MODULE 20.

Acute Malignant Blood Disorders

TABLE OF CONTENTS
COURSE MODULE MAP............................................................................. 1

MODULE RATIONALE................................................................................ 2

MODULE COMPETENCY AND LEARNING OBJECTIVES................. 3

MODULE PREREQUISITES AND CO-REQUISITES............................. 3

LEARNING ACTIVITIES
Connection Activity................................................................................ 4
Objective One: Classification of Acute Leukemias................................ 6
Learner Objectives..........................................................................7
Exploration Activity...................................................................... 8
Experiential Activities.................................................................. 10
Objective Two: Differential Diagnosis of Acute Malignant Blood
Disorders...................................................................................... 11
Learner Objectives........................................................................13
Exploration Activity..................................................................... 14
Experiential Activity.................................................................... 21
Practical Application Activity....................................................................... 22
Objective Three: Cytochemistry............................................................ 20
Learner Objectives........................................................................21
Exploration Activity..................................................................... 21
Experiential Activity.................................................................... 22
Practical Application ....................................................................23
Objective Four: Laboratory Testing of Acute Malignant Blood Disorders.............. 24
Learner Objectives........................................................................24
Exploration Activity..................................................................... 24
Practical Application Activity...................................................... 24

MODULE SUMMARY.................................................................................. 24
MODULE POST-ASSESSMENT................................................................. 25
REFERENCES............................................................................................... 28
APPENDIX A - Experiential and Module Post-Assessment Answers........29

Prof. Dr. Yousif Y. Bilto 1 Acute Malignant Blood Disorders

MODULE RATIONALE

Acute Leukemias follow a much more aggressive clinical course than do the chronic
malignancies. It is important that the Technologist respond appropriately to significant
laboratory test results and morphological changes. This module assumes that you can
differentiate blood cells - particularly blast cells and that you understand the principles
of cytochemistry.

MODULE COMPETENCY AND LEARNING OBJECTIVES

Module Competency
On completion of module competency, the learner will be able to evaluate validity of
laboratory test results, as well as recognize and respond to significant results by
correlating with clinical information about acute malignant blood disorders.

Module Learning Objectives

To successfully complete the module competency, the learner will be able to:

 describe the classification and cellular manifestations of acute leukemias.

 discuss the laboratory tests used in the differential diagnosis of acute malignant
blood disorders.

 apply principles of cytochemistry to perform stains commonly used in the

differential diagnosis of blood cell malignancies.

 recognize and respond appropriately to laboratory test results which could have
diagnostic significance in acute malignant blood diseases.

LEARNING ACTIVITIES

Connection Activity

As the pathophysiology of some common acute malignant blood diseases is

described, you should be able to relate any changes in morphology, numbers or
function of cells to hematology (and in some cases, clinical chemistry) test results
you know. You should be trying to focus on how the results in these disorders
differ from what you would see in reactive or non-malignant responses, eg. to
infection. That's what is meant by a clinically significant finding - something that
could direct a physician to a specific diagnosis.
Prof. Dr. Yousif Y. Bilto 2 Acute Malignant Blood Disorders
OBJECTIVE ONE
Upon completion of the module competency, the learner will be able to describe the
classification and cellular manifestations of acute leukemias.

Learner Objectives
The learner will be expected to:
1. demonstrate knowledge of relevant terminology by defining the following as
they relate to blood diseases:
acute non-lymphatic leukemia (M0-M7)
acute lymphoblastic leukemia (L1 - L3)
gammopathy
lymphoma
2. describe the typical peripheral blood and bone marrow picture in each of M0 to
M7 and for L1 and L2 above. The bone marrow description should include
typical cellularity, M:E ratio, the predominant cell type and stage(s) of
development present and any differentiating features.

Exploration Activity
Refer back to the beginning of Module 2 - Introduction to the Hemopoietic
Malignancies to review the place of "acute" disorders in the scheme.
You may also wish to review a schematic showing specific blood cell development
from the pluripotential stem cell.

ACUTE NON-LYMPHATIC LEUKEMIAS (ANLL)

Read pg. 442-444 from Chapter 34 in your text. These pages cover the definition,
etiology and classification of ANLL. A few highlights from this section are featured
below.
 ANLL include all acute leukemias involving cells other than lymphocytes.
 normal marrow elements are crowded out by the leukemic cells.
 if untreated, pancytopenia is seen as a result of:
1. failure of normal marrow
2. dyserythropoiesis
3. Dysmyelopoiesis
(see Table 34-4 and 34-5 on pg. 447 - for clinical features of ANLL and
morphological abnormalities associated with ANLL)

Prof. Dr. Yousif Y. Bilto 3 Acute Malignant Blood Disorders

Typical patient age group for ANLL is adulthood but it can occur in any age group.
Classification of ANLL - Read the heading Classification on pg. 443 of the text.

 The FAB system is a morphological classification based on examination of

Romanowsky-stained preparations and aided as required by cytochemistry.

Read pg. 443 - Cytochemical Techniques.

pg. 444 - French-American-British System

Define cytochemistry (Glossary in text).

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Cytochemical reactions will be discussed under Objective Two of this module.

The primary distinction to be made when classifying acute leukemia is whether the
malignancy is non-lymphocytic or lymphocytic leukemia.

 Methods available to help confirm a classification include:

1. morphologic

2. cytochemical

3. immunologic

The morphology from Romanowsky-stained preparation is ineffective in identifying

cell lineage in acute leukemia because lymphoblasts, myeloblasts and monoblasts have
the same characteristics using this staining method.

Finding Auer rods in blast cells is significant. They are primary granules fused
together in a rod-like shape and are found in the cytoplasm of leukemic non-lymphoid
blasts or progranulocytes, i.e. myeloblasts, monoblasts or promyelocytes.

The FAB classification system uses both morphology and cytochemistry to define
eight subclasses of ANLL. Specific morphologic criteria used are:

1. direction of cell line differentiation

2. degree of maturation of the proliferating cells.

Why is it necessary to determine the % of blasts present in bone marrow?

Prof. Dr. Yousif Y. Bilto 4 Acute Malignant Blood Disorders

_____________________________________________________________________
There are three types of myeloblasts that the author of the text describes on pg. 306.
Use the blanks below to describe the three types of myeloblasts detectable in
hematologic malignancy.

Describe Type I blasts.

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Describe Type II blasts.

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Describe Type III blasts.

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General Laboratory Findings in ANLL

1. Peripheral Blood

In general, ANLL have some common features related to the replacement of

normal marrow tissue by malignant cells.

 WBC - Leukocytosis secondary to circulating blast cells

 RBC/Hb - decreased

 platelets - decreased

 erythrocyte morphology - normocytic/normochromic

In your text, pg. 444-450 describes peripheral blood changes specific to each FAB
subgroup of ANLL.

Prof. Dr. Yousif Y. Bilto 5 Acute Malignant Blood Disorders

It is best to learn what features differentiate these FAB subgroups.

2. Bone Marrow

KNOW TABLE 34-3 on pg. 445.

In addition to that information, consider, for each classification:

 cellularity
 M:E ratio

Write the names of the eight subclasses of ANLL:

M0
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M1
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M2
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M3
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M4
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M5a
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M5b
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M6
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M7
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ANLLs not included in FAB classification are found on pg. 450 of the text.

Prof. Dr. Yousif Y. Bilto 6 Acute Malignant Blood Disorders

  mixed leukemias
 secondary leukemias

ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

Read pg. 473-475 of Chapter 36 of your text. These two pages describe in general the
characteristics of ALL. Listed below are a few general points about ALL:

 typical patient age is between 2 and 10 years.

Clinical symptoms include:

 pallor and fatigue caused by decreased red cells (anemia).

 bruising, petechiae, epistaxis as a result of thrombocytopenia.
 chills, fever and/or overt infection as a result of granulocytopenia.

FAB Classification of ALL

Read pg. 475-476 about L1, L2, and L2 FAB classifications of ALL. Use that
information to fill in the blanks below. Match the FAB classification with their
descriptive synonym.

List the three FAB classifications with their descriptive synonyms.

L1:

2:

L3:

The morphological differentiation of L1 and L2 is based on four features of the

malignant blasts:
L1 L2
1. cell size: small twice the size of a small lymph
2. nuclear shape regular cleaving, folding and indentations

Prof. Dr. Yousif Y. Bilto 7 Acute Malignant Blood Disorders

3. nucleoli not visible visible
4. amount of cytoplasm scanty more than L1
See Table 36-1 - Features of Acute Lymphoblastic Leukemias

An important clinical differentiating feature is the age of the typical ALL patient

L1 ALL - patient age - 74% of ALL cases occur in children 15 years of age or younger
are L1.

L2 ALL - patient age - 66% of ALL cases in children over 15 years are L2.

Malignant Cells

The lineage and developmental stage of the malignant blasts in ALL affects treatment
selection and prognosis.
 most ALL's are caused by malignant B-lymphocytes.
 in general, the more immature the B lymph is, the better the prognosis.
 Burkitt type or L3 ALL is associated with transformed mature B cells and has a
poor prognosis.
 T cell ALL patients (adults and children) are not commonly seen and generally have
poorer prognosis.
The lineage and developmental stage can be identified using immunological techniques
to identify cell surface markers, This is known as immunophenotyping.

Experiential Activity
Complete the following to see if you have met the Objectives. Try to do it without
reference to information in your notes, your text or this package.

1. For each of the following acute leukemias, write a descriptive synonym.

Example: M1 - acute myeloblastic without maturation.

a. M2 -

b. M4 -

c. M5 -

Prof. Dr. Yousif Y. Bilto 8 Acute Malignant Blood Disorders

 d. L1 -

e. L2 -
2. Compare the morphology of Type I and Type II blasts in ANLL.

3. For each of the following conditions, describe the typical diagnostic bone
marrow to include:

 cellularity
 predominant cell type and developmental stage(s)
 any morphologically differentiating features
 M:E ratio

a. M2 acute leukemia

b. M3 acute leukemia

c. M5 acute leukemia

d. M0 acute leukemia

e. L1 acute leukemia

4. Consider the peripheral blood picture in acute leukemia and complete the
following to describe expected findings:

WBC (, N, ) ________________________________

Hb (, N, ) ________________________________

PLT (, N, ) ________________________________

Prof. Dr. Yousif Y. Bilto 9 Acute Malignant Blood Disorders

 RBC morphology ________________________________

Refer to Appendix A for Answers.

Prof. Dr. Yousif Y. Bilto 10 Acute Malignant Blood Disorders

OBJECTIVE TWO
Upon completing the module competency, the learner will be able to discuss the
laboratory tests used in the differential diagnosis of acute malignant blood disorders.

Learner Objectives

The learner will be expected to:

1. briefly explain the use of the following in the differential diagnosis of malignant
blood disorders:
morphology
cytochemistry
immunological method
electron microscopy
cytogenetics/molecular genetics

2. state the primary use for each of the following cytochemical reactions in
differential diagnosis of acute leukemias ad state/recognize results that provide
diagnostic information:
myeloperoxidase
sudan black B
periodic acid Schiff's
non-specific esterase (with and without fluoride)
specific esterase

3. recognize on stained films/preparations the peripheral blood and bone marrow

changes that would be diagnostic of:
L1 acute leukemia
M1, M2 and M3 acute leukemia

4. explain the significance of seeing L1, M1, M2 and M3 blood pictures in a new
patient and give some examples of appropriate response for a new Technologist.

Prof. Dr. Yousif Y. Bilto 11 Acute Malignant Blood Disorders

Exploration Activity
Read pg. 511-514 in Chapter 40 of the text - Systematic Laboratory Evaluation of
Leukocyte Abnormalities.

Examination of the "Decision Tree" on pg. 512 will explain the rationale for various
laboratory tests used to aid in differential diagnosis of leukocytosis and/or the presence
of abnormal/immature cells.

Note the following decisions that lead to diagnosis.

1. Does the problem involve lymphocytes or non-lymphocytes?

 usually the WBC and differential will answer this question - but what if
the predominant peripheral blood cell is a blast?

2. Is the leukocytosis a "reaction" (and therefore normal, self-limiting and

physiologically useful) or a "disorder"?

 usually the degree of leukocytosis and the actual cells present will
differentiate (plus the clinical picture). What other laboratory evidence
could apply here?

3. If the condition is a "disorder", differential diagnosis is required to separate the

various causes of similar clinical and laboratory pictures. Treatment and
prognosis varies with the cause.

This objective will deal with laboratory aids to differential diagnosis:

a. chronic vs acute malignancy

b. lineage i.e. lymphoproliferative or myeloproliferative (and what specific myeloid
cells)
c. degree of immaturity of cells with "blast" morphology

Prof. Dr. Yousif Y. Bilto 12 Acute Malignant Blood Disorders

Chronic vs. Acute Disorder

At diagnosis, the classification of leukemia as chronic or acute is determined by:

1. the patient's clinical symptoms and demographic factors.

2. the maturity of the affected blood cells and their prevalence (percentage) in the
patient's bone marrow.
3. the total WBC count/hemoglobin/platelet count.

Clinical
Symptoms/factors Chronic leukemia Acute leukemia
1. Clinical onset Insidious Sudden
2. Splenomegaly Becomes prominent Mild
3. Lymphadenopathy Usually mild Mild
4. Weakness/pallor Mild (progressive) Prominent
5. Bleeding/bruising Mild (progressive) Prominent
6. Fever/infections Mild (progressive) Prominent
7. Bone or joint pain Mild (progressive) Prominent
8.Neurological symptoms Mild (progressive) Prominent
9. Prognosis (untreated) 2-6 years 6 months or less
10. Patient age Typically adults All ages
Blood Cell Maturity
1. Peripheral blood cells Mostly mature Many blasts
2. % blasts - peripheral < 10% > 30%
3. Bone marrow cells Mostly mature Many blasts
4. % blasts - BM < 30% > 50%
Laboratory Data
1. Total WBC Count Always increased Variable
2. Hemoglobin mildly decreased Decreased (marked)
3. Platelet count variable (sl  to ) Decreased (marked)

* When correlating results with diagnosis in the laboratory, it is important to

remember the expected effect of treatment.

Prof. Dr. Yousif Y. Bilto 13 Acute Malignant Blood Disorders

Lymphoproliferative vs. Myeloproliferative

If the disorder is an acute leukemia, the first differentiation must be between lymphoid
and myeloid malignancy. The FAB classification relies on morphology and
cytochemistry.
1. Morphology from a Romanowsky-stained peripheral blood film or marrow
preparation is often not definitive. Blast cells often have few features to
differentiate lymphs from other cell lines.

Morphologic Clues for Differentiation of Leukemic Blasts

Myeloblast Monoblast Normoblast Megakaryocyte Lymphoblast
Blast size Large Large Megaloblastoid Small (10-20 Small (10-20 m)
m)
Cytoplasm Mod. amt Much/granular Mod/dark blue Irr. projections Scant/blue
Chromatin Fine, lacy Lacy/folded Denser/multinuc. Dense Dense
Nucleoli Prominent (> 2) Very prominent Not prominent Not prominent Indistinct/< 2
Auer rods Often present Rarely present Absent Absent Absent

2. Cytochemistry can provide more evidence as to the lineage of poorly

differentiated "blast" cells.
Cytochemistry is discussed in Chapter 29 of the text, pg. 393-398.
This Objective is concerned only with the use of cytochemical stains as an aid to
differential diagnosis. The next Objective will deal with performing and
evaluating the cytochemical reactions.
The usual purpose for performing cytochemical techniques is to differentiate
first between ANLL and ALL. To do this we can use:
 myeloperoxidase (MPO) stain
 Sudan Black B (SBB) stain

The choice of technique used will vary with the laboratory. MPO is probably
more frequently used. Some laboratories will use both.

Other stains discussed in this objective include:

 Leukocyte Alkaline Phosphatase (LAP)
 Specific and Non-Specific Esterase
 Peroxidase
 Periodic Acid-Schiff (PAS)

Prof. Dr. Yousif Y. Bilto 14 Acute Malignant Blood Disorders

  Perl’s Prussian Blue (PAB)
 Terminal deoxynucleotidyl transferase (TdT)

Prof. Dr. Yousif Y. Bilto 15 Acute Malignant Blood Disorders

Myeloproliferative - FAB Classification

Once an acute leukemia has been shown to be non-lymphoid, cytochemical reactions

can help identify specific lineage and, therefore, FAB classification.

a. MPO can be demonstrated in neutrophil primary granules but also in areas

of blast cytoplasm where primary granules will develop.

This could help differentiate myeloid or monocytic acute leukemia from

others.

b. Similarly, SBB could show some positivity in the malignant blasts in

granulocytic or monocytic acute leukemia.

c. Specific esterase, also called chloroacetate esterase is found in mature and

immature neutrophils and mast cells.

This technique is generally not considered as sensitive as MPO.

d. Non-specific esterase (NSE, acetate or butyrate esterase) is particularly

useful in monocytes. Positive cells in the marrow would be evidence for a
diagnosis of M4, M5a or M5b acute leukemia.

Esterase stains can be used together and/or with other reagents to provide
more information.

NSE is found in several cell types. However, monocyte NSE is inhibited

by addition of fluoride.

SE and NSE used together could demonstrate precursors of both the

myeloid series and monocytic series in M4 acute leukemia and the
relative numbers of each.

e. Periodic Acid Schiff's (PAS) (pg. 396 - Non-Enzymatic Techniques -

Carbohydrates).

Note that it is the pattern and intensity of staining that is important here.
It can be used to identify lymphoblasts in L1 or L2 acute leukemia.
Another purpose is to support a diagnosis of M6 acute leukemia.

Prof. Dr. Yousif Y. Bilto 16 Acute Malignant Blood Disorders

 Study the following chart on the cytochemical stains taking careful note about the
following:

a. the cell constituent that is stained

b. the cells that usually stain positive
c. the usual use or expected results from each stain

Cytochemical Stain a. Constituent stained b. Positive cells c. Expected results

Leukocyte alkaline LAP enzyme (present in Neutrophils Low score: CML, PNH
phosphatase (LAP) neutrophils) High score: leukemoid rx
pregnancy, polycyth, vera
Myeloperoxidase primary granules - granulocytes M1-M4 - Strong rxn
(MPO) - monocytes M5 - negative or weak rxn

Sudan black B (SBB) phospholipids and - granulocytes M1-M4 - positive rxn

lipoproteins - monocytes M5 - weakly positive
- macrophages
Naphthol ASD stains iso-enzymes - neutrophils M1-M4 - positive rxn
chloracetate esterase 1, 2, 7, 8 and 9 (mature and M0, M5-M7 - negative rxn
(specific) immature)
- mast cells
Alpha-naphthyl stains iso-enzymes - monocytes M4-M7 - positive rxn
acetate esterase 3, 4, 5 and 6 - megakaryocytes M1-M3 - negative rxn
(nonspecific) with
fluoride - plasma cells
- lymphocytes
Alpha-naphthyl stains iso-enzymes - monocytes M1-M3 - negative rxn
butyrate esterase 2 and 4 - megakarycytes M4-M5 - positive rxn
(nonspecific) with
fluoride - lymphocytes M0, M6-M7 - negative rxn

Periodic acid Schiff - glycogen - leukocytes M0-M4 - not done but cells
- mucoproteins show variety of rxns
- high molecular weight M4-M5 - positive
- carbohydrates ALL - granular positivity
TdT - terminal - lymphoid cells ALL - positive
deoxynucleotidyl AML - negative
transferase

It should be noted that sometimes, because of degree of immaturity or abnormality,

leukemic cells will not react with any of the cytochemical techniques. There are other

Prof. Dr. Yousif Y. Bilto 17 Acute Malignant Blood Disorders

methods used in such situations to try to obtain the necessary information.

Prof. Dr. Yousif Y. Bilto 18 Acute Malignant Blood Disorders

3. Immunological testing (Immunophenotyping)

Antibodies are used to detect markers on cell membranes, in the cytoplasm or

the nucleus, that are useful in identifying the lineage and degree of immaturity of
cells in malignant disorders.

a. Cell surface markers are antigens on cell surfaces that are expressed at
different times during maturation.
detected using specific antibodies
detected using peripheral blood or bone marrow cell suspensions and
immunofluorescence often with a flow cytometer.

The monoclonal antibodies used to identify these markers are named

"CD" (cluster designation) with an identifying number.

eg. CD8 monoclonal antibody reacts with an antigen specific to

the membrane of suppressor-cytotoxic T cells.

CD4 reacts with helper-inducer T cells

CD19 reacts with B cells and early B cell precursors.

CD33 reacts with myeloid progenitors.

b. Cytoplasmic marker
the only one used is cytoplasmic immunoglobulin.
it is a marker for pre-B cells
detected using direct immunofluorescence.

c. Nuclear markers
TdT (terminal deoxynucleotide transferase) - a nuclear enzyme present
in lymphoid stem cells and precursor B and T cells
useful in distinguishing ALL from AML
detected using immunocytochemistry (PAP) or immunofluorescence,
or occasionally, radioimmunoassay (text, pg. 397 and 476-478).

4. Electron Microscopy

 used to study ultrastructure of the cells

 may be used to search for organelles or inclusions that can identify
lineage in malignant cells which show no specific morphological features
using standard optics and which react negatively with cytochemistry.

Prof. Dr. Yousif Y. Bilto 19 Acute Malignant Blood Disorders

5. Cytogenetics/Molecular Genetics

 identifies chromosome abnormalities in leukemic cells

 some are strongly associated with a particular leukemia (eg. Ph1); others
are not but detecting the abnormality may affect:
the diagnosis
subclassification
selection of therapy (provides prognostic information)
monitoring effects of therapy

Molecular genetics can detect the locations on the chromosome that are
affected. It is used to:
confirm suspected abnormalities which weren't detected by
cytogenetics
detect residual malignant cells after treatment. The marrow may
appear morphologically normally, but molecular techniques can detect
extremely small numbers of cells.

These procedures are not in routine use as yet.

To summarize, in suspected malignant blood diseases:

1. The laboratory assists with differentiating chronic and acute disease using the

 leukocyte count and differential

 hemoglobin and platelet count
 bone marrow morphology

These tests would also differentiate most non-malignant disorders from

malignant ones.

2. lymphoproliferative and myeloproliferative disease can usually be differentiated

by:

 marrow morphology
 cytochemistry (which two reactions?)

3. The FAB of a myeloproliferative disorder is identified using morphology and

cytochemistry.

4. More information about malignant cell lineage and maturity can be provided by

 immunophenotyping (can you list three types of markers detected?)

 electron microscopy

Prof. Dr. Yousif Y. Bilto 20 Acute Malignant Blood Disorders

  cytogenetics/molecular genetics

Prof. Dr. Yousif Y. Bilto 21 Acute Malignant Blood Disorders

Experiential Activity

1. Complete the following:

Chronic leukemia Acute Leukemia

% blasts in peripheral blood ………………... ………………..
Maturity of marrow cells ………………… ……………….
Hemoglobin level ………………… ……………….

2. Briefly describe at least 3 types of testing used in the differential diagnosis of

acute leukemias when morphology and cytochemistry are not definitive.

3. Which cytochemical reaction could be used on bone marrow to differentiate

between L1 and M1 acute leukemia? Describe expected results.

4. Patients with active acute leukemia typically show petechiae, bruising and often
bleeding from mucous membranes. Explain how the disease process can lead to
these clinical findings.

5. What is demonstrated in a cell by a TdT?

Refer to Appendix A for Answers.

Prof. Dr. Yousif Y. Bilto 22 Acute Malignant Blood Disorders
Practical Application Activity
Attend (a) laboratory session(s) to examine peripheral blood films and bone marrow
preparations from cases of common acute leukemia.

Remember that not all patients will present with the "textbook" picture. It is important
that the significant changes be detected and, when unexpected, be drawn to the
attention of the physician according to lab protocol.

OBJECTIVE THREE
Upon completing the module competency, the learner will be able to apply principles
of cytochemistry to perform stains commonly used in the differential diagnosis of
blood cell malignancies.

Learner Objectives

The learner will be expected to:

1. for each of the following reactions:

 myeloperoxidase
 sudan black B
 periodic acid Schiff's
 non-specific esterase (with and without fluoride)
 specific esterase
select/prepare/describe a suitable preparation.
select/prepare/describe suitable Controls.
prepare reagents as required, following written instructions.
perform the procedure, following written instructions.
evaluate the suitability of the stained preparation for examination.
respond appropriately to unsuitable results.

The students may be asked to explain the rationale for any of the steps involved.

Exploration Activity
Read the details of the methods given on pg. 394-397 in Chapter 29 - Cytochemistry.
A few points for each stain are highlighted below:

1. Myeloperoxidase (MPO) (pg. 396)

 It is an enzyme technique. The myeloperoxidase enzyme is found in the
primary granules of myeloid and monocytic cells.

Prof. Dr. Yousif Y. Bilto 23 Acute Malignant Blood Disorders

  Myeloperoxidase is best done on FRESH peripheral blood films or bone
marrow preparation as it deteriorates during storage.
 The slides may be fixed and stored at 4°C if staining must be delayed.
 The reagent - 3,3-diaminobenzidine tetrahydrochloride used in this stain is
carcinogenic and requires special handling.
 It should be handled in a fume hood with the user wearing gloves and a mask.
 Controls should be a normal donor with neutrophils (positive) and
lymphocytes (negative).
 A specimen with varying degrees of myeloid cells is good as well.
 To assess whether the stain worked the neutrophils should stain positively
(dark brown granules in the cytoplasm) while the lymphocytes stain
negatively (no granules in the cytoplasm).

2. Sudan Black B (SBB) (pg. 397)

 Stains the lipids within monocytes and granulocytes but NOT lymphocytes.
 This technique may be performed on peripheral blood films or bone marrow
preparations.
 The SBB is stable and can be performed on specimens even after several
months.
 SBB is slightly more sensitive to primitive myeloid cells than MPO.
 SBB reagents are not carcinogenic.
 A normal blood film can be used as a control slide.
Results:
- neutrophils - positive
- lymphocytes - negative
 The neutrophil lineage should show increasing positivity as the cells mature.
 If Romanowsky stains are used to counterstain these preparations, the nuclei
will appear red.

3. Periodic Acid Schiff's (PAS) (pg. 396)

 Best done on fresh peripheral blood films or marrow preparations.
 Glycogen is the component being demonstrated and is found in all neutrophils
and their precursors.
Prof. Dr. Yousif Y. Bilto 24 Acute Malignant Blood Disorders
  All normal leukocytes are PAS positive, except lymphocytes and erythrocyte
precursors. They are differentiated using staining intensity and the pattern
of reaction.
 M6 (Erythroleukemia) - erythroid precursors (granular PAS positive - early
forms to diffusely positive - later forms).
 ALL - may show "chunky" granular PAS positivity.
 Gaucher disease - Gaucher cells show strong PAS positivity.
 Normal peripheral blood films can be used as a control with neutrophils being
bright pink.

4. Non-Specific Esterase (NSE) (pg. 395-396)

 Enzyme technique demonstrating iso enzymes 3, 4, 5 and 6.
 Found in monocytes, megakaryocytes and plasma cells.
 This procedure can be done on peripheral blood films or bone marrow
preparations.
 This enzyme remains stable and active after months of storage and can be
stored unfixed for up to 2 weeks in the dark.
 Some of the reagents for this stain must be made up of fresh.
 There are at least two different substrates (butyrate or acetate) used for
demonstrating substrates. Different cell types stain somewhat differently in
the different substrates. The primary purpose for demonstrating NSE is to
identify monocyte precursors.
 Normal peripheral blood smears with monocytes as well as bone marrow
aspirates with monocytes and histiocytes can be used as positive controls.
 Positive results show monocytes and histiocytes with dark red precipitate in
the cytoplasm.
 The NSE of monocytes is inhibited by addition of sodium fluoride to the
staining mixture. This can be used to identify monocyte precursors in a
mixture of cells.

5. Specific Esterase (SE) (pg. 395-396)

 Enzyme procedure demonstrating iso enzymes 1, 2, 7, 8 and 9.
 These iso enzymes are present in neutrophils and mast cells.
 Peripheral blood or marrow preparations can be used for this stain.
 The enzyme is stable and remains active months after storage.
Prof. Dr. Yousif Y. Bilto 25 Acute Malignant Blood Disorders
  This technique is NOT as useful as MPO for demonstrating myeloid cells in
peripheral blood or marrow. It is useful in two areas:
to demonstrate myeloid elements in paraffin embedded sections.
to be used with NSE (combined esterase technique) to show the
presence of both myeloid and monocyte precursors in M4 acute
leukemia.
 A normal peripheral blood film can be run along with the patient specimen.
 Positive activity is seen as bright red granules in the cytoplasm of mast cells,
neutrophils and neutrophil precursors.
(promyelocytes and myeloblasts may also be positive)
occasional monocytes may be weakly positive as well.

Experiential Activity
1. For each of the following stains indicate which cells will stain positively.

a. Myeloperoxidase

b. Non-specific Esterase (with and without flouride)

c. Specific Esterase

d. Sudan Black B

e. Periodic Acid Schiff's

Prof. Dr. Yousif Y. Bilto 26 Acute Malignant Blood Disorders

Refer to Appendix A for Answers.

Prof. Dr. Yousif Y. Bilto 27 Acute Malignant Blood Disorders

Practical Application Activity
Perform the procedure(s) available at your facility or selected by your Instructor.
Follow written instructions, prepare reagents as required, select and include
appropriate controls and evaluate suitability of your results.

Submit finished preparations that you consider to be satisfactory to your instructor for
his/her evaluation.

Finished preparations and controls must be clearly and carefully labelled according to
facility policy and must identify the procedure done.

OBJECTIVE FOUR
Upon completing the module competency, the learner will be able to recognize and
respond appropriately to laboratory test results which could have diagnostic
significance in acute malignant blood diseases.

Learner Objectives

The learner will be expected to:

1. perform related laboratory tests and/or examine results to correlate significant

findings with a given diagnosis.

2. recognize changes in tests results that could have significance in malignant

blood diseases and respond appropriately. (Results could be obtained by
performing the tests or could be given.)

3. detect morphological changes in peripheral blood and bone marrow that could be
significant in the detection and differential diagnosis of blood diseases and
respond appropriately.

Exploration Activity
1. Attend the laboratory sessions scheduled to study examples of peripheral blood
films and bone marrow preparations from patients with the disorders discussed
in this module.

Since few patients present with the "textbook picture" of a disorder, try to
differentiate between findings that would be critical to a diagnosis (clinically
significant) and those that could vary.

Prof. Dr. Yousif Y. Bilto 28 Acute Malignant Blood Disorders

Consider the effects of treatment on the laboratory findings. Specimens seen in a
clinical setting may be for diagnosis, on patients being treated, in relapse or remission.
This definitely affects what you might see.

Practical Application Activity

1. Perform examination of peripheral blood films and/or bone marrow preparations
from patients with a known diagnosis. Identify anomalous/unexpected and
take/describe appropriate action.

2. Perform examination of peripheral blood films and/or bone marrow preparations

from undiagnosed patients. Identify findings that could have important clinical
significance and take/describe appropriate action.

3. Given sets of laboratory test/procedure results from patients with known

diagnoses, identify changes in results that could have clinical significance and
take/describe appropriate action.

4. Given sets of laboratory test/procedure results from undiagnosed patients,

identify results that could have important clinical significance and take/describe
appropriate action.

MODULE SUMMARY
This module describes the blood and bone marrow changes expected in acute
leukemia. The FAB classification identifies 8 types of acute non-lymphoblastic
leukemia (including M0) and 3 types of acute lymphoblastic leukemia. The FAB is a
morphological classification. Often more specific testing is required to correctly
classify non-typical diseases.

The differences in cell maturity between acute and chronic leukemias is usually
marked. Most acute leukemias show a preponderance of blast cells which can make
morphological identification difficult. Cytochemistry is often useful in differentiating
the various acute leukemias and several reactions are in common use. These include
Myeloperoxidase, Sudan Black B, Specific and Nonspecific esterases and Periodic
Acid Schiff's.

In addition, cytoplasmic and nuclear markers are used to determine lineage, clonality
and maturity of malignant cells.

Prof. Dr. Yousif Y. Bilto 29 Acute Malignant Blood Disorders

MODULE POST-ASSESSMENT

Complete the following exercise to help you focus on hematologic findings that are
clinically significant.

1. For each patient shown or on the following chart:

a. examine given results.

b. give the diagnosis with which these results are most consistent. Your
choices are any of the disorders discussed in this module.

c. explain, giving as many reasons as possible from the data:

 why malignant (and not benign) blood disease?

 why the particular diagnosis you suspect?

 Which peripheral blood finding(s), in a new patient, could result in

bone marrow aspiration being ordered?

Prof. Dr. Yousif Y. Bilto 30 Acute Malignant Blood Disorders

LAB TEST Patient A Patient B Patient C Patient D Patient E Patient F
patient data Male: 5 yr Female: 16 y Male: 25 y Female: 45 y male: 25 y Female: 8 y
multiple lethargic, lethargic, lethargic, gingival fatigue, night
bruises, pale fever, urinary headache, splenomegaly, bleeding, sweats,
tract bruised bruised petechiae petechiae,
infection, hepatomegaly
menorrhagia
WBC (x 109/L) 14.5 22.2 82.4 3.2 30.8 109.5
Hb (g/L) 95 75 90 82 85 70
Platelets (x 109/L) 24 60 14 82 22 32
WBC Differential
% Blasts 89 40 68 8 70 49
% Promyelocytes 0 10 0 1 25 15
% 0 10 0 0 0 13
Myelocytes/metas
%Bands 0 8 0 5 0 6
%Neutrophils 2 5 5 15 2 10
%Lymphocytes 9 2 2 66 3 7
%Monocytes 0 25 25 5 0 0
Comments on Normocytic Auer rods, large blasts, 20 NRBCs, Auer rods, Auer rods
peripheral blood normochromic RBCs: normal lacy anis, poik Schistocytes
film chromatin,
many nucleoli
Comments on Hypercellular, Hypercellular, Hypercellular Erythroid Myeloid Myeloid
bone marrow 95% blasts blasts 60% - blasts 90% hyperplasia, hyperplasia hyperplasia
monos 30% ringed blasts 60%
sideroblasts
Myeloperoxidase neg pos neg neg pos pos
stain
SBB stain neg pos neg neg pos pos
SE stain neg pos neg neg pos pos
NSE stain neg pos pos neg neg neg
PAS stain block - neg block - neg
positivity positivity
LAP stain NA - - - -

Refer to Appendix A for Answers.

Prof. Dr. Yousif Y. Bilto 31 Acute Malignant Blood Disorders

REFERENCES

Dacie, Sir John V and S.M. Lewis. Practical Haematology. Churchill Livingstone, New York.
ISBN 0-443-03952-6.
Harmening, Denise M. Clinical Hematology and Fundamentals of Hemostasis. F.S. Davis
Company, Philadelphia.
Rodak, Bernadette F. Diagnostic Hematology. W.B. Saunders, Philadelphia.
Sacher, Ronald A. and Richard A Macpherson. Widmann's Clinical Interpretation of Laboratory
Tests. F.A Davis Company, Philadelphia. ISBN 0-8036-7694-8.

Prof. Dr. Yousif Y. Bilto 32 Acute Malignant Blood Disorders

APPENDIX A
Answers to Experiential Activity - Objective One
1. a. M2 - Acute Myeloblastic Leukemia with Maturation.
b. M4 - Acute Myelomonocytic Leukemia
c. M5 - Acute Monocytic Leukemia
d. L1 - Small cell, Homogeneous
e. L2 - Large cell, Heterogeneous
2. Type I Blasts
- no cytoplasmic differentiation
- no cytoplasmic granules
- prominent nucleoli
- smooth chromatin structure
- high nuclear/cytoplasmic ratio
Type II Blasts
- some cytoplasmic differentiation
- cytoplasmic granules (azurophilic) present (<20)
- central nucleus
- nuclear/cytoplasmic ratio is low
3. a. M2 Acute Leukemia
- hypercellular
- increased M:E ratio
- blasts (Type I and Type II combined) greater than 30%
- 10% of remaining cells are granulocytic
- less than 20% monocytes
- auer rods may be present
b. M3 Acute Leukemia
- hypercellular
- increased M:E ratio
- predominant cell - promyelocyte with heavy granulation
- M3m - a variant of this leukemia is lacking the granules
- bundles of auer rods (faggots) may be present
- nuclei can be kidney shaped or bi-lobed
c. M5 Acute Leukemia (Schilling Leukemia)
- hypercellular
- normal M:E ratio
- promonocyte predominant cell in the marrow
- M5a - more than 80% of monocytes are blasts
- M5b - less than 80% of monocytes are blasts
- all stages of monocyte development present

Prof. Dr. Yousif Y. Bilto 33 Acute Malignant Blood Disorders

 - auer rods may be present
- large blast with delicate lacy chromatin
- one to three prominent nucleoli

d. M0 Acute Leukemia
- hypercellular
- increased M:E ratio
- greater than 30% blasts in marrow
- blasts are usually Type I
- auer rods are NOT found

e. L1 Acute Leukemia
- hypercellular
- normal M:E ratio
- predominant cells are blasts
- blasts are small - up to twice the diameter of a small lymphocyte
- uniform cell (homogeneous)
- fine chromatin structure
- nuclear may cleave, fold or indent
- cytoplasmic vacuoles may be present

4. WBC - increased (Leukocytosis secondary to increased circulating blast cells)

Hb - decreased

Platelet - decreased

RBC Morphology - normocytic/normochromic

Answers to Experiential Activity - Objective Two

1. Chronic Leukemia Acute Leukemia

% blast in peripheral blood < 5% > 30%

maturity of marrow cells all stages little or no

maturation

hemoglobin level decreased decreased

(as condition
progresses)
Prof. Dr. Yousif Y. Bilto 34 Acute Malignant Blood Disorders
2. immunophenotyping - antibodies are used to detect markers on cell membranes

electron microscopy - study the ultrastructure of cells as well as search for

organelles or inclusions

cytogenetics/molecular genetics - detects chromosomal abnormalities - detects

locations on chromosome where genes are affected

3. myeloperoxidase

L1 Acute Leukemia - negative

M1 Acute Leukemia - positive

4. With acute leukemia megakaryocytes are decreased leading to decreased

platelets. The platelets may also be dysfunctional. This will lead to petechiae,
bruising and gingival bleeding.

5. TdT - Terminal deoxynucleotidyl Transferase is an intranuclear DNA

polymerase enzyme.
- TdT can be detected in human thymocytes, primitive lymphocytes.
- 90% of ALL and lymphoblastic lymphomas show strong activity.
- Useful in detection of lymphoblastic transformation of CML.

Answers to Experiential Activity - Objective Three

1. a. MPO - neutrophils - positive

- lymphocytes - negative

b. NSE - without flouride - monocytes positive

- with flouride - monocytes negative

c. SE - neutrophils and neutrophil precursors - positive

- mast cells positive
- occasional monocyte positive
- promyelocytes and myeloblasts - may be positive

d. Sudan Black B - neutrophils (segmented) - strong positivity

- myeloblasts - negative
- positivity increases as myeloid cell matures
- monocytes slight to moderate positivity
- lymphocytes - negative
Note: blasts in AGL can be strongly positive
- auer rods - positive
Prof. Dr. Yousif Y. Bilto 35 Acute Malignant Blood Disorders
 e. PAS - all normal leukocytes show positivity except lymphocytes
and erythroid precursors.
- M6 Erythroleukemia - erythroid precursors show granular PAS
positivity (early forms) and diffuse positivity (later forms).
- ALL lymphoblasts show "chunky" granular positivity
- Gaucher cells - strong positivity

Prof. Dr. Yousif Y. Bilto 36 Acute Malignant Blood Disorders

Answers to Module Post-Assessment

1. a. Patient A - Acute Lymphoblastic Leukemia

Patient B - M4 - Acute Myelomonocytic Leukemia
Patient C - M5 - Acute Monocytic Leukemia
Patient D - M6 - Acute Erythroleukemia
Patient E - M3 - Acute Promyelocytic Leukemia
Patient F - M2 - Acute Myeloblastic Leukemia with Maturation

b. Patient A - malignant (ALL) due to:

- blast in peripheral blood and marrow
- low hemoglobin
- low platelets
- block positivity of PAS (typical for L1 - ALL)
- hypercellular marrow with 95% blasts

All of these factors and the patient's age (5 years) make a diagnosis of
ALL feasible.
- a bone marrow aspirate would be necessary to diagnose the
leukemia and determine the marrow involvement

Patient B - M4 - Myelomonocytic Acute Leukemia - malignant (acute)

due to:
- high number of blasts in the peripheral blood and bone marrow
- increased monocytes in PB and bone marrow also increased
- auer rods
- WBC, Hgb and PLt

Patient C - M5 - Acute Monocytic Leukemia - malignant:

- 90% of marrow cells are blasts
- 68% of PB cells are blasts
- greater than 20% of PB cells are monocytes
- blasts description matches leukemic monoblasts
- WBC, Hgb and PLt all support acute leukemia
- NSE positive indicates monocytes
** cytochemical stains for myeloid cells are negative

Patient D - M6 - Erythroleukemia - malignant

- pan-cytopenia
- increased nucleated RBC's
- > 5% blasts in PB

Prof. Dr. Yousif Y. Bilto 37 Acute Malignant Blood Disorders

 - dyserythropoiesis (aniso and poik)
- block positivity for PAS
- pancytopenia

Patient E - M3 - Acute Promyelocytic Leukemia - malignant

- increased WBC and 70% blasts in PB
- 25% promyelocytes
- auer rods present
- cytochemical stains indicate myeloid line
- presence of schistocytes indicate DIC which is a symptom of M3

Patient F - M2 - Acute Myeloblastic Leukemia with Maturation - malignant

- 49% blasts in PB and 60% in bone marrow
- auer rods present
- WBC, Hgb, PLt
- some maturation of myeloid cell line
- cytochemical stains indicate myeloid cell line

Prof. Dr. Yousif Y. Bilto 38 Acute Malignant Blood Disorders