Hematopathology Lab 2

5/22/2018 Hematopathology Lab 2

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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 answersAfter 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 ofmicrographs 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).
http://www.med.umich.edu/digitallab/m2pathlabs/hemepath/lab2.htmlhttp://www.med.umich.edu/digitallab/m2pathlabs/hemepath/virtual.htmlhttp://www.med.umich.edu/digitallab/m2pathlabs/hemepath/virtual.htmlhttp://www.med.umich.edu/digitallab/m2pathlabs/hemepath/virtual.htmlhttp://www.med.umich.edu/digitallab/m2pathlabs/hemepath/virtual.htmlhttp://www.med.umich.edu/digitallab/m2pathlabs/hemepath/lab2.html


2/13

Normal smears

Blood smear stack Bone marrow stack

Virtual Slide Normal Blood Smear (Web viewer) Virtual Slide Normal Marrow Smear (Web viewer)
http://141.214.65.171/M2%20Pathology/Hemepath/H002.svs/http://141.214.65.171/M2%20Pathology/Hemepath/H_ST.svshttp://141.214.65.171/M2%20Pathology/Hemepath/H_ST.svshttp://141.214.65.171/M2%20Pathology/Hemepath/H002.svs/


3/13

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)
http://141.214.65.171/M2%20Pathology/Hemepath/H002.svs/http://141.214.65.171/M2%20Pathology/Hemepath/smear100x03S.svshttp://141.214.65.171/M2%20Pathology/Hemepath/smear100x03S.svshttp://141.214.65.171/M2%20Pathology/Hemepath/H002.svs/


4/13






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)


5/13






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 (largelyunknown) 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.


6/13

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)
http://141.214.65.171/M2%20Pathology/Hemepath/smear100x04S.svshttp://141.214.65.171/M2%20Pathology/Hemepath/smear100x02S.svshttp://141.214.65.171/M2%20Pathology/Hemepath/smear100x02S.svshttp://141.214.65.171/M2%20Pathology/Hemepath/smear100x04S.svs


7/13

Case #2









Many (>20%) of the WBC are mononuclear cells that differ from both normal monocytes and activated lymphocytes . Most havenuclei with lighter staining, finely divided, less clumped chromatin and occasional nucleoli (some multiple). These fit the generalcategory of "blast" forms. Blast is an imprecise term that refers to cells with nuclear characteristics shared by early stages ofdifferentiation in the bone marrow.

Blasts have an "active" appearing nucleus (finely dispersed chromatin, nucleoli). There may be evidence of cytoplasmicdifferentiation that can help identify lineage (e.g. granules). Normal bone marrows have 1-2% blasts that include the earliest stagesof myeloid (non-lymphoid/non-erythroid) and lymphoid (pro-, pre-B and pro-, pre-T) differentiation. Any blasts in a blood smear isabnormal 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 progenitorcells in the bone marrow (What are the major groups? How do they differ from one another? See schematic of hematopoiesis in Atlasor Dr. Stoolmanslecture). The neoplastic stem cells in these cases both self renew and give off progeny that differentiate to a limiteddegree. In acute leukemias, maturation of the neoplastic cells arrests at a relatively early stage of lymphoid or non-lymphoiddifferentiation. The abnormal cells accumulate in the bone marrow, suppress normal hematopoiesis (what does this cause?) and mayspill 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) fromleukemia based on the appearance of the atypical cells in the smear alone. The history, CBC and clinical findings are crucial pieces ofinformation. Patients with acute leukemia generally have the signs, symptoms and laboratory findings indicating bone marrowinsufficiency 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 additionalimmunologic studies (flow cytometry primarily), cytochemical stains, cytogenetics and molecular studies are used to establish aprecise classification in acute leukemias (see questions below). You should be able to identify Auer rods and know the clinical

significance of the finding.


8/13

Case #2








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 basisof these cancers.


9/13

Case #2








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% 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 guidelinesemphasize 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.


10/13

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)
http://141.214.65.171/M2%20Pathology/Hemepath/H002.svs/http://141.214.65.171/M2%20Pathology/Hemepath/BS0023b_100X.svshttp://141.214.65.171/M2%20Pathology/Hemepath/BS0023b_100X.svshttp://141.214.65.171/M2%20Pathology/Hemepath/H002.svs/


11/13







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)


12/13

Flow cytometry results


13/13






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.

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Hematopathology Lab 2