]




 :: Results Top


The following were the observations in the cases studied.

Romanowsky stained smears

Details of nucleus and cytoplasm were studied in blast cells in the peripheral blood smears and bone marrow smears. The nucleus was rounded in majority of the blast cells in acute lymphoblastic and acute myeloid leukaemia, as well as acute myelo-monocytic leukaemia, while in acute monocytic leukaemia, the nucleus had an irregular shape due to indenta­tions and lobulation. However the nucleus had lobulations in 10%, of cases of acute myeloid leukaemia. Nuclear chromatin was fine in blast cells of acute myeloid leukaemia, and coarser in acute lympho­blastic leukaemia. However in 32%, of cases of acute myeloid leukaemia, the chromatin was coarse and as the nuclear cytoplasmic ratio was also high, they were indistinguishable from lymphoblasts (on cytochemistry they proved to be myelo­blasts). In acute myelomonocytic leukae­mia, the blast cells resembled myeloblasts and were indistinguishable from those cases of acute myeloid leukaemia, where the nuclei showed lobulations.  Auer rods More Details were present in only 40% of cases of acute myeloid leukaemia. They were also observed in one case of acute myelomono­cytic leukaemia Cytoplasmic budding was present in 10% cases of acute myeloid and in 60% of cases of acute myelomonocytic leukaemia and in 40% of cases of chronic myeloid leukaemia. Rieder cell formation was only observed in acute lymphoblastic leukaemia. In acute lymphoblastic leukae­mia group, there were 4 cases with vacuoles in the cytoplasm in Ramanowsky stained preparations, the cytoplasm being deep blue.

Cytochemical observations

Cytochemistry revealed definite stain­ing patterns in different types of leukaemia.

Sudan Black B Staining

In myeloid leukaemias a diffuse overall or a localised positivity was observed of dense black staining in the cytoplasm of blast cells see [Figure 1] on page 230A. The blast cells of lymphoblastic series were negative. In monoblasts see [Figure 2] on page 230A a very faint black granular positivity was seen. In cases of acute myelomonocytic leukaemia, there was a mixed pattern of staining see [Figure 3] on page 230A observed in the blast cells and consisted of black coarse granular as well as a diffuse black positivity in the blast cells. The auer rods showed a black staining. The granules of myeloid series were stained intense black.

Perioxidase staining

The pattern of staining observed was the same as for Sudan Black stain. There was a localised or diffuse positivity in the myeloid group; the lymphoblasts showed a negative reaction. The monoblasts show­ed a granular positivity. However the percentage of positive cells showing the. characteristic positivity was less than that seen in the Sudan Black stained smears.

Periodic Acid-Schiff reaction

The stain showed a diffuse pink posi­tivity in the cytoplasm of myeloblasts. This was also seen with more intense staining in the cytoplasm of promyelo­cytes and mature granulocytes. Mono­blasts had a faint pink fine granular posi­tivity. In cases of acute myelo-monocytic leukaemia, the blast cells had both a granular positivity as well as a diffuse positivity. In cases of acute lymphoblastic leukaemia, the blast cells were either negative or showed a corase dark pink granular positivity while some cases had blocks of PAS positive material in their blast cells see [Figure 4] on page 230A. None of the cases of acute lymphoblastic leukaemia showed diffuse pink positivity in their cytoplasm.

Alkaline phosphatase stain

This was of help in distinguishing bet­ween chronic myeloid leukaema and leukaemoid reactions. Five cases of leukaemoid reactions diagnosed a leukaemia were thus excluded and ij these cases the blood picture returned to normal after their causes were treated In leukaemoid reactions a very high score was noted-140 and above (Normal established zone for our laboratory being 15-92).


 :: Discussion Top


In our study it was seen that examination of Romanowsky stained smears alone was not sufficient to diagnose the cell type involved in acute (blastic) leukaemias Supplementary cytochemical stains were helpful in diagnosing the cell type involved, as by staining sub-cellular structures, they help in recognising the precursor cells, because they indicate the specific cellular differentiation which the precursor cells are undergoing. Distinct cytochemical patterns were observed in different types of leukaemia (in accordance with earlier observations of some w orkers. [3],[4],[5],[8]

(a) In acute myeloid leukaemia the blast cells showed a diffuse localised to heavy overall positivity of black coloured staining in Sudan black stained smears In peroxidase stained smears, a similar pattern was observed. The more mature granulocytes showed a heavies staining. In Periodic Acid Schiff stain, the blast cells showed a diffuse pink staining of the cytoplasm with some blast cells also showing fine pink granules. The mature granulocytes showed a more in­tense positivity. The alkaline phosphatase stain showed a score between 20-60.

(b) In acute lymphoblastic leukaemia group, considering the appearance of the blast cells in Romanowsky stains and other cytochemical stains, they were sub­divided into three groups.

  1. Group I: Blast cells with appear­ance of typical lymphoblasts on Roma­nowsky stained preparations and Peroxi­dase and Sudan black negative. P.A.S. stain on these smears showed typical pattern of magenta pink coarse granules or blocks of P.A.S. positive material in the cytoplasm of blast cells.
  2. Group II: Appearance of typical lymphoblasts on Romanowsky stained preparations and Peroxidase and Sudan Black negative. P.A.S. also showed a negative reaction (undifferentiated group) .
  3. Group III (Burkitt cell type) : On Romanowsky stained preparations, Blast cells had dark blue cytoplasm with vacuoles. Sudan black and Peroxidase Stains were negative. P.A.S. reaction showed a variable reaction. These consti­tuted 10 ; ~ of the cases of A.L.L. (see [Figure 5] on page 230B).


In the group of acute lymphoblastic leukaemia, there are varying reports of the relation of P.A.S. positivity, to the duration of first remission and survi­val. [2],[8],[10] However Bennet et al [1] found contradictory results.

In our study we did find a relationship between P.A.S. positivity (above 50% of Blast cells being P.A.S. positive) and longer duration of first remission. This was however related to only the P.A.S. positive group (Group I).

(c) In acute monocytic group, the blast cells showed fine pink dusty granules in the cytoplasm in the PAS stain. Sudan black and peroxidase stain showed dusty black, granular positivity in the blast cells.

(d) In the acute myelo-monocytic group, the Sudan black and the peroxi­dase stains showed a mixed pattern of diffuse or localised positivity of black colour in the cytoplasm of blast cells while the Periodic Acid Schiff reaction showed pink granular positivity of mode­rately coarse granules as well as diffuse pink staining in the cytoplasm of blast cells.

These distinct cytochemical patterns in the four different groups of leukaemia, helped to identify the blast cells and in some cases where, on Romanowsky stain­ed smears alone the diagnosis of the cell type involved was in dispute, the cyto­chemical pattern helped to differentiate the blast cells.

In 30% of cases labelled as lympho­blastic leukaemia, on Romanowsky stains alone, cytochemistry revealed a myeloid pattern in Peroxidase, Sudan black and PAS stains.

Among 5 cases in which cytochemistry revealed a myelomonocytic pattern, 3 cases were diagnosed as acute myeloid leukaemia, on Romanowsky stained smears. Cases of acute monocytic leukae­mia, were separated from acute-myelo monocytic leukaemia on the basis of cyto­chemistry. The alkaline phosphatase re­action helped to separate out, leukaemoid reaction, from cases of chronic myeloid leukaemia as in these leukaemoid re­actions the score was very high, compared to the low score of chronic myeloid leukaemia.


 :: Acknowledgements Top


We wish to thank the Dean, K.E.M. Hospital, for his kind permission to carry out the study. Acknowledgements are also due to The Research Society, K.E.M. Hospital as a part of the scheme was aid­ed by their grant. We also thank the Authorities of The Tata Cancer Hospital for permission to collect material from their hospital.

 
 :: References Top

1.Bennett, J. M., Catosky, D., Daniel, Nl. T., Flandrin, G., Galton, D. A. G., Grannick, H. R. and Sultan, C.: Proposal for the classification of acute leukaemias. Brit. J. Haematol. 33: 451-458, 1976.  Back to cited text no. 1    
2.Feldges, A. J., Aur, R. J. A., Verzosa, M. S. and Daniel, S.: Periodic acid Schiff re­action-a useful index of duration of com­plete remission in acute childhood lym­phocytic leukaemia. Acta. Haematol. 52: 8-13, 1974.  Back to cited text no. 2    
3.Flandrin, G. and Daniel, M. T.: Practical value of cytochemical studies for the classification of acute leukaemias. Recent Results in Cancer Research, 43: 43-56, 1973.  Back to cited text no. 3    
4.Fraumeni, J. F. Jr., Manning, M. D. and Mitus, W. J.: Acute childhood leukaemias. Epidemiologic studies by cell type of 1263 cases at the Childrens' Cancer Research Foundation of Boston, 1947-1965. J. Nat. Cancer Insti. 46: 461-470, 1971.  Back to cited text no. 4    
5.Hayhoe, F. G. J., Quaglino, D. and Doll, R.: Material and Methods of Study. In, "The Cytology and Cytochemistry of Acute Leukaemias". MRS special report series No. 304. Her Majesty's Stationary Office, London, 1964, p. 11-23.  Back to cited text no. 5    
6.Kaplow, L. S.: A histochemical procedure for localizing and evaluating leucocyte alkaline phosphatase activity in smears of blood and marrow. Blood, 10: 1023-1029. 1955.  Back to cited text no. 6    
7.Kaplow, L. S.: Cytochemistry of leucocyte alkaline phosphatase: use of Complex Naphthol AS Phosphates in Azo-dye coupling technics. Amer. J. Clin. Path. 39: 439-449, 1963.  Back to cited text no. 7    
8.Kurz, R. and Haas, H.: Value of the com­bined cytological and cytochemical classi­fication in the management of acute child­hood leukaemia. Acta. Haematol. 52: 1-7, 1974.  Back to cited text no. 8    
9.Sheehan, R. L. and Storey, G. W.: An improved method of staining leucocyte granules with Sudan Black. Brit. J. Path. & Bact. 59: 336-337 1 1947.  Back to cited text no. 9    
10.Vowels, M. R. and Willoughby, M. L. N.: Cyclic chemotherapy in acute lymphblastic leukaemia of childhood. Acta. Dis. Child. 48: 436-440, 1973.  Back to cited text no. 10    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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