HEMATOLOGY 2
FINALS: NEOPLASTIC ALTERATIONS OF LEUKOCYTES (WBC)
2ND SEMESTER | S.Y. 2021-2022
LECTURER: REY ANTHONY PEREZ
TOPIC
SUBTOPIC
INTRODUCTION
Leukemia – These are abnormal, uncontrolled
proliferation and accumulation of not just one or
more hematopoietic cells. Leukemias major
symptoms are fever, weight loss and increased
sweating
NEOPLASTIC DISEASES OF WBC
➢ Acute Myeloid Leukemia (AML)
➢ Acute Lymphoid Leukemia (ALL)
➢ Chronic Myeloid Leukemia (CML) and other
Chronic Myeloproliferative Disorder (CMD)
➢ Myelodysplastic Syndromes (MDS)
➢ Chronic Lymphocytic Leukemia (CLL)
➢ Plasma Cell Neoplasms (PCN)
➢ Waldenstrom’s Macroglobulinemia (WM)
MAJOR TYPES
A. LEUKEMIAS
1. Acute Leukemias (Usually bone pain from
large leukemic cell mass in the bone marrow)
✓ Acute Myeloid Leukemia
✓ Acute Lymphoblastic Leukemia
2. Chronic Leukemias (Usually enlargement,
splenomegaly, hepatomegaly, and lymph
adenopathy)
✓ Chronic Myeloproliferative Disorders
✓ Chronic Lymphoproliferative Disorders
B. LYMPHOMAS
1. Hodgkin’s Disease
2. Non-Hodgkin’s Lymphoma
C. PLASMA CELL DISORDERS/DYSCRASIAS
➢ Like multiple myeloma these conditions are
uncommon
D. HISTIOCYTIC DISORDERS
➢ For example: Sea-blue Histiocytosis
TRANSCRIBED BY: LOUWENCE A. LAPITAN
LABORATORY TESTS
A. MORPHOLOGY (Abnormality happening in the
cell. This is a very helpful test)
✓ Wright-Giemsa Stain
✓ Blood Smear
✓ Bone Marrow Biopsy
B. ELECTRON MICROSCOPY
✓ Not routinely used, on rare occasions but can
be very helpful.
C. CYTOCHEMISTRY (Microscopic study and
identification of chemical constituents within
individual cells. We will be able to identify malignant
cell types based on cytochemistry results)
✓ Enzymatic Techniques (peroxidase)
✓ Non-enzymatic Techniques (Lipids or
glycogen for example is SUDAN BLACK B, will
stain sterols, neutral fats or phospholipids
which are commonly found on the secondary
granules or in the lysosomal granules of
monocyte)
ENZYMATIC TECHNIQUES
A. MYELOPEROXIDASE (MPX)
✓ Stains neutrophilic/eosinophilic cells
monocytes (weak); lymphocytes (negative)
✓ Observing Dark brown granules
(in monocytes have scattered activity)
✓ Deteriorate very fast, that is why a fresh
sample is needed
✓ Eosinophil: Activity will be seen in the
presence of SODIUM CYANIDE
✓ Marker for Primary granules and Auer rods
(pink or red rod shape structures, usually
fused primary granules)
✓ Neutrophil and Monocyte: Diaminobenzidine
method
 ✓ Eosinophil: Cyanide-resistance peroxidase 2. Alpha-naphthyl acetate ssterase (ANAE)
stain
✓ Marker for monocytes, megakaryocytes and
plasma cells and some lymphocytes
✓ Monocytes Stain RED BROWN (it reveals
AUR ROD
strong esterase activity in the monocytes,
however it can be inhibited with the addition
of SODIUM FLUORIDE)
✓ This is useful particularly in Chronic
Promyelocyte
Lymphocytic Leukemia (CLL)
From a patient ✓ Letter b
With acute Addition of
Promyelocytic Inhibitor
Leukemia (Sodium
Fluoride)

ESTERASES
➢ Specific Esterase (SE) – stains myeloid cells like
3. Alpha-naphthyl butyrate esterase
neutrophil (isoenzymes 1,2,7,8,9) (The substrate
✓ Identifies monocytes, promonocytes, and
is Chloroacetate for neutrophil)
monoblasts
➢ Non-Specific Esterase (NSE) – stains monocytes
✓ DARK RED precipitates in cytoplasm (positive
(isoenzymes 3,4,5,6) (The substrate is Butyrate
reaction)
or acetate)
✓ Non-specific
1. Naphthol AS-D chloroacetate esterase
✓ Patient with acute

✓ Markers for mature and immature Monoblastic or

neutrophils and mast cells Monocytic Leukemia

✓ Bright red granules in cytoplasm of

neutrophils, neutrophil precursors, and mast
cells
PHOSPHATASES
✓ This is used in demonstrating myeloid
➢ Acid Phosphatase – Present in all hematopoietic
elements on paraffin embedded section such
cells and found in lysosomes
as GRANULOCYTIC SARCOMA or MAST CELL
Activity indicated by PURPLE TO RED GRANULES
in SYSTEMIC MAST CELL DISEASE.
- Positive for this is T cell Leukemia and Hairy Cell
✓ Positive for GRANULOCYTES and negative for
Leukemia
MONOCYTES
- Negative for non T-cell Leukemia
 ISOENZYME OF ACID PHOSPHATASE LAP CYTOCHEMICAL STAIN SCORING
CHARACTERISTICS
ISOENZYME POSITIVE CELLS
0 Gaucher cells
1 and 4 Neutrophil and
Monocyte
3a Lymphocyte and
Platelet
3b Primitive cells and
Blasts
5 Hairy Cells
(The number of cells counted in each category 0-4

Tartrate-Resistant Acid Phosphatase (TRAP) will be multiplied by the value for that category)

✓ Marker for Hairy Cell Leukemia FOR EXAMPLE: 50 cells with 4 rating = 50X4 =200

✓ Activity indicated by purple to red granules in 30 cells counted and 3 rating = 30X3 = 90
the cytoplasm 20 cells counted and 2 rating= 20X2 = 40
✓ Endotheliosis
TOTAL = 330
✓ The acid phosphatase isoenzyme band 5 is
FAST BLUE = 30-182 normal range
the only isoenzyme that is resistant to
VIOLET B = 12-180 normal range
TARTARIC ACID. Most useful in leukemic
patient
IMMUNOCYTOCHEMICAL TECHNIQUES
Terminal deoxynucleotidyl transferase (TDT)

✓ Marker for immature lymphocyte

✓ Stains DNA polymerase

Leukocyte-Alkaline Phosphatase (LAP)
✓ Present in 90% cases of Acute Lymphoid
✓ Also known as Kaplow Count
✓ Specific for Neutrophil (because these are the Leukemia (ALL)

only leukocytes that contains various amount ✓ Acute Myeloid Leukemia (AML): NEGATIVE
of alkaline phosphatase)
✓ Acute Lymphoid Leukemia (ALL): POSITIVE
✓ This enzyme test we will differentiate CML
from leukemoid reactions (Leukemia like NON-ENZYMATIC TECHNIQUES
conditions)
Periodic Acid-Schiff (PAS)
✓ CML for LAP decrease activity
✓ Marker for glycogen, glycoproteins,
✓ 100 cell count is done = scored from no activity
✓ Capillary blood is most commonly preferred mucoproteins and high molecular weight
and heparin is used (EDTA is avoided for LAP
carbohydrates
staining)
✓ Activity result in BRIGHT FUSCHIA-PINK

✓ All blood cells (+) except erythroblast

✓ M6 Leukemia positive for PAS

✓ Pattern of staining varies each cell type Toluidine Blue

✓ Lymphoblastic Leukemia: ✓ Recognition of mast cells and Tissue Basophils

Blocky/Chunky pattern = REDDISH VIOLET

✓ L1 and L2: Block Pattern ✓ Binds with acid mucopolysaccharide in blood

✓ L3: Negative cells

✓ Erythroblast in M6 Leukemia: positive ✓ Strongly metachromatic (the reaction

product color is insignificantly different from

the color of the dye itself)

✓ This is helpful in recognition with MASS CELL

DISEASE and ACUTE OR CHRONIC

BASOPHILIC LEUKEMIA

Sudan Black B (SBB)

✓ Marker for phospholipid and lipids
✓ Neutrophil precursors: PURPLE-BLACK
GRANULES CYTOCHEMISTRY IN ACUTE LEUKEMIA
✓ Same pattern as MYELOPEROXIDASE (TABULAR)
(MPX): more sensitive to younger cells
(this can be done on store samples)
✓ The positivity will increase with the
maturity of the granulocytic cells (The
more mature the more positive it is)
✓ Lymphoblast: NEGATIVE
✓ Acute Myeloid Leukemia (AML): POSITIVE
✓ Acute Lymphoid Leukemia ALL: NEGATIVE
D. IMMUNOPHENOTYPING IMPORTANT MONOCLONAL ANTIBODIES USED IN
THE DIAGNOSIS OF ACUTE LEUKEMIA
✓ Look for antigens or markers on the cell
BLASTS
surface ✓ Anti-TdT
✓ Identifies more than 95% of all Acute Myeloid
✓ T and B Lymphoblasts
Leukemia (AML) and Acute Lymphoid
✓ Less in myeloid blasts
Leukemia (ALL)
✓ Rapid identification on the T cell Leukemia ✓ HLA-DR

1. Some Useful Markers: (Detected via Flow ✓ Myeloid blasts and B-Lymphoblasts
Cytochemistry)
✓ CD34
✓ CD5 – Expressed on all T cells Myeloid
✓ CD10 – Expressed on developing B cells
✓ CD13
✓ CD19/20 – Expressed on mature B cells
✓ CD33 – Expressed on myeloid precursors and ✓ CD33
monocytes ✓ Anti-myeloperoxidase

2. Most Helpful in Lymphoid Leukemias Monocytic

✓ CD11b

IMMUNOPHENOTYPING OF ACUTE ✓ CD14

LEUKEMIAS
Megakaryocyte
5 MAJOR GROUPS IDENTIFIED
✓ CD41
Acute Lymphoid Leukemia (ALL) (3 subgroups)
✓ CD61
✓ Precursor B-ALL
✓ Burkitt B-ALL Erythroid

✓ Precursor T-ALL ✓ Anti-glycophorin

Acute Myeloid Leukemia (AML) B-lymphoid

✓ Provide correlation with the FAB ✓ CD10

morphologic subtype of AML
✓ CD19
✓ Diagnosis of AML-M0 and AML-M7
✓ CD20
Acute Leukemias of Ambiguous Lineage
✓ CD22

✓ Surface immunoglobulins

T-lymphoid

✓ CD3

✓ CD5

✓ CD7
E. CYTOGENETICS

✓ Examination of chromosomes under a

microscope (only detect abnormalities that
cause BIG CHUNKS on the chromosomes)
✓ Gives a rough overall look at all the patient’s
chromosomes
✓ Sometimes confirms diagnosis; often more
useful for prognosis

F. MOLECULAR STUDIES

✓ Examination of DNA using polymerase chain

reaction (PCR), Southern Blotting
✓ Very sensitive for specific chromosomal
abnormality (know the exact DNA sequence)
✓ Can often detect abnormalities that don’t
show up on cytogenetics