B Kon 2020 Souvenir

B - Kon 2020 - DIAGNOSTIC DILEMMAS IN WBC DISORDERS
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B-Kon 2020
DIAGNOSTIC DILEMMAS IN WBC DISORDERS
Organised by:
Department of Pathology
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DEPARTMENT OF PATHOLOGY MEDICAL COLLEGE KOTTAYAM
CHAIRMAN - DR. S. SANKAR

ORGANISING SECRETARY - DR. SHEEJA S.
JOINT ORGANISING SECRETARY - DR. LEKSHMIDEVI P.
TREASURER - DR. DEEPA S.
SCIENTIFIC COMMITTEE TRANSPORT AND ACCOMODATION

DR. PRIYA.P.V DR. ABHILASH.K.K
DR. RENU THAMBI DR. NIMMI ANDREWS
DR. VEENA R NAIR DR. ARUN JOSE
DR. ATHEETHA R. DR. ROSHAN A MATHEW
DR. JASEELA .T.K DR. RINKU SUSAN
DR. SREELEKSHMI.S.N DR. ALEESHA TERESA
DR. LIZ MARIA JOSEPH SRI. PRAMOD.S
SRI. GIREESH K.THYKOODAN SMT. USHA .S
SRI.UNNIKRISHNAN
RECEPTION FOOD AND REFRESHMENTS
DR. LETHA V DR. CICY.P.J
DR. LEKSHMI DEVI P DR. SAJI MARY VARGHESE
DR. ASWATHY SHIBU DR. DEEPTHI MARY SOBHA
DR. ANN MILI KURIAKOSE DR. PARVATHY.V
DR. HIBU YAMI DR. JISHA RAJ
DR. REJILA. R SMT. CHINCHU RAJAN
DR.SILPA .S RAJ SMT. SARIGA SIVADAS
DR. ANN MARIA SUNNY SMT. USHAKUMARI.P.C
DR.ASWATHY.P SMT. SOUDA BEEVI.
SMT. PREETHA.P.NAIR MODERATOR PANEL DISCUSSION
SMT. ANITHA ELIZABETH DR.JAYALEKSHMI P L
SMT. SUJA K.P QUIZ
SMT. SUSAN VARGHESE DR.LILLYKUTTY POTHEN
SMT. SOBHANA.P.S DR.NEETHA UNNIKRISHNAN
SMT.KUMARI.N.C FINANCE COMMITTEE
SMT. SHEEJA CHACKO DR. RAJAN G
VENUE & AUDIO VISUAL AIDS DR. DEEPA.S
DR.ANJU.C.K DR. ANJU.S
DR. SAJI MARY VARGHESE DR. SEENA SUSAN
DR.VEENA R NAIR SMT. SARAMMA JOSE
DR. SURYA GAYATHRI .V SMT.SUSAN VARGHESE
DR. ALVIN VINCENT COMPARING
DR. ANN ROSE THOMAS DR. LIZ MARIA JOSEPH
SRI.ANILKUMAR DR ANN MARIA SUNNY
SRI.AKHINKUMAR.T.K. DR ANJU S.
SMT. LISSY JOSEPH DR ALEESHA THERESA
VALEDICTORY FUNCTION
DR.ANN ROSE THOMAS
DR. LIZ MARIA JOSEPH
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PRINCIPAL’S MESSAGE
It gives me immense pleasure and pride to be a part of the development, growth and
academic achievement of Government Medical College, Kottayam. The annual CME
on haematopathology - The Annual CME on haematopathology
B-KON 2020 organized by the Department of Pathology has great
significance in the context of changing trends in haematological diseases in our
country. I hope this CME will appropriately direct the Post Graduates and practicing
Pathologists in understanding haematological diseases. It needs special mention that
the staff and students take great effort in bringing out the handout of the topics being
discussed. I congratulate the department of pathology for their effort and wish them
the very best all the success of the programme.
PRINCIPAL
GOVT. MEDICAL COLLEGE,
KOTTAYA M
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MESSAGE FROM ORGANISING DESK
Dear Colleagues,
We are honoured and pleased to welcome you to B-Kon 2020 - the annual CME on Hematopathology,
organized by Department of Pathology, Government Medical College, kottayam. The objective of the CME
is to improve the competence of post graduates and practising pathologists in the diagnosis of hematological
disorders.
PRINCIPAL’S
We are extremely happyMESSAGE
to announce that this is our third CME of the series. B - kon 2018 dealt with
basic concepts of hematology and B-Kon 2019 about disorders of red blood cells. This year we are dealing
with the diagnostic problems in WBC disorders. Each topic will be presented and discussed by eminent
academicians and experienced hematopathologists. B-kon 2020 is conducted as a webinar.
We whole heartedly welcome all the delegates. Hope all of you will benefit from this academic feast.
The theme for B-Kon 2021 is platelet and coagulation disorders . Looking forward to see you for the
same.
Dr. S. Sankar Dr. Sheeja S.

Organizing Chairperson Organizing Secretary
Professor and HOD Associate Professor
Dept. of Pathology Dept. of Pathology.
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INDEX
Leucocyte Morphology in Blood Smears : Dr. Joy Augustine
Interpretation of WBC Histograms and Scattergrams : Dr. Feroze M.
WBC Disorders - when to study and what to look : Dr. K. Gayathri
in the marrow
The basic Principles of Flowcytometry : Dr. Geetha Vidyadharan
Approach to B-Cell Lymphomas - Flow cytometry : Dr. Linda Rose
How to make a good Smear : Smt. Vijayalekshmi S.
Cytochemical Studies in the Study of Acute Leukemia : Smt. Nisha Kumari K.R
Microvessel Density Among the Morphological : Dr. Parvathy V.

Types of Multiple Myeloma
Latent Membrane Protein 1 Expression in : Dr. Jaseela T.K.

Non Hodgkin’s Lymphoma
Panel Discussion
Snippets
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LEUCOCYTE MORPHOLOGY IN BLOOD SMEARS
Dr Joy Augustine MD, DCP.

Professor and Head, Pathology,
AIMS, THRISSUR, Kerala.
Introduction
Not every complete blood sample that is tested will require a blood smear examination. The most common
reasons for a blood film to be made are given below: A blood film is made when morphologic features of cells
are detected and flagged as abnormal by the automated counter. Or sometimes the clinician suspects some
specific condition eg: suspected leukaemia or malaria.
Basic Principles of Smear preparation have to be followed in terms of :
1) Blood sample collection 2) Preparation of the slide 3) Staining of the slide
The smear can now be reviewed microscopically with a thin film of oil for lower magnifications and a
larger drop for oil immersion magnifications. Alternatively, a cover slip can be mounted on the slide either
temporarily with oil or permanently with mountant (recommended).
Microscopic assessment of WBCs
When discussing white blood cells, 5 normal subtypes are involved: neutrophils, lymphocytes, monocytes,
eosinophils, and basophils. Their precursors including blasts have to be identified and quantified. Morphologi-
cal heterogenicity of these immature forms may pose difficulty in categorization. Atypical lymphocytes may
sometimes mimic other types of cells including blast cells.
There are a large number of artefacts that may mislead the pathologists. It would be appropriate to recognize
them.
Neutrophils
Size - Neutrophils are approximately 12 to 15 µm in diameter and generally do not vary in size. Variation in
size may be seen in sepsis and is usually associated with toxic granules and vacuolation. Some of the newer
cell analysers have incorporated this as a new parameter (NDW and MDW= Neutrophil and Monocyte
Distribution Width respectively). Large hypersegmented neutrophils are seen, called macropolycytes and can
be seen in megaloblastic anemias, rarely in normal individuals and as a medication effect. Neutrophils have
multilobed nuclei connected by thin chromatin bridges. The chromatin pattern is clumped and normally there
are 3 to 5 nuclear lobes. Two lobes or less are considered hypolobulated or pelgerized, which is known as
Pelger Huet Anomaly(inherited). Acquired or Pseudo Pelger Huet forms may be seen but can be distin-
guished from the inherited variant, by a smaller percentage of neutrophils showing the characteristic abnor-
mal features, a possible associated neutropenia with a reduction in neutrophil granules, and the absence of
mononuclear Pelger Huet forms. The acquired or pseudo Pelger-Huet anomaly has been noted in the
myelodysplastic syndromes, acute myeloid leukemia, chronic myeloid leukemia, idiopathic myelofibrosis, and
myeloma. Six or more lobes, or 3% to 5% with 5 lobes are considered hyperlobated, and indicates B12/folate
deficiency, MDS, and is rarely seen in reactive neutrophilia.
Cytoplasm-The cytoplasm of neutrophils is pinkish with fine reddish granules. They are MPO, CAE, PAS
and NAP positive. Toxic granules (Primary granules, dark red and purple), along with vacuolization and
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Dohle bodies, can be seen in toxic states like pyogenic infections, inflammation, trauma and G-CSF adminis-
tration. Vacuolization is more significant than toxic granules in diagnosis of sepsis. It is important to be aware,
however, that artifactual vacuolization may occur in older (stored) blood specimens collected in EDTA. This
vacuolation and pyknosis of neutrophils are degenerative changes that may be mistaken for toxic changes,
and a diagnosis of infection or inflammatory condition may be suggested. In these cases, the absence of dark
staining granules points to the correct interpretation. Dohle bodies without the other toxic changes is seen in
May Hegglin Anomaly and pregnancy. Granules, very similar (but larger and darker) to toxic granules are
seen in Mucopolysaccharidoses and termed Alder Reilly anomaly. Abnormal large granulation can be seen in
hereditary disorders such as Chediak–Higashi syndrome. Pseudo Chediak-Higashi like granules, has been
described in MDS and acute myeloid leukemia (AML M2 variant) with giant granules within the granulo-
cytes, morphologically similar to those of the hereditary form. A variant type of promyelocytic leukemia with
‘giant Chediak-Higashi’ like granules has also been described. Hypogranulation is the absence of granulation,
and in fact the cytoplasm may have the same color as the background. This can be seen in MDS. It is
important, not to mistake light staining of neutrophils for hypogranulation. In some cases, the granules of
lightly stained neutrophils will not be seen but the cytoplasm will have a pinkish color. Some neutrophils will
have a corner of hypogranular cytoplasm on a background of normally granulated cytoplasm, which is not
pathological.
Other inclusion bodies- Aside from the inclusion bodies such as Dohle bodies and the large granulation in
Chediak-Higashi syndrome, infectious inclusion bodies can be seen in neutrophils and Monocytes. This could
include bacteria (Meningococci, Clostridia) and fungal elements (Histoplasmosis, Candida), Rickettsiaceae
(Ehrlichia) and protozoans (Malaria, Leishmaniasis). The presence of intracellular organisms on a blood
smear usually portends a grave prognosis, because the finding correlates with overwhelming sepsis. ‘Howell-
Jolly body’ like inclusions are dark, round, uniform structures that exactly mimic the red cell inclusions for
which they are named, except that they are present in neutrophils. They have been described in 12% of
patients with AIDS, and are nearly specific for this condition.
Critical green inclusions, aka green neutrophilic inclusions . They are amorphous blue-green cytoplasmic
inclusions found in neutrophils and occasionally in monocytes. They are most frequently seen in cases of
acute hypoxic and ischaemic hepatitis and have been observed after uncomplicated liver transplantation.
They are also associated with lactic acidosis. Predicts high mortality. Other pigments, that may be seen are
the malarial pigment (Falciparum malaria - a clue to check for the parasite, more diligently), Bile pigment and
rarely melanin pigment (in disseminated melanoma).
Left shift - Immature forms of neutrophils can be seen in the peripheral blood. Starting from segmented
neutrophils and progressively becoming more immature are bands, metamyelocytes, a few myelocytes, and
promyelocytes. This may represent a reactive process such as infection or marrow infiltration (when NRBCs
are present). A bone marrow study should always be suggested when larger numbers of myelocytes,
promyelocytes or blasts are seen along with nRBCs(Leucoerythroblastosis). An increase in band forms alone
has been used in pediatric populations as an indicator for infection but does not seem to hold for adult patients.
Distribution on the slide: Neutrophils are occasionally seen in aggregates in blood samples which have
been stored for a long period prior to making the blood films. This feature can also be seen in an infectious
process and immune disorders. It is not generally clinically significant. Pseudoleukopenia may be seen when
blood has been subjected to prolonged storage at room temperature or has undergone cell lysis for other
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reasons. Lysis has been associated with leukemia (especially chronic lymphocytic leukemia), uremia, and
immunosuppression.
Lymphocytes
Size- Lymphocytes are approximately 1 to 1.5 times the size of normal red blood cells, 10-12µm. Larger
lymphocytes (12-16 µm) can be seen in atypical or reactive lymphocytosis or large granular lymphocytosis.
Nuclei- Lymphocytes are mononuclear. Mature lymphocytes have a round nucleus with clumped chromatin
and generally have a uniform shape. Atypical lymphocytes having significant pleomorphism in nuclear size
and shape with altered cytoplasmic staining and contours are seen in viral infections notably IMN. Nucleoli
may be seen. Nucleoli alone are not pathognomonic for malignant lymphoid cells because such cells may be
seen in small numbers in reactive states. Nuclear clefting is unusual in normal lymphocytes and should
arouse suspicion of a lymphoproliferative disorder; however, smokers may have cleft or bilobed lymphocytes.
Complex nuclear folding or nuclear lobation should arouse suspicion of lymphoproliferative disorders. Nuclei
are generally found centrally in lymphocytes. Large Plasmacytoid Lymphocytes with eccentric nucleus and
deep blue cytoplasm is often seen in Malaria and in viral infections. Plasmacytoid lymphocytes may be seen
in large numbers in high-protein states like Waldenstrom’s Macroglobinaemia.
Cytoplasm-Mature lymphocytes generally have scant, pale, purple–blue cytoplasm and have a high nuclear:
cytoplasmic (N: C) ratio. Reactive lymphocytes may have a slightly lower N: C ratio and a darker blue
cytoplasm. These lymphocytes can be seen in infections and inflammatory states. Atypical lymphocytes
generally have a low N: C ratio with a spread out, dark blue or water clear cytoplasm. The cytoplasm is
denser in the periphery in some cells (skirting). Several types of atypical lymphocytes are described (often
referred to as the ’Downey’ type cells). These generally represent viral infections, classically infectious
mononucleosis (EBV), or CMV and rarely an acute seroconversion of HIV infection. The minimal morpho-
logic criteria for IMN as proposed by Horwitz et al are (1) mononuclear cells (lymphocytes and monocytes)
comprise greater than 50% of leukocytes (2) reactive lymphocytes represent greater than 10% of leukocytes
(not of lymphocytes); and (3) there is marked lymphocyte heterogeneity. When these criteria are met, most
patients (90%) are found to have EBV infection. Frequently, atypical lymphocytes have a spread out cyto-
plasm that invaginates around adjacent red blood cells (Molding); however, this phenomenon can be seen
occasionally in lymphoproliferative disorders as well.
Note: Atypical lymphocytes in significant numbers, in patients above 40 should be viewed with suspi-
cion and at the very least; the patient should be followed up. Large granular lymphocytes (LGL), both
reactive and clonal, have a pale gray cytoplasm with low or moderate N: C ratio, with a few scattered reddish
fine granulation. Significant numbers may be seen in hyperactive immunological states (SIRS) and in some
autoimmune diseases. Hair-like projections may be seen in euplastic lymphoid cells. Note: Unidirectional
polar cytoplasmic projections on the majority of lymphocytes may be an artifact. Lymphocytes are
MPO, and CAE negative. ANAE is positive in T Lymphocytes.
Smudge cells: Smudge cells represent disintegrated cells. Although these may represent lymphocytes, other
cells may smudge as well. Smudge cells can be seen in small numbers on normal blood films.
Lymphoproliferative and leukemic disorders and occasionally reactive processes may have increased num-
bers of smudge cells as well, but should have other features suggestive of these processes.
Inclusion bodies: Immunoglobulin deposits may be seen in the cytoplasm of plasmacytoid lymphocytes in
high-protein states. Inclusion bodies can be seen in the cytoplasm of lymphocytes in hereditary disorders such
as Chediak–Higashi syndrome. Vacuolization of the cytoplasm could be seen in inherited metabolic disorders.
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Monocytes
Size-Monocytes are the largest normal leucocyte and measures approximately 12 to 20 µm in diameter. They
generally do not have significant size variation. Nuclei - Monocytes are mononuclear. However, their nuclear
shape is generally a kidney shape, horseshoe-like, thick band. The chromatin is finely clumped. In some cases
the nucleus folds in on itself, giving the appearance of a mononuclear shape. Dysplastic monocytes may have
abnormal nuclear segmentation or lobation. Monocytes with more open chromatin, round/ oval, folded or
convoluted nuclei, and nucleoli should be precursor of monocytes, which are counted as blasts in monocytic
leukemias.
Cytoplasm-The monocyte cytoplasm is grayish-purple and spread out. A low N: C ratio is seen. Cytoplasmic
vacuoles are noted with occasional fine reddish granules. Inclusions may be seen such as infectious agents
(bacteria-Meningococci, Clostridia, and fungal elements-Histoplasmosis, Candida, Rickettsiae and protozo-
ans like Malaria, Leishmaniasis ) vacuoles in inherited metabolic disorders, and occasionally erythrocytes.
Sometimes Histiocytes and macrophages are seen in a peripheral smear. They are larger than Monocytes
and may contain prominent cytoplasmic vacuoles or phagocytized cellular elements, debris, or organisms.
However monocytes may also contain phagocytized material, and therefore cannot be used as a distinguish-
ing feature between monocytes and histiocytes. Monocytes demonstrating phagocytic activity in blood, how-
ever, probably carry the same significance as circulating histiocytes. Circulating macrophages are usually
restricted to the terminal (feathered) edge of the blood smear because of their large size. Circulating histio-
cytes have been described in as many as 20% of patients with subacute bacterial endocarditis, and this
finding used to be touted as being highly specific for the diagnosis. They are ANAE and PAS positive. MPO
and CAE are negative.
Eosinophils
Size - Eosinophils are approximately 12 to 16 µm in diameter. They generally do not have significant size
variation. Giant Eosinophilic precursors are documented in Megaloblastic anaemia.
Nuclei- Eosinophils normally have 2 to 3 nuclear lobes. The nuclei demonstrate clumped chromatin con-
nected by chromatin bridges. Hyposegmented eosinophils have 1 lobe and can be seen in Pelger Huet anomaly,
MDS, and myeloproliferative disorders. Hypersegmented eosinophils have 4 or more lobes. This feature can
be seen in both reactive (Megaloblastic Anaemia) and neoplastic conditions. Occasionally, ring nuclear forms
can be seen and occurs in both reactive and neoplastic disease. Cytoplasm: Eosinophil cytoplasm contains
large, chunky, orange granules. They are MPO positive. PAS and CAE are negative in normal eosinophils.
Eosinophils may degranulate in the peripheral blood. This feature can be seen rarely in reactive and fre-
quently in neoplastic states. Purplish/orange granulation can be seen in eosinophils present in AML-M4Eo
associated with inversion/translocation of chromosome 16. PAS and CAE are positive in these eosinophils.
Abnormal inclusion bodies can be seen in hereditary disorders such as Chediak–Higashi syndrome.
Basophils
Size- Basophils are approximately 10 to 14 µm. They generally do not have significant size variation.
Nuclei- Basophils normally have 2 to 3 nuclear lobes. The nuclei demonstrate clumped chromatin connected
by chromatin bridges and are commonly obscured by the heavy basophil granulation.
Cytoplasm- Basophil cytoplasm contains large, dark purple/black granules. Basophils may degranulate in the
peripheral blood. This feature can be seen in both reactive (eg, allergic reactions) and neoplastic states.
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Degranulating basophils can sometimes be mistaken for neutrophils with toxic granulation. Unlike neutrophils,
they are MPO negative. PAS and CAE are positive.
Morphology of Neoplastic WBCs
Blast cells may be Myeloblasts, Monoblasts, Megakaryoblasts, Early Erythroblasts, Proerythroblasts
Lymphoblasts, Plasmablasts and Other abnormal cells/mononuclear cells including abnormal Lymphoid cells.
No single characteristic identifies a blast. In general, blasts are cells that have a large nucleus, immature
chromatin, a prominent nucleolus/nucleoli. They may have, abundant/moderate/scant cytoplasm and few or
no cytoplasmic granules. Blasts will have these features in varying combinations.
Cell size - Blasts are often medium to large cells. They are usually larger than a lymphocyte and at least the
size of a monocyte. NOT ALWAYS. L1 type blasts may look like lymphocytes and Monoblasts may be very
large. Also remember that, Megakaryoblasts may be small and can resemble Lymphoblasts.
Large nucleus - Most of the cell is taken up by the nucleus (sometimes a high nuclear to cytoplasmic ratio).
Immature chromatin - The nuclear chromatin looks as if it composed of fine dots. One can visualize this
chromatin as many tiny points (of light shining through) made by the tip of a sharp pencil on a piece of paper.
Monocyte chromatin is more linear and sometimes dark, looking like smudged pencil lines. Lymphocyte
chromatin looks to be done in heavy crayon. The point to remember is that the blast cell nucleus is not usually
opaque and allows faint light to pass through between the chromatin materials.
Prominent nucleoli may be seen in blasts. Not always in lymphoblasts, especially in the lymphoblasts with
L1 morphology. Punched out nucleoli are frequently the feature in Myeloblasts and Monoblasts. Nucleoli with
indistinct margins are frequently seen in Lymphoblasts. A single central nucleolus is a feature of prolymphocytes
in PLL.
Conformable nuclear membrane - The nuclear membrane often conforms to the shape of the cytoplasmic
membrane. The nucleus appears squishy. A normal lymphocyte nucleus appears dark, sharp and rigid.
Scant to moderate cytoplasm is the rule. Not always. Monoblasts may have moderate to abundant cyto-
plasm. Granules may be seen in Myeloblasts. Few to no cytoplasmic granules - blasts usually lack the numer-
ous granules seen in mature granulocytes. Occasionally fine dark granules may be present. Acute
promyelocytic leukemia is one exception. In Promyelocytic Leukaemia (APML/ APL), the cells may have
granules in plenty and may even mask the nuclei. The variant APML may appear agranular, but are strongly
positive with MPO. Auer rods - orange-pink, needle-like cytoplasmic structures in blasts of myeloid lineage.
These may be several in acute promyelocytic leukemia (APL). Prominent granules may be seen in other
myeloid leukaemias. Eg: AML M2, AML associated with MDS. In AML M2 variant, large Chediak Higashi
like granules may be seen. Multiple Auer rods may be seen but are usually very thin.
Other types of leukaemias may be associated with nuclear and cytoplasmic alterations. Hairy processes are
seen in Hairy cell Leukaemias. Cells are small / intermediate size cells with moderate pale gray cytoplasm,
cells have circumferential hair-like and short, blunt cytoplasmic projections, round / oval nuclei with smooth
nuclear borders, stippled chromatin, occasional nucleoli. They are TRAP positive.
In SLVL -When the peripheral blood is involved in Splenic Marginal Zone Lymphoma (SMZL), the neoplastic
cells usually, although not always, have short villous projections localized to one or both poles. They are
TRAP Negative. Sometimes lymphoplasmacytoid features may also be seen.
Lymphomas sometimes spill over into the peripheral blood. The cells may not have the features as described
relating to blast cells. They may resemble lymphocytes (CLL/SCLL), may be larger or very large, with deep
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clefts (referred to as “Butt cells”) seen in Follicular Lymphomas. The chromatin is however usually dark and
‘mature’ looking. Lymphoid cells with anisocytosis and abnormal lobulations (“Florette” cells) may be seen in
ATCL. So also, cerebriform type of nuclei is seen in Sezary syndrome. In the Large cell lymphomas, circulat-
ing cells may have resemblance to the cells in the original Lymphoma, and may have bizzare nuclear contours
with blastoid chromatin. Rarely Carcinoma cells may be seen. Neuroblastoma cells may sometimes be seen
and may mimic a Leukaemia. Other alien cells seen in a blood smear includes endothelial cells and squamous
cells.
A recent useful article is given below; good to read and is open access
Morphological and Immunophenotypic Clues to the WHO Categories of Acute Myeloid Leukaemia
Bain B.J. Béné M.C. Acta Haematol 2019; 141:232–244
Artifacts in Blood Smears

Morphological alterations can occur on storage, Smearing and Staining. As the EDTA blood stands in the test
tube changes in leukocyte morphology begin to take place. The need of the hour is to have knowledge about the
artifactual changes which can mimic various mild to serious disorders hence giving false/biased information to
the clinician.
Nuclear Changes: Initially lobulations (when lymphocytes show nuclear lobulation, it can be misinterpreted as
Lymphoma cells) were observed as early as 2 hrs followed by nuclear degeneration, karyolysis, nuclear vacu-
olations and nuclear rupture. The nucleus of the neutrophil may become clumped and appear round; and this
may mimic a NRBC.
Cytoplasmic Changes: cytoplasmic vacuoles. Membrane morphology may be altered: hairy projections, blebs.
Hairy projections may be circumferential or polar. In case the hairy projections are polar, check if it is unidirec-
tional (artifact). Cytoplasmic vacuolation of neutrophils and Monocytes mimics infection/sepsis (Pseudotoxic
changes). A Variety of “PSEUDO” changes have been documented in Literature: Pseudo Pelger-Huet anomaly,
Pseudo Chediak-Higashi granules, pseudoleukopenia, Pseudoleukocytosis etc.
IMPORTANT: Please remember that the WBC morphology and count should never be interpreted in isola-
tion, as a ‘STAND ALONE’ feature. In the smear, RBC and platelet morphology should be used in tandem in
the final opinion.
A Morphologic feature or multiple features in the Blood Smear sometimes point to specific diag-
noses. Below is a limited list of some of them:
1.Nucleated red blood cells and left-shifted neutrophils (myelocytes and more immature forms):
Leukoerythroblastic picture : This may represent a reactive picture in extreme blood loss, hemolysis, trauma,
or infection or could represent marrow infiltration. When combined with teardrop cells,
this may represent the blood picture in Primary Myelofibrosis.
2. Nucleated red blood cells and Pancytopenia – Acute Bone Marrow Neoplastic disease - AML,
Acute Myelofibrosis, MDS. Check for large spleen. May be seen in Hypersplenism
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Pancytopenia with circulating Blast cells points to Leukaemia or MDS

Leukopenia with selective neutropenia may be seen in Viral infections, Kikuchi Disease
3. Neutrophilia with significant left shift, eosinophilia, or basophilia, with or without thrombocyto-
sis -Think of a Chronic Myeloproliferative neoplasm . If Basophilia is striking CML should be considered
seriously in the Differentials.
4. Oval macrocytosis with hypersegmented neutrophils: B12/folate deficiency, medications affecting
folate metabolism, and rarely in myelodysplastic disorders.
5. Mild–moderate thrombocytopenia with large platelets and neutrophils with Dohle bodies and
no other toxic changes- Hereditary thrombocytopenic disorders- May–Hegglin anomaly.
6. When large numbers of bacteria are seen extracellularly – Sometimes the bacteria may be intrac-
ellular; check if the blood sample is old or stored before making a decision on it. Intracellular bacteria
are relevant in rare situations only: Meningococcemia, Septicemias, and certain specific rare infections.
7. Atypical Lymphocytes; As per Literature (DC)12% Atypical Lymphocytes and above in the Total
Count, is considered abnormal. However, this may not hold true, for older patients. Atypical lymphocytes in
patients above 40, should be viewed with suspicion; above 60 years, very seriously. In an older patient if,
Malaria, Dengue and Chikungunya are excluded, the possibility of a lymphoma/Leukemia has to be consid-
ered and excluded. In younger individuals, if afebrile, ideally a connective tissue disorder has to be excluded.
The Final Report
Once a Smear has been assessed morphologically, a report is required to be issued. There are different ways
to approach this task. The major emphasis of issuing a report is to be able to impart the significant findings of
the blood film in words to those individuals reading the report, who have not seen the blood film themselves.
The report should be clearly worded and concise. It should not be assumed that the reader of the report has
knowledge of the clinical significance of all the morphologic changes being described and, therefore, attach-
ing a comment on the possible clinical significance becomes extremely important.
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INTERPRETATION OF WBC HISTOGRAMS AND SCATTER GRAMS
Dr.Feroze M
Professor Pathology,
Govt Medical Col-
lege, Kozhikode
Blood cell counters have become an essential part of any standard medical laboratory. The primary advantage
of a counter is that it gives accurate blood counts. Visual counts, in addition to technical errors, have always
had an element of inherent error which can be eliminated only by counting large number of cells. The inherent
error in an RBC count is 4.3% when the usual number of 450 red cells is counted in a Neubeuer counting
chamber. It is > 10% in WBC and platelet counts. This error is less than 1% in a blood cell counter, as it counts
more than 10,000 cells.
Differential count is the real challenge in a blood cell counter. It is made possible by noting the cell volume using
aperture impedance principle. It also looks at white cell internal constituents by a variety of methods like light
scatter at different angles, high frequency alternating current, and fluorescent signals. Various combinations
are also used by different blood cell counters to increase the accuracy of differential count.
Most blood cell analyzers are 3 part differential analyzers that differentiate WBCs into three subpopulations. 5
part differential analyzers differentiate WBCs into 5 major subpopulations.
Principle: The 3 part blood cell counters work in two ways.
1) Aperture impedance method: Blood is diluted in a buffered electrolyte solution and passed between two
electrodes. There is a continuous flow of current. Blood being
a poor conductor of electricity, it produces impedance of electric flow when blood cells pass through the
aperture. This is counted electronically.
2) Light scatter method: Laser beam is used as the light source and cells pass through focused light. The
light scattered by the cells as they pass is detected by the photo detector and counted electronically.
The size of the pulse is proportional to the size of the cell. The size of the pulse is used to discriminate the
cells of interest from the other cells by a process called thresholding. The thresholds are set as follows:
platelets 2 to 25fl; red cells 25 to 250fl and white cells 30 to 300fl. Red cells and platelets are counted
together in the same chamber while white cells are counted in a separate chamber after lysing the red
cells. This is done because the thresholds of red cells and white cells overlap.
In a 3-part differential blood cell counter, WBCs are discriminated into small, middle and large WBCs using
4 discriminators. They are the lower and upper discriminators at 30fl and 300fl respectively, and the two
troughs called T1 and T2. The small cells represent lymphocytes, middle cell population includes monocytes,
eosinophils and basophils, and large cells are neutrophils. Some instruments, however, count all granulocytes
together as large cells which make it difficult to differentiate eosinophils from neutrophils. The normal
histogram
of a 3 part differential analyzer is given below.
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In a 5-part instrument, in addition to aperture impedance, the principle of ow cytometry is employed to differentiate
WBC into their five major sub-populations. The cells are forced to ow in a single file through the aperture by
a sheath uid, created by a fast- moving diluent that surrounds the slow moving sample as shown in the figure
below
A laser beam is passed through the sample and the scattered light is measured by a photoconductor. The
number of impulses generated correlates with the number of cells, whereas the light scatter is used to determine
cell size, shape and granularity.
3D analysis is done using information from scatter of laser light and fluorescence signals. Low-angle light
scatter or forward scatter reflects cell size while the high angle light scatter or
side scatter reflects intracellular granularity. Intensity of fluorescent signal reflects degree of cell staining.
The fluorescent signal strength is proportional to individual cell RNA/DNA content. By sensing the difference
in signals in 3 dimensions of the cells, the WBCs are differentiated into 5 major subpopulations.
Lymphocytes are small, have high nucleocytoplasmic ratio, low nucleic acid content and so occupy lower
position in the direction of fluorescence as well as side scatter. Monocytes are larger in size, have high
nucleocytoplsamic ratio and high nucleic acid content, and less complex in structure. They are at a higher
position in the direction of fluorescence, and have stronger side scatter. Neutrophils and basophils are
larger in size, have medium nucleocytoplasmic ratio and low nucleic acid content. They are at a lower
position in the direction of fluorescence, but have stronger side scatter. Eosinophils are similar to neutrophils,
but contain a lot of alkaline grains, so have very strong side scatter.
In a 5 part differential blood cell counter the WBCs are counted in two separate channels. In the first one,
WBCs are separated as lymphocytes, monocytes, eosinophils and neutrophils plus basophils. The normal
scatter gram of this channel is given below.
16
Basophils are then counted in another channel using light scatter only.
Blast cells, atypical lymphocytes and immature granulocytes have high nucleic acid content, and so are at a
higher position in the direction of fluorescence on the scatter gram. Fluorescence flow cytometry enables the
identification of immature cells on the basis that they have higher nucleic acid content in comparison with their
mature counterparts. This has enabled the generation of a 6-part differential count by addition of immature
granulocytes which includes promyelocytes, myelocytes and metamyelocytes but not band cells.
Nucleated red cells occupy a position below mature lymphocytes and the lysed red cells are represented as
ghost cells as shown in the figure below.
Histograms and scatter grams Histograms are representation of separation of RBCs,

WBCs and platelets calculated by using the range of thresholds as mentioned earlier which
includes the differential count of WBCs into 3 subpopulations. Analysis of the histograms gives us an
opportunity to detect the abnormalities in the respective cell groups. Scatter grams represent the counts of
blood cells and the differential count of WBCs into 5 major subpopulations. The normal histogram and
scatter gram has already been discussed along with the principle of the respective instruments.
Abnormal WBC histograms and scatter grams
Two commonest abnormalities of immense clinical significance are neutrophilia and lymphocytosis with
atypical lymphocytes. This will answer the question of a viral infection or a bacterial infection, the latter
being treated with antibiotics. In neutrophilia, WBC histogram shows the neutrophil peak overlapping with
the mixed cells and the range of T2 is above the normal frequency. This signifies neutrophilia with a shift
to left as is shown below.
17
The scatter gram in such a case will show an increase in neutrophil fraction with cells extending to the left shift
region as is shown below.
In a case of atypical lymphocytosis, there is a high range of T1 with additional lymphocyte peak. T2 usually
cannot be determined. Even when such a histogram, as shown below, is highly suggestive of atypical
lymphocytosis, it can also be found in eosinophilia or monocytosis.
In such cases, the scatter gram gives a much better picture. There is an increase in lymphocyte fraction with
atypical lymphocytes having a higher position in the direction of fluorescence, since it has high nucleic acid
content, as is shown below
.
Unlike the above histogram, there can be situations in which the T2 cannot be determined and the mixed cell
peak overlaps with the large cells. This is the appearance in cases of monocytosis, eosinophilia, basophilia,
plasmacytosis or presence of blasts as is shown below. A 3-part differential analyzer is unable to differentiate
between the above conditions which is considered as a major limitation of such instruments.
18
A 5-part differential counter can identify many such cases as is shown in a case of eosinophilia.
Normoblasts in the peripheral smear can interfere with the WBC count and so is important to be identified
in the histogram. Normoblasts increase the frequency of lower discriminator and appears as debris below
the lower discriminator.
A 5-part differential analyzer counts normoblasts separately in the NRBC channel and automatically
corrects the WBC count and 5 part differential counts. It can also be picked up in a scatter gram in which
it occupies a position below the lymphocytes, as shown below.
The classical histograms of acuteleukemia and chronic myeloid leukemia in a 3 part differential counter are
given below. Acute leukemias show a monotonous population of cells in the region of lymphocytes without a
distinct T1 or T2. It is not possible to differentiate acute lymphatic leukemia from acute myeloid leukemia.
In chronic myeloid leukemia, the scatter gram shows an increase in neutrophils with many immature
granulocytes having a higher position in the direction of fluorescence, as is shown below.
19
Chronic myeloid leukemia shows increase in neutrophil peak overlapping with middle cell population because of
the presence of a significant population of immature cells. There is an absence of T1 and T2.
The scatter gram of acute leukemia will show marked increase in cells occupying the lymphocyte and monocyte
region. The blast cells, which contain an increase amount of nucleic acids, are identified by its higher position in
fluorescent scale as shown below.
In chronic myeloid leukemia, the scattergram shows an increase in neutrophils with many immature granulocytes
having a higher position in the direction of fluorescence, as shown below
The 5 part scatter grams can be very contributory in cases of myelodysplastic syndrome with excess
blasts. The 3 part histogram will show only an increase in large cells that overlap with mixed cells with the
absence of T2. This is the same picture as seen in monocytosis, eosinophilia or basophilia. However, in the
scatter gram, presence of immature granulocytes can be identified by its higher position in the fluorescent
scale as shown below
20
From the above illustrations, it can be made out that the histograms of the 3 part differential analyzers can
detect many abnormalities, but have limitations in a few other cases. The 5 part differential analyzers can
diagnose abnormalities in the counts of 5 major subpopulations of WBCs and presence of normoblasts, and
also give important clues towards other abnormalities including presence of immature granulocytes.
3 - part or 5 - part?
Although information on all 5 subtypes of WBCs helps in the diagnosis of blood related disorders, a 3-part
differential analyzer would be sufficient for basic illnesses coming to a primary health center or for general
screening and pre surgical screening. For specialty clinics, however, a 5-part differential analyzer would
provide a more detailed and targeted assessment of the blood status.
The cost of a 5-part instrument can be five times higher than that of a 3-part instrument. A 5-part differential
blood cell analyser requires more reagents than a 3-part differential, thus increasing the cost per test. Also, a
3- part differential instrument, which is based on impedance technology, might require less maintenance than
a 5-part instrument, which uses a more sensitive laser-based and fluorescent measurement
technology. Hence, it is evident that while 5-part analyzers can offer more detailed information on the white
blood cells, 3-part instruments can offer great cost benefits.
Even when 3-part differentials provide excellent precision for general
screenings, accuracy is improved with 5- part differentials for abnormal samples, reducing the number of
manual blood smears. An important limitation of a 3 part instrument is that it is unable to differentiate
eosinophilia from monocytosis or basophilia, all of which showing an increase in middle cell population.
And in instruments that count all granulocytes as large cells, a blood smear would have to be studied to
diagnose eosinophilia. The presence of atypical lymphocytes or immature granulocytes also will flag the
sample, making a blood smear examination mandatory. A 5-part differential instrument can give unflagged
results in many of such cases.
It is said that about 30% of the total number of samples generates a suspicious ag with a 3-part instrument,
necessitating a blood smear examination. This may go to above 50% in specialty clinics. The more detailed
information provided by a 5-part instrument can reduce this number to less than 20%, ultimately decreasing
laboratory time and cost.
However, for most cases, the same decisions can be made with a 3-part analyzer as with a 5-part analyzer,
particularly in general clinics. If abnormal values are obtained, microscopy is still required, irrespective of
instrument type. So the answer to the question of 3-part or 5-part analyzer would finally depend on the
setting in which the instrument is used and its affordability. Regardless of the choice of a 3-part or a 5-part
instrument, of utmost importance is the ability of the analyzer to detect and flag the abnormal samples.
21
WBC DISORDERS - WHEN TO STUDY AND WHAT TO LOOK FOR IN THE MARROW
K. Gayathri.
Lifeline Tapadia Diagnostic Centre.
Hyderabad
The normal source of cellular components of the blood is the marrow in the adult, supported by the
lymphoreticular system that provides lymphocytes. Quantitative and qualitative disorders of white blood cells
(WBC), namely the granulocytes, lymphocytes and the macrophages are therefore reflected in the marrow.
Peripheral blood and smear provide vital clues to underlying haematologic disorders and must be utilised in
order to raise the index of suspicion of such disorders.
Bone marrow is readily accessible and is well studied through multiple methods and is therefore often used
for diagnostic purposes.
The study of marrow in WBC disorders can be effected by:
· Morphologic evaluation
· Immunologic assessment
· Cytogenetic studies
· Culture studies
Indications for marrow evaluation in WBC disorders:

· Numerical abnormalities: Leucopenia as well as leucocytosis along with abnormalities of red cells/
platelets will both warrant marrow evaluation especially when persistent and /or not explained by
systemic pathology.
· Abnormal WBCs: These are definite reasons for further evaluation in the direction of establishing
diagnosis –
Lymphocytosis
Blasts
Dysmorphic granulocytes
Basophilia
Myelocyte prominence with/ without leucocytosis
Monocytosis
Plasma cells circulating in blood
· Accompanying abnormalities: Red cell abnormalities as well as platelet abnormalities provide useful
pointers to marrow pathology and may be surrogate pointers to underlying WBC disorders –
Red cell abnormalities – tear drop cells, nucleated RBC in peripheral smear, with or without accompanying
WBC abnormalities can be seen in primary marrow pathology.
Platelet abnormalities - thrombocytopenia, thrombocytosis or dysmorphic platelets, with or without WBC
abnormalities, in appropriate clinical setting provide useful clues to underlying marrow pathology.
Methods for study of WBC disorders: WBC abnormalities can be picked up on peripheral blood even
when their counts are not high, by careful peripheral smear examination. When the number of abnormal
WBC is not sufficiently high, marrow evaluation is essential. Marrow can provide a larger sample of abnor-
22
mal cells and hence is valuable material for morphologic as well as other evaluations that are essential to
establish diagnosis.
· Morphologic evaluation: This is the mainstay of all WBC evaluations and can establish the broad
nature of underlying disorder. It forms the basis of choice for subsequent investigation. Marrow
cytologic and histologic evaluation is critical in identifying the nature of pathology. In addition to
WBCs marrow morphology reveals accompanying cell line and stromal morphology thus setting the
baseline for further study. Marrow cytology on aspirate smears and on biopsy imprint smears is a
reliable and rapid step to planning the next level of investigations. This must be put to robust use for
every diagnostically important situation. Marrow biopsy histology while providing support to cytologic
evaluation, adds critical information on the other cell lines as well as on the marrow stroma, not to
mention the bony trabeculae. Non haematopoeitic infiltrates, granulomas, vascular abnormalities can
be picked up on trephine biopsy sections. Staging of lymphoreticular and solid organ malignancies is
possible on the biopsy material.
· Immunologic evaluation: A cell rich suspension is ideal for the identification of an abnormal cell
population and characterise their lineage. Marrow aspirate which is such a cell suspension is highly
informative for the detection of clonal WBC disorders. Flowcytometric evaluation is the most
appropriate investigation in the diagnosis and in monitoring residual disease for clonal WBC disorders
and is employed in the management of acute leukemias. PNH is another disorder that affects all cell
lines for which flowcytometry is of diagnostic use. Trephine biopsy material is another source of
immunologic evaluation through immunohistochemical staining.
· Cytogenetic studies: Karyotyping and chromosomal studies are useful in categorising and classifying
several disorders and are required as per current diagnostic criteria. Marrow aspirate provides the
best cell population for chromosomal studies.
· Culture studies: PUO associated with WBC abnormalities indicate the usefulness of marrow aspirate
in establishing the nature of infection. In an appropriate clinical situation with leucopenia/ pancytope-
nia this is useful in the investigation of chromosomal breakage/ repair disorders such as Fanconi
anemia.
Summary: There are several indications as well as several manners in which marrow evaluation is
relevant, mandatory and useful in WBC disorders.
However marrow evaluation does not provide useful information in the evaluation of functional
abnormalities of WBC.
Relatively easy access of marrow, the simplicity of the procedures and the multiple manners in
which the material obtained can be studied, are some of the reasons why the marrow is the most
studied tissue. Also the familiarity with this tissue among pathologists makes it a useful diagnostic
material. It is easy to handle, is amenable to use with several techniques and is a rich source of
diagnostic and prognostic information.
Marrow continues to be a fascinating cell rich tissue that gives diagnostically critical information.
23
THE BASIC PRINCIPLES OF FLOWCYTOMETRY
Dr. Geetha Vidyadharan

Associate Professor
Dept. of Pathology
Amrita Institute of Medical Sciences
Kochi
The basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be
detected, counted, very similar to the process to all the new age routine blood cell counters. Additionally here
the cells are majorly interrogated further for presence of antigens by demonstrating an antigen-antibody reac-
tion and visualising it with the help of fluorescence. The antibody fluorescence combinations being determined
by the type of laser the particular flow cytometer possesses.
Flow cyometer can measure upto thousands of particles per second, which would produce an accurate count
and the data derived therefore would be precise. The most important parameter which are captured and analysed
in flow cytometry are the forward scatter(FSC) and side scatter (SSC) of the incident laser light which falls on
the cell of interest and then the respective expression of fluorescence and by that way the type and the amount
of antigen over a cell surface a well as within the cytoplasm. Generally, FSC can detect the cell volume
whereas the SSC reflects the inner complexity of the particle such as its cytoplasmic granule content or nuclear
structure. Particles or cell sizes which could be analysed range from 0.2 to 150ìm.
Light Scatter
Forward scattered light (FSC) is proportional to cell-surface area or size. FSC is a measurement of mostly
diffracted light and is detected just off the axis of the incident laser beam in the forward direction by a photodiode.
FSC provides a method of detecting particles greater than a given size independent of their ûuorescence and is
therefore often used in immunophenotyping to trigger signal processing.
Side-scattered light (SSC) is proportional to cell granularity or internal complexity. SSC is a measurement of
mostly refracted and reflected light that occurs at any interface within the cell where there is a change in
refractive index. SSC is collected at approximately 90 degrees to the laser beam by a collection lens and then
redirected by a beam splitter to the appropriate detector.
24
Optics:
Fluorescence:
A fluorescent compound absorbs light energy over a range of wave lengths that is characteristic for that
compound. This absorption of light causes an electron in the fluorescent compound to be raised to a higher
energy level, which decays and emits excess of energy in form of photon. This transition of energy is called
fluorescence. The range over which a fluorescent compound can be excited is termed its absorption spectrum.
As more energy is consumed in absorption transitions than is emitted in fluorescent transitions, emitted
wavelengths will be longer than those absorbed.
The argon ion laser is commonly used in flow cytometry because the 488-nm light that it emits excites more
than one fluorochrome, giving a variety of antibodies and fluorescent molecule combinations available for use.
The signals emitted are picked up by system of various photodiode and many times the weak signals are
amplified by photomultiplier tube arrays situated within the flow cytometer. All of these constitute the optics.
Electronics
The processing unit which converts the optical information into digital informations for our interpretation. They
are accompanied in many instruments by sofisticated software which help in analysis of complex data .
Applications:
Diagnosis of acute leukemia
Categorisation of lymphoproliferative disorders/lymphomas and plasma cell disorders
Lymphocyte subet analysis
PNH analysis
Erythrocyte membrane study-EMA
DNA Ploidy study
HLA typing
Cytological ample-detection of malignant cells
Residual disease detection in haematological malignancies
25
Analysis for Acute leukemia:Blasts exhibit dim CD 45 and therefore a gate is constructed in this area.The
gated population is further analysed for immature markers like CD34 and or HLA-DR/Tdt.
This population is then examined for expression of lineage markers including surface markers, eg CD 13/33/117
of myeloid lineage. CD14/64 used commonly to interrogate presence of monocytic blasts.
CD 10/19/20/22 are used for detection of B-ALL.
CD 1/ 2/3/4/5/7/8 analysis helps in assigning T cell lineage. Also loss of T antigens and abnormal CD4/8 ratio
are noted.
Lineage specific markers which are of cytoplasmic nature are also used for confirmation of lineage eg: CD3/
CD79a/CD 22/MPO are most important.
Other interfering cell population like erythroid cells, plasma cells, metastasis as well as cell debris need exclusion.
Diagnosis – AML – M2
AML – M5a&b
26
Diagnosis – AML – M3 (APML)
Diagnosis of Lymphoma
Identification of increased lymphocytic population
Characterisation of B/T/NK/Plasma cell lineage
Establishment of clonality
Figure : Immunophenotypic differentiation between CLL/SLL and mantle cell Lymphoma
27
APPROACH TO B-CELL LYMPHOMAS – FLOW CYTOMETRY
Dr Linda Rose
MBBS, MD, DNB, FRCPA
Haematologist,
Dorevitch Pathology Melbourne
B-cells go through various stages of maturation as they differentiate from Pro-B cells to mature B-cells and
then to plasma cells.
The resemblance of B-cell lymphomas to the various stages of normal B-cell differentiation is a major basis for
their classification and nomenclature.
The stages of B-cell differentiation include pro-B cells, pre-B cells, immature B-cells and mature naïve B-cells.
Upon antigen stimulation, naïve B-cells proliferate and ultimately transform to plasma cells and memory B-
cells.
Pro B-cells are positive for CD34, TdT, CD10 and CD19 and are negative for surface light chains. As they
mature, they become negative for CD34, TdT and CD10 and express surface light chains. (Note: CD10 later
get expressed in germinal centre B-cells, so not restricted to lymphoblasts).
Assessment of immunoglobulin light chain (i.e., kappa or lambda) expression by flow cytometry is a key com-
ponent in the diagnosis and monitoring of B cell lymphoid neoplasms. Normal and reactive B cell lymphocytes
exhibit expression of both kappa and lambda light chains at an expected ratio (polyclonal), while neoplastic cells
express either kappa or lambda (monoclonal). Light chain monoclonality is the cornerstone of the diagnosis of
B cell lymphoproliferative disorders.
Normal: K:L ratio is considered to be between 3:1 and 0.3: 1. [Monoclonal: K : L ratio >3:1 (kappa light chain
restricted) or <0.3:1 (lambda light chain restricted)].
Mature B-cell lymphomas

Based on flow cytometry, mature B-cell lymphomas can be subdivided into those with specific phenotypes and
those without.
Lack of CD20, bright CD10, lack of surface light chain and dim CD45 raises possibility of B-ALL and requires
additional testing for CD34 and TdT.
The flow chart below is for a general outline only and one should be mindful of exceptions and variations and
should be interpreted in conjunction with morphology.
References:
1. WHO revised 4th edition 2017
2. Flow Cytometric Testing for Kappa and Lambda light chains (by ICCS Quality and Standards Committee,
Written by: Melanie O’Donahue et al, Jan 2018)
3. Flow cytometry in neoplastic haematology by Wojciech Gorczyca, second edition
Few tips to keep in mind while reporting flow cytometry:
• Make sure that lympho-sum (sum of % of T-cells, B-cells and NK cells) - should be close to 100%
(which means the lymphoid gate is good and includes only lymphocytes).
28
• Check if the total of CD4+ and CD8+ T-cells is close to total CD3+ T-cells. If the sum is lower, that
means there is a CD4 and CD8 dual negative population. A higher sum indicates and CD4 and CD8
dual positive T-cell population.
• Check for 4+/8+ co-expression
• Check 4:8 ratio
• Compare the percentage of CD19-, CD5+ cells (should be T-cells) with the percentage of CD3 + cells.
This should be roughly equal. A lower CD5% indicates CD5 deletion in T-cells.
• Any CD10/CD19 co-expression
• Any CD5/CD19 co-expression
• Check if the sum of percentages of CD19+ Kappa+ cells and the CD19+ Lambda+ cells roughly
equals total CD19+ (if the sum is lower, that indicates a population of sIG negative B-cells)
Negative
DLBCL
lymphoma
29
HOW TO MAKE A GOOD SMEAR
Vijayalekshmi S
Senior laboratory technician, Central lab
Government medical college, Kottayam.
Examining a stained blood smear is a routine part of the complete blood count.
Purpose:
• To find the appropriate numbers of each of the 3 cellular elements
• To study the morphology of these.
• To detect blood parasites.
Slides: Glass slides should be clean and grease free. They should be well washed and treated with alcohol or
ether and polished with a clean soft cloth.
Spreader: The slide used as a spreader slide should have a smooth edge and should be narrower in breadth.
If the edge of the spreader is rough, the film will have ragged tails containing many leucocytes.
Blood: use an appropriate sized blood drop
Key points: Make the smear immediately after putting the drop of blood in the slide. Any delay can cause
uneven distribution of blood cells.
Move the spreader slide confidently. Jerky movement of the spreader slide will give poor smear. The
angle of holding the spreader slide decides the thickness of the smear. Increasing the angle above 50° gives a
thick smear whereas small angle will give a thin smear. The spreader slide should be pushed completely across
the spreading slide in order to have even distribution of the smear.
Blood smear should be dried quickly by waving rapidly in air. This prevents distortion of blood cells.
When the moisture is too high, dry the film by waving it rapidly about 5cm from the spirit lamp.
Qualities of an ideal smear:
• An ideal smear should be tongue shaped
• It should be thick at one end and thin at the other
• It should occupy the central portion of the slide with clear margins on all the sides
• An ideal smear should have a head portion, body and tail and it should be around 3cm in length.
Staining : Using Leishmans stain/ Giemsa stain. Staining time range from 10-15 minutes and should be adjusted
on a trial and error basis depending on the quality of the stain.
BONE MARROW ASPIRATION & SMEAR PREPARATION

Different types of needles are available for bone marrow aspiration. The Klima needle is one of the
most common. This is a stout needle with a short beveled tip and a wide bore with a close fitting stillet. There
is an adjustable guard on the needle which provides adequate penetration. It can be adjusted for the desired
distance.
The usual sites of aspiration are sternum, the iliac crest and the posterior spinous processes of the
lumbar vertebrae. In infants and children below 2 years of age, the front upper end of tibia is selected. The
doctors usually give a small local anaesthetic for the skin and the periosteum covering the bone. Only 0.2-0.5ml
of bone marrow is required for analysis.
30
A 20 ml syringe is attached and the marrow is aspirated quickly by applying continuous suction. The
syringe and the needle are removed. The area of aspiration is kept under pressure with a gauze swab and an
adhesive dressing is applied.
After disconnecting the needle from the syringe, the contents of the syringe are emptied into a glass
petridish. The yellow particles of the marrow are picked up with a glass slide and are spread on a clean glass
slide. 10-12 smears of the bone marrow aspirate are prepared quickly avoiding marrow clotting. The remaining
bone marrow aspirate sample is send to the lab along with the smears.
If the volume is less than 0.2ml, all the marrow is expressed onto a glass slide and then with a capillary
pipette, some of the small irregularly shaped bone marrow fragments with the surrounding blood is picked up
and smears are prepared in the same way by which peripheral smears are made.
Other methods to smear the bone marrow are- particles can be crushed with the corner of another
slide and spread out with a gentle smearing motion. Alternatively a crush preparation is made by placing another
slide over the first slide with the particles.
The smears are then air dried and stained with Leishman stain or Wright stain or May Grunwald and Giemsa
stain with increased staining timing than a blood smear. Preferred staining time for bone marrow smears ranges
from 15-20 minutes. Marrow aspiration smears can be fixed with 100% methanol and stored for later use.
Check for the quality of smear and stain before applying oil immersion in case the smears are under
stained, it has to be de-stained with methanol and then re-stained giving (usually 5-10 minutes) extra time.
31
CYTOCHEMICAL STUDIES IN THE STUDY OF ACUTE LEUKEMIA
Mrs. Nisha Kumari K R

Lab Technician, Central Lab, MCK
1. MYELOPEROXIDASE( MPO):
Myeloperoxidase is located in the primary and secondary granules of neutrophils and their precursors, in eosi-
nophil granules and in the azurophilic granules of monocyte. This test is used to differentiate acute myeloid
leukemia from acute lymphoblastic leukemias. Auer rods are better visualised with MPO than Romanowsky
stain.
Principle: MPO splits H2O2 and in the presence of a chromogenic electron donor, forms an insoluble reaction
product which is brown and granular.
Reagents
Fixative - Buffered formal acetone
Substrate - 3,3’- diaminobenzidine (DAB)
Buffer - Sorensen phosphate buffer PH 7.3
Hydrogen peroxide
Counter stain - Aqueous haematoxylin
Working substrate solution- 30 mg DAB in 60 ml buffer, add 12µl H2O2 and mix well
Method
• Fix air dried smears for 30s in cold BFA
• Rinse thoroughly in gently running tap water and air dry
• Incubate for 10 min in working substrate solution. Thoroughly mix 30 mg DAB in 60 ml buffer, add 12µl
H2O2 and mix well
• Counter stain with haematoxylin for 1-5 min, rinse in running tap water and air dry
Technical consideration
• Can be done with Heparin/ oxalate/EDTA blood(does not inhibit MPO)
• Done with unfixed smear and also on smears >3 weeks old
• MPO sensitive to light, heat and methanol.
• Permanent mounting media – fading of stains
Results and interpretation
Control- Neutrophil
Reaction product - Brown granular
Myeloblasts-except primitive ones which are negative
Promyelocyte and myelocytes -most strongly staining cells
Eosinophils - larger granules positivity than neutrophil
Monocyte and monoblasts- sometimes positive, small granules diffusely scattered throughout cytoplasm
Eosinophils- cyanide resistant
Neutrophils and monocytes- cyanide sensitive
Pathological Variation
• MPO negative neutrophils-Congenital deficiency of neutrophil MPO, Dysplastic neutrophil
• Pseudo peroxidase reaction / Lepehne reaction- Erythroid cell stain for MPO due to non enzymatic
32
interaction between reagent and Hemoglobin.
2. SUDAN BLACK B (SBB)

Principle: SBB is a lipophilic dye that binds irreversibly to an undefined granule component in granulocytes,
eosinophils and some monocytes
Reagents
Fixative - 40% formaldehyde vapour, Buffered formol acetone
Stain - SBB working stain -40 ml buffer to 60 ml SBB solution
Buffer - phenol buffer – 16 gm crystalline phenol + 30 ml absolute ethanol
Add to 100 ml D.H2O in which 0.3 gm Na2HPO4 .12H2O has been dissolved.
Counter stain - Giemsa and Leishman
Method
• Fix smear in formalin vapour.
• Immerse the slides in working stain solution for 1 hour in coplin jar with a lid on
• Transfer slides to a staining rack and immediately flood with 70% alcohol. After 30seconds, tip
the 70% alcohol off and flood again for 30 seconds. Repeat 3 times in total.
• Rinse gently in running tap water and air dry.
• Counter stain without further fixation with Leishman or Giemsa
Technical consideration: Bone marrow films with fatty particles containing lipid soluble SBB benefit from a
5second swirl in xylene followed by rinsing in running tap water and air drying prior to counter stain.
Interpretation:
Reaction product - black and granular
Cells positive - same as MPO
Exceptions
Eosinophil granules have clear core
Basophil may show bright red/ purple metachromatic granules
1-2% ALL- Smudgy positivity.
3. PAS ( PERIODIC ACID SCHIFF REACTION )
Principle:
Periodic acid Schiff’s reagent
1,2 glycol group Dialdehyde Pink to bright red product
Positivity in hematopoetic cells is due to glycogen
Reagents
• Fixative - methanol, BFA , formalin ethanol
• 1% Periodic acid
• Schiff reagent:
Dissolve 5gm of basic fuchsin in 500 ml of hot distilled water. Filter when cool .Saturate with SO2 gas by
bubbling 1-12 hr in a fume cupboard. Shake vigoursly with 2 gm activated charcoal for 1 min in a conical flask
in a fume cupboard filter immediately through Whatman no.1 filter into a dark bottle.
• Counter stain- Aqueous haematoxylin
Technical considerations
• Can be done with prefixed /Perl stained/ Romanosky stained smears
33
• Quality of staining depend on Schiff reagent

• Assessed by staining intensity of neutrophils
Method
• Fix smears for 15 minutes in methanol
• Rinse in running tap water and air dry
• Flood slides with 1% periodic acid for 10 minutes
• Immerse in Schiff reagent for 30 minutes in coplin jar with lid
• Rinse in running tap water with 10 minutes and air dry
• Counter stain for 5-10 minutes
Interpretation
- Reaction product is red with intensity ranging from pink to bright red
- Lymphoblast block positivity in a clear background
Control Reaction product Positive cells
Neutrophil Pink to bright red Lymphoblast, abnormal erythroid precursors
Pattern of positivity in normal cells
Neutrophil Intense confluent granular

Granulocyte precursors Diffuse weak cytoplasmic
Eosinophil Negative, Diffuse Cytoplasmic
Lymphocytes 10-40% granular positivity
Monocytes and precursors Variable diffuse positivity and fine granular positivity at periphery
Megakaryocytes and platelet Intense diffuse with fine and coarse granules
RBC, normal erythroid precursors and basophils- Negative

Uses-
• To differentiate ALL from AML
• To differentiate AML and MDS
• APML promyelocyte – cytoplasmic blush
4. ESTERASES- Specific esterases and non specific esterases

• Leucocyte esterase causes hydrolysis of alpha naphthol
• 9 isoenzymes
• 1,2,7,8,9 – specific esterases ( chloro acetate esterase)
• 3,4,5 – Non specific esterases (ANA E, ANBE)
Naphthol AS-D chloroacetate esterase

Reagents
• Fixatives – Buffered formal acetone
• Buffer – 66mmol/ l phosphate buffer , PH – 7.4
• Naphthol AS-D chloroacetate in 40 ml N,N – dimethyl formamide
34
• Working substrate solution- Add 2ml of naphthol AS-D chloroacetate stock solution to 38 ml of 66mmol
/l phosphate buffer, Ph 7.4. mix well . Add 0.4 ml of freshly prepared hexazotised New Fuchsin. Mix
well
• Coupling reagent-
Hexazotised New fuchsin . Dissolve 4 gm of new fuchsin in 100 ml of 2 N HCl
Sodium nitrite solution 0.3mol/l. Dissolve 2.1gm of sodium nitrite in 100 ml H2O
Immediately prior to using add 0.2 ml of the hexazotised New Fuchsin to 0.4ml sodium nitrite, Mix well
and leave for 1 minute before adding to substrate.
• Counter stain – Aqueous hematoxylin
Method
• Fix air dried smears in cold buffered formal acetone for 30 s
• Rinse in gently running tap water and air dry
• Immerse the slides in the working substrate solution in a coplin jar for 5-10 minutes
• Rinse in running tap water and air dry.
• Counter stain in aqueous hematoxylin for 1 minute
• ‘Blue ‘in running tap water for 1 minute and air dry.
Results & interpretation
Reaction product – Bright red
Positivity starts with promyeloctes to neutrophils
More mature cell – stronger staining
APML and AML- M2 t ( 8,21) Auer rods- positive staining with hollow core
Alpha naphthyl butyrate esterase
Reagents
• Fixative – Buffered Formol Acetone
• Substrate – ANBE
• Coupling reagent- Fast Garnet GBC- reaction product dissolve mounting media and oil. Aqueous mounting
media used (glycerine/gelatin), Hexazotised pararosaniline
• Counter stain –aqueous haematoxylin
Method
• Fix air dried smears in buffered formal acetone 30 minutes
• Rinse in water and air dry
• Add the fast garnet GBC to a 50 ml buffer and mix well.
• Add 0.5ml of the alpha napthyl butyrate /acetone and mix well.
• Pour the incubation medium with a coplin jar containing the fixed slides and incubate for 20- 40 minute
• Rinse thoroughly by running tap water
• Air dry and counter stain in aqueous Fast Garnet GBC for 1-5 minutes
Interpretation
• Brown granular reaction product
• > 80% monocytes- strong positivity
• T lymphocyte – variable positivity
• Bone marrow monocytes and precursor macrophages-stain strongly.
• More specific than ANAE
35
MICROVESSEL DENSITY AMONG THE MORPHOLOGICAL TYPES OF

MULTIPLE MYELOMA
Dr Parvathy V, Dr Sheeja S, Dr.Renu Thambi, Dr Sankar S
Introduction:
Multiple myeloma is a malignant plasma cell dyscrasia which is characterised by bone marrow plasma-
cytosis. Multiple myeloma causes 1 % of all cancer deaths in western population.
Objectives:
1. To describe the various morphological types of myeloma in bone marrow examination and its
kappa and lambda expression
2. To assess the microvessel density using CD 34 in the bone marrow biopsy of patients with
multiple myeloma
Methods:
Study Design : Descriptive study
Study Population : Study sample includes all the cases of multiple myeloma
received in the Department of Pathology during the study period.
Sample Size : 64
Sampling Procedure : Continuous sampling
Study Procedure: Microscopic examination of H& E sections of all cases followed by Immunohistochemical
staining for CD 34, kappa and lambda
Analysis: The morphological types of multiple myeloma and its relationship with microvessel density was
analysed. The relationship of plasma cell burden with the morphological types of myeloma and microvessel
density were also analysed.
Results:
The mean age of the study group was 61 years. The study group consisted of 31 females (48.44% ) and
33 males (51.56% ) with female to male ratio of 1.06:1. In our study normocytic normochromic anaemia was
the most commonly observed peripheral smear pattern. 47 patients showed exaggerated rouleux formation in
the peripheral smear. Bone marrow aspirate study showed that normal hematopoiesis was suppressed in 62
cases (96.87 %).In our study the most common morphological type of myeloma was plasmacytic which consti-
tuted 57.82 % of the cases. Plasmablastic morphology was seen in 42.18 % of the cases. Bone marrow
36
trephine study showed that the cellularity was increased in 96.87 % of the cases whereas it was normocellular
in the rest of the cases. The major pattern of infiltration in biopsy was a diffuse pattern which was seen in 43
cases. While assessing the histological stage of multiple myeloma it was found that majority of the patients
(48.44 %) were in stage III at the time of diagnosis in which the plasma cell percentage was >50%.In all the
cases the monoclonality was proved by kappa and lambda light chain restriction. The cut off for microvessel
density was calculated to be 18. The study showed that 28 patients (43.75 %) had microvessel density above
this cut off value and 36 patients (56.25 %) had MVD below the cut off value. Statistical analysis showed that
plasmablastic morphology was having a higher microvessel density compared to plasmacytic morphology. The
analysis also showed that having plasma cell percentage above 50 % was more likely to have a higher microvessel
density.A high microvessel density was observed in diffuse pattern of infiltration of bone marrow.
Conclusion:
In our study, myeloma with plasma cell morphology constituted 57.82% and plasmablastic morphology
constituted 42.18 %.A high microvessel density above the cut off value was seen in 28 cases (43.75 %) and a
low MVD in 36 cases (56.25 %).In all the cases monoclonality was proved by kappa and lambda light chain
restriction.In the present study ,plasmablastic morphology was found to have a higher microvessel density
compared to plasmacytic type.It was also suggested that a higher microvessel density was seen in cases with
plasma cell percentage of more than 50 % and diffuse plasma cell infiltration in the bone marrow .
Reference Link
https://dx.doi.org/10.18535/jmscr/v8i2.167
37
LATENT MEMBRANE PROTEIN 1 EXPRESSION IN

NON HODGKIN’S LYMPHOMA
Dr Jaseela TK, Dr. Letha V, Dr. S. Sankar ,Dr. Jayalakshmy.P.L
Background
Non Hodgkin’s Lymphoma is a haematological malignancy with various etiological factors and one
among them is Epstein Barr Virus. The expression of Epstein-Barr virus in Non-Hodgkin’s lymphoma can be
identified by immunohistochemistry for detection of Epstein-Barr virus latent membrane protein (LMP-1). In
our study we are trying to clarify the extent of expression LMP-1 in our population. This can be used as a
prognostic marker and for therapeutic interventions targeting EBV encoded proteins.
Objectives
1. To determine the proportion of LMP1 (EBV marker) expression in Non Hodgkin’s Lymphoma.
2. To identify association of LMP1 expression in B cell and T cell type of NHL.
Materials and methods
A descriptive study was conducted on 67 cases of histopathologically diagnosed Non Hodgkin’s Lymphoma
specimens received in the department of Pathology, Govt. Medical College, Kottayam during the study period
of 18 months (December 2017 to May 2019). NHL cases were subtyped into B cell type and T cell type and
LMP-1 immunohistochemistry was done on all cases to assess its expression.
Results
• Mean age of Non Hodgkin’s lymphoma is 57.82 in this study. Minimum age is 2 years and maximum
age is 89 years. The present study had 10.4 % LMP 1 positive cases. Of which there were 6% moderately
stained positive cases, 3% of weakly stained cases and 1.5% intensely stained cases. Among NHL 86.6%
cases were B cell lymphoma and 13.4% cases of T cell lymphoma. Although 6 out of 58 cases(10.3%) of
B cell lymphoma were positive for LMP 1 and only 1 out of 9 cases (11.1%) of T cell lymphoma showed
positivity for LMP-1, there was no significant association between LMP 1 positivity and cell type according
to our study. This may be due to small sample size.
Conclusion
The present study was done to determine the proportion of LMP 1 expression in Non Hodgkin’s Lym-
phoma and to find out whether there is any association between LMP1 expression in B cell and T cell type of
NHL. LMP 1 was positive in 10.4% of NHL and there was no association between LMP 1 positivity in B cell
and T cell Lymphoma. This suggest that EBV might play a role in pathogenesis of NHL.
38
Panel Discussion
Case 1
A 58 year old female presented with fatigue, loss of weight and appetite – 1 month, on and off fever - 2 weeks
and excessive bleeding after tooth extraction. On examination- pallor +. No lymphadenopathy / HSM. Labo-
ratory work up: Hb- 9.8 g%, TC- 50,500/mm3, PL- 43,000/ mm3, ESR- 80mm/1st hr. LDH- 12,995 IU/L.
Peripheral smear: Acute leukemia. Blasts show cytoplasmic vacuolation. MPO negative.
Flow cytometry
Positive markers - CD19, CD79a, CD20, CD10, HLADR
Negative markers - CD34, CD13, CD33, CD117, MPO, CD64 , CD11c, CD36, CD3, CD4, CD5, CD7, CD8
Final diagnosis - B lineage neoplasm. Acute lymphoblastic leukemia/ Burkitt leukemia.
39
Case-2
65 year old male presented with breathlessness – 1 week, facial puffiness and decreased urine output – 3
weeks. Known case of Type 2 diabetes, hypertension, COPD. On examination- bilateral pedal edema, in-
creased JVP. No lymphadenopathy/ HSM.
Laboratory work up: Hb- 11.6 g%, TC- 29,800/mm3, PL- 2,34,000/mm3, ESR - 86mm/1st hr, S. urea-
114mg%, S. creatinine-7.9mg%, S. calcium-12mg%, uric acid 14.8mg%. AG reversal. 24 hour urine
protein - 154 mg /day. USG abdomen- kidneys show increased echo texture with normal corticomedullary
differentiation. SPEP- no M band.
Peripheral smear- 22% plasma cells. Bone marrow biopsy- 80% plasma cells. IHC – kappa light
chain restriction +.
Final diagnosis - Plasma cell leukemia
40
Case 3
32 year old female presented with lower abdominal pain and distension- 6 months, fatigue -2 months, palpita-
tion- 2 months. On examination- pallor, generalised lymphadenopathy, hepatomegaly, and massive splenom-
egaly present.
Laboratory work up: Hb- 8.3g%, TC – 19,500/mm3, uric acid- 7.7mg%, LDH- 2,238IU/L.
Peripheral smear- Acute leukemia. MPO negative. LEBP with tear drop poikilocytosis.
Basophil – 2%.
Bone marrow aspirate – Dry tap
Bone marrow biopsy- Fibrosis with megakaryocyte hyperplasia. Reticulin stain - grade 3 fibrosis.
Differential diagnosis- 1. CML in blast crisis
2. Primary myelofibrosis with transformation to acute leukemia.
Flow cytometry
Positive markers – CD34, CD13, CD33, HLADR, CD36, CD11c dim, CD4, CD7 dim.
Negative markers – CD117, CD14, CD64, CD10, CD19, CD20, CD79a, CD3, CD5, CD8.
Cytogenetics - Philadelphia chromosome positive.
Final diagnosis – CML in blast crisis.
Acute myeloid leukemia (AML M4)
41
Case 4
17 year old female presented with on and off fever- 1 month, generalised body ache-1 month, loss of weight and
appetite- 1 month. On examination – No pallor/lymphadenopathy /HSM.
Laboratory work up: Hb- 12.7g%, TC- 31,100/mm3, PL- 8,02,000/mm3. Absolute eosinophil count-
1,946/mm3, LDH- 599 IU/L. USG abdomen – normal study.
Peripheral smear- Neutrophilic leucocytosis with shift to left, basophilia (8%) and thrombocytosis.
Bone marrow aspirate- Myeloid hyperplasia
Bone marrow biopsy- Myeloid and megakaryocyte hyperplasia.
Quantitative real time PCR- Major (p 210) BCR-ABL1 fusion gene transcript- 82.4% detected.
Final diagnosis- Chronic myeloid leukemia.
42
SNIPPETS
• Leukemia with eosinophilia
• Prognostic and predictive factors of ALL
• Variant translocations in Acute Promyelocytic Leukemia
• Acute leukemia of ambiguous lineage
• Leukemia with BCR-ABL fusion gene
• Causes of Imatinib resistance in CML
• Additional clonal abnormalities in CML
• Idiopathic hypereosinophilic syndrome
• Chromosomal abnormalities in Myelodysplasia
• Hypoplastic myelodysplastic syndrome
• International Prognostic Scoring System in de novo MDS
• Stress cytogenetics
• ALIP vs ALPS
• Prognostic factors of myelofibrosis
• Telomeropathies affecting leucocytes
• POEMS TEMPI
• Prognostic markers in myeloma
• Bone marrow plasmacytosis
• Grey zone lymphoma
• Non lymphoid neoplasms mimicking lymphoma
• Double hit lymphoma versus double expressor lymphoma
• Hematologic malignancies in post transplant patients
• Pathogenesis of post transplant lymphoproliferative disorder
• Hematogones
• Post therapy marrow changes
• Acquired bone marrow failure syndromes
• Bone marrow changes in HIVinfection
• Neutrophil inclusions
• IT ratio and its relevance
• Hematologic abnormalities in Down syndrome
• Pathogenesis of transient abnormal myelopoiesis
• Congenital defects of phagocytosis
• Neutrophil function tests
• Syndromes associated with Neutropenia
• Syndromes associated with pancytopenia
• WHO revision (2016) for classification of myelodysplatic syndrome
• Syndromes associated with acute leukemia
• Flow cytometry algorithm in B cell lymphomas
• Flow cytometry algorithm in T cell lymphomas
• Flow cytometry algorithm in Acute leukemia
43
LEUKEMIA WITH EOSINOPHILIA

1. Myeloid and lymphoid neoplasm with PDGFRA, PDGFRB and FGFR rearrangements
2. Myeloid neoplasm with PCM1-JAK2, ETV6-ABL, ETV-FLT3
3. Chronic eosinophilic leukemia NOS
4. Chronic Myelomonocytic leukemia with eosinophilia(CMML-Eo)
5. Atypical chronic myeloid leukemia
6. Chronic myeloid leukemia
7. Acute myeloid leukemia with t(8,21) or inv (16) AML-Eo
PROGNOSTIC AND PREDICTIVE FACTORS OF ALL

FAVOURABLE UNFAVOURABLE
PROGNOSIS PROGNOSIS
3
WBC count <10,0000/mm >2,00,000/mm3
Age 2-6 yearsAdult: < 30 years <1 year, > 10 yearsAdult: > 30 years
Mediastinal mass Poor prognosis
Liver, Spleen, LN
involvement Poor prognosis
CNS leukemia Poor prognosis

FAB type L1 L2, L3
T-ALL Poor prognosis
Day 7 Blast in BM Blast> 25% on day 7 of induction therapy
Downs Syndrome Poor prognosis
BCR-ABL fusion gene Poor prognosis
t(4,11), t(1,19) Poor prognosis
Hyperdiploidy Favourable prognosis
Immunophenotype B-ALL with CD10+ CD19+
MLL gene Poor prognosis
Sex Girls Boys
VARIANT TRANSLOCATIONS IN ACUTE PROMYELOCYTIC LEUKAEMIA

Classic translocation: t (15;17) (q24.1;q21.2) giving rise to PML-RARA gene fusion product.
Cryptic or masked t (15;17) (q24.1;q21.2)- submicroscopic insertion of RARA into PML.
Secondary cytogenetic abnormalities
• Gain of chromosome 8(10-15%)
• Mutations involving FLT3-FLT3-ITD &FLT3-TKD(30-40%)
Note : FLT3-ITD mutations are most common and are associated with a higher WBC count, microgranular
morphology & involvement of bcr3 breakpoint of PML.
Variant RARA translocations in acute leukaemia
ZBTB16-RARA and STAT5B-RARA fusions-are resistant to tretinoin.
Others – NUMA1, NPM1.
44
ACUTE LEUKAEMIA OF AMBIGUOUS LINEAGE

• Acute undifferentiated leukaemia(AUL)
• Mixed phenotype acute leukaemia(MPAL) with t(9;22)(q34;q11.2);BCR-ABL1
• Mixed phenotype acute leukaemia with t(v;11q23);MLL rearranged
• Mixed phenotype acute leukaemia B/myeloid, NOS
• Mixed phenotype acute leukaemia T/myeloid, NOS
LEUKAEMIA WITH BCR-ABL1 FUSION GENE

• Acute myeloid leukaemia with BCR-ABL 1
• Mixed phenotype acute leukaemia with t(9;22)(q34.1;q11.2);BCR-ABL 1
• B lymphoblastic leukaemia/lymphoma with t(9;22)(q34.1;q11.2);BCR-ABL 1
• Chronic Myeloid Leukaemia, BCR-ABL 1 positive
CAUSES OF IMATINIB RESISTANCE IN CML
• Decreased intracellular drug levels

• Plasma binding by á-1 acid glycoprotein
• Drug efflux from P-glycoprotein (MDR-1) overexpression
• Increased expression of BCR-ABL kinase from genomic amplification
• Clonal evolution (non-BCR-ABL–dependent mechanism)
• Mutations in ABL kinase of BCR-ABL affecting drug interaction or kinase activity
Ref : Mark R. Litzow, Imatinib Resistance Obstacles and Opportunities, Arch Pathol Lab Med. 2006;130:669–679
ADDITIONAL CLONAL ABNORMALITIES IN CML
• Second Ph chromosome
• Trisomy 8
• Isochromasome 17q
• Trisomy 19
• Complex karyotype
• Abnormalities of 3q26.2
45
IDIOPATHIC HYPEREOSINOPHILIA SYNDROME

• Diagnosis of exclusion.
• Persistent eosinophilia (eosinophil count > 1.5 x 10 9 /L for atleast 6 months) & not meet the criteria for
chronic eosinophilic leukemia or a familial eosinophilia (Autosomal dominant disorder).
• Pathogenesis unknown.
• There is no evidence of clonality /T cell abnormality.
• Presence of organ damage.
• Rx : I st line –corticosteriods
• Regular follow up is needed to rule out Leukemia
CHROMOSOMAL ABNORMALITIES IN MYELODYSPLASIA
CHROMOSOMAL FREQUENCY
ABNORMALITIES MDS overall (%) Therapy related MDS (%)
UNBALANCED
Gain of chromosome 8 10
Loss of chromosome 7 or del (7q) 10 50
Del (5q)
Del (20q) 10 40
Loss of Y chromosome 5-8
Isochromosome 17 q or t (17p) 5
Loss of chromosome 13 or del (13q) 3-5 25-30
Del (11q)
Del(12p) or t (12p) 3
Del(9q) 3
Idic(X)(q13) 3
BALANCED
t(11;16)(q23.3;p13.3) 1-2
t(3;21)(q26.2;q22.1) 1-2
t(1;3)(p36.3;q21.2) 1 3
t(2;11)(p21;q23.3) 1 2
inv(3)(q21.3q26.2)/ 1
t(3;3)(9q21.3;q26.2) 1
t(6;9)(p23;q34.1)
References: WHO Classification of Tumours of Hematopoietic and Lymphoid Tissues, 2017
46
HYPOPLASTIC MYELODYSPLATIC SYNDROME

• FAB group initially described MDS having a hypercellular or normocelluar bone marrow.
• Subsequently it became apparent that approximately 15% in all had hypocellular marrow.
• Diagnosed by severe cytopenia and hypocellular marrow (marrow cellularity <30% in those who are
<60 years of age OR <20% in those >60 years of age.)
• Females > Males
• Prognosis does not differ from MDS in general.
• D/D – Hypocellular AML , Aplastic anaemia.
IPSS ( International Prognostic Scoring System) denovo MDS
Prognostic factors 0 0.5 1 1.5 2 3 4

Marrow <2 >2 -<5 5-10 >10%
blast (%)
Hb >10 8-<10 <8
Platelets >1lakh 50000- <50000
100000
ANC >0.8 <0.8
Cytogenetics Very good good intermediate Poor Very
poor
Cytogenetics
• Very good - -Y, del (11q)
• Good - normal ,del (5q), del (12p),del (20q) double including del (5q)
• Intermediate – del(7q), +8,+9 I (17q), any other single or double independent clones
• Poor - -7 inv(3)/ t (3q)/ del (3q), double including -7/del (7q), complex :3 abnormalities
• Very poor – complex:> 3 abnormalities
STRESS CYTOGENETICS
• Diagnostic molecular test of Fanconi anemia
• Fanconi anaemia is an inherited bone marrow failure syndrome due to impaired response to DNA
damage leading to chromosomal instability resulting in decreased production of all types of blood cells
• This test is based on demonstration of breakage of abnormally sensitive chromosomes with absence
of DNA repair mechanisms on exposure to oxidant stress.
• Oxidant stress is created by using either Mitomyin C or Diepoxybutane (DEB).
47
ALIP VS ALPS
Abnormal Localisation of Immature Precursors (ALIP)
• Usually in MDS patients- detected in bone marrow biopsy
• Blasts cells are increased. Clusters of > 5 blasts are abnormally located in the central parts of the
marrow ALIP (normally blasts are located in paratrabecular location).
• Preponderance of promyelocytes and myelocytes in the marrow.
• There may be increase in monocytoid forms.
• The higher the number of such ALIPs, the worse is the prognosis.
• To be distinguished from the clusters of early erythroid precursors ( regular chromatin with 2-4 linear
nucleoli near nuclear membrane in erythroid precursors).
• Blasts of the ALIP are CD34 positive while early megaloblasts are not.
Autoimmune LymphoProliferative Syndrome (ALPS)
• ALPS is an inherited disorder of lymphocyte homeostasis seen in children.
• Characterized by non -malignant lymphoproliferation
• Lymphadenopathy, hepatosplenomegaly with/without hypersplenism, IgG/IgA hypergammaglobulinemia
• Autoimmunity mostly directed towards blood cells
• Increased risk of lymphoma.
• ALPS is characterised by accumulation of polyclonal population of T cells called double negative
(CD4 and CD8 negative) T cells (DNT).
POOR PROGNOSTIC FACTORS OF MYELOFIBROSIS

Anemia <10gm/dl
Age >65years
Leukocytosis >25x109/L
Circulating blast >/= 1%
Constitutional symptoms
Risk stratification based on poor prognostic factors:

Risk group Number of factor Median survival
a) Low 0 135 months
b) Intermediate 1 1 95 months
c) Intermediate 2 2 48 months
d) High >3 27months
TELOMEROPATHIES AFFECTING LEUCOCYTES

• Dyskeratosis congenita: BM failure , MDS, AML
• Fanconi anemia: AML
• Schwachman-Diamond syndrome: Acute leukemia• Bloom syndrome: Leukemia & lymphoma
• Ataxia telengiectasia: leukemia & lymphoma
48
POEMS TEMPI
P- Polyneuropathy T- telengiectasia
O- organomegaly E- Elevated erythropoietin levels, Erythrocyoisis
E- endocrinopathy M- monoclonal gammopathy
M- M protein P- perinephric fluid collection
S- skin abnormalities I - intrapulmonary shunting

PROGNOSTIC MARKERS IN MYELOMA
Good prognostic markers: Poor prognostic markers:

• Stage I disease • Stage III disease
• Serum beta 2 microglobulin < 3.5 mg/L • Serum beta2microglobulin > 5.5 mg/L
• Low CRP • High CRP
• Nodular pattern of Bone marrow • Plasmablastic morphology
involvement in bone marrow biopsy • Diffuse bone marrow involvement in
• Plasma cell labelling index < 1% bone marrow biopsy
• Normal Serum creatinine • High serum LDH
• Low IL6 and IL6 receptor levels • Plasma cell labelling index > 3%
•Low angiogenesis on bone marrow biopsy • High serum creatinine
• Low serum free light chain level • Deletion 13q, 11q, 17p
• Hypodiploidy & Monosomy 13
• High IL6 & IL6 Receptor levels
• High angiogenesis in bone marrow biopsy
• High serum free light chain levels
BONE MARROW PLASMACYTOSIS

Reactive increase in polyclonal plasma cells Bone marrow infiltration by neoplastic
– 10-20% plasma cells plasma cells
•HIV Infection •Multiple myeloma

•Leishmaniasis •Immunocyte derived amyloidosis
•Chronic inflammatory disease Eg: Rheumatoid Arthritis •Systemic light chain disease
•Haemopoietic & non-haemopoietic malignant disease •Monoclonal gammopathy of
•Angioimmunoblastic lymphadenopathy undetermined significance
•Systemic Castleman’s disease
•Cirrhosis
•Diabetes mellitus
•Iron deficiency
•Megaloblastic anemia
•Hemolytic anemia
49
GREY ZONE LYMPHOMA

B cell lymphoma with features intermediate between DLBCL & Classical Hodgkin lymphoma
Demonstrates overlapping clinical, morphological and/or immunophenotypic features between DLBCL & CHL.
Most common in young men (20-40 years)
Most common presentation: Anterior mediastinal mass
Microscopy: Areas resembling both CHL and DLBCL within the same tumor. Marked cellularity, sheet like
growth of pleomorphic tumor cells in a diffusely fibrotic stroma with sparse inflammatory cells in the back-
ground. Necrosis is frequent. Due to variation in histology in different areas, core needle biopsy is not recom-
mended for diagnosis.
Immunophenotype: Most cases show strong expression of CD45, CD 20,CD 30, MUM1
A smaller subset resemble primary mediastinal large B cell lymphoma with loss of CD 20
Prognosis: More aggressive and poor outcome than either CHL or DLBCL
NON LYMPHOID NEOPLASMS MIMICKING LYMPHOMA
• Myeloid sarcoma
• Carcinoma
• Melanoma
• Germ cell tumours(can be CD 30 +)
• Small blue cell tumours- neuroendocrine carcinoma, neuroblastoma, rhabdomyosarcoma
• Thymoma
Reference: CMC vellore presentation on lymphoma
DOUBLE HIT LYMPHOMA VS DOUBLE EXPRESSOR LYMPHOMA
DOUBLE HIT LYMPHOMA DOUBLE EXPRESSOR LYMPHOMA
•High grade lymphoma with MYC and BCL2 •Co-expression of MYC and BCL2 without
and/or BCL6 rearrangement underying rearrangements
•High LDH •Multiple extranodal site
•Extranodal diseases •Intermediate/High risk to high risk IPI score
•High risk IPI score •Advanced stage diseases
•Advanced stage diseases (stage III/IV) •Higher Ki67 proliferation index
•Ki67 proliferation index variable •R-CHOP low response
•R-CHOP better response •Diagnosis- IHC
•Intensified R-EPOCH regime
•Diagnosis- FISH analysis/ advanced
genomic technology
50
HEMATOLOGICAL MALIGNANCIES IN POST TRANSPLANT PATIENTS:

Higher risk for a variety of cancers is the major complication of post transplant immunosuppression.
Post Transplantation Lymphoproliferative Disorder(PTLDs) comprise the great majority of hematologic can-
cers, represent a spectrum of conditions. Oncogenic EBV is the main culprit in many PTLDs.
Lymphomas Plasma cell neoplasm Leukemia
Hodgkin’s lymphoma Plasma cell myeloma Acute myeloid Leukemia

DLBCL-MC type of NHL Plasmacytoma Chronic myeloid leukemia
after organ transplantation Acute promyelocytic leukemia
Burkitt’s lymphoma
PATHOGENESIS OF POST TRANSPLANT LYMPHOPROLIFERATIVE DISORDER

• Impaired immune Surveillance of tumor cells
• Medication causing direct DNA damage
• Oncogenic virus such as EBV
Diagnosis- PTLDs are stratified in 4 groups based on H&E characteristics
• Early lesion
• Polymorphic PTLDs
• Monomorphic PTLDs
• Classical Hodgkin lymphoma like PTLD
Treatments- Reduction of immunosuppression -1st line
Anti B cell antibody-Rituximab, Chemotherapy –CHOP
Surgery/ radiation.
Reference: Vikas R Dharnidharka. Comprehensive review of post organ transplant hematologic cancers.
American journal of transplantation. 2017 vol 18,issue3
HEMATOGONES
• Lymphoid progenitor cells, usually < 1% of bone marrow cells
• Represent normal immature B-cells
• Seen in bone marrow of infant during the first month of life
• Associated with childhood cytopenias, neoplasms, viral infections, ITP and regenerative marrow after
chemotherapy and marrow transplantation.
• Microscopy- Scant cytoplasm , high N/C ratio, highly condensed chromatin , inconspicuous
nucleoli.(Morphology is variable)
• Difficult to differentiate hematogones from lymphoblasts based on morphology & phenotype.
• CD 10+, CD 38 positive
Reference: Mc Kenzie, Pathology outlines
51
POST THERAPY MARROW CHANGES: Bone marrow biopsies and aspirate from patients undergoing
various treatments show variety of histological and cytological changes.
TIME POINT CHANGES

1 WEEK Marrow Aplasia, Loss of Adipocyte, Fibrinoid necrosis, Edema, Dilated Sinuses
2 WEEKS Fibrinoid Necrosis,
Cellularity composed of stromal cells, hemosiderin laden macrophages,
lymphocyte and plasma cells, Return of adipocyte, Loose network
of reversible reticulin
fibrosis, Hematogone Hyperplasia
4 WEEKS Erythroid precursor return, Granulopoiesis, Clusters of megakaryocytes,
Reticulin fibrosis diminishes
Reference: Jason H Kurzer and olga k Weinberg Atlas of Bone marrow Pathology 2018 pp 27-32
ACQUIRED BONE MARROW FAILURE SYNDROMES

• Idiopathic
• Drugs:Chloramphenicol,Sulfa drugs,antithyroid,Tetracycline,Penicillin,Methylphenyl hydantoin
• Chemical agents: Benzene, insecticides, hair dye, carbon tetra chloride, Chemotherapeutics (vincris-
tine, busulfan), arsenic.
• Ionising radiation
• Biological agents: Parvovirus, infectious mononucleosis, measles, influenza, errors of aminoacid
metabolism, starvation
• Pregnancy
• Paroxysmal nocturnal hemoglobinuria
• Myelophthisis - infiltration by granulomatous or neoplastic cells and fibrosis
BONE MARROW CHANGES IN HIV INFECTION
• Morphologic changes tend to be more pronounced in more immunosuppressed patients and increase in
frequency as disease progresses. All lineages can be involved.
• Morphologic abnormalities: Hypercellular or hypocellular marrow, myelodysplastic changes, fibrosis,
granulomas, lymphoid aggregates.
• Hyperplasia involving the granulocytic and erythroid lineages
• Myeloid: Erythroid ratio varied from 2:1 to 5:1.
• Increase of bone marrow lymphocytes, plasma cells and eosinophils; macrophages with or without
haemophagocytosis (as in viral infections).
• Megaloblastic changes and ringed sideroblasts are frequent.
• Dysplastic changes increase with disease progression.
• Fungi in severely immunocompromised patients (HIV infection or following bone marrow transplanta
tion) include Candida albicans, Aspergillus fumigatus, Histoplasma capsulatum, Cryptococcus neoformans,
Penicillium marneffei, Blastomyces dermatitidis & Coccidioides immitis.
52
• In AIDS lack of T- cell function may result in failure to produce granulomas in response to infection
with organisms that stimulate granuloma formation in normal individuals.
• Multicentric Castleman disease- bone marrow shows lymphoid follicles with depleted or hyalinized
germinal centres & mantle zones in which there are scattered HHV8 - positive plasmablasts .
• NHL and Hodgkin lymphoma occur with increased frequency in patients infected with HIV. Three
types of lymphomas are now recognized as AIDS defining by the CDC: Burkitt lymphoma, immunoblastic
lymphoma, and primary lymphoma of the brain.
• IHC for p24 capsid protein positive in bone marrow.
References: 1. Wintrobe’s Clinical Hematology12th Edition
2. Bone marrow pathology Barbara. J. Bain
NEUTROPHIL INCLUSIONS
• Toxic granules: Bacterial infections, a/c liver failure, neutrophilic leukaemoid reactions, aplastic anemia
and following administration of G-CSF.
• Vacuoles: Severe sepsis, alchohol toxicity and a/c liver failure
• Bacteria & Fungi: Overwhelming septicaemia (meningococcal & pneumococcal ),
staphylococcocal septicaemia in children, patients with infected central line.
• Dohle bodies –Round /oval, small, blue grey structures found at the periphery of neutrophils, seen in
bacterial infection, burns, following administration of G-CSF, neutrophilic leukaemoid reactions.
• Cyroglobulin
• Bilirubin crystals
• Erythrocytes
• Malarial pigment
I/T RATIO AND ITS RELEVANCE

I/T RATIO Bands+ Metamyelocyte + Myelocyte/ all neutrophils
< 0.2 Normal
0.2 – 0.25 May be suggestive of infection
> 0.8 Carries higher risk of infection
• I/T ratio is one of the early diagnostic tool of neonatal sepsis. Blood cultures are a gold standard for
diagnosis of neonatal sepsis but it takes a long time delaying the management.
• I/T ratio has higher level of effectiveness and sensitivity
• Less expensive
• Its usefulness is enhanced when considered in conjunction with other sepsis screen markers like CRP
and WBC count in ruling out sepsis
53
HEMATOLOGICAL ABNORMALITIES IN NEWBORNS WITH DOWN SYNDROME

Neonates:
Transient abnormal myelopoeisis
Neutropenia, thrombocytopenia, erythroblastosis ,polycythemia
Myelodysplasia- abnormal myeloid cell granulation, giant platelets
Infants and children:
Myeloid leukaemia of Downs syndrome
Myelodysplasia
Acute myeloid leukemia
PATHOGENESIS OF TRANSIENT ABNORMAL MYELOPOIESIS

Individuals with Down have increased risk of leukemia, estimated at 10-100 fold. They also have increased
risk of developing Transient Abnormal Myelopoesis(TAM),which is a unique disorder of Down syndrome.
Newborns with TAM have clinical and morphological findings indistinguishable from AML. Blasts are of mega-
karyocytic lineage. Thrombocytopenia is the most common feature at presentation. There may be marked
leucocytosis with an increase in blast. Hepatosplenomegaly may be present. In majority,spontaneous remission
occurs within first three months of life. In most cases,blasts are positive for
CD34,CD56,CD117,CD13,CD33,CD7,CD4,CD41,CD42 and CD61.In addition to trisomy 21, acquired GATA
1 mutations are present in blast cells of TAM. Children with Down have 50fold increase in acute leukemia in
first 5year of life. About 1-2%children with Down develop AML by this time. Bone marrow core shows
increase in reticulin fibrosis resulting in dry tap. In addition to trisomy21, somatic mutations of gene encoding the
transcription factor GATA1 are considered pathognomonic of TAM or MDS/AML of Down syndrome.
CONGENITAL DEFECTS OF PHAGOCYTOSIS
1. Chronic granulomatous disesase of childhood(CGD)
2. Chediak- Higashi syndrome (CHS)
3. Job’s syndrome (Hyperimmunoglobulin E- recurrent infection syndrome)
4. Myeloperoxidase deficiency (MPO)
NEUTROPHIL FUNCTION TESTS

Useful in diagnostic workup of patients with suspected Chronic Granulomatous Disease.
1. Evaluation of NADPH Oxidase function in neutrophils
-Nitroblue Tetrazolium Test(NBT)
-Dihydrorhodamine Flow Cytometric Test(DHR Test)
2. Chemotaxis assays
-Rebuck skin window
-Invitro assay with patient control sera
3. Myeloperoxidase stain
4. Flow cytometry to measure CD11/CD18 surface glycoproteins on neutrophils
5. Quantitative ingestion assays(patient and control sera as opsonins)
6. Flow cytometry to measure selectin on neutrophils
7. Rolling on L-selectin ligand
Reference-Williams hematology 8th edition
54
SYNDROMES ASSOCIATED WITH NEUTROPENIA:
Disorders of production Disorders of RNA Disorders of metabolism Disorders of

synthesis and vesicular
processing transport
•Cyclic neutropenia (AD) •Cartilage hair •Barth syndrome •ChediakHigashi

•Fanconi pancytopenia (AR) hypoplasia(AR) (AR,XLR) syndrome(AR)
•Reticular dysgenesis (AR) •Dyskeratosis congenita •Glycogen storage •Cohen
•Severe congenital neutrophilia (XLR,AD,AR) disease,type 1b(AR) syndrome (AR)
(AD,AR) •Schwachman- Diamond •Pearsons syndrome •Griscelli
•Wiskott Aldrich (mitochondrial) syndrome(AR)
syndrome (XLR) •Hermansky
Pudlak
syndrome
type 2 (AR)
•P14 deficiency
(probable AR)
SYNDROMES ASSOCIATED WITH PANCYTOPENIA.
Aplastic anaemia a) Fanconi anaemia(AR) b) Familial aplastic anaemia c) Dyskeratosis congenita-inherited

bone marrow failure syndrome d) Congenital amegakaryocytic thrombocytopenia-isolated thrombocytopenia
due to absent or reduced megakaryocytes. e) Schwachmann Diamond syndrome-have exocrine pancreatic
dysfunction & malabsorption syndrome.
Myelophthisic anaemia-myelophthisis signifies marrow replacement or infiltration by fibrotic,granulomatous
or neoplastic cells. Most characteristic findings are leucoerythroblastic reactions and a moderate to marked
poikilocytosis.
Myelodysplastic syndromes-group of hematological disorders with propensity to terminate in acute leuke-
mia. Peripheral blood shows pancytopenia,bicytopenia or isolated cytopenias with reticulocytopenia.
Congenital dyserythropoetic anaemia-rare familial refractory anaemia characterised by both abnormal and
ineffective erythropoiesis. Bone marrow is normocellular or hypercellular but peripheral blood show pancy-
topenia.
55
WHO REVISION (2016) FOR CLASSIFICATION OF MYELODYSPLASTIC SYNDROME
56
SYNDROMES ASSOCIATED WITH ACUTE LEUKEMIA
• Rothmund Thomson syndrome • Diamond Blackfan Syndrome

• Seckel syndrome • Kostmann syndrome
• Shwachman syndrome • Down syndrome
• Werner syndrome (Progeria) • Dubowitz syndrome
• Holf- Hirschhorn syndrome • Naxos syndrome
• WT syndrome • Noonan syndrome
• Poland syndrome
Reference:Williams Hematology (8 th edition)
57
58
Acknowledgements
JAYPEE PUBLISHERS
CBS PUBLISHERS
NANDA BIOTECH
GIRIJAS LAB
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Best Complements from
Roche
Diagnostics
64
65
THANK YOU FOR YOUR PARTNERSHIP IN THE PURSUIT OF CONTINUED INNOVATION IN THE SPECIMEN
MANAGEMENT PATHWAY. For patients around the world, the breakthroughs in care that leaders like you pioneer and
champion hold the promise of better outcomes. Because tomorrow’s cures are rooted in the work you’re doing and
supporting today. Thank you for letting BD Vacutainer® specimen collection products be a part of the journey with
you, through every stage of research, every step forward in discovery, and every moment of truth. We look forward
to an even brighter future advancing the world of health, together.
Learn more at bd.com/70YrsHealthy
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B - Kon 2020 - DIAGNOSTIC DILEMMAS IN WBC DISORDERS

DEPARTMENT OF PATHOLOGY MEDICAL COLLEGE KOTTAYAM

CHAIRMAN - DR. S. SANKAR

SCIENTIFIC COMMITTEE TRANSPORT AND ACCOMODATION

MESSAGE FROM ORGANISING DESK

Dr. S. Sankar Dr. Sheeja S.

Leucocyte Morphology in Blood Smears : Dr. Joy Augustine

Interpretation of WBC Histograms and Scattergrams : Dr. Feroze M.

WBC Disorders - when to study and what to look : Dr. K. Gayathri

The basic Principles of Flowcytometry : Dr. Geetha Vidyadharan

Approach to B-Cell Lymphomas - Flow cytometry : Dr. Linda Rose

How to make a good Smear : Smt. Vijayalekshmi S.

Microvessel Density Among the Morphological : Dr. Parvathy V.

Latent Membrane Protein 1 Expression in : Dr. Jaseela T.K.

LEUCOCYTE MORPHOLOGY IN BLOOD SMEARS

Dr Joy Augustine MD, DCP.

Artifacts in Blood Smears

Pancytopenia with circulating Blast cells points to Leukaemia or MDS

INTERPRETATION OF WBC HISTOGRAMS AND SCATTER GRAMS

Histograms and scatter grams Histograms are representation of separation of RBCs,

Indications for marrow evaluation in WBC disorders:

THE BASIC PRINCIPLES OF FLOWCYTOMETRY

Dr. Geetha Vidyadharan

Diagnosis – AML – M3 (APML)

Figure : Immunophenotypic differentiation between CLL/SLL and mantle cell Lymphoma

APPROACH TO B-CELL LYMPHOMAS – FLOW CYTOMETRY

Mature B-cell lymphomas

HOW TO MAKE A GOOD SMEAR

BONE MARROW ASPIRATION & SMEAR PREPARATION

CYTOCHEMICAL STUDIES IN THE STUDY OF ACUTE LEUKEMIA

Mrs. Nisha Kumari K R

interaction between reagent and Hemoglobin.

2. SUDAN BLACK B (SBB)

3. PAS ( PERIODIC ACID SCHIFF REACTION )

• Quality of staining depend on Schiff reagent

Pattern of positivity in normal cells

Neutrophil Intense confluent granular

RBC, normal erythroid precursors and basophils- Negative

4. ESTERASES- Specific esterases and non specific esterases

Naphthol AS-D chloroacetate esterase

MICROVESSEL DENSITY AMONG THE MORPHOLOGICAL TYPES OF

Dr Parvathy V, Dr Sheeja S, Dr.Renu Thambi, Dr Sankar S

Study Design : Descriptive study

received in the Department of Pathology during the study period.

Sampling Procedure : Continuous sampling

LATENT MEMBRANE PROTEIN 1 EXPRESSION IN

Dr Jaseela TK, Dr. Letha V, Dr. S. Sankar ,Dr. Jayalakshmy.P.L

2. To identify association of LMP1 expression in B cell and T cell type of NHL.

Materials and methods

Final diagnosis- Chronic myeloid leukemia.

LEUKEMIA WITH EOSINOPHILIA

PROGNOSTIC AND PREDICTIVE FACTORS OF ALL

CNS leukemia Poor prognosis

VARIANT TRANSLOCATIONS IN ACUTE PROMYELOCYTIC LEUKAEMIA

ACUTE LEUKAEMIA OF AMBIGUOUS LINEAGE

LEUKAEMIA WITH BCR-ABL1 FUSION GENE

CAUSES OF IMATINIB RESISTANCE IN CML

• Decreased intracellular drug levels

ADDITIONAL CLONAL ABNORMALITIES IN CML

IDIOPATHIC HYPEREOSINOPHILIA SYNDROME

References: WHO Classification of Tumours of Hematopoietic and Lymphoid Tissues, 2017

HYPOPLASTIC MYELODYSPLATIC SYNDROME

IPSS ( International Prognostic Scoring System) denovo MDS

Prognostic factors 0 0.5 1 1.5 2 3 4