Cytochemical  Markers  in Acute  Leukemias

THESIS

Submitted in Partial Fulfillment of the Requirements for the

Master Degree in Clinical Pathology 

 

 

By

 Elham Omar Hamed

M. B. B. Ch. Assiut  University

 

Supervised by

 

Prof.  Zeinab M. M. Diab

Professor  & Head  of  Clinical  Pathology dep.

Sohag Faculty  of  Medicine

South  Valley  University

 

Prof. Mohammed Yousef Al-Kabsh   

 Professor of  Clinical  Pathology

Assiut  University

 

 Dr. Mohammed  Ebrahiem Ahmed

Lecturer of Clinical  Pathology

Sohag  Faculty of  Medicine

South  Valley  University

 

 

Sohag Faculty of Medicine

South Valley  University

1998

 

 

 

 

 

 

ACKNOWLEDGEMENTS

            I would like to express  my  deep  gratitude  to  professor         Zeinab M. M. Diab professor & head  of Clinical  Pathology dep., Sohag Faculty of  Medicine , South  Valley  University for her  valuable  supervision , continuous encouragementand  help kindly and  generously offered throughout  the  whole  work  that  could  never  be  forgotten.

 

         I am  deeply  indebted to  Prof.  Mohammed  Y.  Al-Kabsh     professor  of  Clinical Pathology, Assiut  University , for  his  expert comments  and  skilful  and  very  helpful  advises.

 

               I acknowledge  with  deep  appreciation  the  supervision  of  Dr. Mohammed  Ebrahiem Ahmed, lecturer  of  Clinical  Pathology, Sohag Faculty  of  Medicine,  South  Valley  University , for  his  sincere    help  and  continuous  encouragement  to  accomplish  this  work 

 

          Elham   Omar  Hamed

                                       1998

 

 

 

 

 

 

 

 

 

الدلالات  الكيميائية للخلية لمرضى سرطان  الدم  الحاد

رســالة

مقدمة  تمهيدا  للحصــول  على  درجــة

  المـاجستير  فى  الباثولوجيا الإكلينيكية

من  الطبيبة

الهـــام  عمر  حامد

بكالوريوس الطب  والجراحة – جامعة أسيوط

تحت إشراف

أ0 د0زينب محمد محمود دياب      أ.د0 محمد  يوسف  الكبش

أستاذ و رئيس قسم الباثولوجيا  الإكلينيكية               أستاذ الباثولوجيا  الإكلينيكية

كلية طب  سوهاج - جامعة جنوب الوادى                  كلية الطب - جامعة أسيوط

د0محمد ابراهيم  أحمد

مدرس الباثولوجيا  الإكلينيكية

كلية طب  سوهاج - جامعة جنوب الوادى

 

 

كلية طب  سوهاج – جامعة  جنوب  الوادى

1998 

 

 

 

 

 

 

 

 

 

 

 

 

CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introduction and aim of the work

Leukemias are a group of malignant disorders characterized by progressive, uncontrolled proliferation of hemopoietic cells, at different stages of cell maturation. Leukaemias were classified by the FAB (French-American-British) group according to the clinical course of the disease (acute and chronic) and according to type of cell involved lymphoid, myeloid, monocytic, erythroid, or megakaryocytic. Besides morphology of the cells involved, and because sometimes, it is very difficult to discriminate between the different types of acute leukemia, various laboratory procedures have been developed in order to reach the proper diagnosis of the case. These procedures include various cytochemical techniques.

This study aims at application of various cytochemical techniques for identification and classification of acute leukemias. Also these  cytochemical  tests  help prediction of prognosis and planning of proper chemotherapy.

 

          

Definition and Classification of leukemias

           Leukemias are a group of diseases in which the common manifestation is a malignant unregulated proliferation of cells endogenous to the bone marrow. Leukemia   may involve any of the blood forming cells or their precursors. Although each type of leukemia progresses differently, the unregulated proliferating cells:

1- Usually replace normal marrow.

2- Eventually interfere with normal marrow function.

3- May invade other organs.

4- Eventually cause death if not treated.

          The leukemias were first described in 1845. The chronic leukemias were first to be characterized. Bennett in 1945 and Wirchow in 1946 noticed large spleens and white blood at autopsy of patients who had died of an uncharacterized chronic disease of 1-2  years duration. Wirchow’s description of white blood was Subsequently translated into the Greek word leukemia (Gunz; 1980). Acute leukemia was described about 25 years later when patients with white blood were noted to die rapidly, not after a long debilitating illness  (Friedreich, 1957 and Ebstein, 1889). It was not until 1877, when Ehrlich, 1891, developed a stain that allowed microscopic evaluation of white blood cells, that it was realized that white blood was caused by increase number of white corpuscles. Later in 1900’s, it was established that acute and chronic leukemias involved different types of white blood cells (Gunz 1980). In the chronic leukemias white blood was made up of mature cells while acute leukemia involved immature cells, or blasts. Since 1900’s, leukemias have been studied with the microscope, cytochemical stains, electron microscopy, immunologic, nuclear and surface markers, and cytogenetic techniques.

 

                 Classification of leukemias

1- Hayhoe (1960 ) and Dameshek  (1964))  classification

          Leukemia being subjected to considerable variations in the growth pattern and in the growth rate may be classified ;

1st) According to the course of the disease :

          Despite continued studies and subclassification, leukemias are still generally divided into :

 

Acute leukemia ;

    Large number of undifferentiated primitive cells in the bone marrow and blood resulting from either maturation arrest or an increased growth with little time for differentiation or maturation (Lee et al, 1961) . It is characterized by abrupt onset of clinical symptoms (fever, hemorrhage, weakness), and death occurs within months if treatment is not instituted. Acute leukemia occurs both in children and in adults. Thrombocytopenia is usually found in patients with acute leukemia. Anemia and neutropenia are also hallmarks of the acute leukemias. Blasts or immature cells populate the bone marrow and constitute at least 30 percent of all nucleated bone marrow cells (Bennett, et al , 1985).

 

Chronic leukemia :

          It is characterized by the insidious onset of symptoms (weakness, pallor, enlargement of spleen and liver), and the death occurs years after the diagnosis.  Chronic leukemia usually occurs in adults. In patients with chronic leukemia, thrombocytopenia is rare until late in the course of the disease. Initially platelets seem normal or increased. Leucocytosis and anemia  are usually present. Bone marrow is infiltrated by an increased number of mature cells or cells with recognizable counter part of normal maturation ( Simone, 1978.)

 

B- According to the leucocytic count : we may get:

1- Leukemic leukemia: i.e. with high leucocytic counts  representing the classical leukemic picture.

2- Subleukemic leukemia: i.e. with normal or low leucocytic counts, but with abnormal differential      

counts showing primitive leucocytes.

3- Aleukemic leukemia: in which there is a normal or  usually  low leucoytic counts but with no abnormal  or primitive cells in differential counts.

Classification of acute leukemia:

  On the basis of the presumed cell of origin,acute  leukemia  was classified by  John N. Lukens (1993) into:

1- Acute lymphocytic leukemia (ALL)

     - Early pre-B-cell ALL

     - Pre -B-cell ALL

     - B-cell ALL

     - T-cell ALL

2 - Acute nonlymphocytic leukemia (ANLL)

    - Acute myelocytic leukemia (AML)

    - Acute promyelocytic leukemia (APL)

    - Acute myelomoncytic leukemia (AMMoL)

    - Acute monocytic leukemia (AMoL)

    - Acute erythroleukemia (AEL)

    - Acute megakaryocytic leukemia (AMegL)

3- Acute undifferentiated leukemia: -

 

II :  French - American - British (FAB) classification: -

A uniform classification system for the acute leukemias and myelodysplastic syndromes was developed 1976 by an international group of investigators in 1976 (Bennett, et al, 1976).  

Known as the FAB classification, this system is based on the morphologic appearance of bone marrow and blood leukemic  blasts in Romanovsky  stained smears, supplemented when necessary with cytochemical stains. Modifications for the assessment of lymphoblasts were introduced in 1981 to improve reproducibility and interobserver concordance (Bennett et al, 1981, Miller, et al., 1981). In 1985 criteria for the diagnosis of acute megakaryocytic leukemia were adopted  (Bennett et al, 1985). Although not defined by the FAB cooperative group, a myeloid leukemia variant lacking features of myeloid differentiation was proposed in 1987 (Lee et al, 1987). 

The FAB classification has been adopted widely to permit comparison of treatment results and to take advantage of biologic differences between morphologic subtypes.

 

    Acute lymphocytic leukemia -:

Three subtypes of ALL are distinguished on the basis of cell size, nuclear shape, number and prominence of nucleoli, and the relative amount and appearance of cytoplasm (Benedetto et al, 1980, Behm. 1990) (Table )

Table(1). Morphologic (FAB) classification of acute lymphoblastic leukemia

 

 

 

 

 

 

 

 

Acute Non lynphoblastic leukemia :                           

 ( Bennett et al 1976, 1985, 1991 )

          The expanded FAB classification defines eight variants of ANLL that differ with respect to cell line and degree of differentiation,table(2) and  (3).

Table(2) classification of Myelogenous leukemias

 

 

 

 

Table(3);Characters of Myelogenous   leukemias

Mo - Large , agranular blasts without cytologic maturatien ( resemble ALL - L2 ) .

Myeloperoxidase  negative or < 3 percent positive .

 

M1 - Blast cell, agranular and granular types   ( Types I and II)   > 90  percent of non-erythroid cells . At least  3 percent of these  are peroxidase or Sudan black positive.

       - remaining 10 percent (or less) of cells are maturing granulocytes on monocytes .

 

M2  1. Sum of agranular and granular blasts is from 30 to 89  percent of non-erythroid cells .

       2. Monocytic cell < 20 percent.

       3. Granulocytes from promyelocytes to mature polymorphs > 10 percent

 

 M3 - 1. In the marrow , blasts > 30 percent of non-erythroid cells

         2.  Majority of cells are abnormal promyelocytes with heavy  

                granulation.

       3.  Characteristic cells containing bundles of Auer rods.

       4.  Micro granular variant also occurs .

 

M4- 1. In the marrow, blasts > 30 percent of non-erythroid cells.

        2. Sum of myeloblasts, promyelecytes, myelocytes and  later granulocytes is between 30 and 80 percent of  non-erythroid cells.

       3. > 20 percent of non-erythroid cells are monocyte lineage

       4. If monocytic cells exceed 80 percent, diagnosis is M5.

     

 

 

 Note :

        a) If marrow findings as above and peripheral blood monocytes  ( all types) are > 5.0 x 10 ⁹/L diagnosis  is  M4

        b) If monocyte count < 5.0 x 10 ⁹/L , M4 can be confirmed on basis of serum lysozyme , combined esterase .

        c) Diagnosis  of M4 confirmed if  >  20 percent of marrow  precursors are monocytes (confirmed by special stains )

M4 with eosinophilia :

        1-Eosinophils    >  5percent  of  non-erythroid    cells in marrow   

        2-  Eosinophils are abnormal

        3- Eosinophils are chloroacetate   and PAS  positive .

M5 

       1. 80 percont of marrow  non-erythroid cells  are monoblasts , promonocytes or monoecytes.

      2. M5a , 80 percent   of  monocytic cells are monoblasts .

      3. M5 b , < 80 percent of monocytic cells are  monoblasts, remaimder are predominantly promonocytes and monocytes.

 

M6

       1. the erythrid component of the marrow exceeds 50 percent of all nucleated cells.

     2. 30  percent of the remaining non-erythroid cells are agranular   or granular blasts ( types I and II)

 

M7

     1. 30 percent at least of nucleated cells   are blasts

     2. Blasts identified by platelet peroxidase on electron microscopy, on by monoclonal antibodies.

     3. Increased reticulin is common.

 

 

 

 

Cytochemical tests

        Cytochemistry is the application of chemical processes to microscopical preparations, and by its use an attempt is made to reveal the chemical composition of cells, usually by the development of a colour reaction.

These chemical elements may be enzymatic (e.g.  peroxidase) or non-enzymatic (e.g.  lipids and glycogen).

Since the early 20th century, cytochemical staining of cells has been found to be a useful tool in  differentiating  hematopoietic diseases.

Over the years, these stains have been , if not the , most important laboratory test for the differentiation of acute and chronic leukemias .

In 1905 and 1906 Opie drew attention to the existence of a proteolytic enzyme in neutrophils in inflammatory exudates.

This initial observation of leucocytic proteolytic enzyme was followed in 1907 by Winkler‘s description of an oxidase in the leucocytes of blood films, oxidation metabolism of leucocytes in leukemic blood was observed by Grafe (1911) and the intense glycolytic activity of normal leucocytes was described by Levene  &   Meyer in 1912 . This interest in the biochemistry of white cells was maintained, and  by  1923 , Fiessinger  was able to produce a monograph including discussions of leucocyte lipase, alkaline lecithinase , amylase  and  proteolytic  ferments (Rebuck, 1947). With the development of new technologies such as the evaluation of cell surface markers by flow cytometry and improved high-resolution techniques for cytogenetics, however, cytochemical studies are used mostly in conjunction with these new technologies and not as the sole diagnostic tool.

The cytochemical stains widely used in haematological laboratories are Periodic Acid Schiff  (PAS) , Sudan black  B.,( SBB), Peroxidase, Specific and Non-specific esterase and terminally  Acid Phosphatase         ( Catovsky   1979 and Snower ,     1991).  A few others not so commonly  used  are   B-glucuronidase  (BG),Nuramidase, 5-Nucleotidase ( 5-NT ) , diaminopeptidase  IV  (DAPIV), tetrahydrofolate dehydrogenase (H₄FDH), Oil  Red  O  (ORO), and toluidine blue stains (Nano , 1982) .

Most of these stains can be  applied to air-dried blood and bone marrow aspirate films,(Catovesky ,et al, 1979 and   Li  1984), and some (e.g. peroxidase and esterase ) can be applied to whole blood and  cells in suspension (D’Onofrio , et al, 1984).

None of these is in any way leukemia-specific. However , each may serve as an adjunct to morphologic investigation of individual cases.

 

 

(1) Sudan Black B

Principle:

          Sudan black B  stains intracellular lipids, such as sterols, neutral fats and phospholipids, because of the solubility of the dye in lipid particles     ( Hayhoe  et al, 1964). These lipid are found in the primary and secondary granules of neutrophils, in the lysosomal granules of monocytes and in the specific eosinophil granules. (Hayhoe 1953). The precise mechanism of the reaction is not known . One possible view is that Sudan black  B stains the lipid membrane of the granules which contain the enzyme myeloperoxidase . Another is that the dye stains through on enzymatic mechanism, perhaps linked to myeloperoxidase and not just by physical solution in the lipids . (Hayhoe , et al 1980)  It also stains some cellular components that are not lipid, as evidenced  by the failure to extract the dye completely with lipid solvents such as acetone (Lillie et al. , 1953). In granulcytic cells , sudanophilia parallels the peroxidase reaction and may be linked to it (Hayhoe , et al., 1988).

Interpretation     (Hayhoe et al., 1988).

Granulocytes are Sudan black B positive from the myeloblast through the maturation series. The staining is more intense the more mature cells become as a result of increase in the numbers of the primary and secondary granules , creating black colored cytoplasmic granules. Monocytes may be negative , may show diffuse activity or may contain a few discrete sudanophilic granules. Lymphocytes are not stained with Sudan black. Eosinophil granules are Sudanophilic, especially at the rim of the granules with a hollow center.

Basophil granules are negative or occasionally show positive or metachromatic staining. Megakaryocytes and platelets are negative. Erythoblasts are not stained with Sudan black.

       In ALL, less than   3 percent of the blast cells are positive for Sudan black B. (Bennett, et al. , 1985). Blast cells that are positive for Sudan black B and completely negative for peroxidase do occur , however. This has been found in  1.5 to 2 percent of cases and appears to be due to lipid material (rather than primary granules of the neutrophil series ) . (Staas   et al , 1984 and Ngan  et al , 1992 ). In AML, acute myeloblastic (M₁) and acute myeloblastic with maturation (M₂) as well as acute promyelocytic (M₃), more than 85 percent of blast cells are Sudan black B positive , In a cute myelo monocytic (M₄) and acute monocytic (M₅) , the monocytic precursor cells of in stain weakly positive . Acute erythroleukemia (M₆) , acute      megakaryocytic leukemia (M₇) and undifferentiated from of AML are negative for Sudan black B . Sudan black B staining greatly facilitated the recognition of Auer rods , which were found in 67% of specimens on the Sudan black B  stain as compared with 32 % on the  Romonowsky stain . Auer rods are virtually  pathognomonic of an acute myeloid leukemia with granulocytic component. (Comley & Hayhoe , 1973). In general the Sudan black B stain is similar to the peroxidase reaction in distinguishing acute lymphocytic leukemia from acute myelogenous  leukemia.

 

 

(2) Peroxidase

         Myeloperoxidase  (MPO)  is a  lysosomal enzyme localized in the primary azurophil granules of neutrophils and monocytic (Baintan et al. , 1971 and Nichols , 1971). Peroxidase can also be demonstrated in the specific granules of eosinophils and basophils.

In eosinophils the specific granules are not newly formed but derive from primary granules which are also myeloperoxidase positive. The eosinophil peroxidase has been shown by chemical, cytochemical and immunological methods to be different from that of neutrophils and probably to be under separate  genetic control (Archer, et al. , 1965).

 

Principle :

          Peroxidase oxidizes dye substrates, when hydrogen peroxide is present causing its disposition, creating black to red brown, depending on the substrate, at the site of the activity. Benzidine used to be the substrate most often used, but because of its carcinogenic properties , other substrates such as3, 3 diaminobenzidine (DAB) (Hanker  et al, 1979;      de Salvo Cardullol , et al , 1981 ; Hayhoe  et al, 1988 ) or 2,7 fluorenediamine (FDA) are currently used (Inagaril , et al, 1976 ; Benavides, et al, 1978 ).  Sodium nitroprusside serves as a catalyst for this reaction.

 

Interpetation : 

Douglas  , Nelson Flederick  and   Davey  (1995).

       Myeloperoxidase enzymes are present in all stages of granulocytic maturation and are located in primary azurophilic granules . Monocytes do not stain as intensely as granulocytes and show either tiny colored granules or faint diffuse cytoplasmic staining. Lymphocytes and erythrocytes do not show peroxidase activity at any stage of maturation     ( Hayhoe et al. 1972 ; Zucker-Franklin , et al 1988). Eosinophil granules show an intense peroxidase reaction.

The enzyme in eosinophil is different from that of other cells in  that it remains active in the presence of cyanide,   whereas the peroxidase     of   neutrophils  is inhibited by    cyanide (Archer , et al, 1963 ; Yam et al, 1971). Peroxidase is not demonstrable in mature basophils from normal individuals but may be found in basophils in patients with myelogenous leukemias (Zucker, et al.  1988). Megakaryocytes and platelets do not stain for peroxidase when visualized with light microscopy ; however, a platelet peroxidase can be demonstrated by electron microscopy. Functional MPO is totally absent in cells of the lymphocyte lineage , be they normal or leukemic. Thus , a positive MPO reaction excludes lymphoid disease         ( Jean  et al , 1995).

 In acute  myelogenous  leukemias  (AML;M₁ through M₃) , three  percent or more of the blast cells are positive , and in many cases the great majority of blasts are positive.

 Auer  rods found in leukemic blasts and promyelocytes are strongly peroxidase positive. Because of their strong peroxidase positivity, many Auer   rods that could not be seen with just a Wright-Giemsa stain can be seen with  peroxidase  stain .

         In minimally differentiated acute myelogenous leukemia  (AML;Mo), some  monocytic (M₄ , M₅ ) , also in erythroid (M₆ ) and megakaryocytic (M₇) , the blast cells are negative for myeloperoxidase .

Peroxidase activity may be decreased or even absent in mature neutrophils in acute myelogenous leukemia, myelodysplastic syndromes and infections (Hayhoe et al, 1988 ) .  So the main value of  peroxidase  reaction is in the distinction between acute myeloid and acute lymphoblastic  leukemias.

 

 

 

(3) Esterases

         Esterases are a group of hydrolases with a wide range of pH activity. Li et al (1973) described   nine esterase  iso-enzymes in  leucocytes.

 

Specific esterase : represents  1, 2 , 7 , 8 and 9  present in mature and immature granulocytes , which can be demonstrated by means of naphthol  ASD-chloracetate.

Non-specific esterase : represents 3 , 4 , 5 and  6  which   are sensitive to sodium fluoride ( Na F ) and are found in monocytes, lymphocytes, megakaryocytes  and platelets and are demonstrated by     µ-naphthyl esters (acetate and butyrate) as substrate. (Yam et al, 1971).

Principle :           (Hayhoe , et al 1994).

    Esterases are enzymes that hydrolyze  esters of carboxylic  acids.

Ester + water ® acid + a1coho1.

 

1- Non specifie esterase (µ -naphlhyl acetate ) :

        Esterase splits µ-naphthyl acetate with the release of               µ-naphthyl which combined with the hexazonium salt, pararosaniline forming insoluble orange, brown amorphous deposit in the cell at the sites of esterase activity.

2- Specific  esterase (Naphthyl ASD chloroacetate):-

Naphthyl ASD chloroacetate is hydrolyzed by   esterases  with the liberation of   naphthol ASD   which combines with the diazonium salt, Fast Blue BB to produce azo dye (Li et al, 1973)

 

3-Double esterase :

The chloroacetate  esterase reaction and the µ-naphthyl acetate,or butyrate,  esterase reaction can be used in combination. The advantage of a double esterase technique is that individually visualized leukemic cells can be evaluated for commitment to granulocytic or monocytic lineages , or both.

Interpretation :   Douglas  ,  Nelson , et al, 1995

Non specific esterase :

The reaction for nonspecific esterases using the substrates              µ-naphthyl acetate  or  µ-naphthyl  butyrate  is  diffusely positive in monocytes and negative or focally positive in the neutrophil series.         µ-Naphthyl acetate esterase is strongest in monocytes, macrophages, megakaryocytes, and platelets. It is also positive in basophils and plasma cells, focally positive in the cytoplasm of resting   T-lymphocytes, and weakly positive in the erythroblasts  of  some normal individuals                ( Rosenszajn , et al., 1962  and  Davey  et al, 1980). The activity in monocytes , megakaryocytes , platelets and plasma cells  is  inhibited by sodium fluoride but that in lymphocytes and granulocytes is not (Li et al, 1973 ; de - Olive et al , 1987). However, Fluoride inhibition studies usually are not necessary to differentiate granulocytes from  monocytes because granulocytes very rarely  give  a  diffusely positive reaction with µ-naphthyl acetate . µ-Naphthyl butyrate esterase is also strongly reaction in monocytes and macrophages; it is somewhat less sensitive than            µ-naphthyl acetate esterase but has the advantage of being more specific for these cells. Megakaryocytes and precursors are distinctly positive for   µ-naphthyl acetate esterase but only weakly positive or negative for       µ-naphthyl butyrate esterase. Nonspecific esterase  are often applied to study normal and leukemic samples. In normal samples it help to distinguish between T-lymphocytes and monocytes.

In acute myeloblastic leukemia  (AML; M₁ to M₃), nonspecific esterase are negative. In approximately one third of cases of acute promyelocytic leukemia, however, leukemic cells may be strongly positive for µ-naphthyl acetate esterase      ( Davey  et al, 1989 ).  Although such diffuse positively is inhibited by sodium flouride it is not associated with a monocytic immunophenotype        (Das Gupta et al , 1989).

 In acute myelomonocytic leukemia (AML; M₄) , more than 20 percent of non-erythroid cells are positive  . In acute monocytic leukemia  (AML; M₅), typically 80 percent or more of the non-erythroid cells are positive.

        In erythroleukemia (AML; M₆), the cells may be positive and in acute megakaryocytic leukemia (AML;MM₇) , they  are  weakly  positive with    µ-naphthyl butyrate esterase, and positive with     µ-naphthyl acetate esterase ( Hayhoe , 1984 ) . The µ-naphthyl acetate esterase reaction is focally positive in the cytoplasm of blasts in a small percentage of cases of acute lymphocytic leukemias or leukemic lymphomas.

 

 

The specific esterase

       It is present in the  non specific  granules of   granulocytes.The positive reaction is found in mature and immature granulocytes and is insensitive to fluoride inhibition  ( Li  , et al,1973).

           Chloroacetate esterase has an optimum pH between 7.0   and 7.6 . Eosinophils, lymphocytes, plasma cells, erythroblasts, megakaryocytes, and mature monocytes are negative , but tissue mast cells are positive . The reactions of chloroacetate esterase, are parallel to those of peroxidase and Sudan black B, both in normal granulcytes and in the acute leukemias. Peroxidase and Sudan black B positivity appears earlier than chloroacetate esterase positivity in the maturing cells   of the granalocytic series . Normal myeloblasts are negative. but myeloblasts in some cases of acute myelogenous leukemia are positive. Auer rods are usually positive (Rosenszajn    et al,1962 ) .

 

          In acute myeloblastic leukemia (AML; M₁),  the  chloroacetate esterase may be positive in a  proportion of the blast cells . In the acute myeloblastic leukemia with maturation (AML; M₂),it usually is positive in at least 20 percent of the nonerythroid , non-lymphoid cells.

 In acute promyelocytic leukemia (AML; M₃) the leukemic cells are strongly positive for chloroacetate esterase. In acute myelomonocytic leukemia (AML; M₄ )  more than 20 percent of the non-erythroid cells are positive,whereas eosinophils typically are negative  for  chloroacetate esterase.

There exists a variant of AML;M₄ , acute  myelomonocytic leukemia  with  eosinophilia  (AML ; M₄E)  in which abnormal eosinophils are positive  .

In acute monocytic leukemia (AML; M₅), erythroleukemia (AML; M₆) and acute megakaryocytic leukemia,(AML;M₇ ) the blastic cells are negative.

                       

 

 

 (4) Periodic acid Schiff (PAS)

PAS is a specific stain for all cells containing glycogen

Principle :

Many different cell types contain glycogen. Periodic acid oxidizes glycogen, mucoproteins, and other high-molecular weight carbohydrates to aldehydes. These aldehydes react with the colorless Schiff reagent (Leuko-fuchsin), staining it a bright red-pink. The intensity of the staining depends on the number of aldehyde groups liberated by the periodic acid (Hayhoe & Quaglino  et al,1988). This can be confirmed by demonstrating that the positive reaction disappears when the film is treated with saliva or diastase before it is stained (Yam, et al 1971).

Interpretation :

Freberick et al , 1995.

Granulocytes at all stages of maturation are PAS positive, the intensity of staining increases as the cell matures, the cytoplasm of polymorphonuclear leukocytes stains intensely pink or red, with a granular appearance in some cells. Monocyte cytoplasm stains faintly pink and may contain fine or coarse granules. Easinophils cytoplasm is PAS-positive but the large specific granules are PAS-negative. Basophil granules are heavily positive against negative or weakly stained background. Erythrocyte and erythrocyte precursors in the normal bone marrow do not stain, whereas platelets stain intensely.

Megakaryocytes stain diffusely and intensely pink or red. Lymphocytes contain much less staining material than granulocytes but a few fine or even coarse granules may often be demonstrated. Digestion with diastase removes the staining material from the cytoplasm of all types of blood cells (Hayhoe et al , 1988).

Lymphocytes in the B-lymphoproliferative disorders often contain an increased number of positively-staining granules. Lymphoblasts of all may also be PAS-negative, especially for the subtype L3. In acute myeloid leukemia the degree of PAS reactivity in the blasts is variable. The blasts are generally negative, although a diffuse  tinge with or without fine granules may be present, coarse or black reactivity may seen in some type of  AMonL ( acute monocytic leukemia ) .

In erythroleukemia, erythroid precursors are strongly PAS positive. Erythroid precursors may be PAS positive in thalassemia and, occasionally, in iron deficiency anemia, so PAS may be helpful in diagnosis of FAB  M6  type of  AML and in diagnosis of some cases of acute lymphoblastic leukemia.

 

(5) Neutrophil Alkaline  Phosphatase  (NAP)

 

NAP is an enzyme found in secondary granules of neutrophils , mature cells and bands only(Bainton et al, 1971). The enzyme is not demonstrable in other blood leucocytes, but fibroblast-like reticulum cells, part of the bone marrow stroma, react strongly (Bennett  et al 1985). 

Recently , small amount of NAP activity have been demonstrated in the membrane of some lymphoid cells ( Catovsky  et al,1976 ) , and shown  histochemically in a type of B-cell lymphoma  (Moloney et al,1960).Recent studies by  Rustin et al (1979)  have shown that NAP is associated with a membranous component of the cytoplasm identified as an irregularly shaped tubular structure distinct from primary or secondary granules or other cytoplasmic organelles.

Principle :      Erenest Beutler ,1995.

        The substrate  naphthol AS-Bi-phosphate is hydrolyzed by the enzyme at an alkaline phosphatase , hydrolyzed to phosphate and an aryl naphtholamide. The aryl naphtholamide is coupled to diazoniam salt , such as fast red-violet  LB or fast blue BB produces a colored insoluble dye, precipitate at the site of enzyme activity .

 Scoring:  ( sir John et al, 1991)

          NAP activity is scored in mature polymorphonuclear cells and bands only. The activity is indicated by a precipitate of bright blue granules , the cell  nuclei are stained  red . The activity scores range      from 0 to 40:

0 : negative , no granules .                                

1: positive but very few blue granules .

2: positive with few to moderate number of granules .

3: strong positive with numerous granules .

4: very strong positive with cytoplasm crowded with granules .

 

The scores are  performed of 100 cells . Because scoring is  so subjective , it is recommended that two slides from an individual patient be scored by two people. The scores should agree within 10% of each other and if  the scores do not agree, third slide from the patient must be scored . for example:

score     number of cells     score  x  number of cells

0                     20                          0

1                     45                          45

2                     25                         50

3                     5                           15

4                     5                           20

 

total               100                       130= NAP score   

 

 

 

Interpretation   ( Rosner  et al, 1972 ):

The  normal NAP score should be between 20 and 100 , but because of the scoring subjectivity , it is very important that every laboratory establish its own normal values . The score is higher in women and children than in men and in new born infants  the range  is  150-300 .

High scores ( which  lie  in  the  upper  portion  of the  normal  range  or elevated )   are found in : 

1- neutrophilia of infection

2- leukemoid reactions

3- liver cirrhosis

4-Down’s syndrom

         5 - polycythaemia vera

6- pregnancy as the enzyme influenced by estrogens and                          

corticosteroids.

7- active Hodgkin’s  disease

8- aplastic  anemia   (Okun  et ql , 1978 ) and

9- idiopathic  myelofibrosis .

 

Low scores are found in:

1 -chronic  granulocytic leukemia which  usually 

         has  score between  0  and  20 .

2 -paroxysmal nocturnal hemoglobinuria  (lewis  et al 1965).

3 - development of   P N H  with  aplastic  anemia  .

      So estimation of leukocyte alkaline phosphatase activity is useful in the differentiation of chronic myelogenous leukemia from neutrophilic  reaction as seen in severe  infections , polycythaemia  vera , and idiopathic myelofibrosis .

 

 

(6)  acid phosphatase

Acid phosphatase isoenzymes are present in the lysosomes of many cells. All cells contain seven nonerythroid isoenzymes of acid   phosphatase   ; 0 , 1 , 2 , 3, 3b , 4  , and 5 . ( Li     et at 1973 ) .

 

Princple;  ( Li   et al  ,1970 )

Acid phosphatase hydrolyzes the substrate napthol AS biphosphoric  acid . Once  hydrolyzed , this substrate couples with a dye such as fast garnet-GBC and produces  a  red precipitate at the site of the enzyme activity .

     When L(+) tartaric acid is added , all isoenzymes except isoenzyme5 , are inhibited . Isoenzyme5  is  tartrate resistant .

 

Interpretation (Frederick , et al ., 1995 )

   Acid phosphatase is demonstrable in most cells of the hemopoietic system (Janckila et al 1978 ) .

Intense activity is present in osteoclasts, and some macrophages. Moderate staining is seen in plasma cells, megakaryocytes, and monocytes.

Weak reactions are noted in neutrophils, band, metamyelocytes, myelocytes and promyelocytes.

Very little acid phosphatase activity is present in normal lymphocytes and erythroblasts . Increased acid phosphatase activity is observed   in  lymphoblasts  from patients with T-cell leukemias, abnormal mononuclear cells of patients with hairy-cell leukemia, lymphocytes from patients with macroglobulinemia, atypical lymphocytes from infectious mononucleosis and some tissue sections infiltrated with Hodgkin disease. In leukemic  cells from patients with T-acute leukemia, T-prolymphocytic leukemia, and T-chronic  lymphocytic leukemia, acid phosphatase is characteristically strong and localized to the Golgi zone      ( Sun et al,1985 ). Since a  similar focal globular positive reaction may be observed in non-T-cell malignancies, other T-cell marker studies should be employed to confirm the lineage of the neoplastic cells  (Sun  et al,1985 ) .

The reaction in T-ALL is inhibited by tartaric acid.The activity is inhibited because  isoenzyme5 is lacking .

 Hairy cell leukemia produce isoenzyme5 in abundance so it remain positive with the addition of L(+) tartaric acid , so tartrate-resistant acid phosphatase reaction diffusely prominent in the cytoplasm of neoplastic cells is highly characteristic of hairy-cell leukemia (Chang , et al , 1992 ) .

 

 

Cytochemistry of Acute Leukemias

Cytochemistry of Acute Lymphoblastic Leukemia (ALL)

 

According to the defining criteria of Loffler , et al (1994) peroxidase activity and sudan black B staining of leukemic cells are indicators for myeloid nature of blast cells and negativity of these tests is a prerequisite for the diagnosis of acute lymphoblastic leukemia. However, Sudan black B staining of blasts has been demonstrated in rare cases of ALL , and less than 3 percent of the blast cells . Sudan black B positivity was found in six out of 350 patients with immunologically  proven ALL ( Tricot, et al , 1982 ; Stass, et al , 1983) .

 Consequently, they prefer the peroxidase  reaction and use Sudan black B just in case of peroxidase deficiency , but others prefer  Sudan     black B stain as sudanophilia is slightly more sensitive than is peroxidase activity , even the DAB-peroxidase method. An advantage of the Sudan black B method is that the air dried films retain their reactivity for several weeks to months, where as their reactivity for peroxidase often deteriorates within two weeks  (Douglas  et al, 1995).

The non-specific esterase with  µ-naphthyl acetate as substrate  shows dot-like positivity in some cases of T-cell ALL . The main intention of using this cytochemical test is to exclude poorly differentiated monoblastic leukemia which shows diffuse esterase activity . Peroidic acid Schiff positivity of acute lymphobastic leukemia is seen as coarse granules or blocks (dots) against a negative cytoplasmic background (Snower , et al, 1991) . Among ninety  percent of ALL have been said to demonstrate some degree of  positivity ,  only 10 to 15 percent will have strong block-like reaction.

 In ALL the PAS reaction never shows diffuse pattern or fine granules, On the contrary , diffuse PAS positivity is an indicator of the myeloid nature of the leukemia . The positive reaction is found in cases from the L1 & L2 (FAB classification ) subtypes of acute lymphoblastic leukemia . The L3 subtype, however , is generally negative (Humphrey , et al,1974 ; Schmalzl  et al , 1974). It should be noted that PAS reactivity is an indicator of low priority which is overruled by positive results of any other of cytochemical tests mentioned . Blasts being positive for peroxidase as well as for PAS , belong to the myeloid lineage independent of the PAS-reaction product . 

The diagnosis of ALL is strongly suggested when , blast cells  are negative for myeloperoxidase , Sudan black B ,  and fail to react in an intense diffuse pattern with µ-naphthyl butyrate esterase, yet exhibit a positive PAS stain (Snower , et al , 1991).

Focal acid phosphatase positivity is highly correlated with ALL  of T-phenotype and to a  lesser  degree  with ALL of pre-T-phenotype ; 70 versus 30 percent of the respective cases are focally positive . However  , this type of acid phosphatase positivity can also be found in 15 percent  of B-lineage ALL (Loffler, et al , 1987)  , and  6  percent of AML cases, so that the finding is not so specific as to be diagnostic , but is never the less strongly suggestive of T-cell ALL and is of particular value as a first  line test , when immunophenotypic studies are not readily available .           Yet another study ( Inevernizzi , et al , 1992) on 531 cases of childhood ALL revealed sixteen cases with more than one percent and seven cases with more than ten percent blasts containing cytoplasmic azurophilic granules , positive for µ-naphthyl acetat esterase and acid phosphatase , but negative to  Sudan black B  and  myeloperoxidase.

 

 

Cytochemistry of Acute Myeloid Leukemia (AML)

Many cells of acute myeloid leukemias are peroxidas and Sudan black B  positive , at least 3 percent of the blast cells (Bennett   et al, 1985). In some cases of acute myeloblastic leukemia (AML. ; M₁ ) and many cases of acute myeloblastic leukemia  with   maturation (AML. M₂)   as  well as  more than 85 percent of blast cells of acute promyelocytic leukemia (AML;M₃) are myeloperoxidase (MPO) and Sudan black B positive. Undifferentiated forms  (AML; M₀) are MPO and Sudan black B  negative .

 The positivity  to peroxidase and Sudan black B stains is generally very strong in APL as might be expected from the heavy azurophilic granulation of the predominant cells  (Hayhoe , et al , 1964) . The blast cells may fail to show these reactions if they have failed to show any development of azurophilic granules , on the other hand , in many AML cases with visible azurophilic granules with Wrights stain, MPO activity and Sudan black  can be demonstrated. Auer rods are positive with these staining reaction  (Cowley & Hayhoe , 1973) .

The periodic acid Schiff reaction in AML shows a range from negativity to diffuse cytoplasmic staining with or without fine superimposed granules .(Hayhoe , et al , 1964 ; Hayhoe  , 1984  &  Snower , et al , 1991). The reaction being generally stronger in cases of  APL . Coarse granules or block positivity are rare , but may occasionally be seen in few blast cells .Where the usual pattern is diffuse cytoplasmic    reaction with some localized clumping  of moderate positivity  and some scattered granules , the specific esterase activities are  usually  positive , with   increasing positivity   with increasing maturity ,  but  non- specific esterase  activities  are usually negative .

 

The leucocyte alkaline phosphatase  (LAP ) score is usually low  (Hayhoe , et al ,1964 ) , perhaps  representing a deficiency of the tertiary specific granules in which  alkaline phosphatase is probably located            ( Cawlley  & Hayhoe  , 1973 ) ,  or possibly  an  isolated  defect  in the production  of the enzyme   .

Acid phosphatase   shows only weak scattered  positivity .

 

 

Cytochemistry  in Acute Myelomonocytic  (AMMoL )

and Monocytic Leukemia  ( AMoL  )

 

          In acute myelomonocytic  (AML ;M4 ) and  acute monocytic leukemia  ( AML ;  M5   ) , Sudan black  B and peroxidase positivity are conspicuous in the granulocyte precursors  whereas   most  leukemic  monocytes  show negative reaction to peroxidase and occasionally weak discrete scattered granule type positivtiy  to Sudan  black B .  Acid phosphatase and lysozyme are strongly positive   especially in  monocyte  precursors ( Drexler , et al ,1984 )  .

  Concerning  PAS in acute myelomonocytic leukemia, some blast cells are entirely   negative , others show a reaction like that found in myeloblastic  leukemia , with weak tingeing  of cytoplasm and perhaps fine granulation , while others give fine to moderately coarse granulation  against  a background of diffuse cytoplasmic staining . Rarely much stronger positivity may be encountered  in leukemic  monocytes precursors , with coarse granules and blocks like reaction pattern with background of diffuse positivity  which distinguishes  this type of positivity from ALL. So PAS staining is a poor  discriminator of various cell types but may be useful as part of battery of stains , particularly when many reaction are negative or non specific . specific  esterase is positive in granulocytic component but is negative in monocytic cells , non specific esterase is  positive in monocytic cells and is inhibited by sodium fluoride , mixed reactions may occur (Li  et al , 1986 ).  Even when only  acetate esterase reactions are studied , the positivity may vary in depth and pattern very considerably. Wrotnowski et al, (1987)  found, for example, that six of fourteen cases of acute monocytic leukemia classified as M₅ had finely granular diffuse cytoplasmic positivity for acetate esterase, whereas eight had dense focal staining against a weak cytoplasmic background reaction, although no differences were found among the cases in clinical features or in the coarse of the disease.

 

 

Cytochemistry of Erythroleukemia

 

Erythroleukemia as defined in the FAB classification (M₆) with erythroblasts making up 50% or more of the nucleated cells in the bone marrow, is an uncommon form of AML  (Swirsky et al, 1986).  In a study of 619 AML cases, only 15cases met this criterion. There is virtually always evidence that neoplastic proliferation of erythroblasts, are accompanied by parallel involvement of other myeloid precursors (Scott  et al, 1964; Hayhoe et al, 1964) when granulocyte precursors are involved cytochemical staining with Sudan black B, peroxidase, PAS and chloroacetate esterase show the granulocyte type of reaction.

When monocyte precursors or mixture of monocyte and granulocyte precursors are present, the cytochemistry shows the mixed reactions to be expected in these all lines. The neoplastic erythroblasts themselves show commonly but not always, strong PAS positivity  (Baldini, et al, 1959; Quaglino & Hayhoe , 1960; Elghetany, et al, 1990).

 The intensity of the reaction and the percentage of erythroblasts showing positivity vary considerably in different patients and also in the same patient during the course of the illness.

 PAS positivity, granular or diffuse is present in red cell series at all stages of maturity. In a study of 20 patients with erythroleukemia, Cuneo et al, (1990) reported that 15 of them are PAS positive and 5 are PAS negative.

Sudan blacks B and MPO reactions are negative in erythroblasts.    µ-Naphthyl butyrate and acetate are strongly positive whereas specific esterases are negative.

They also show strong paranuclear unipolar acid phosphatase activity (Leder, 1967).

 

 

Cytochemistry of Acute Megakaryocytic Leukemia

 

The frequency of promegakaryoblastic leukemias is much  higher than has been thought, especially in transforming CML; M1, and in  patients with secondary leukemias. Breton-Gorius et al, (1985) collected 27 cases; three appeared to be primary leukemias; two showed marked myelofibrosis; eight represented the megakaryoblastic crisis of CML and six cases were in patients previously treated with alkylating agents and/or radiotherapy. Other cases have occurred with widely variable background: in Down’s syndrome, following preleukemic states and in other myeloproliferative disorders. AML cases with a megakaryoblastic, megakaryocytic component, or even predominance have come increasingly to be recognized with the use of ultra-structural platelet peroxidase and immunological markers. The main cytochemical diagnostic features can be summarizod in the following paragraph.

          The frequently mixed myeloid character of cases with predominating megakaryoblasts gives a  mixed cytochemistry , but the salient features of the megakaryoblast are Sudan black B  and peroxidase negativity at the light microscope level . Acid phosphatase is usually strongly positive and predominantly localized in the Golgi zone (Oliveira et al, 1987). PAS reaction shows diffuse and granular positivity in many blasts, with coarse peripheral granules and sometimes block posivity is conspicuous (Hayhoe   1984; Davey, et al, 1989). Chloroacetate esterase activity is negative,     µ-naphthyl butyrate esterase is usually weak or negative, but µ-naphthyl acetate esterase is positive. (Li, et al., 1973; Oliveira, et al., 1987).

 

Diagnostic value of cytochemistry in other

Malignant diseases

 Cytochemistry of chronic myeloid leukaemia CML :

The most striking established abnormality of cytochemistry in CML concerns the low alkaline phosphatase activity of mature neutrophils, which helps in the differential diagnosis of CML from non-leukemic myeloproliferative states where alkaline phosphatase scores are generally high. The lowest scores in CML are found in untreated cases and those with high peripheral leukocyte counts. The low levels in full relapse tend to return towards normal as remission is established although scores seldom rise above the lower ranges of normal even in full remission (Hayhoe et al, 1994).

 Other cytochemical studies in CML have revealed less consistent changes.  PAS gives strong diffuse staining in later granulocytes, which makes it difficult to assess changes in intensity unless they are gross.         ( Gahrton 1966 ; Gahrton et al., 1969 ) .  Peroxidase usually gives strong positivity especially in cases in relapse and with high peripheral leukocyte counts. Indeed, an inverse relationship exists between peroxidase and alkaline phosphatase levels in neutrophils (Quaglino, 1961).

 On the other hand, there may very occasionally be neutrophils in CML negative to Sudan black B and peroxidase as well as to chloroacetate esterase and alkaline phosphatase. Basophils may sometimes be numerous in CML, and their characteristic cytochemical reactions need to be recognized and distinguished from those of the neutrophil series. PAS reaction in basophils at all stages of maturity may be much increased in CML and other myeloproliferative states, with coarse granularity or block positivity, the reaction is due to glycogen present between the basophil granules rather than in them (Kaung, 1969).

  PAS appearance is very characteristic especially in earlier basophils where positivity forms irregular pools or lakes of reaction product, clearly distinguishable from the more regular, round, oval or paracrystalline appearance of blocks in ALL. Sudan black B gives reddish metachromatic hue, with only occasional black granules of normally reacting sudanophilic material. The stain is often stronger in basophil precursors then in mature cells and in the basophils of CML and other myeloproliferative states than in those of normal blood. (Parwaresch, 1976). In blastic transformation, cytochemical reactions unlike those usually found in AML, have long been noted to occur in some cases (Hayhoe  et al, 1964).

Apart from the common elevation of LAP scores to normal or higher than normal levels, peroxides and Sudan black B give weak or negative reactions and PAS gives coarse granular positivity in blast cells occur in more than 50% of cases.

Cytochemically these cases of blastic crisis may resemble acute lymphoblastic leukemia.

 

Cytochemistry of Chronic Lymphatic Leukaemia (CLL)

          In T-cell CLL which   accounts  for no more than 5 % of all CLL cases , there is generally a strong localized positivity for one or more of the hydrolases , acid phosphatase and butyrate or acetate esterase , while in B-cell  diseases  including the great majority of CLL cases , these hydrolases are negative or show only weak scattered reactivity  ( Cawley & Burns , 1980 ; Feller et al ,1982; Crockard et al,1982 ; Boesen et al  , 1984 ) . These reactivity may show different patterns , some the crescentic positivity of hairy cells and others the null cell pattern of scattered granules ( Crockard et al, 1985 ) , noting that the expression of acid hydrolases may be a marker of cell maturity especially in T-cells .

Basso et al, (1980) examined hydrolase cytochemistry in 30 patients with B-CLL, counting cells showing any clear  reaction  , however  weak, as positive,  they found five cases with less than 50 % cells positive for both acid phosphatase (ACP) and acetate esterase (AE) , eleven cases with more than 50 % cells ACP positive but less than 50% AE positive , seven with the reverse of that finding and seven with more than 50% positive for both enzymes . Mature neutrophils in CLL show normal or increased amounts of alkaline phosphatase  scores usually being higher in untreated cases or those in relapse and lower in well controlled cases  ( Hayhoe & Quaglino 1958).  The lymphocytes in CLL show a much richer content of glycogen than do normal lymphocytes but this pattern is not peculiar to CLL but occurs in Hodgkin disease, non-Hodgkin lymphoma and in other non-leukaemic lymphoproliferative diseases (Quaglino & Hayhoe, 1959).

In CLL there is a relationship between the level of PAS scores and the effect of treatment. When suitabletherapy induces a progressive fall in the leukocytic count, decreasing levels of glycogen are found in the lymphocytes, while leukemic relapse is associated with rise to high levels of glycogen once more. However , the occasional transformation to an immunoblastic proliferation  associated with weak PAS positivity                 ( Hayhoe  et al , 1994 ).

 

Cytochemistry in Prolymphocytic leukaemia PLL

          This variant of CLL is characterized clinically by substantial splenomegaly and high lymphocytic count ( usually over 100 x 109  /L ) and cytologically by lymphocytes  with a conspicuous central nucleolus and a larger size and greater amount of cytoplasm than is usual in CLL . The cytochemistry of PLL  in both  the common       B-cell variant and the rarer  T-cell variant has been described by  Costello et al (1980), Crockard et al ( 1982 )   and Matutes et al          ( 1983 ) .  In general, the reactions for acid  hydrolases  are negative or show no more than weak scattered positivity in B-PLL , as in      B-CLL , while more variable positivity is found in T-PLL . Strong positivity for µ-naphthyl acetate esterase occurs in T-PLL contrasting with weak or negative reactions in T-CLL.

Acid phosphatase was also more variable in T-PLL than in T-CLL, although Tsai et al (1984) reported strong paranuclear ACP activity in all of seven T-PLL cases tested for this enzyme among 19 cases collected from the literature. These findings were initially thought to reflect a predominance of helper-cells-among-T-PLLs, as against a predominance of suppressor cells among T-CLLs,  but  the cytochemical reactions on further study appeared to be  unrelated to membrane phenotype (Crockard  et al , 1982 ) .

Cytochemistry of hairy cell leukemia (HCL)                 

       Hairy cell leukemia (HCL) has a number of distinctive cytological and cytochemical  features  which may be used as diagnostic aids in separating the condition from CLL  or chronic myelofibrosis or even  ALL  or malignant lymphoma, since confusion may sometimes arise with any of these disorders . These features of   HCL include the presence of tartrate-resistant acid phosphatase (TRAP) isoenzyme 5 (Yam et al, 1971)  in the  cells. But this activity may by weaker or   absent in a small  proportion of cases ( Higgy et al, 1978;  Catovsky,1977). TRAP staining concluded that  when  fast garnet was used as capture agent, and intense TRAP positivity was taken as the criterion, the test was effectively diagnostic of HCL (Yam et al., 1987 ).  Negative TRAP reactions occurred in only 2 of 200 HCL cases included in the review, while positive TRAP reactions were seen in only 3 of 800 non-HCL cases.     The raised leukocyte alkaline phosphatase (LAP) score in H‎CL, which was first noted by  Hayhoe  et al , (1977 ) has generally been confirmed, although a minority  of  cases with  low  scores  have  since  been  reported .

Aiba  et al, (1980 ) found high LAP (leucocytic alkaline phosphatase ) scores in  17  of  23  patients , but normal scores in the remaining six, the scores appearing to show as inverse correlation with the peripheral blood neutrophil count , but no relation to age , splenectomy or other clinical features . These authors thought the high LAP score to be secondary to decreased marrow granulocyte  reserves . Hairy cells are Sudan black negative.     A peroxidase reaction can  be demonstrated at EM level . PAS staining usually shows  quite strong positivity , with both diffuse and granular reactions.   Non specific esterase with  µ-naphthyl butyrate is positive and shows a  crescentic disposition at one or more peripheral sites (Higgy et al ., 1978) . These characteristic pattern of positivity was  , however, invariably found in a high proportion of the cells of HCL and appears to offer not only a simple and reliable diagnostic aid but also a means of estimating the numbers of circulating  leukemic hairy cells . The particular hairy cell pattern of crescentic positivity is only very rarely encountered in normal lymphocytes, and among other lymphoproliferative states   and  found  only in a few examples of CLL.

 

Cytochemistry of Multiple Myeloma

          Cytochemically, myeloma cells do not differ sharply in their reactions from normal plasma cells. Varying patterns of PAS positivity may be seen, but reactions are usually negative or weak and diffuse, with sometimes  fine granularity. Russell bodies are commonly weakly  PAS  positive , probably because of mucoprotein content rather than glycogen , since the material is not digested by diastase ( Pearse, 1949) . Intense enzymatic activity of acid phosphatase, non-specific esterase is generally found in myeloma cells ( Quaglino et al , 1967; Suzuki et al , 1969) .

 Hayhoe   and  Neuman  (1976) from a study of 195 cases of multiple myeloma divided according to immunoglobulin patterns, found no significant differences between the uniformly high acid phosphatase scores, the generally weak PAS reactions, and the normal to high LAP scores in the separated  IgG ,  IgA  or  Bence jones variations of the disease. Nor were differences to be found in relation to the nature of the light chains. Confirmation of these findings and detection of a prognostic correlation between acid hydrolase levels and survival in myeloma were reported by (Saeed et al , 1991) ; the higher scores the longer the survival.

Cassuto et al, (1977) compared the acid phosphatase scores in myeloma cells with those in normal plasma cells and in polyclonal plasmacytosis and found the myeloma scores to be markedly higher.       In non-myelomatous monoclonal dysglobulinaemia, lower and normal scores were found in the 10 cases studied. These findings were partially confirmed by Tortatolo et al, (1981) who recorded a mean score for acid phosphatase of  343 (range   0  -  400)   in 30 cases of myeloma, as compared with  204  in  21  non-myelomatous monoclonal paraproteinemias, however he  found a considerable  overlap between the patient groups, as did Johansen & Krogh-Jensen (1980) and                      Miliani et al, (1982). Alkaline phosphatase scoring in neutrophils may be more helpful in differentiating myeloma and benign monoclomal gammopathy than is the acid phosphatase score in the plasma cells.

Johansen & Krogh.-Jensen (1980) found the LAP score higher in the malignant group, with a positive correlation between the   M-band level and the LAP score in myeloma cases. (Majumdar et al ., 1991) measured LAP scores in  20  patients with myeloma and in 18 with MGUS(monoclonal gammopathy of unspecified significance) ; the mean score in the first group was 180 (range 169 - 218)  and in the second 92 (range 64 – 120 ) . In this study no correlation was found with the plasma cell percentage in the bone marrow, with paraprotein level, or with peripheral blood count. The finding of a raised LAP score is strongly suggestive rather than pathognomonic of the more malignant form of paraproteinemia.

 

Cytochemistry of Polycythaemia and Myelofibrosis

          They are both myeloproliferative states. Cytochemically they show similarities, probably the most important, and certainly the most useful in differentiating forms of the diseases with high leucocyte counts from CML, is the level of leucocyte alkaline phosphatase, which is usually considerably above normal (Wachstein, 1946; Wiltshaw & Moloney, 1955; Hayhoe & Quaglino, 1958). When treatment is successful in inducing some degree of remission there is a tendency for high scores to diminish, but they usually remain above the normal range. Scores within the normal range have sometimes been reported in polycythaemia vera       (Meislin et al, 1959; Anstey et al, 1963), but such cases form a small minority .

           Similarly in myelofibrosis, while most patients have raised LAP scores, normal or even low scores may sometimes be found especially when there is marked cellular immaturity in the peripheral blood (Valentine et al, 1952; Mitus et al, 1958; Koler et al, 1958; Mitus and Kiossoglou 1968). The high levels of LAP found in polycythaemia vera are not present in the neutrophils from patients with erythrocytosis secondary to chronic pulmonary disease, congenital heart disease or primary renal lesions (Mitus et al, 1959), although the presence of infection may elevate the score   in these conditions and this reduces the diagnostic value of the test. Nevertheless, the finding of a normal LAP score in a patient with erythrocytosis strongly points to the condition being a secondary erythrocytosis and not polycythaemia vera.

              Other cytochemical reactions do not differ markedly from normal in polycythaemia or myelofibrosis, although the glycogen content of the polymorphs has long been known to be high in the former (Wagner , 1947) and there is occasional PAS positivity to be seen in a small proportion of the erythroblasts in the latter (Quaglion & Hayhoe, 1960).

 

 

Cytochemistry of Sezary Syndrome (SS)

          This but not always of helper subset. Cytochemical studies of sezary disease is the leukemic phase of mycosis fungoides and is of T-cell origin, chiefly cells have been reviewed by (Flandrin & Daniel, 1984). The PAS reaction ranges from negative to variably positive with a finely granular pattern but occasionally strong and coarse positivity may be seen. The acid hydrolases show unevenly scattered granular reactions of moderate to strong intensity but only rarely solitary large granules or dots as seen in some T-cell populations.

 

 

 

 

Prognostic value of cytochemistry

in acute leukemias

      Cytochemical markers are a practical ancillary method for identification and classification acute leukemias and are helpful in prediction of  prognosis and planning of proper chemotherapy.

Acute lymphoblastic leukaemia (ALL)

          Laurie (1968) first reported  a correlation between extent of PAS positivity and survival , frequent PAS positivity in the blast cells being associated with better prognosis. This finding has  been supported by many independent studies (Vowels & Willoughby , 1973 ; Feldges  et al, 1974 ; Ascari et al, 1975; Paolucci et al , 1977 ) , but denied by others       ( Bennett & Henderson , 1969 ; Berrebi et al , 1973 ; Humphrey et al,1974; Show et al . 1977) .

         The differences are not easy to explain , but a study by Lilleyman and his associates  (1979)  suggested that the distribution of other features carrying poor prognosis , such as age over 14 years or high presenting  leukocytic  count , may have influenced the comparative figures.  In their series, actuarial survival studies showed that patients with more           than  20% PAS positive blast cells survived longer than those with less , but that the difference was statistically significant only after the exclusion of other adverse factors .

            From the prognostic point of view Basso et al (1984) , in an interesting study of 120 childhood non-T , non-B ALLs , correlated the duration of first remission with initial cytochemical positivity  of the  blast    cells for four cytochemical reactions , acid  phosphatase ,   µ-naphthyl acetate esterase , B-glucuronidase and     N-acetyl-B-glucosaminidase. The percentage in complete remission up to 6 years was always higher for children whose blasts lacked these enzymes .

         At 6 years 89 % of ALLs with no cytochemical positivity for the four enzymes studied were in  complete remission , as  were 59 %  of those with one positive reaction , but less than    39 % of those with two or more positive reactions . The probable interpretation of this finding is that the least mature cell type among the non-B, non-T ALL variants, with least marker enzyme  expression  respond  best  to  therapy .

 

Acute myeloid leukemia AML

          Attempts to determine the prognostic relevance of cytological and cytochemical findings have been made in recent years (Swirsky et al, 1985; Hayhoe 1985 ; Swersky et al , 1986) .The studies involved patients with de novo  AML , entered into the Medical  Research  Council`s  8th  and 9th trials of therapy. The patients were  all   treated with similar remission-induction regimen , and pre-treatment variables comprising age, clinical status (Performance index), hematological parameters (hemoglobin, leukocyte total and differential counts and platelet count ) and a detailed cytological and cytochemical assessment of bone marrow aspirates were analyzed. As has repeatedly been found in earlier large-scale studies, the most important single prognostic feature was age, with significantly poorer remission rates in patients over   60  years old .

           Other poor prognostic features at presentation included low performance index , and platelet counts below 25 × 109 ­/L. . Account had to be taken of these features, especially age, in comparing cytological and cytochemical findings in different prognostic groups . These studies showed that cases of AML with cytological  maturation  along  either granulocytic or monocytic lines, or both, evidenced by granules, Auer rods, more  than 5 % Sudan black positive blast cells and morphological and cytochemical abnormalities of neutrophils were associated with a good response to treatment .

              Another study of (Hoyle et al, 1991), multivariate analysis suggested that patients having fewer than 50 % blasts with Sudan black    positivity had a poorer rate of complete  remission, duration of remission, and survival compared    with patients having  50   %  or more blasts with Sudan black  positivity .

                Myeloperoxidase activity may have prognostic significance (Bennett,  1981;  Matsuo et al, 1989). For example, of 72 patients with the FAB M1 phenotype studied for MPO activity, two groups could be distinguished by the proportion of MPO-positive blast cells (less than or more than  50 % ).  When MPO occurred in fewer than 50  %  of blast cells (immature group = 38 cases) , the complete remission ( CR ) rate was 53 %, in contrast to a  CR  rate of  85  %  for 34 patients with more than 50  %  MPO-positive blasts.

 

 

Material  and methods

This study included  30  patients with acute  leukemia randomly selected  , aged  from  2.5  to  60   years  , 17 males  and  13 females . In addition 10 of healthy individual are included  as control at the same age group.  Patient and controls were subjected to the following:                               

*Complete history and clinical examination ,

*Laboratory investigations which include :

        1- complete hemogram ,

        2- bone marrow aspiration ,

        3- cytochemical markers

                     

Complete hemogram 

preparation  of  sample :  3ml venous blood  are drown from patients and delivered into tube containing k-E.D.T.A as anticoagulant for blood picture  .

 Method : The tube containing 3ml blood was adapted to gentle shaking about 20 times before processed by Coulter device T.890  (Coulter,1956 ; Dacie , 1994) .

 

bone marrow aspiration

Bone marrow  samples  can be aspirated from the sternum , iliac crest  or anterior or  posterior iliac spines and  from the spinous processes of the lumbar vertebrae , and in children aged less  than two years from the upper end of the tibia. By the use of Salah or Klima  needles  ( Dacie 1994) .

 

Cytochemical markers

Collection and preparation of samples :

Freshly prepared whole EDTA-treated  or heparinized blood or bone marrow films are used. Fix as soon as possible . In neutrophil alkaline phosphatase ,  fresh blood or bone marrow  films or samples anticoagulated  with  heparin ,  and  not   EDTA , may  be used  .  Blood smears should be stained for enzyme activity within  8  hours after preparation  . However, if this is not possible , gradual loss of alkaline  phosphatase activity may be delayed by fixation and storage over night in freezer , films should be dried at least one hour prior  to  fixation and three hours post fixation before freezing .The  following  cytochemical  tests  were  done  :

 

1_   Sudan black B staining

Principle of test

Several lipids including  phospholipids , neutral fats and sterols are stained intensely by Sudan black B  the leukocyte Sudan black B  staining  pattern usually parallels that of myeloperoxidase . Cells committed along lymphoid pathways display  a  negative  reaction  ,  whereas  myeloid  and  monocytoid  forms  display characteristic positive reactions , thus Sudan black B is considered a  useful  adjunct in the identification of myelocytic and myelomonocytic  leukemias.

Reagents ( provided in kits)

  1-Sudan black B staining reagent (Catalog number 380-1 )

      Sudan black B,  0.18% (w/v) , in 69 %  ethanol and containing  phosphate .buffered  phenol .Store at room temperature (18-26 C).

 2- Hematoxylin solution  , Gill  number  3     catalog  number  GHS - 3 certified  hematoxylin   6 gm /L ,  sodium  iodate  0.6 gm / L ,     Aluminum sulfate   52.8 gm/L  ,  and stabilizer . Store at room temperature.

 3-Glutaraldehyde  solution  catalog no.  380-2

    Glutaraldehyde,  o.4  %  in borate buffer, pH  7.6

    Store in  refrigerator  ( 2 - 6  C) .

5- : Glutaraldehyde fixation  solution is  prepared  by  adding  25 ml       

      reagent grade  acetone  to  75 ml  glutaraldehyde solution ,

      catalog  no. 380-2 .

      Solution is stable for  several months when stored tightly capped

      in a  glass bottle  in refrigerator .

Reagents :  required but not provided in kits

1-Acetone ,  reagent grade

2-Ethanol   70 %

3-Xylene  based  mounting  medium

Procedure:

1- Cool glutaraldehyde fixation solution in refrigerator .

2- Smears or films of  blood or bone marrow  preparation are   

     fixed for one minute at   2 - 6  C  with gentle agitation  

     followed by thorough rinsing in de-ionized  water .

3- Stain in Sudan black B staininig reagent by immersion for  5 minutes with intermittent agitation.

4- Rinse three or  mote times  in  70 %  ethanol until no more dye washes out .  This is  followed by thorough rinsing in distilled  water .

5- Counter stain in  hematoxylin  solution , for  5 minutes followed by thorough rinsing in tap water

 6-  After air drying , slides may be mounted  in a  xylene based mounting medium.

 Expected  observations:

Neutrophils  and  their  precursors show blue Black intracellular granulation .  Monocytes  stain less intensely  and lymphocytes do not stain with Sudan black B .

 

 Periodic acid Schiff  ( PAS )

Back ground and principle of test

Whe­­n treated with periodic acid , glycols are oxidized to aldehyde . After reaction with Schiff’s  reagent (a mixture of pararosaniline and sodium metabisulfite ) a  pararosaniline adduct is released that stains the glycol-containing cellular components. The test may be helpful in recognizing some cases of   erythroleukemia and acute lymphoblastic leukemia.

Reagents

1- periodic acid solution , catalog no. 395.2 periodic acid  1g/dl      

2-    Schiff ‘s reagent , catalog no. 395-2  pararosaniline Hcl 1%and sodium metabisulfite, 4%, in hydrochloric acid , 0.25 mol/l

3-    Hematoxylin solution, Gill  no. 3 , Catalog no. GHS-3 certified hematoxylin , 6 g/l, sodium iodate , 0.6 g/k, aluminum sulfate, 52,8 g/l and stabilizer.

   The reagent are supplied ready for use.

4-    Formalin -ethanol fixative solution is prepared by mixing 5 ml of Formaldehyde 37% with 45 ml of 95% ethanol. Prepare fresh daily and keep tightly capped

Procedures

Standard procedure

1-Fix air-dried blood films for  1 minute at room temperature in formalin-      

     ethanol fixative solution.

2-Rinse slides 1 minute in slowly running tap water.

3-Immerse slides in periodic acid solution ,for 5 minutes at room

     temperature

4-Rinse slides in several changes of distilled water.

5-Immerse slides in Schiff’s  reagent , for 15 minutes at room timperature

6-Note :immediately after use cap Schiff’s  reagent and return to

    refrigerator [2-8ْ c].

7-Wash slides in running tap water for  5 minutes.

  Counterstain slides in hematoxylin solution , for 90  seconds

8-Rinse slides in running tap water for 15-30 seconds, air dry and examine

   microscopically under oil immersion lens . Slides may be mounted in

    toluene or  xylene  based mounting media.

      

Microwave procedure

1-    Fix air-dried blood films for 1minute at room temperature in formalin -ethanol fixative solution.

2-    Rinse slides 1 minute in slowly running tap water.

3-    Place slides in plastic coplin jar  containing  40 ml  periodic acid solution , and microwave on high for 10 seconds. Discard periodic acid solution after use in microwave . (periodic acid solution may be reused if slides are immersed for 5minutes in room temperature  periodic acid solution )

4-      Rinse slides in several changes of distilled water Immerse slides in 40 ml Schiff’s   reagent , contained in plastic coplin  jar

5-    Microwave on high for  15 second , stir with applicator stick and let stand for 20 second .

6-    Wash slides in Running tap water for 5 minutes .

7-    Place slides in 40 ml  hemotoxylin solution , contained in plastic coplin jar and microwave on high for 5-10 seconds.

8-    Rinse in tap water and air dry .

9-    Films may be mounted in toluene or xylene based mounting media

 

Expected observations

Normal :

In normal bone marrow the earliest myeloid precursors do not  stain but diffuse and granular staining increases as function of maturation along myeloid pathways . Erythrocytes precursors do not stain while megakaryocytes and platelets stain intensely , monocytes stain faintly and may display fine granules.

Erythroleukemia  :

Intense cytoplasmic granular PAS staining may be observed in early erythroid precursors . Diffuse staining may be present in more mature nucleated cells.

Acute lymphoblastic leukemia  :

PAS  activity is highly variable , in most cases, some precursor cells show coarse granules or block-like posititivity.

Acute granulocytic leukemia :

Myeloblasts  are usually negative , although a faint-diffuse reaction product may occasionally be observed.

 

 

 

 

Naphthol AS-D chloroacetate esterase

and  µ-Naphthyl acetate esterase

Background and principle of test ;.

          The slides are incubated with either naphthol AS-D chloroacetate or µ-naphthyl acetate in the presence of freshly formed diazonium salt . Enzymatic hydrolysis of ester linkages liberates free naphthol compounds. These couple with diazonium salt, forming highly colored deposits at sites of enzyme activity.

 Most recent procedures, employ stablediazonium salts. These are formed by reacting an arylamine with sodium nitrite in an acid medium . The resulting diazonium chloride (usually unstable) can then be treated with compounds such as zinc chloride, zinc sulfate or naphthalene 1-6 disulfonate, forming stablesalts. These stabilizers may exert marked inhibition on some enzymatic systems, where as the diazonium chlorides are less inhibitory. For this reason, stablesolutions for fast red violet LB base, fast blue BB base and sodium nitrite for esterase cytochemistry . To further simplify these methods, stablesolutions of naphthol AS-D chloroacetate and µ napthyl acetate are included.

 

Reagents

1-Naphthol AS-D chloroacetate solution, catalog No . 91-1 Napthol AS-D chloroacetate, 8 mg/ml, and stabilizer.

2-Fast red violet LB base solution , catalog No . 91-2 fast red violet LB base , 15 mg/ml , in 0.4 mol/L hydrochloric acid with stabilizer .

3-Trizmal^TM 6.3 concentrate , catalog No . 91-3 Trizmal maleate, lmol/L , with surfactant        pH 6.3 + 0.15 at 25  C .

4-Sodium nitrite solution , catalog No.91 - 4 sodium nitrite , 0.1 mol /L

5-Citrate solution , catalog No . 91 - 5 citric acid , 18 mmol /L, Sodium citrate , 9 mmol /L , sodium chloride , 12 mmol /L, with surfactant               pH 3.6 + 0.1 at  25   C . 

6-µ - Naphthyl acetate solution , catalog No . 91 – 6   - µ-Naphthyl acetate , 12 . 5 mg/ml , in methanol solution with stabilizers .

7-Fast blue BB base solution , catalog No . 91 -7 fast blue BB base , 15 mg/ml in 0.4 mol/L hydrochloric with stabilizers .

8-Trizmal^TM 7.6 concentrate , catalog No . 91 - 8 Trizmal maleate , 1 mol/L , with surfactant  pH 7.6 + 0.15 at 25  C .

9-Hematoxylin solution Gill No .3 , catalog No . GHS - 3 certified hematoxylin , 6.0 g/l , sodium iodate , 0.6 g/L, and aluminum sulfate , 52.8 g/L , with stabilizers .

10- Sodium Fluoride Solution , catalog No.91-9 - Esterase reagents are provided ready for  use .

11-Citrate - Acetone - formaldehyde  fixative.

      To 25  ml citrate solution , add 65 ml  acetone  (reagent grade)    and 8 ml 37% Formaldehyde . Place in glass bottle and cap tightly. Store in refrigerator (2-8 C ). Bring to room temperature prior to use . Stableup to 4 weeks if stored tightly capped in refrigerator .

   Procedures

     The described procedures are performed at 37 C.

    µ- Naphthyl Acetate esterase procedure

1-prewarm sufficient deionized water for substrate use to 37ْ   C.   Check temperature before use .

2-Immediately prior to fixation , add I mL sodium nitrite solution to I mL fast blue BB base solution in a test tube . Mix by inversion and allow to stand at least 2 minutes .     The color will change from dirty brown to deep  yellow . Active evolution of gas bubbles should be  avoided .

3-Add solution from step 2 to 40 ml prewarmed  deionized water .

4-Add 5 ml Trizmal ^TM 7.6  buffer concentrate .

5-Add ImL µ-Naphthyl acetate solution . The solution should turn  greenish . Mix well and pour into coplin jar .

6-Bring citrate-acetone-formaldehyde (CAF) solution to room temperature (23 – 26  C  ) . Fix slides by immersing in CAF solution for 30 seconds . Agitate slides vigorously for the last 5  seconds.

7- Rinse slides thoroughly in running deionized water for 45- 60 seconds, then place in solution from step 5. Do not allow slides to dry.

8- Incubate for 30 minutes at  37  C . Protected from light.

9-After 30 minutes, remove slides and rinse thoroughly for at least 2 minutes in running deionized water.

10- Counterstain 2 minutes in hematoxylin solution.

11- Rins in tap water and air-dry.

12- Evaluate microscopically. If coverslipping is required use only an aqueous mounting media.

 

Notes:

1- For use with Columbia jars, divide reagent volumes by 5.

2- If substrate (See step 5) appears turbid bring to room temperature (23- 26  C  ) and mix well .

3- If slides have been prefixed and stored, skip fixation (steps 6 and 7) and begin staining of dry, prefixed slides at step 8.

 

Naphthol AS-D chloroacetate esterase procedure

1- Prewarm sufficient deionized water for substrate use to 37ْ  C.Check temperature before use.

2- Immediately prior to fixation, add I mL Sodium nitrite solution to I mL fast red violet LB base solution in a test tube.

   Mix gently by inversion and allow to stand 2 minutes.

   Active evolution of gas bubbles should be avoided .

3-Add solution from step 2 to 40 ml prewarwed  deionized water.

4- Add 5 ml Trizmal^TM 6.3 Buffer concentrate.

5-Add I mL Naphthol AS-D chloroacetate solution .

The solution should turn red . Mix well and pour into coplin   jar.

6-Bring citrate-acetone-formaldehyde (CAF) Solution to room

    temperature (23- 26ْ C  ) . Fix slides by immersing in (CAF) solution for 30 seconds .

7-Rinse Slides thoroughly in running deionized water for 45-60 seconds then place in solution from step 5 . Do not  allow slides to dry.

8- Incubate for 15 minutes, at 37  C .,    protected from light.

9- After 15 minutes , remove slides and rinse  thoroughly indeionized water for at least 2 minutes.

10- counterstain  2  minutes in hematoxylin solution.

11- Rinse in tap water and air dry .

12-Evaluate microscopically  . if coverslipping is required use only an aqueous mounting media.

 

 

Double staining Esterase Procedure.

   1.   Perform µ-Naphthyl Acetate esterase test as described in procedure. Do  not  counterstain .

   2.   Rinse slide 5 minutes in deionized water.

   3.   Perform Naphthol AS-D chloroacetate esteras test as described in procedure step 1-12 . Omit step 6

 

 

µ- Naphthyl acetate esterase with fluoride inhibition   

procedure.

          Although µ- naphthyl acetate esterase is found primarily in cells of monocytic lineage when performed as describes, it should be recognized that megakaryocytes and erythroid precursors are positive for this enzyme. Lymphocytes and some mature granulocytes also show occasional positivity.

To differentiate these cells conclusively from monocyte, sodium fluoride is incorporated with the incubation system. The monocyte enzyme is inactivated in the presence of this compound. The following procedure may be used to perform the fluoride inhibition test.

1- To 2 ml fast blue BB base solution add 2 ml sodium nitrite solution . Mix gently by inversion.. Allow to stand 2 minutes.

2- Label 2 beakers A and B, and add the following:

 

 

 

3- Mix  well and pour into coplin jars labeled A and B.

4- Proceed as described in step 6-12 of µ- naphthyl acetate esterase procedure.

Expected observations.

          Naphthol AS-D choroacetate esterase activity will appear as bright red granulation, µ naphthyl acetate esterase as black granulation

1- Naphthol AS-D chloroacetate esterase (fast red violet LB) - Enzyme is usually considered specific for cells of granulocytic lineage. Sites of activity show bright red granulation. Activity is weak or absent in monocyles and lymphocytes

2- µ Naphthyl acetate esterase (fast blue BB) - Enzme is detected primarily in monocytes, macrophages and histiocytes, and is virtually absent in granulocytes. Monocytes should show black granulation.Lymphocytes may occasionally exhibit enzyme activity.

3- µ Naphthyl acetate esterase with fluoride inhibition.

All cells of monocytic lineage will be negative for enzyme activity, with the exception of differentiated histiocytes or specialized macrophages in tissue which may also be resistant to sodium fluoride.

4- Double staining esterase.

Specimens taken through the double staining procedure will demonstrate the granulocytes with red granulation and monocytes with black granulation.

NB : Fast blue BB base solution, catalong No. 91-7 may be substituted for Fast red violet LB base solution, catalog  No. 91-2, if blue granulation is preferred for Naphthol AS-D chloroacetate esterase.

 

 

                                  4 ) Leukocyte acid phosphatase

Principle

      A variety of hydroxy naphthoic anilides. releasing insoluble naphthols that couple efficiently at acidic pH with acid phosphatase in hemopoietic cells can hydeolyze a diazonium salts . The resultant product forms a colored precipitate and gives good microscopic enzyme localization.

 

Reagents

Fixative.

          Methanol, 10 ml; acetone, 60 ml; water, 30 ml; Citric acid , 0.63 gm. This solution should be adjusted to pH 5.4 with 1 mol / L  Na OH and controlled weekly.

Stock Solutions

1-  Buffer, pH 5.0  sodium acetate, trihydrate, 19.5 gm; sodium    barbiturate, 29.5 gm ; water to one  liter                   ( Michaeli’s veronal acetate buffer ) . Stored at . 4 0C

2-   Substrate. Napthol AS- Bi-Phosphate dissolved  in           N,N-dimethyl formamide, 10 mg / ml ( i.e 25 mg in 2.5 ml )

_3-      Sodium nitrite ( NaNo₂ ) 4 % aqueous solution ( i.e. 4 gm  dissolved in 100 ml D.W. ) . This solution should be freshly made each time or can only be stored at 4^oC for up to one  week .

_4-      Pararosanilin chloride. 2 gm in 50 ml of 2 mol / L HCL. Heat gently, without boiling; Cool down to room temperature and filter.       Stored at 4 C

Working solution

          Add 5 ml of Michaelis buffer ( solution 1 ) and 1 ml of substrate ( solution 2 ) to 13 ml of distilled water . In a separate container, mix 0.8 ml of 4 % NaNo_2­ ( solution 3 ) with 0.8 ml of  pararosaniline  ( solution  4 ) to obtain 1.6 ml of hexazonium pararosanilin. This is then added to the first mixture. The pH of the final solution is a adjusted to 5.0 with 1 Normal  Na OH .

 Countrestain :

·       Harris hrmatoxylin solution or

·       Methyl green solution : use 1 % methyl green in veronal or phosphate buffer, pH 4.0.

Method :

1-   Air-dry blood or bone marrow smears for 10 – 20 minutes.

2-   Fix for 10 minutes at 4 C in The fixative solution.

3-   Incubate smears in incubation solution for 2 hours at room temperature or for 1 hour at 37ْ C . Preferably the incubation should be done in the dark.

4-   Rinse well with distilled water.

5-   Counter stain with either of the following reagents :

a-    Counter stain with Harris hematoxylin for 2 minutes. Or

b- Counter stain with 1 % methyl green in veronal or phosphate. Buffer, pH 4.0 for 30 to 60 seconds. Rinse slides again. Then quickly dip in alcohol. Rinse again.

6-   Dry slides. Most mounting media are unsatisfactory because they can adversely affect the stain. Unmounted stained slides usually remain unchanged for years.

Note : Enzyme activity sites are identified usually as paranuclear deposits of amorphous red azo dye (red granular precipitant ) .

Interpretation :

          Acid phosphatase activity is indicated by a red granular precipitate and is demonstrable in most cells of the hemopietic system. Intense activity is present in osteoclasts and some macrophages. Moderate staining is seen in plasma cells.megakaryocytes, and monocytes. Weak reactions are noted in neutrophils, bands, metamyelocyles , myelocytes, and promyelocytes. Very little acid phosphatase activity is present in normal lymphocytes and erythoblasts. In leukemic cells from patients with T- acute lymphoblastic leukemia, acid phosphatase is strong and localized to the Golgi  zone.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Results

         Of 30 patients with acute leukemia, 10 cases were diagnosed as acute lymphoblastic leukemia (ALL) and 20 cases were acute non-lymphoblastic leukemia (ANLL) by peripheral  blood and bone marrow morphology . table(4) – diagram (1)

I – Acute  Lymphoblastic  Leukemia

               In ALL patients, the diagnosis is based on clinical data, blood picture, blast  morphology  and cytochemical studies. In most cases the bone marrow is hypercellular and nearly  all  the  cells  are lymphoblasts. Most of the blasts were small to medium sized with a rather low grade of cell to cell variability. The nucleocytoplasmic ratio is high with just a small cytoplasmic rim in many cases. The cytoplasm tends to be moderately basophilic. The nuclei were regular, some of these showing narrow clefts or indentations. The chromatin is condensed and nucleoli tend to be indistinct.

By cytochemistry the characteristic findings were:

1-   Periodic acid Schiff:

         Seven cases were periodic acid Schiff ( PAS ) negative and 3 cases show coarse granules with block positivity of at least 3 percent of the lymphoblasts, Fig.(1) , and according to FAB classification we found 8 cases classified as L₁ and 2 cases as L₂.  Two cases of   FAB type L₁ and one case of L₂ subtype are positive to PAS . This is  in comparison with normal controls in which, the  granulocytic series are stained and the staining increased with increase maturity of the granulocytic cells, but earliest granulocytic precursors did not stain. Monocyte stained faintly, erythrocytes and erylhrocytic precursors did not stain. Megakaryocytes stained diffusely and intensely.          Fig.(2),Table(5)    diagram (2)

            2- Sudan black B:

        Nine cases were negative to Sudan black ( SB ) , Fig.(3), and one case showed less than 3 percent of blast cells positive with SB, this in comperison with to normal controls in which granulocytic series were stained and sudanophilia corresponding to the granules present. Also eosinophils  stained. Monocytes may be unstained or may contain a few discrete sudanophilic granules. Lymphocyte and erythroblasts did not stain. Megakaryocytes and platelets were negative for SBB. Fig.(4). Table(5) diagram (2)

3       – Alpha-Naphthylacetae esterase;

              All cases of ALL were negative to alpha-naphthyl acetate esterase ( ANAE), in comparison with normal controls in which monocytes, macrophages, megakaryocytes, and platelets were stained, whereas  the neutrophilic series were regative or focally positve. Also the test is positive in basophils and plasma cells, focally positive in resting T-lymphocytes and weakly positive in the erythroblasts.Fig(6) , Table(5) diagram (2)

4- Acid phosphatase (A C P )

    Seven cases were acid phsphatase positive, Fig (7), and 3 cases were negative, in comparison with normal controls in which granulocytic series are weakly stained. Very little acid phosphatase activity is present in normal lymphocytes and erythroblasts. Moderate staining is present in plasma cells, megakaryocytes and monocytes. Intense activity is present in osteoclasts and some macrophages.     Fig (8), Table (5) diagram (2)

 

      In our study, we found that there is correlation between positivity of PAS and the age of the patient, positivity occurs mainly in children. But in acid phosphatase reaction we found the reverse, positivity mainly occurs  in adults.

II   Acute  NonLymphoblastic  Leukemia

          In ANLL, the “FAB” classification was based upon bone marrow morphology and cytochemical reactions, in the bone marrow we found 30% or more of all nucleated cells blasts, and myeloid to erythroid ratio >1. Myeloblasts were large cells with large nuclei.  The cytoplasm was moderate and gray with uniform fine chromatin, and two or more prominent nucleoli. Auer rods may be seen in the cytoplasm. Monoblasts were large cells with ground glass cytoplasm and irregularly folded nuclei with 2 or more prominent nucleoli. In the present study, according to FAB classification we found one case M₁, 8 cases as M₂, 2 cases as M₃, 7 cases as M₄ and 2 cases as M₅. Table(4) diagram(3)

By cytochemistry and in comparing to normal controls the characteristic findings were as follows ; 

*[اال1]  FAB Type M1, One case were negative to both PAS and ANAE   but positive to SB in which more than 3 percent of blast cells were positive with positive Auer rods. Fig(9), Table( 7) diagram(4).

FAB type M2, Six cases were PAS negative and 2 cases were PAS positive with diffuse positivity and   superimposed fine granules. All  cases were SB positive, Fig(10), more than 50 percent of blast were positive. Seven cases was ANAE negative and one case was positive with diffuse staining of granulocytic series. Fig(11),  Table( 7) diagram(5).

·       FAB type M3, Two cases were SB positive, most of blast cells and promyelocytes are strongly positive. Fig (12), The cases were negative to PAS and ANAE staining. Fig (13), Table ( 7) diagram(6).

·       FAB type M4, Five cases were PAS negative while 2 were PAS positive, one with diffuse staining and superimposed fine granules and other in addition showed block positivity. Fig(14), Four cases were SB positive, Fig(15), and 3 cases were negative. Fig(16), Four cases were weakly positive to ANAE and 3 were negative. Fig(17),Table(7) diagram(7)

·       FAB type M5 , Two cases were positive to ANAE, more than 80 percent of blast  cells were positive Fig (18).  The 2 cases were negative to both PAS and SB   staining. Fig (19), Table (7) diagram(8)

In this study, by application of morphology alone, 18 cases were difficultly diagnosed , while when used morphology combined with  cytochemical tests, we could reach to definite diagnosis in 24 cases. Table(8) diagram (9)

 

Table(4)  : Morphological classification of acute leukemias

 

 

      Table(5) :

                Cytochemical findings in ALL

 

Table(7) :

                    Cytochemical findings in AML

 

Table(6) : Correlation between total white cell counts, blast count in peripheral blood film and BM cytochemical findings in patients with acute lymphoblastic leukemia

 

 

 

Table(8) :No. & % of Cases diagnosed by morphology only and in combination with cytochemical studies in acute leukemias.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table( 9) :  Qualitative Results of cytochemical reactions in   

             normal and abnormal Blood Cells

 

 

 

 

 

 

 

 

 

		Diagram 3: Percent of FAB classification of AML

	Diagram 5 :  Percent of positive and negative cases by cytochemical  stains in  FAB Type M2

		Diagram  7 : Percent of Positive and negative cases  by cytochemical  stains  in  FAB Type M4

 

Discussion

             Morphologic examination and cytochemical staining of air-dried bone marrow and peripheral blood smears continue to form the basis for diagnosis and classification of acute leukemias. In this study, we found that : by morphological study alone we could reach definite diagnosis in 18 cases ( 60 percent ) while with the application of various cytochemical tests beside morphological study, proper diagnosis and classification of type of leukemia could be reached in 24 cases ( 80 percent).  So cytochemical markers are very useful and simple methods for identification and classification of acute leukemias

            The use of cytochemistry has greatly facilitated the discrimination of acute lymphoblastic leukemia from the various types of acute myeloblastic leukemia.

            In this study 10 cases were classified as ALL (33%).  The finding of PAS reaction are agreed with those of Hayhoe et al, (1988) who found, in 50 percent of cases, at least 5 percent of the lymphoblasts contain coarse granules or large blocks of PAS-positivity. A coarsely positive reaction and not a fine, diffuse stain is characteristic of ALL. A positive reaction is found in cases from the L1 and L2 (FAB classification) subtypes of acute lymphoblastic leukemia. In our study, we found that there is correlation between positivity of PAS and the age of the patient, positivity occurs mainly in children. But in acid phosphatase reaction we found the reverse, positivity mainly occurs  in adults.

          No correlation could be found between total white cell count, blast count in peripheral blood or B. M. aspiration and positivity of cytochemical stains. Table (6)           

  Our study showed the reactivity of ALL to SBB and ANAE , as follows;  most of cases are negative to SBB and all cases are negative to ANAE these results are consistent with those of Hayhoe, et al , (1994) who found that SBB reaction are almost always negative, however atypical coarse sudanophilia has rarely been recorded, but probably indicates bi-phenotypic disease. µ-Naphthyl aceate are negative or weakly positive with scattered granules in null cell ALL, however, localized granules may be seen in T-cell ALL.  Yet another study (Invernizzi et al., 1992) on 531 cases of childhood ALL revealed 16 with >1% and 7 with >10% blasts containing cytoplasmic azurophil granules  positive for ANAE, but negative to SBB.  Tricot et al, (1982), Bennett, et al, (1985), found SBB staining of less than 3% of blasts has been demonstrated in rare cases of ALL.  Stass et al., (1984) found SBB positivity in six out of  350 patients with immunologically proven ALL.  Thus, the diagnosis of ALL is strongly suggested when blastic cells are negative for Sudan black B, and ά alpha-naphthyl acetate esterase, yet exhibit a positive PAS stain (Snower  et al, 1991).

                  The finding of acid phosphatase are agreed with those of Hayhoe et al, (1994), who found that acid phosphatase shows a localized cytoplasmic reaction often in T-cell but rarely in other forms of ALL. McKenna et al., 1979; Savage et al., 1981 Boesen et al., 1984; Lilleyman & Scott (1989) found that acid phosphatase positivity occurred as focal paranuclear deposits more frequently, (in about 87% of cases), and strongly in T-cell, so the finding is strongly suggestive of   T-cell ALL and is of particular value as a first line test when immunophenotypic studies are not readily available.α

 

             Acute  myeloid leukemia frequently shows cross-lineage overlap, pure forms of AML with a single cell lineage, as in the case in ALL, are the exception rather than the rule, so the morphological and cytochemical criteria basically used for subdividing AML are concerned chiefly with;

(One) defining the predominant cell type and determining its position in the maturation sequence of that cell lineage, and

(Two)                        assessing the extent of involvement of other myeloid cell lineages  in the leukemic process.  (Hayhae, et al. 1994).

     

                   In this study 20 cases classified as acute myeloid leukemia (66%). The finding of M1 and M2 subtype (FAB classification) are agreed with those of Snower et al, (1991) and Hayhoe et al, (1994), who found cases with M1 subtype are negative to PAS, but some cases show diffuse tinge with or without superimposed fine granules .  Alpha-Naphthyl acetate activity are also usually negative  while SBB reaction are positive with positive Auer rods.  SBB staining greatly facilitated the recognition of Auer rods, which were found in 67% of specimens on the SB stain as compared with 32% on the Romanowsky stain.  Auer rods are virtually pathognomonic of an acute myeloid leukemia with agranulocytic component.

           In this study, M3 subtype finding is confirmed by Swirsky      et al (1984),  who found that of 30 patients with M3 subtype (FAB classification), strong sudanophilia with 20 to 100% of leukemic cells showing positivity mostly as a localized densely staining clump of deposits to one side or in a cleft of the nucleus, often so coarse as to obscure Auer rods buried within it.  Positivity to Sudan black is generally very strong in APL, as might be expected from the heavy azurophilic granulation of the predominant cells.  Sudan black positivity is usually more marked than peroxidase in acute granulocytic  leukemia and may sometime be present where peroxidase activity appears absent by orthodox cytochemical techniques (Hayhoe et al, 1994).  So Sudan black staining is very important in diagnosis of acute granulocytic leukemia (FAB classification M1, M2, and M3).  Buccheri et al., (1992),   found of 103 AML cases, 90 were SB positive (>3% blasts positive) by light microscopy cytochemistry, the remaining 13 cases,  six showed 3 – 7% blasts immunocytochemically SB positive and the remaining cases were two M0, three M5, one M6  and one M7 by FAB classification.   This   study show the reactivity of M3 subtype to            µ-naphthyl acetate esterase in which all cases were negative.  This finding is different from Scott et al., (1989), who found atypical esterase cytochemistry of many hypergranular   promyelocytes of APL, not to reflect mixed lineage but to be a consequence of increased granulation.

           µ-Naphthy acetate and butyrate esterase reactions are usually negative in granulocyte precursors, but this is not always true.  They have seen already that normal precursors may show mixed reactions, and in leukemic proliferations.  Myeloblasts    and      promyelocytes, clearly recognizable as such from other criteria (  presence of Auer rods, coarse sudanophilia, peroxidase positivity, weak diffuse PAS reaction, positive chloroacitate esterase ) may also sometimes show acetate or butyrate esterase positivity. 

          In this study, the finding of M4 subtype are consistent with these of Hayhoe et al, (1994),   who found that the PAS reaction in acute myelomonocytic leukemia vary, some blast cells are entirely negative, other show a reaction like that found in myeloblastic leukemia, with weak tingeing of the cytoplasm and perhaps fine granulation, while others give the PAS reaction typical of many normal monocytes, with fine to moderately coarse granulation against a background of diffuse cytoplasmic staining.  Rarely, much stronger positivity may occur in leukemic monocyte precursors, with coarse granules and even blocks of glycogen found, but the coarse granules or blocks of PAS positivity are commonly found against back ground of fines granules or diffuse staining in most of the positively reacting primitive cells.

          Sudanophilia is conspicuous in the granulocyte precursors whereas most leukemic monocytes show occasionally weak discrete scattered granular type positivity to SBB.  Esterase activities   against µ-naphthyl acetate are positive in monocytic cells, varying from weak to intense, while a variable proportion, sometimes high , of the leukemic monocyte precursors in some cases of M4 may show weak or even negative reactions for monocytic esterase.   The cytochemical heterogeneity in esterase expression was again confirmed by Scott and his colleagues (1985).  Li et al., (1986) reported five cases of AMML, all of which showed many leukemic cells positive for ANAE.   In our study M5 finding is confirmed with Wrotnowski et al, (1987), who found, for example that six of 14 cases of acute monocytic leukemia classified as M5 had finely granular diffuse cytoplasmic positivity for acetate esterase, whereas eight had dense focal staining a weak cytoplasmic background reaction.  PAS reaction is variable and SBB completely negative.

 

Summary and Conclusion

        This study was conducted in  University Hospital  of   Sohag  Faculty  of  Medicine

          It was performed on 30 patients with acute leukemia, randomly selected, aged form 2.5 : 60 years, 17 males and 13 females. In addition 10 of healthy individuals are included as control at the same age group.

          The patients and controls were subjected to the following:

·    Complete history and clinical examination.

·     Laboratory investigations which include;

1-   Complete hemogram

2-    Bone marrow aspiration

3-   Cytochemical markers which include periodic acid Schiff, Sudan black B and µ-naphthyl acetate esterase, leukocte acid phosphatase.

      - Complete hemogram and bone marrow aspiration films demonstrate the morphological criteria of the case which initially based upon clinical findings, hematological picture, morphological appearance of blast cells and the nature of the more differentiated cells when present. Cytochemistry was then chosen according to the data presented .

 

-         Cytochemical tests which demonstrate the following :

-          

(One)                        Periodic acid Schiff  (PAS)

           PAS is a useful marker for lymphoblasts in acute lymphobastic leukemia, especially FAB L₁ and L₂ morphology. It showed that at least 5 percent of the lymphoblasts contain coarse granules or large block positivity with clear back ground of the cytoplasm while in acute myeloid leukemia, showed diffuse tinge staining of the cytoplasm with or without superimposed fine granules. In myelomonocytic leukemia ( FAB M₄ ), rarely showed much strong positivity, with coarse granules and even blocks of glycogen against back ground of finer granules or diffuse staining .

 

(Two)                       Sudan black B

    SBB is a useful marker for acute myeloid leukemia. It showed positive reaction in FAB classification M_1,  M₂ and M₃ and were very strong in APL . In myelomonocytic leukemia, sudanophilia were conspicuous while in monocytic leukemia, leukemic monocyte were negative or showed occasionally weak discrete scattered granules. In acute lymphoblastic leukemia SBB was almost always negative , atypical sudonophilia < 3% were rarely recorded.

 

(Three)                µ - Naphthyl acetate esterase

          ANAE is a useful marker in identification of the monocytic lineage. It showed positivity in monocytic precursors of acute myelomonocytic and monocytic leukemia. Atypical esterase positivity would found in hypergranular promyelocytes of APL . In acute lymphoblastic leukemia, it were negative or weakly positive with scattered granules.

 

(Four)                    Leukocyte acid phosphatase

                     Acid phosphatase is a useful marker in identifying the T-cell subset of acute lymphobastic leukemia , particularly when other methods of identifying  T-cells are not available. It also may assist in confirming the diagnosis of hairy-cell leukemia . Its reaction is strong and localized to the Golgi zone .

         

                      In this study, we found that : by morphological study alone we could reach definite diagnosis in 18 cases ( 60 percent ) while with the application of various cytochemical tests beside morphological study, proper diagnosis and classification of type of leukemia could be reached in 24 cases ( 80 percent).  So cytochemical markers are very useful and simple methods for identification and classification of acute leukemias. However  in our study we conclude that cytochemistry is the best method for diagnosis of  granulocytic leukemias while for lymphocytic leukemias , it is limited and need immunophentyping.  So immunophenotyping tests are recommended for accurate diagnosis, classification and therapy of lymphoblastic leukemias.

 

 

 

 

 

 

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