Hematopathology Lab 2

Start Slide Show

Read history , review image stacks (space bar, arrow, mouse click)
Space bar, arrow, mouse click to reveal question
Space bar, arrow, mouse click to reveal answers
After working through case using PPS, open virtual slide (links in PPS
or LAB 2 Web page) and review
This PowerPoint show (PPS) uses simple space-bar/arrow/mouse-click triggered animations to present the case
history, targeted micrographs (from virtual slides), relevant questions and a uniform set of "answers". Your instructor
will briefly introduce each case then ask students to work through the questions, answers and virtual slides using the
PPS on their laptops. The micrographs are displayed adjacent to micrographs of a "normal" or "look-alike" abnormal
smear at the same magnification. The micrographs should serve as a guide for viewing the virtual slides. The virtual
slides for this exercise can be accessed from the links embedded in the PPS (if you are using a workstation or laptop
on the Medical School Campus) or the LAB 2 web page. The final exam has 10 lab questions. Five from the set of
micrographs you studies during week 1 (review Atlas of normal and abnormal Hematology 2013) and five from Lab
1 and Lab 2 virtual slides (self-test tool).
Normal smears
Blood smear stack Bone marrow stack
Virtual Slide Normal Blood Smear (Web viewer) Virtual Slide Normal Marrow Smear (Web viewer)

Case #1: The patient is a 55-year-old female who presented to her local physician with increasing
fatigue, night sweats, weight loss, and abdominal "fullness." On physical examination, the patient
was noted to have a firm, nontender spleen, 10 cm below the left costal margin. The liver was
slightly enlarged to 2 cm below the right costal margin. No lymphadenopathy was identified. The
CBC showed: WBC = 105,000 cells/mm3, Hb = 10.5 gm/dl, and platelets = 85,000/mm3.
Normal smear stack
Patient stack
Virtual Slide Normal Blood Smear (Web viewer) Virtual Slide Case#1 (Web viewer)

Case #1: The patient is a 55-year-old female who presented to her local physician with increasing
fatigue, night sweats, weight loss, and abdominal "fullness." On physical examination, the patient
was noted to have a firm, nontender spleen, 10 cm below the left costal margin. The liver was
slightly enlarged to 2 cm below the right costal margin. No lymphadenopathy was identified. The
CBC showed: WBC = 105,000 cells/mm3, Hb = 10.5 gm/dl, and platelets = 85,000/mm3.
What are the major abnormalities on the smear?
Markedly elevated WBC, much higher than sepsis case examined previously. Neutophilia with many precursors (see virtual slide of
bone marrow aspirate for comparison). Percentage of precursors higher than in most reactive causes of leukocytosis. Also more
immature forms (myelocytes, prograns and a few blasts) in this smear compared to usual "reactive left shift". Basophils easy to spot
(usually very few in normal or reactive leukocytosis). The cells may not be markedly abnormal appearing, just too many.

What is the differential diagnosis based on the CBC and blood smear?
Chronic myeloid leukemia (CML), chronic phase, versus leukemoid reaction (excessive reactive leukocytosis).

Would flow cytometry help with the diagnosis?
No. We know from the morphology that the proliferative process involves relatively normal appearing granulocytes and granulocytic
precursors. There are no antigenic findings that can separate chronic phase CML from a reactive leukocytosis.

Would molecular or cytogenetic tests help with the diagnosis?
Absolutely since the acquired structural genetic defect that causes CML has been identified. The Philadelphia chromosome t(9;22) can
be detected in the neoplastic cells using classical cytogenetics or FISH. The translocation creates a new gene, the bcr:abl transgene,
that can be detected using molecular (PCR) techniques. Demonstration of either the Philadelphia chromosome or the bcr:abl
transgene are required for diagnosis. (more questions on next slide)


Case #1: The patient is a 55-year-old female who presented to her local physician with increasing
fatigue, night sweats, weight loss, and abdominal "fullness." On physical examination, the patient
was noted to have a firm, nontender spleen, 10 cm below the left costal margin. The liver was
slightly enlarged to 2 cm below the right costal margin. No lymphadenopathy was identified. The
CBC showed: WBC = 105,000 cells/mm3, Hb = 10.5 gm/dl, and platelets = 85,000/mm3.
What is the natural course of this disease? Are there changes in the blood or bone marrow smears that one can use to monitor
progression?
The bcr:abl transgene (formed by t(9;22)) encodes a chimeric fusion protein with a constitutively active protein tyrosine-kinase
domain that affects many downstream signaling cascades. Clonal expansion and immortalization of the pleuripotential
hematopoietic stem cell occurs (common to lymphoid and non-lymphoid or myeloid lineages). In most individuals, the mutant stem
cells continue differentiating primarily down the granulocyte lineage. The bone marrow fills up with normal looking granulocytic
precursors that spill out into the bloodstream and accumulate in the spleen. Over time, additional mutations accumulate (largely
unknown) that result in loss of differentiation, and an increase in the percentage of minimally differentiated cells (blasts). Ultimately,
a lymphoid or myeloid "blast crisis" is likely to occur (conversion to acute leukemia).
The loss of maturation in the neoplastic clone results in a gradual increase in the percentage of cells with the morphologic features of
immature bone marrow cells called "blast forms". These cells have a variety of microscopic appearances. It may be difficult to tell
whether they are myeloid or lymphoid blasts based on appearance in the microscope. Flow cytometry (detects lineage-specific
antigens on cell surface) and cytochemical stains (detects enzymes associated with granulocytic and monocytic maturation) can help
determine lineage.
When the percentage of blast forms in the blood or bone marrow is > 10% but < 20% then the patient is in an "accelerated" phase of
CML. When the percentage of blast forms in the blood or bone marrow is 20% or greater the patient is in "blast crisis" or blast
phase. Blast crisis or blast phase is a conversion to an acute leukemia.
Case #2
History #1: 58-year-old male with increasing weakness, fatigue, malaise, and weight loss over the
previous six weeks. He sought medical attention for several unexplained lower extremity bruises.
Petechiae and bruises noted. WBC = 155,000 cells/mm3, Hb = 9.0 gm/dl, platelets = 11,000/mm3.
History #2: Four-year-old female with a recent onset of pharyngitis and otitis, unresponsive to
antibiotics. The patient was febrile to 101.3 degrees F with sudden onset of fatigue, malaise, and
nondescript bone and joint pains. Physical exam revealed conjunctival pallor and petechiae on
her lower extremities. WBC = 55,000 cells/mm3, Hb = 7.6 gm/dl, platelets = 5,000/mm3.
History #1 stack
History #2 stack
Virtual Slide History #1 (Web viewer) Virtual Slide History #2 (Web viewer)
Case #2
History #1: 58-year-old male with increasing weakness, fatigue, malaise, and weight loss over the
previous six weeks. He sought medical attention for several unexplained lower extremity bruises.
Petechiae and bruises noted. WBC = 155,000 cells/mm3, Hb = 9.0 gm/dl, platelets = 11,000/mm3.
History #2: Four-year-old female with a recent onset of pharyngitis and otitis, unresponsive to
antibiotics. The patient was febrile to 101.3 degrees F with sudden onset of fatigue, malaise, and
nondescript bone and joint pains. Physical exam revealed conjunctival pallor and petechiae on
her lower extremities. WBC = 55,000 cells/mm3, Hb = 7.6 gm/dl, platelets = 5,000/mm3.
What are the major abnormalities on the smear?
Many (>20%) of the WBC are mononuclear cells that differ from both normal monocytes and activated lymphocytes . Most have
nuclei with lighter staining, finely divided, less clumped chromatin and occasional nucleoli (some multiple). These fit the general
category of "blast" forms. Blast is an imprecise term that refers to cells with nuclear characteristics shared by early stages of
differentiation in the bone marrow.
Blasts have an "active" appearing nucleus (finely dispersed chromatin, nucleoli). There may be evidence of cytoplasmic
differentiation that can help identify lineage (e.g. granules). Normal bone marrows have 1-2% blasts that include the earliest stages
of myeloid (non-lymphoid/non-erythroid) and lymphoid (pro-, pre-B and pro-, pre-T) differentiation. Any blasts in a blood smear is
abnormal and, if unexplained, may require a bone marrow aspiration/biopsy to evaluate for a possible stem cell neoplasm.
The disorders in this case illustrate two types of stem cell neoplasms. All stem cell neoplasms arise from stem and/or early progenitor
cells in the bone marrow (What are the major groups? How do they differ from one another? See schematic of hematopoiesis in Atlas
or Dr. Stoolmans lecture). The neoplastic stem cells in these cases both self renew and give off progeny that differentiate to a limited
degree. In acute leukemias, maturation of the neoplastic cells arrests at a relatively early stage of lymphoid or non-lymphoid
differentiation. The abnormal cells accumulate in the bone marrow, suppress normal hematopoiesis (what does this cause?) and may
spill out into the bloodstream (few or many).
Are these histories and smears from patients with acute leukemias, viral infections or sepsis ?
Both are acute leukemias. It can be difficult for the novice to distinguish a reactive lymphocytosis (e.g. florid mononucleosis) from
leukemia based on the appearance of the atypical cells in the smear alone. The history, CBC and clinical findings are crucial pieces of
information. Patients with acute leukemia generally have the signs, symptoms and laboratory findings indicating bone marrow
insufficiency or failure in addition to the presence of blast forms. Patients with reactive lymphocytosis should not have such findings.
Once you have determined that there are a large percentage (>20%) blasts in blood or bone marrow, know how additional
immunologic studies (flow cytometry primarily), cytochemical stains, cytogenetics and molecular studies are used to establish a
precise classification in acute leukemias (see questions below). You should be able to identify Auer rods and know the clinical
significance of the finding.
Case #2
History #1: 58-year-old male with increasing weakness, fatigue, malaise, and weight loss over the
previous six weeks. He sought medical attention for several unexplained lower extremity bruises.
Petechiae and bruises noted. WBC = 155,000 cells/mm3, Hb = 9.0 gm/dl, platelets = 11,000/mm3.
History #2: Four-year-old female with a recent onset of pharyngitis and otitis, unresponsive to
antibiotics. The patient was febrile to 101.3 degrees F with sudden onset of fatigue, malaise, and
nondescript bone and joint pains. Physical exam revealed conjunctival pallor and petechiae on
her lower extremities. WBC = 55,000 cells/mm3, Hb = 7.6 gm/dl, platelets = 5,000/mm3.
Look carefully at multiple abnormal cells. Does their appearance provide any clues to that help refine the diagnosis?
Patient with history #1 has one of several types of Acute Myeloid Leukemia (AML). Suspect the diagnosis when blasts have
cytoplasmic granularity. Finding a few blasts with Auer rods confirms the diagnosis but occurs in small percentage of cases.
Additional studies (see questions below) needed to confirm and further sub classify these neoplasms.
Patient with history #2 most likely has one of two types of Acute Lymphoblastic Leukemia (ALL): Acute B-lymphoblastic leukemia or
Acute T-lymphoblastic leukemia (synonymous with acute precursor B- and precursor T- lymphoblastic leukemias, respectively).
Suspect the diagnosis when blasts have a high nuclear/cytoplasmic ratio and agranular cytoplasm. Cannot determine whether B- or
T-blasts without additional studies. Some poorly differentiated AMLs have similar blasts.
Lymphoblastic neoplasms may present as leukemias, bone marrow and blood involvement primarily, or lymphomas, thymic, nodal
or extra-nodal infiltrates primarily (can you explain this behavior ?). You may see them referred to as B-lymphoblastic or T-
lymphoblastic leukemia/lymphoma as a result.
The relative frequencies of different types vary in children and adults (e.g. ALL > AML in children, AML > ALL in adults). The clinical
presentations and the approach to diagnosis are similar, regardless of age.
Would flow cytometry help with the diagnosis?
Absolutely. One can usually determine the lineage of an acute leukemia using a panel of antibodies that react with stem cell,
granulocytic, monocytic, T and B cell antigens. Currently part of the initial diagnostic work-up for all suspected acute leukemias.
Cytochemical stains for enzymes associated with granulocytic (myeloperoxidase, MPO) and monocytic (non-specific esterase, NSE)
differentiation is helpful in diagnosing AML. No cytochemical stains help with ALL.
Would molecular or cytogenetic tests help with the diagnosis?
Absolutely. Necessary for confirmation of acute promyelocytic leukemia (t15;17; APL), identification of de novo acute leukemias
associated with Ph1 translocation and detection of the recurrent genetic abnormalities used by the WHO system to classify acute
leukemias. Likely that impact of cytogenetic, FISH and molecular genetic studies will grow as we learn more about the genetic basis
of these cancers.


Case #2
History #1: 58-year-old male with increasing weakness, fatigue, malaise, and weight loss over the
previous six weeks. He sought medical attention for several unexplained lower extremity bruises.
Petechiae and bruises noted. WBC = 155,000 cells/mm3, Hb = 9.0 gm/dl, platelets = 11,000/mm3.
History #2: Four-year-old female with a recent onset of pharyngitis and otitis, unresponsive to
antibiotics. The patient was febrile to 101.3 degrees F with sudden onset of fatigue, malaise, and
nondescript bone and joint pains. Physical exam revealed conjunctival pallor and petechiae on
her lower extremities. WBC = 55,000 cells/mm3, Hb = 7.6 gm/dl, platelets = 5,000/mm3.
Is a bone marrow aspirate and biopsy necessary to establish the diagnosis?
If the differential count shows a blast percentage that equals or exceeds 20% on the peripheral blood smear then the diagnosis of
acute leukemia is made. However, bone marrow aspiration and biopsy may be needed to collect enough material for additional
diagnostic studies. If a patient has <20% blasts then a bone marrow aspirate and biopsy are required for diagnosis since other stem
cell neoplasms may also increase the blast percentage (e.g. myelodysplastic syndromes).

What is/are the minimum criteria for diagnosis and what additional information is needed for precise classification?
The presence of > 20% blast forms in the blood or bone marrow smear is sufficient for a diagnosis of acute leukemia. However, more
information required for treatment decisions and precise classification. Treatments for acute myeloid leukemias and acute
lymphoblastic leukemias (B and T cell types) are different. Also there are treatments targeted at some of the recurrent genetic
abnormalities that contribute to leukemogenesis. One example is ATRA-containing regimens for acute promyelocytic leukemias
(diagnosed by the presence of the t(15;17) translocation) but others are under development.
The current classification of acute leukemias was developed by an International panel under the auspices of the World Health
Organization and published in 2008. It replaced the French-American-British [FAB] classification system. The WHO guidelines
emphasize cytogenetic (large structural chromosomal abnormalities, e.g. translocations) and molecular features (small mutations,
e.g. FLT3, NPM1 and CEBPA) then supplement with morphologic, immunologic and clinical characteristics where needed.
In some cases, the older FAB classification is still used. This system separates leukemias into acute myeloid (AML) and acute
lymphoblastic (ALL) types based on immunologic, cytologic (appearance in microscope) and cytochemical features (e.g. detection of
lineage-specific enzymes myeloperoxidase [granulocytic] or non-specific esterase [monocytic]). AMLs are subdivided based on degree
and lineage(s) of differentiation (M0-M7, details not important) and the ALLs are subdivided into precursor-B and precursor-T types
based largely on antigenic profiles defined by flow cytometry.


Case #3: The patient is a healthy 65-year-old male who was noted to have slightly enlarged
axillary and cervical lymph nodes on an annual physical examination. The patient offered no
complaints and was given a "clean bill of health" one year previously. No organomegaly was
noted. The CBC showed: WBC = 45,000 cells/mm3, Hb = 13.5 gm/d., platelets = 185,000/mm3.
Compare the blood smear in this patient to the normal and abnormal smears from other cases.
Normal smear stack
Patient stack
Virtual Slide Normal Blood Smear (Web viewer) Virtual Slide Case#3 (Web viewer)
Case #3: The patient is a healthy 65-year-old male who was noted to have slightly enlarged
axillary and cervical lymph nodes on an annual physical examination. The patient offered no
complaints and was given a "clean bill of health" one year previously. No organomegaly was
noted. The CBC showed: WBC = 45,000 cells/mm3, Hb = 13.5 gm/d., platelets = 185,000/mm3.
Compare the blood smear in this patient to the normal and abnormal smears from other cases.
What are the major abnormalities on the smear?
Too many small lymphocytes with soccer-ball/cracked-mud/ginger-snap nuclear chromatin (absolute lymphocytosis). Otherwise
unremarkable.

What is the differential diagnosis based on the CBC and blood smear?
Reactive lymphocytosis versus chronic lymphocytic leukemia or peripheralized lymphoma (lymphoid neoplasms that arise in any
lymphoid organ may involve the peripheral blood). Need test that can help determine whether lymphoid cells are primarily
monoclonal B-cells, aberrant T-cells or a mixture of polyclonal T and B cells without any abnormalities.

How does the flow cytometry study help with the diagnosis? (click to see histograms)
Flow cytometry results
Case #3: The patient is a healthy 65-year-old male who was noted to have slightly enlarged
axillary and cervical lymph nodes on an annual physical examination. The patient offered no
complaints and was given a "clean bill of health" one year previously. No organomegaly was
noted. The CBC showed: WBC = 45,000 cells/mm3, Hb = 13.5 gm/d., platelets = 185,000/mm3.
Compare the blood smear in this patient to the normal and abnormal smears from other cases.
How does the flow cytometry study help with the diagnosis?
Can identify a monoclonal B-cell population (generally, but not always, means neoplasm) and identify antigens that are specific for
some specific types of lymphoid leukemias and lymphomas (e.g. low level CD20, low-level monoclonal surface Ig, CD5 and CD23 in
classic CLL/SLL).
The flow data in this case demonstrate that most of the lymphocytes express a single light chain type on their surfaces (i.e. kappa
or lambda); therefore, they are monoclonal. Normally there are many more T-cells than B-cells in the blood and the B-cells are a
mixture of cells expressing kappa and lambda light chains (K:L ratio = 0.5-2, may be slightly higher or lower in reactive conditions
but rarely less than 0.1 or greater than 10).
The large number of monoclonal cells in this case confirms that this is a B-cell neoplasm but there are many different types. In this
case, the cells express several antigens that nail the diagnosis: when low-levels of CD20, CD5 and CD23 are co-expressed on small,
monoclonal B-cells then the diagnosis is chronic lymphocytic leukemia/small lymphocytic lymphoma in >95% of cases.