By Dr.

Deeksha Sikri MBBS, DNB (Pathology)

Associate Professor
St. George’s University, Grenada, West Indies

Points in red: UPDATES FROM 10th EDITION

Points in green: PREVIOUS YEAR QUESTIONS IN AIIMS/PGI/JIPMER/NEET PG
• Deficiency or defect- Glanzmann
thrombasthenia
• Autosomal recessive
• Bleeding time increased gp IIb/IIIa
• Normal platelet size and count • Deficiency or defect- Bernard
• Aggregation in response to all agonists Soulier syndrome
abnormal except to ristocetin • Autosomal recessive
• Bleeding time increased
gp IIb/IIIa • Normal to low platelet count
Fibrinogen • Giant platelets
• Aggregation in response to
ristocetin abnormal
Platelet gp Ib/IX

• Most commonly autosomal dominant

Deficiency or defect in subendothelial Subendothelial collagen • Bleeding time increased; aPTT maybe increased
and circulating vWF- Von Willebrand • Normal platelet count and size
disease • Aggregation in response to ristocetin abnormal
All alpha granules have “F” in them (“alFa” have “F”)
Epinephrine, ADP and
Fibrinogen- for fibrin clot Alpha granules ATP- for activation of
formation platelets to bind with
fibrinogen, and aggregate
Fibronectin, TGF β and PDGF- Dense (δ) granules
Ionized calcium- for
for wound healing and fibrosis
activation of coagulation
factors
Von Willebrand Factor- for
platelet adhesion and carrier Serotonin- for
for Factor VIII vasoconstriction

Platelet factor 4 (PF4)- heparin binding

cytokine and target in HIT (next slide)

Factor V- for activating prothrombin • Deficiency of alpha granules- “Grey Platelet syndrome”
along with Factor X • Deficiency of dense granules- “Storage pool deficiencies like
Hermansky Pudlak syndrome”
 Review From Robbins 10th Ed

• Serious, potentially life-threatening disorder following the administration of

unfractionated heparin- UFH
• Mechanism: Formation of antibodies that recognize complexes of heparin and
PF4 on the surface of platelets + complexes of heparin-like molecules and PF4-
like proteins on endothelial cells
• PF4 in platelet alpha granules → released on activation of platelets → binds to
heparin → undergoes a conformational change → results in the formation of a
neoantigen against which IgG antibodies are formed
• PF4-IgG immune complex attaches to and cross-links the Fc receptors on the
platelet surface →platelet activation and aggregation → release of more PF4 →
creating more target antigen for HIT antibodies
• Activation of endothelium by binding of HIT antibodies to PF4-like proteins on
their surface → augments the pro thrombotic state
• Macrophages remove the platelets with HIT antibodies → thrombocytopenia
• Thrombocytopenia is the most common manifestationMCQ
• Thrombosis is the most serious complicationMCQ → approximately 50% of cases
and affects both veins and arteries (don’t confuse thrombosis and thrombocytopenia)
• Necrosis of the skin, gangrene of the limbs, stroke, and myocardial infarction are
some of the sequelae
 CD 19 PAX 5
TdT
CD 20
CD 34
CD 22

HLA DR CD 79a CD 38
BLASTS B CELLS CD 138

CD 3
PLASMA CELLS
CD 2
CD 16
CD 5 CD 56

CD 7
NK CELLS
T CELLS
 CD 11c
CD 14

CD 64
CD 45
CD 13
Leucocyte common antigen
CD 19
CD 33
CD 20
Monocytes
CD 22
CD 10
CD 11c
BCL 6
CD 13
All Leucocytes Germinal
centre B cells
CD 33

CD 15

Neutrophils CD 10- also called Common ALL Antigen (CALLA) seen in ALL
CD 19- B cell lineage specific marker
CD 3- T cell lineage specific marker
 Cells shown in Cells shown in
yellow here are blue here are
negative for A positive for
and positive for B both A and B
Antigen B being checked on cells

antigen (behind antigen (ahead

on X axis and on X axis and
high on Y axis) high on Y axis)

Cells shown in Cells shown in

red here are green here are
negative for positive for A
both A and for B and negative for
antigen (behind B antigen (ahead
Every dot is a on X axis and low on X axis and low
cell we are on Y axis) on Y axis)
trying to check
antigen
expression of Antigen A being checked on cells

The cells in this graph fall into what was
the blue quadrant in the previous pic.
Which means cells are positive for both
the markers. Hence, these cells are TdT
positive- Immature cells and CD 22
positive- B cells
• Both graphs show the
same cells
• We are checking the
expression of the four CD
markers mentioned in
the graphs on these cells
• Always focus on the
BULK of the cells, not the
outlier
• Check which quadrant
they lie in
TdT positive- immature cells
The cells in this graph also fall into what (blasts)
was the blue quadrant in the previous pic. CD 19 and CD 22 positive- B
Which means cells are positive for both cell markers
the markers. Hence, these cells are CD 10 CD 10 positive- CALLA
positive- CALLA positive and CD 19 → B- Acute lymphoblastic
positive- B cell lineage specific leukemia (B-ALL)
Lymphoma Cytogenetics CD 5 CD 10 BCL 6 Extra points
Mantle cell lymphoma t(11;14) + - - CD 23 -, Cyclin D1 + (if -, SOX 11 is positive)
Chronic lymphocytic Trisomy 12 + - - CD 23+, Cyclin D1 – ; Del 13q: good prognosis
leukemia/lymphoma and Deletion Del 17p and Del 11q, ZAP 70, CD 38 and CD 49d: poor
13q prognosis
Precursor- Monoclonal B cell lymphocytosis
Follicular lymphoma t(14;18) - + + BCL 2 +; KMT2D (previously called MLL) gene in 90% cases
Burkitt lymphoma t(8;14)- 75% - + + C-myc +, BCL 2 –
t(2;8) Mutations in TCF3 or E2A gene (transcription factor)
t(8;22)
Diffuse large B cell Translocations - + + C-myc and BCL 2 can be positive;
lymphoma of BCL 2, BCL 6 High level expression of both MYC and BCL2 proteins is
and c-myc seen in some cases and may predict more aggressive
behaviour
CAR T therapy is now available for relapsed refractory
DLBCL
Clonal bone marrow plasma cell percentage ≥10% or biopsy proven plasmacytoma ALONG WITH ≥1 of the following
myeloma defining events (MDEs) (adapted from International Myeloma Working Group 2014 criteria)
1. End organ damage (“CRAB” criteria- hypercalcemia, renal insufficiency, anemia, bone lesions ≥1 lesions on imaging)
2. ≥1 of the following malignancy biomarkers
- Clonal bone marrow plasma cell percentage ≥60%
- Involved to uninvolved serum free light chain ratio ≥100
- >1 focal lesion on MRI

So, diagnosis of plasma cell myeloma is possible even without CRAB criteria if plasma count is more than 60% or other
markers of malignancy are present

RISK STRATIFICATION OF PLASMA CELL MYELOMA BY CYTOGENETICS

STANDARD (LOW) RISK INTERMEDIATE RISK HIGH RISK
t(11;14) t(4;14) Deletion 17p
t(6;14) Deletion 13q t(14;16)
Hyperploid Hypoploid t(14;20)
Myeloproliferative Major criteria Minor criteria
neoplasm
Polycythemia vera 1. Hb >16.5 g/dL in men and >16 g/dL in women OR Hematocrit Subnormal serum erythropoietin
(all 3 major or first 2 >49% in men and >48% in women OR increased red blood cell level
major and minor) mass
2. Bone marrow biopsy- hypercellularity with panmyelosis with
pleomorphic, mature megakaryocytes (differences in size)
3. Presence of JAK2 V617F or JAK2 exon 12 mutation
Essential 1. Platelet count ≥ 450 x 109/L Presence of a clonal marker or
thrombocytosis 2. Bone marrow biopsy with proliferation of mainly absence of evidence of reactive
(all major or first 3 megakaryocytic lineage with increased numbers of mature, thrombocytosis
major and minor) enlarged megakaryocytes with hyperlobulated nuclei
3. WHO criteria for other MPNs are not met
4. JAK2, CALR or MPL mutation
• Primary myelofibrosis occurs in two stages- prefibrotic and fibrotic with different criteria for each.
• Mastocytosis is NOT included under MPNs anymore; it is a separate category under WHO classification.
• Other MPNs are- chronic neutrophilic leukemia; chronic eosinophilic leukemia, NOS and myeloproliferative neoplasm,
unclassifiable.
• All MPNs have growth factor independent tyrosine kinase overactivity.
Peripheral smear Bone marrow
cytology (biopsy is
not mandatory)

LAP score is low (leukemoid reaction has high score)

CRITERIA FOR ACCELERATED PHASE OF CHRONIC MYELOID LEUKEMIA- WHO 2017
 Karyotype FISH

CYTOGENETIC TECHNIQUES TO DIAGNOSE CML RT PCR

Karyotype helps to Fluorescent in situ hybridisation Reverse transcriptase (RT)

visualise the chromosomes (FISH) helps to visualise the genes PCR is a molecular
(structural and numerical (not read them; just see them). technique to read the
problems seen easily) Fusion between BCR (green) and mRNA (hence RT) made
Translocation between chr. ABL1 (red) due to t(9;22) can be from the fusion of BCR-
9 and 22 seen with identified by seeing the fused ABL1 due to t(9;22). Copies
formation of Philadelphia sequence (yellow) and counting the of fusion transcripts are
chromosome (changed chr. signals in a significant number of compared with ABL1 to
22 with BCR ABL1 fusion). cells to diagnose CML. use for follow up.
Type Most dominant cells MPO NSE PAS CD 34/ Name of the type Morphology
HLA DR
M0 Blasts with little evidence of - - - Low or AML with minimal No Auer rods or
differentiation (<3%) absent differentiation granules
M1 Maturing granulocytic cells are ≥3% - - ~70% AML without maturation Few Auer rods in
<10%; rest all myeloblasts granular cells
M2 Maturing granulocytic cells are + - - + AML with maturation Auer rods +
≥10%; some dysplasia +
M3 Promyelocytes (blast ++ Low: - Low or Acute promyelocytic leukemia Many Auer rods
equivalents) and myeloblasts 25% absent
M4 Monocytic cells (monoblasts + in + - + AML with myelomonocytic Convoluted nuclear
and promonocytes (blast myelo differentiation contours in
equivalents) ≥20%; myeloblasts blasts monocytic cells
M5 Monocytic cells ≥80%;rest are + in ++ - CD 34- AML with monocytic Convoluted nuclear
myeloblasts myelo 30% differentiation contours in
blasts monocytic cells
M6 Erythroid cells >80% - - ++ - Pure erythroid leukemia Vacuoles +
M7 Megakaryoblasts >50% - Focal ++ - AML with megakaryocytic Pseudopods or
differentiation blebs +
Hemolytic anemia Inherited/Acquired Intrinsic/Extrinsic Extravascular Peripheral smear and other
cause defect /intravascular hemolysis findings
Hereditary Autosomal Intrinsic Extravascular Spherocytes
spherocytosis dominant- M/C in
Ankyrin
Glucose 6 phosphate X linked Intrinsic Extravascular and Bite cells and spherocytes
dehydrogenase (recessive) intravascular Supravital- Heinz bodies
deficiency
Sickle cell disease Autosomal Intrinsic Extravascular > Sickle cells
recessive Intravascular Target cells
Beta thalassemia Autosomal Intrinsic Extravascular Target cells
recessive Minor: uniformly microcytic
Major: more variations
Paroxysmal nocturnal Acquired mutation Intrinsic Intravascular Normal looking cells
hemoglobinuria in PIGA gene Flow cytometry is diagnostic
Autoimmune hemolytic Acquired Extrinsic Extravascular and Spherocytes
anemia intravascular Coomb’s test positive
Microangiopathic Acquired Extrinsic Intravascular Schistocytes and
hemolytic anemia microspherocytes
Examples of supravital stains
1. Crystal violet
2. New methylene blue
3. Brilliant cresyl blue
4. Azure B
 Exclusive! NEW!!

TOPIC UPDATE
Myelodysplastic Most common cytogenetic change in India is complex karyotype
syndrome
Beta thalassemia The five most common mutations are
(IVS stands for intervening sequence, meaning these mutations are
in introns, not exons. The letter ahead convey the change)
1. IVSI-5 (G>C)
2. 619 bp deletion
3. IVSI-1 (G>T)
4. Codon 41/42 (-TCCT)
5. Codon 8/9 (-G)
Sickle cell anemia Asian Indian haplotype is less severe than the African haplotype
Indian sickle cell anemia- increased HbF, increased Hb and lower
reticulocyte count