Hematologic Pathology p24-35

7/30/2019 Hematologic Pathology p24-35

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Eosinophils and basophils are rare in the blood.

LEUKEMIA:AcuteALL and AMLHi/Low WBC (unpredictable)Rapidly fatal if untreated (matter of weeks); anemic andthrombocytopenic (almost always. Patient is very sick.)Curable

`

ChronicCLL and CMLAlways high WBCSlowly progressive- patient lives many yearsDifficult to cure

Above: SLL/CLL, lymph node. Leukemias start in the bonemarrow; lymphomas start in lymph nodes. Small lymphocyticlymphoma = chronic lymphocytic leukemia, but they have differentappearances. SLL goes from the lymph nodes blood, and CLLgoes from the blood lymph nodes.

Acute leukemia. This is a40x microscope field. Should

~25 platelets, but see nonehere. This patient will have ahigh WBC count and a lowplatelet count.


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Above: CML. Lots of blasts plus AML: All blasts, no platelets.different forms of neutrophils.

Auer rod = precipitatedmyeloperoxidase


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M3 = treated w/retinoic acid. M4 = myelomonocytic. M5 = monocytic.

M6 = red cells. M7 = megakaryocytic.

Rouleaux formation: RBCs stack on top of each other. Seen inmultiple myeloma make amyloid. See halos due to exaggeratedGolgi.

Lymphomas:

Hodgkin (CD15, CD30) Non-HodgkinRS Cells Low grade- slowly progressive, rare cureOften curable High grade- rapidly progressive; a minority are curableStage important Grade importantFew malignant cells Mostly lymphoma< cell-mediated < humoralLocalized radiation Systemic chemotherapy


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Above: Nodular Sclerosis Hodgkin Lymphoma, lymph node. Above: Hodgkin Lymphoma. Eosinophils, lymphocytes,Fibrous bands (sclerosis), nodules. Hodgkins cells.

Above: Hodgkin Lymphoma, spleen. Lymphocytes, Above: Follicular hyperplasia, lymph node. Mantle zone,Reed-Sternberg cells (identifies a sample as Hodgkins). light zone, dark zone, tingible body macrophages (inside

macrophages are retractable bodies). See rim of blue cells.

Above: Follicular hyperplasia, lymph node. Mantle zone, Above: Malignant follicular lymphoma, lymph node. Seelymphocytes, tingible body macrophages. follicles.

Follicular lymphoma, lymph node. See follicles. Norim of blue cells.


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Hematology Overview Mon. 10/18/10

Objectives: Overview of hematopoiesis (excluding

detailed discussion of immune system,addressed in previous lectures)

Review of evaluation of hematologic patient Blood exam Bone marrow exam Lymph node evaluation Spleen evaluation

Discussion of integrative approach tohematopathology diagnosis Morphology Flow cytometry Cytogenetics/molecular studies

Components of hematopoietic system:

Hematopoiesis: the formation and developmentof blood cells. Pictured here Sites are bone marrow, thymus, lymph nodes,spleen.

Hematopoiesis: Myeloid Development:

Figure: Myeloid DevelopmentGoal: formation of mature granulocytes andmonocytes for non-specific immune responses.Site: bone marrow.

Stages are defined by specific markers:CD34, HLA-DR, CD117 immature markers define acute leukemic processes (involving blasts).CD13, CD33 lineage-specific markers.

DONOT

MEMORIZE


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Erythroid development: Megakaryocyte development and platelet formation:

Goal: formation of RBCs oxygen transport. Goal: formation of platelets for hemostasisSite: bone marrow Site: bone marrow

B-cell development:

Goal: formation of diverse B-cell population for humoral immunity.

Site: bone marrow, lymph nodes.

These stages are not as discrete morphologically as monocytic development. Developmentof immature B cells also occurs in bone marrow, but all are quite similar morphologically (soit is not helpful to define stages of maturation).

T-cell development:

DO NOT MEMORIZE!

Goal: formation of diverse T-cellpopulation for cellular immunity.

Site: bone marrow, thymus.

Only very early T cells actuallyreside in the bone marrow.Can stain for particular antigensto differentiate stages ofmaturation.

DO NOT MEMORIZE


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Hematopoiesis: mature cells present in blood.

Diagnosis of hematologic disorders:- Morphology plays an important role in the initial evaluation

- Due to the overlap in morphologic features, certain hematologic disorders can only be distinguished by thepresence of specific genetic lesions or the expression of specific proteins- Treatment is strictly based on the pathologic diagnosis and increasingly targeted at specific protein/gene

expression (e.g. Imatinib for CML and Ph+ ALL)

Integrated approach to the hematopathology diagnosis: Integration of morphology withimmunophenotype and genetic signature:

With the use of cytogenetics, could this be the end of the morphology era?

In appropriate clinical and laboratory context - Morphology- Flow cytometry, immunohistochemistry- Cytogenetic and molecular studies

- Precise, clinically relevant classification- Diagnostic support in morphologically challenging cases- Follow-up of the disease

Precursor lymphoid neoplasms (acute lymphoblastic leukemias):- B lymphoblastic leukaemia/lymphoma, NOS- B lymphoblastic leukaemia/lymphoma with recurrent genetic abnormalities

- B lymphoblastic leukaemia/lymphoma with t(9:22)(q34;q11.2); BCR-ABL1- B lymphoblastic leukaemia/lymphoma with t(v;11q23); MLL rearranged- B lymphoblastic leukaemia/lymphoma with t(12;21)(p13;q22); TEL-AML1 (ETV6-RUNX1)- B lymphoblastic leukaemia/lymphoma with hyperidipoloidy- B lymphoblastic leukaemia/lymphoma with hypodiploidy (Hypodiploid ALL)

- B lymphoblastic leukaemia/lymphoma with t(5;14)(q31;q32); IL3-IGH- B lymphoblastic leukaemia/lymphoma with t(1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1)

- T lymphoblastic leukaemia/lymphoma

Very few entities are actually defined by morphology alone.

Evaluation of a patient with hematologic condition: Starting point: complete blood count (CBC) and blood differential Additional serum studies: iron, B12, folate, Coombs test etc + bone marrow exam: morphology (biopsy, smear), flow cytometry, cytogenetics, molecular studies + biopsy of lymph node or extranodal site: morphology, immunophenotyping, molecular studies

Although there are multiple sites forhematopoiesis (bone marrow, thymus, lymphnodes, spleen), the end effect is the same all the different types of cells are in the blood.


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Evaluation of a patient with hematologic condition (contd): Clinical pathologist:

CBC and peripheral blood differential Serum studies (iron, B12, folate, Coombs test etc)

Hematopathologist bone marrow exam (biopsy, smear, flow cytometry, cytogenetics, molecular studies) biopsy of lymph node or extranodal site (including morphology, immunophenotyping, molecular studies)

Complete Blood Count (CBC): Starting point of hematologic evaluationPurpose:

- initial evaluation of the status of hematopoietic system- Quantitative: increase or decrease in specific cell types- Morphologic changes through review of blood smear- reflects both production in bone marrow and modification by organs/factors external to bone marrow (e.g.

sequestration by spleen)

CBC Normal Values: Peripheral blood evaluation:

Peripheral blood smear

Indications for bone marrow examination:Abnormal CBC or blood smear

- Unexplained cytopenias (low blood counts)- Unexplained leukocytosis or abnormal WBCs on blood smear- Unexplained thrombocytosis

Systemic disease- Unexplained lymphadenopathy, splenomegaly, hepatomegaly- Tumor staging: solid tumors, lymphomas- Monitoring response to chemotherapy- Fever of unknown origin

Bone marrow examination:- Site: posterior iliac crest, in special circumstances sternum- Instrumentation: Jamshidi needle- Local anesthesia- Most commonly both biopsy and aspirate are obtained (aspirate

only when from sternum)

Purpose of bone marrow examination: Morphology. Assessment ofcellularity: best performed on the biopsy (%

hematopoietic cells vs. adipose tissue) Cellularity is usually50%

Test q:A 45F undergoes a bone marrow biopsy due to fever of unknown origin. Thecellularity of the bone marrow is approximately 50%. This is considered to be: Normal.Test q:A 50M would be expected to have a bone marrow cellularity of: 50%.

Test q:A 50F undergoes bone marrow biopsy to evaluate a peripheral pancytopenia. Themarrow shows 50% adipose tissue and 50% hematopoietic cells. The interpretation is:normal.


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Bone marrow aspirate smear: Morphology Evaluation of maturation and composition of hematopoietic elements.

- Initial evaluation on the biopsy- Detailed evaluation using bone marrow aspirate smear.

- Evaluation of iron stores: best seen on Prussian blue stain of bone marrow aspirate smear- Other: detection of infections (bone marrow can be cultured)

Above: Acute Leukemia.Heterogeneity is somewhat lost.

Purpose of bone marrow examination: Flow cytometry. Evaluation of maturation Identification of specific cell types Specific diagnosis Immunophenotypes suggestive of specific genetic lesion (order additional

molecular studies)

Principle of flow cytometry:Stain cell surface markers different colors. Use antigens/antibodies.

Figure:Cellularity innormal bone marrow,acute leukemia, andaplastic anemia.

In acute leukemia,cellularity can increase toabove 90%. In aplastic

anemia, cellularity can bedown to 10%.

Above: Normal bone marrow. Veryheterogeneous.


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Applications of flow cytometry:

Diagnosis and subclassification of leukemias and lymphomas Detection of minimal residual disease before the overt relapse of acute leukemia T-cell subset determination for diagnosis and follow-up in HIV-positive patients Determination of deficient cell-types in congenital immunodeficiencies Enumeration of hematopoietic stem cells for bone marrow transplantation Diagnosis of platelet disorders Detection of fetal hemoglobin in e.g. feto-maternal hemorrhage

Purpose of bone marrow examination:Cytogenetics and molecular studies Identification of cytogenetic/genetic abnormality

Cytogenetics conventional karyotype In situ hybridization Polymerase chain reaction (PCR)

Summary: Purpose of bone marrow exam Morphology:

Assessment of cellularity Evaluation of maturation and composition of hematopoietic elements Evaluation of iron stores Other: detection of infections (bone marrow can be cultured)

Flow cytometry Evaluation of maturation Identification of specific cell types Specific diagnosis Immunophenotypes suggestive of specific genetic lesion (order additional molecular studies)

Cytogenetics and molecular studies Identification of cytogenetic/genetic abnormality

Above: Flow cytometry. Inject cells into sheath fluid positions cells into single file. Sends them in path of laserbeam. Antibodies attach to fluorochromes tells you aboutpresence of antigens. SSC = side scattered detector.

Above: Flow cytometry in hematology. Expansion of one cell typewith same immunophenotype can lead to a specific diagnosis.


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Components of lymph node examination: Morphology:

Assessment of overallarchitecture

Evaluation of expected lymphnode compartments

Other: detection of infections(lymph node can be cultured)

Flow cytometry Identification of specific cell types Specific diagnosis Immunophenotypes suggestive of

specific genetic lesion (orderadditional molecular studies)

Cytogenetics and molecular studies Identification of

cytogenetic/genetic abnormality

Above: Architecture of normal lymph node. (both figures)

Benign lymph node enlargement (lymphadenopathy)

Most common pattern: Follicular Hyperplasia Infections Autoimmune disorders Non-specific

Benign lymphadenopathy: Paracortical hyperplasia Viral infections Skin disease Non-specific

Benign lymphadenopathy: Sinus histiocytosis LN draining limbs, abdominal organs Inflammatory lesions Malignancies

Follicular Hyperplasia.Many follicles scattered Paracortical Hyperplasia. Expanded area of Sinus histiocytosis.throughout entire lymph node not just in cortical paracortex area containing T-cells. Folliclesarea. See many empty spaces within. displaced to the side.

DZ = dark zone Normal lymph nodes have a very orderly organization.LZ = light zone Mantle zone is a small layer of lymphocytes. DarkMZ = mantle zone zone (DZ) mostly centroblasts. Can see polarization

DZ looks darker than LZ.

Test q:A 25F is evaluated for generalized lymphadenopathy.Histologic exam reveals follicular hyperplasia. She most likely

suffers from: bacterial infection.

Test q:A 36y/o rancher lacerated the skin of his upper arm while

stringing barbed wire. Subsequent infection produced an area ofsubcutaneous suppuration. He then developed an enlarged,tender axillary lymph node. Microscopic exam of the lymph node

would most likely reveal histologic features of: hyperplasia.


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SpleenSplenic Architecture

Function: Removal of damaged red cells, bacteria, cell debris Immune response (follicles, periarteriolar T-cells zones) Temporary storage of non-circulating blood elements such as

granulocytes, platelets, RBCs Site of hematopoiesis if bone marrow compromised (ex: malignant

neoplasm)

Spleen can be very valuable source of info in not only myeloid disorders butalso lymphomas. Spleen has identical composition/architecture of germinalcenters as in the lymph node.

Causes of splenomegaly (palpate LUQ!):

Integrative approach to diagnosing patient with hematologic condition :

Whatever site inspected include: Morphology Immunophenotyping Cytogenetics/molecular studies

Test q: For the past 6mo, a 35F has experienced an excessively heavy menstrual flow each month. She also has noticed increasing numbers ofpinpoint hemorrhages on her lower extremities in the past month. Phys exam shows no organomegaly or lymphadenopathy. CBC shows Hgb 14.2g/dL, hematocrit 42.5%, MCV of 91 m

3, platelet count of 19,000/mm

3, and WBC count of 6950/mm

3. On admission to the hospital, she has melena and

is given a transfusion of platelets, but her platelet count does not increase. An emergency splenectomy is performed, and her platelet count increases.

Which of the following describes the most likely basis for her bleeding tendency? Destruction of antibody-coated platelets by the spleen.

Robbins: This patients bleeding tendency is caused by a low platelet count. She most likely has idiopathic

thrombocytopenic purpura (ITP) ,in which platelets are destroyed in the spleen after being coated w/antibodies toplatelet membrane glycoproteins IIb-IIIa or Ib-IX affecting both the patients platelets and the transfused platelets.Because the spleen is the source of the antibody and the site of destruction, splenectomy can be beneficial.

Directly translates to choice oftreatment (including new targetedapproaches) and patients prognosis

Test q: Causes of splenomegaly include all

the following except: Sickle cell anemia.(Other choices: infections, congestive statesrelated to portal hypertension, Hodgkin

lymphoma, and Non-Hodgkinlymphoma/lymphocytic leukemias)

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Hematologic Pathology p24-35