Cytochemical studies in leukaemiasNeelam R Batra, CK Deshpande, BC Mehta, SH Advani
Department of Haematology, K.E.M. Hospital, Bombay 400 012, India
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.
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. ,,,, We present below our experience with 100 cases of leukaemia studied by various cytochemical stains.
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 , ) . Sudan Black B stain,  peroxidase stain, and Periodic acid Schiff stain
The bone marrow and peripheral smears were fixed in different fixatives for different stains. The fixatives used were as follows:
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.  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. 
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.
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.
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).
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. ,,,
(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.
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. ,, However Bennet et al  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.
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.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]