Eosinophils and basophils are rare in the blood. LEUKEMIA:
Acute ALL and AML
Hi/Low WBC (unpredictable)
Rapidly fatal if untreated (matter of weeks); anemic and thrombocytopenic (almost always. Patient is very sick.) Curable ` Chronic CLL and CML Always high WBC
Slowly progressive- patient lives many years Difficult to cure
Above: SLL/CLL, lymph node. Leukemias start in the bone
marrow; lymphomas start in lymph nodes. Small lymphocytic lymphoma = chronic lymphocytic leukemia, but they have different appearances. SLL goes from the lymph nodes blood, and CLL goes from the blood lymph nodes.
Acute leukemia. This is a
40x microscope field. Should ~25 platelets, but see none here. This patient will have a high WBC count and a low platelet count.
Above: CML. Lots of blasts plus AML: All blasts, no platelets. different forms of neutrophils.
Auer rod = precipitated myeloperoxidase
M3 = treated w/retinoic acid. M4 = myelomonocytic. M5 = monocytic.
M6 = red cells. M7 = megakaryocytic.
Rouleaux formation: RBCs stack on top of each other. Seen in multiple myeloma – make amyloid. See halos due to exaggerated Golgi.
Lymphomas:
Hodgkin (CD15, CD30) Non-Hodgkin
RS Cells Low grade- slowly progressive, rare cure
Often curable High grade- rapidly progressive; a minority are curable Stage important Grade important
Few malignant cells Mostly lymphoma < cell-mediated < humoral
Above: Nodular Sclerosis Hodgkin Lymphoma, lymph node. Above: Hodgkin Lymphoma. Eosinophils, lymphocytes, Fibrous bands (sclerosis), nodules. Hodgkin’s cells.
Above: Hodgkin Lymphoma, spleen. Lymphocytes, Above: Follicular hyperplasia, lymph node. Mantle zone, Reed-Sternberg cells (identifies a sample as Hodgkin’s). 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. See lymphocytes, tingible body macrophages. follicles.
Follicular lymphoma, lymph node. See follicles. No rim of blue cells.
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 to hematopathology diagnosis
– Morphology – Flow cytometry
– Cytogenetics/molecular studies Components of hematopoietic system:
Hematopoiesis: the formation and development of blood cells. Pictured here
Sites are bone marrow, thymus, lymph nodes, spleen.
Hematopoiesis: Myeloid Development:
Figure: Myeloid Development
Goal: formation of mature granulocytes and monocytes 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.
DO NOT MEMORIZE
Erythroid development: Megakaryocyte development and platelet formation:
Goal: formation of RBCs – oxygen transport. Goal: formation of platelets for hemostasis
Site: 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. Development of immature B cells also occurs in bone marrow, but all are quite similar morphologically (so it is not helpful to define stages of maturation).
T-cell development: DO NOT MEMORIZE!
Goal: formation of diverse T-cell population for cellular immunity. Site: bone marrow, thymus. Only very early T cells actually reside in the bone marrow. Can stain for particular antigens to differentiate stages of
maturation.
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 the presence 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 with
immunophenotype 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 for hematopoiesis (bone marrow, thymus, lymph nodes, spleen), the end effect is the same – all the different types of cells are in the blood.
Evaluation of a patient with hematologic condition (cont’d): • 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 evaluation
Purpose:
- 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 of cellularity: best performed on the biopsy (% hematopoietic cells vs. adipose tissue) Cellularity is usually 50%
Test q: A 45F undergoes a bone marrow biopsy due to fever of unknown origin. The cellularity 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. The marrow shows 50% adipose tissue and 50% hematopoietic cells. The interpretation is: normal.
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 in
normal bone marrow, acute leukemia, and aplastic anemia.
In acute leukemia, cellularity can increase to above 90%. In aplastic anemia, cellularity can be down to 10%.
Above: Normal bone marrow. Very heterogeneous.
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 laser beam. Antibodies attach to fluorochromes – tells you about presence of antigens. SSC = side scattered detector.
Above: Flow cytometry in hematology. Expansion of one cell type
Components of lymph node examination: • Morphology:
– Assessment of overall architecture
– Evaluation of expected lymph node 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 (order additional 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. Follicles area. 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. Dark MZ = 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 of subcutaneous suppuration. He then developed an enlarged, tender axillary lymph node. Microscopic exam of the lymph node would most likely reveal histologic features of: hyperplasia.
Spleen
Splenic 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 but also lymphomas. Spleen has identical composition/architecture of germinal centers 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 of pinpoint hemorrhages on her lower extremities in the past month. Phys exam shows no organomegaly or lymphadenopathy. CBC shows Hgb 14.2
g/dL, hematocrit 42.5%, MCV of 91 µm3, platelet count of 19,000/mm3, and WBC count of 6950/mm3. 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 patient’s 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 to platelet membrane glycoproteins IIb-IIIa or Ib-IX affecting both the patient’s 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 of treatment (including new targeted approaches) and patient’s prognosis
Test q: Causes of splenomegaly include all the following except: Sickle cell anemia. (Other choices: infections, congestive states related to portal hypertension, Hodgkin lymphoma, and Non-Hodgkin lymphoma/lymphocytic leukemias)
