Peripheral smear – WBC

PERIPHERAL SMEAR – WBC

• What does DLC means

  • DLC means “Differential leukocyte count”.

  • DLC is visual counting of WBC and their percentages in 100 cells

 

• What are methods used for DLC

  • Longitudinal strip, Battle-ment, Zig Zag

 

• What is the  normal range of DLC

 

• What are granulocytes and agranulocytes

  • Granulocytes are the cells with granules in cytoplasm. eg. Neutrophils, Eosinophils and basophils

  • Agranulocytes are lymphocytes and monocytes

 

• What is the color of the granules in WBC s

  • Neutrophils    –    fine pink granules

  • Eosinophil     –    coarse Crimson red granules

  • Basophil         –    Purple to bluish black granules

 

• Describe the morphology of WBC’s

 

• What are toxic granules?

  • These are coarse deep blue to purple basophilic granules seen in neutrophils( strongly peroxidase positive)

  • Observed in bacterial infections. ex: pyogenic meningitis and enteric fever

 

• What other abnormalities can be seen in neutrophils

  • Toxic granules – as above

  • Large reddish granules – in Aplastic anemia and myelosclerosis

  • Large dark granules – Alder abnormality (genetic disorder where granules can also be seen in lymphocytes and monocytes

  • Vacuoles (Phagocytic vacuoles) – severe bacterial infection and toxemia

  • Dohle bodies – Blue coloured round to oval patches of 2 -3 μm diametre in the periphery of cytoplasm. Seen in bacterial infections

 

• What other information is provided by neutrophils ?

  • Hypersegmentation of the nucleus – in megaloblastic anemia, Uremia and methotrexate drug usage

  • Drum stick on the nucleus is indicative  of second X chromosome (corresponding to Barr body in buccal smear) in females

 

• What is Arneth count?

  • In 1904 Joseph Arneth graded neutrophils from 1 to 5 according to the number of lobes in their nuclei

    • Grade I – Unlobated deeply indented nuclei

    • Grade II – 2 lobes connected by thin segment

    • Grade III – 3 lobes connected by two thin filaments

    • Grade IV – 4 lobes connected by thinfilaments

    • Grade V – Five or more lobes connected by 5 or more thin filaments

  • 100 neutrophils are counted and the number of each type are expressed as percentage

• • If there is increase in younger cells (group I and II) in smear then it is called as “shift to left”, seen in acute infections

  • If there are hypersegmented neutrophils, then it is termed as “shift to right”, seen in megaloblastic anemia

 

• What are virocytes

  • Lymphocytes that are transformed and have blastoid/ plasmacytoid or monocytoid features are called virocytes

  • Eg in Measles, Flu, Infectious mononucleosis

 

• What are “Virgin cells”

  • Mature B lymphocytes that are not exposed to antigen are called “vigin cells”. They express CD 19 and CD20 surface antigens

 

• What is normal range of total leukocyte count?

  • • 4000-11000 cells/cumm

 

• What is leukocytosis

• • Increase in leucocyte count of more than  11000 cells/cumm

 

• What is leukopenia

  • Decrease  in leukocyte count below 4000 cell/cumm

 

• What are the causes of neutrophilia

  • Increase in neutrophil count above 7,500/µl is called neutrophilia

  • Causes are

    • Acute infections –  By bacteria, fungi, parasites and some viruses. Eg.  Pneumonia, Acute cholecystitis, Acute appendicitis,  Salpingitis, Abscess, Peritonitis, Acute tonsillitis, Actinomycosis, Furuncle, Carbuncle

    • Tissue damage causing inflammation – Burns,  Ischemic necrosis, Gout, Hypersensitivity reaction

    • Intoxication –  Uraemia, Diabetic ketosis, Poisoning by chemicals, Eclampsia

    • Acute hemorrhage

    • Neoplastic conditions – Myeloid leukemia(CML), Polycythaemia vera, Myelofibrosis, Disseminated cancers

    • Miscellaneous conditions – Administration of corticosteroids, Idiopathic neutrophilia

 

• What is the difference between Leukemoid reaction and Leukemia

 

• What is NAP score and how to assess it?

  • The neutrophil alkaline phosphatase (NAP) also called as Leukocyte alkaline Phosphatase (LAP) is present in  cytoplasm and secondary granules of neutrophils

  • As the maturity of cell increases NAP also increases

  • EDTA blood should not be used as enzyme activity is reduced by EDTA

• Assessment: 

  • Based on intensity of staining 5 grades ranging from 0 – 4 have been given

• •  100 neutrophils are counted. Neutrophils activity grade is assessed

  • Each activity grade is multiplied by number of neutrophils in that grade

  • By adding up all the activity grades, total score is available

 

• What are the causes of neutropenia

  • Decrease in neutrophil count below 2000/µl

  • seen in conditions like

    • Infections eg. Typhoid, Brucellosis, Measles, Malaria, Kala azar, Miliary tuberculosis

    • Drugs and chemicals

      • Antimetabolities, Benzene, Nitrogen, mustard

    • Physical agents – Irradiation

    • Haematological diseases – Pernicious anemia, Aplastic anemia

    • Other diseases – SLE, Gauchers disease

       

• What are the causes of lymphocytosis

  • When absolute lymphocyte count is more than 4000/µI it is termed as lymphocytosis

  • Causes

    • Acute infections –  Infectious mononucleosis, Pertusis, Viral hepatitis, Chronic infections like Tuberculosis,  Secondary syphilis, Brucellosis 

      Haematopoietic Disorders – CLL, NHL

 

• What are the causes of lymphopenia

  • When absolute lymphocyte count below  is 1500/µI, it is called lymphopenia

  • Lymphopenia is seen in

    • Aplastic anaemia

    • AIDS

    • Hodgkins disease

    • High dose of steroid administration

    • Irradiation

 

• What are causes of monocytosis?

  •  Increase in absolute monocyte count above 800/µl

  • Monocytosis is seen in –

    • Bacterial infections

      • Tuberculosis

      • SABE

      • Syphilis

    • Protozoal infections

      • Malaria

      • Kala azar

      • Trypanosomiasis

    • Haematopoietic disorders

      • Monocytic leukaemia

      • Hodgkin’s disease

      • Multiple myeloma

      • Myeloproliferative disorders

    • Miscellaneous conditions

      • Sarcoidosis

      • Cancer of ovary, breast, stomach

 

• What are the causes of basophillia

  • Increase in the absolute basophil count above 100/µI is called as  basophilia

  • Basophilia is seen in conditions like –

    • Chronic myeloid leukemia

    • Polycythaemia vera

    • Myxoedema

    • Ulcerative colitis

    • Hodgkin’s disease

    • Urticaria pigmentosa

 

• What are the causes of Eosinophilia?

  • Increase in absolute eosinophil count above 400/µI is called eosinophilia

  • Eosinohillia is seen in conditions like –

    • Allergic disorders

    • Bronchial asthma

    • Hay fever

    • Urticaria

    • Drug hypersensitivity

    • Parasitic infestations

      • Roundworm

      • Hookworm

      • Tapeworm

      • Echinococcosis

• • • Skin disease

      • Pemphigus

      • Dermatitis herpetiforms

      • Erythema multiforme

• • • Pulmonary diseases

      • Loeffler’s syndrome

      • Tropical eosinophilla

    • Haematopoietic diseases

      • Chronic myeloid leukaemia

      • Pernicious anaemia

      • Polycythaemia vera

      • Hodgkin’s disease

• • • Miscellaneous conditions

      • Rheumatoid arthritis

      • Sarcoidosis

      • Irradiation

      • Polyarteritis nodosa

 

• What are dohle bodies

  • Dohle bodies are pale light blue-gray, basophilic oval inclusions (1-3µm in diametre) located at the periphery part of the cytoplasm in neutrophils

  • Named after Karl Gottfried Paul Dohle (1855-1928) , German pathologist

  • They are found in conjunction with toxic granulation and are thought to be remnants of rough endoplasmic reticulum

 

• What are Pelger-Huet cells

  • Pelger-Huet anomaly is a condition where nuclei of neutrophils (also eosinophils) show abnormal shapes like bilobed, dumbell shaped or peanut shaped and have coarse chromatin

  • Pelger, a Dutch hematologist (in 1928) first described Pelger-Huet anomaly in neutrophils

  • This anomaly can be inherited as autosominal dominant disorder or can be in individuals with myeloid and lymphocytic leukemias and in myelodysplasia

 

• What are turk cells

  • Turk cells are transformed lymphocytes (immunoblasts) seen in bacterial and viral infection

  • These cells have round eccentrically placed nucleus, large nucleous and abundant basophilic cytoplasm

  • Usually seen in lymphoid tissue diseases

 

• What is “Ballerina Skirt cell” and in which condition these cells are seen in smear

  • These are atypical lymphocytes seen in infectious mononucleosis.

  • These cells have abundant agranular pale cytoplasm which shows dark basophilia at the point of contact with other cells and shows indentation of borders at the site of RBC contact

 

• What is Lazy leucocyte syndrome

  • Condition associated with severe neutropenia and abnormal   mobilization of neutrophils

  • Etiology is not known but bone marrow shows normal number of granulocytes and their precursors

 

• What is LE CELL?

  • LE cell is a neutrophil which has phagocytosed denatured nuclear mass (LE body)

 

• What is Tart cell

  • Tart cell is a monocyte /phagocyte which has ingested nucleus of other cell whose structure is preserveda and unaltered

 

• What is Leukemia

  • Leukemia is malignant condition of hematopoietic tissue characterized by uncontrolled proliferation of leukopoietic cells in the bone marrow with or without thw appearance of premature leukocytes in peripheral blood

 

• Classify Leukemia

  • On the basis of cell origin and rapidity of clinical course, Leukemias are broadly classified into

    • Acute Myeloid Leukemia

    • Acute Lymphoid Leukemia

    • Chronic Myeloid Leukemia

    • Chronic lymphoid leukemia

 

• What is the difference between Myeloblast and lymphoblast

 

• Cytochemistry of AML blasts and ALL blasts

 

• What is aleukemic leukemia and how is it diagnosed

  • Aleukemic leukemia is a condition characterized by normal or decreased number of WBC’s in peripheral blood, but more than 20% of blast cells are seen in bone marrow which is diagnostic

 

• What is subleukemic leukemia

  • Subleukemic leukemia is a condition characterized by abnormal WBC’s in peripheral blood, but the total number of WBC’s are normal

 

• Which type of acute leukemia has better prognosis

  • Acute Lymphoblastic leukemia has better prognosis than Acute Myeloid Leukemia

 

• Which chronic leukemia has worse prognosis

  • Chronic Myeloid Leukemia has worse prognosis than Chronic Lymphoid Leukemia

 

• What is juvenile CML.

  • Juvenilr CML is a condition which occurs in children younger than 4 years of age and has following features

    • Philadelphia chromosome negative

    • spleenomegaly

    • Monocytosis with immature granulocytes  and nucleated RBC’s  in peripheral blood

    • less than 20% of blasts in peripheral blood and bone marrow

    • chrmosomal 7 monosomy

    • Mutation in RAS or PTPN11

    • Increased Hemoglobin F

 

• What are basket/smudge/smear cells

  • Smudge cells are remnants of cells that does not have any identifiable nuclear structure or cytoplasmic membrane 

  • They also called basket cells

  • Seen mostly in CLL

  • Higher percentage of smudge cells indicate poor prognosis

 

• FAB classification of ALL

 

• WHO classification of lymphoid neoplasm

  • Precursor B-cell neoplasms

    • B-cell acute lymphoblastci leukemia/Lymphoma (B-ALL)

  • Peripheral B cell neoplasms

    1. Chronic Lymphocytic leukemia/ small lymphocytic lymphoma

    2. B-cell prolymphocytic leukemia

    3. Lymphoplasmacytic lymphoma

    4. Splenic and nodal marginal zone lymphomas

    5. Extranodal marginal zone lymphoma

    6. Mantle cell lymphoma

    7. Follicular lymphoma

    8. Marginal Zone lymphoma

    9. Hairy cell Leukemia

    10. Plasmacytoma/Plasma cell myeloma

    11. Diffuse large B-cell lymphoma

    12. Burkitt lymphoma

  • Precursor T cell neoplasms

    • T-cell acute lymphoblastic leukemia/Lymphoma (T-ALL)

  • Peripheral T-cell and NK cell neoplasms

    1. T cell Prolymphocytic leukemia

    2. Large granular lymphocytic leukemia

    3. Mycosis fungoides/ Sezary syndrome

    4. Peripheral T cell lymphoma unspecified

    5. Anaplastic large cell lymphoma

    6. Angioimmunoblastic T cell lymphoma

    7. Enteropathy – Associated T cell lymphoma

    8. Panniculitis like T-cell lymphoma

    9. Heaotosplenic γδT- cell lymphoma

    10. Adult T cell leukemia/ lympoma

    11. Extranodal NK/ T- cell lymphoma

    12. NK – cell Leukemia

  • Hodgkin lymphoma

    • Classical Subtypes

      • Nodular Sclerosis

      • Mixed cellularity

      • Lymphocyte rich

      • Lymphocyte depletion

    • Lymphocyte predominance

 

• FAB classification of Acute Myeloid Leukemia

 

• WHO Classification of Acute Myeloid Leukemias

  • AML with Genetic aberrations

    • AML with t(8;21)(q22;q22); RUNX1/ETO fusion gene

    • AML with inv(16)(p13;q22); CBFB/MYH11 fusion gene

    • AML with t(15;17)(q22;11-12); RARA/PML fusion gene

    • AML with t(11q23;v); diverse MLL fusion genes

    • AML with normal cytogenetics and mutated NPM

  • AML with MDS like features

    • With poor prognosis

    • AML with multilineage dysplasia

    • AML with MDS like cytogenetic aberrations

  • AML, therapy related

  • AML, Not otherwise specified

    • AML, minimally differentiated

    • AML without maturation

    • AML with myelocytic maturation

    • AML with myelomonocytic maturation

    • AML with monocytic maturation

    • AML with erythroid maturation

    • AML with megakarycytic maturation

 

• What is the difference between WHO and FAB classification of AML

  • Cut off percentage of blasts in bone marrow for diagnosing AML in FAB classification is 30% while in WHO classification is 20%

  • WHO classification is based on cytogenetic abnormalities and indicates prognosis which is not present in FAB classification 

References

• Kumar, Abbas, Aster. Diseases of WHite Blood Cells, Lymphnodes, Spleen and Thymus. In: Robbins and Cotran Pathologic Basis of Disease. 9th edition 2017;Volume 1; Chapter 13:579-628.

• Dr.Tejindar Singh, Dr.K Uma Chaturvedi. Practical pathology, Third edition, 2015.

• P. Chakraborthy, GargiChakraborthy. Practical Pathology, 2002.

• Dr.Ganga S.Pilli. Practical Pathology, 2007

• Sabitri Sanyal, Aparna Bhattacharya. Clinical Pathology. A Practical Manual. Third edition, 2017

• Harsh mohan. Text book of Pathology, 8th edition.

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