2.

Bone marrow exam (aspirate smear):

Blasts in acute myeloid leukemia:

Normal bone marrow

Acute myeloid leukemia (20% blasts required for Dx)

Homogeneous proliferation of immature cells with high nuclear-cytoplasmic ratio, fine chromatin pattern and visible nucleoli. Sometimes have Auer rods

3. Flow cytometry:

Above: Normal bone marrow

Acute leukemia

Above: Myeloid leukemia More mature granulocytic lineage. Below: CD7 lymphoid lineage, abnormal expression.

CD45 is an antibody that stains hematopoietic cells. In normal bone marrow, see many cell types high granulocytes, low lymphocytes. In acute leukemia, normal hematopoietic cells (granulocytes, lymphocytes, etc.) are displaced by blasts.

AML: Dx based on flow cytometric evaluation. o low density CD45 o CD33 o CD13 o HLA-DR o CD34 o CD117 o CD7 o CD11b o negative for CD15
Test q: Flow cytometry cell markers consistent w/AML are: CD33, CD13.

4. Cytogenetics and molecular studies: Normal male karyotype: 46,XY [20] Molecular testing negative for NPM1 and CEBPA mutations Final diagnosis: AML, without maturation Correlation of morphology, flow cytometry and cytogenetics Example: Acute promyelocytic leukemia with t(15;17)(q22;q12); PML-RARA
Test q: A 23F presents w/mucosal bleeding and fatigue. CBC shows a microangiopathic, hemolytic anemia and numerous myeloblasts w/Auer rods. You suspect: AML (M3).

Above: AML (M3) aka Acute Promyelocytic Leukemia. Promyelocytes with numerous azurophilic granules and frequent Auer rods (myeloperoxidase+).

Acute promyelocytic leukemia [AML with t(15;17)]:

O
High CD33 not normal.

Above: Normal bone marrow

AML with t(15;17) See granulocytes.

Highly proliferative.

Myeloperoxidase+

Above: CD117 marker for immature myeloid cell.

Acute promyelocytic leukemia [AML with t(15;17)]: Characteristic morphology and immunophenotype o CD33 bright + o HLA-DR o CD34 (Note: HLA-DR and CD34 immature.) o CD15 And presence of t(15;17) by cytogenetics or RAR/PML fusion gene by molecular studies Acute Myeloid Leukemia Summary: o Most common in adults o Abrupt presentation with pancytopenia and its complications o Bone marrow and peripheral blood show proliferation of blasts (several types of blasts may be present) and lack of normal maturing hematopoietic elements o Subclassified by stage of differentiation and genetic lesions, which correspond to prognostic groups o Only a proportion of patients can be cured with currently available treatments

Leukemias: Myeloid neoplasms: Acute myeloid leukemia Chronic: Myelodysplastic syndromes Myeloproliferative neoplasms Lymphoid leukemias: Acute Chronic Myelodysplastic syndromes: Clonal stem cell disorder characterized by actively proliferating hematopoietic cells with maturation defect (=ineffective hematopoiesis=cytopenias) Bone marrow is hypercellular, however patient presents with pancytopenia (BM not efficient) Morphology is heterogeneous; all lineages are represented in the bone marrow and show dysplastic features Primary (idiopathic) Develops insidiously usually in older patients (mean age 70) Secondary (therapy-related) Complication of chemotherapy or radiation therapy Clinical course: Pancytopenia and its complications Transformation to acute leukemia (10-40% of cases)

Approach to diagnosis of myelodysplastic syndrome: Integration of morphology, clinical history, ancillary techniques, cytogenetic and molecular features.

MDS Morphology: Milestone Features

Hypercellular BM
(Pic #1: only a few fatty cells)

Dysplastic features
(Pic #2: yellow circle around normal cells, green circles around irregular cells do not have round nuclei.)

Dysplasia: Number of blasts

Higher number = more aggressive disease and higher risk of transformation to acute leukemia (e.g. disease is less aggressive in a patient with 1% of blasts as compared to a patient with 15% blasts) MDS = ALWAYS LESS THAN 20% BLASTS

O O

Ring sideroblasts
(Pic #3: atypical nucleated erythrocytes with granules of iron accumulated in perinuclear mitochondria) Above: Granulocytic lineage. See improper segmentation only two lobes. Above: Megakaryocytes. See loss of lobation only one nucleus.
Test q: Acute leukemia is most reliably distinguished from chronic leukemia on the basis of: number of blast cells in peripheral blood and/or bone marrow. REPEATED x2

Classification of myelodysplastic syndromes:

Disease RCUD RARS RCMD Common blood findings Anemia No blasts Anemia No blasts Bicytopenia or pancytopenia No or rare blasts, no Auer rods
9

Bone marrow findings Dysplasia involving one lineage <5% blasts, <15% ring sideroblasts >15% ring sideroblasts, <5% blasts Dysplasia involving one lineage Dysplasia in >2 lines <5% blasts in marrow, no Auer rods

<1x10 /L monocytes RAEB-1 Cytopenias, <5% blasts

9

<1x10 /L monocytes RAEB-2 Cytopenias, 5-19% blasts

9

Unilineage or multilineage dysplasia 5-9% blasts, no Auer rods Unilineage or multilineage dysplasia 10%-19% blasts, + Auer rods Normal to increased megakaryocytes with hypolobated nuclei, <5% blasts, isolated del(5q), no Auer rods

<1x10 /L monocytes MDS with isolated del(5q) Anaemia Usually normal or increased platelet count, <5% blasts

Higher # of blasts = more aggressive disease. MDS with isolated del(5q): Refractory macrocytic anemia with low degree erythroid dysplasia, characteristic hypolobated megakaryocytes and <5% blasts; normal or elevated platelet count Indolent course with low rate of transformation to AML Isolated deletion of chromosome 5q32-33.3 90% response rate to lenalidomide (normalization of morphology, correction of anemia and high rate of cytogenetic remission) Myelodysplastic Syndromes Summary: Elderly patient presenting with pancytopenia and hypercellular bone marrow In contrast to homogeneous population of blasts in acute leukemia, bone marrow in myelodysplastic syndrome is heterogeneous with all lineages present and showing dysplastic features Majority of patients succumb to complications of pancytopenia and transformation to acute leukemia Clinical course dependent on % blasts and genetic features Myeloproliferative neoplasms (MPNs): Transformation of pluripotent stem cell and bone marrow replacement by a heterogeneous neoplastic population with adequate differentiation Four subtypes: Chronic myelogenous leukemia granulocytic variation Polycythemia vera Essential thrombocytosis proliferation of megakaryocytes Chronic idiopathic myelofibrosis Common features: Increased proliferation of select cells in bone marrow High incidence of extramedullary hematopoiesis Transformation to spent phase with cytopenias or acute leukemias Chronic myelogenous leukemia: Presentation May be asymptomatic Elevated white blood cell count with mature white blood cells and granulocytic precursors Anemia Splenomegaly Figure: Peripheral blood in a patient with chronic myelogenous leukemia

Chronic myelogenous leukemia: Diagnosis 100% cellular bone marrow with myeloid predominance and suppression of erythropoiesis Elevated white blood cell count with maturing myeloid precursors t(9;22) (so called Philadelphia chromosome) -mandatory for diagnosis as differential diagnosis may include other myeloproliferative disorders and even myelodysplastic syndromes t(9;22) = Philadelphia chromosome translocation involving oncogenes BCR and ABL1 produces fusion protein with uninhibited tyrosine kinase activity involved in the development of CML BCR-ABL1 fusion protein is a target for therapy (Imatinib/Gleevec)

Chronic Myelogenous Leukemia Summary: - Adult with: - elevated white blood cell count - hypercellular bone marrow with myeloid predominance and suppression of erythropoiesis - splenomegaly - Philadelphia chromosome: t(9;22) = (BCR-ABL1 fusion protein) - Chronic indolent phase, transformation into acute leukemia within 5 years - Treat w/Imatinib (Gleevec tyrosine-kinase inhibitor)
Test q: A 69M present due to fatigue and a full feeling in his abdomen. Physical exam reveals a huge spleen. The PBS (peripheral blood smear) shows a mixture of granulocytes and granulocyte precursors. You would expect to find: t(9;22). Test q: A 40F shows a total WBC count of 85,000. A left shift of myeloid cells is present w/rare blasts and promyelocytes. t(9;22) is present. Diagnosis? CML. Test q: Treatment of leukemias are increasing based on specific genetic and immunologic diagnosis. For example, Imatinib (Gleevec) is a treatment of choice for: CML. Test q: A 50F has had easy fatigability and noted a dragging sensation in her abdomen for the past 5 months. Phys exam reveals that she is afebrile. 3 She has marked splenomegaly but no lymphadenopathy. Lab studies show her total WBC count is 250,000mm , w/WBC differential count showing 68% segmented neutrophils, 11% band neutrophils, 6% metamyelocytes, 5% myelocytes, 5% myeloblasts, 3% lymphocytes, and 2% monocytes. A bone marrow biopsy is performed, and karyotypic analysis of the cells reveals a t(9;22) translocation. Medical treatment w/a drug having which of the following modes of action is most likely to produce a complete remission in this patient? Inhibiting tyrosine-kinase activity. REPEATED x2 Test q: Successful targeted therapy for CML and GIST tumors has been achieved with the signal-transduction inhibitor GLEEVEC.

Chronic Myelogenous Leukemia Clinical Course: Initially indolent chronic phase Virtually all patients transform into acute leukemia within 5 years from diagnosis The progression may be slow or rapid Myeloid Neoplasms Summary: AML - proliferation of the most immature marrow elements blasts MDS - proliferation of all marrow elements, residual but inadequate differentiation, marked morphologic abnormalities (dysplasia), pancytopenia MPN - proliferation of all marrow elements, adequate differentiation, subtle morphologic abnormalities, often elevated peripheral blood counts Leukemias: Myeloid leukemias: Acute Chronic myeloid neoplasms

Lymphoid leukemias: Acute Chronic

Lymphoid leukemias: Acute: proliferation of blasts at an early stage of lymphoid maturation (similar to myeloid) Chronic: proliferation of mature lymphocytes Lymphoblastic leukemia/lymphoma (a.k.a. acute lymphoblastic leukemia): Most commonly seen in children (also most common neoplasm of childhood) Presentation similar to acute myeloid leukemia: Fatigue (anemia), fever (infections due to neutropenia) and bleeding (thrombocytopenia) Bone pain Lymphadenopathy, organomegaly and CNS manifestations due to meningeal involvement are more common in acute lymphoblastic than in acute myeloid leukemias
Test q: ALL and AML share in common: anemia and thrombocytopenia.

Lymphoblastic leukemia/lymphoma: Replacement of hematopoietic elements by a homogeneous proliferation of lymphoid blasts Hypercellular bone marrow with replacement of hematopoietic elements by a homogeneous proliferation of lymphoid blasts (lymphoblasts) Negative for myeloperoxidase and non-specific esterases Positive for B- or T-cell markers by flow cytometry Lymphoblastic leukemia/lymphoma WHO classification: according to cell of origin (B- or T- lymphoblast) recognizes importance of cytogenetic/genetic lesions
Test q: Acute lymphoblastic leukemia: Independent of immunophenotype is positive for TdT. (Other choices: Includes cases positive for nonspecific diseases; Has a very poor prognosis w/the majority of children experiencing relapse of the disease; In the majority of cases is of precursor T-cell immunophenotype.) Test q: A 4y/o boy has appeared listless for about one week. He now complains of pain when he is picked up by his mother, and he exhibits irritability when his arms or legs are touched. In the past two days, several large ecchymoses have appeared of the right thigh and left shoulder. CBC shows Hgb 3 3 3 10.2 g/dL, hematocrit 30.5%, MCV 96 m , platelet count 45,000/mm , and WBC count 13,900/mm . Exam of the peripheral blood smear shows blasts that lack peroxidase-positive granules but contain PAS-positive aggregates and stain positively for TdT. Flow cytometry shows the phenotype of blasts to be CD19+, CD3-, and slg-. Which of the following is the most likely diagnosis? Acute lymphoblastic leukemia. (REPEATEDx3 but once, the answer was: Acute Lymphocytic Leukemia)

B lymphoblastic leukemia/lymphoma:

Above: Normal bone marrow

Leukemic Precursor B-cells

Normal B-cell Precursors

Leukemic Precursor B-cells

Lymphoblastic leukemia/lymphoma Classification by cell of origin (B- vs. T-immunophenotype): B lymphoblastic leukemia/lymphoma T lymphoblastic leukemia/lymphoma Majority of cases (approximately 80%) Older children Best prognosis Higher white blood cell count Mediastinal mass
Test q: An 18M had a mediastinal lymph node biopsy which showed a malignant lymphoma. At a subsequent bone marrow biopsy, the bone marrow was found to be extensively involved w/lymphoma. Flow cytometery revealed expression in the lymphoma cells of TdT and CD3. The most likely diagnosis is: lymphoblastic lymphoma. (Other choices: mantle cell lymphoma, follicular lymphoma, Burkitt lymphoma, and small lymphocytic lymphoma.)

Due to their clinical relevance, cytogenetic abnormalities are incorporated into WHO classification For example: B lymphoblastic leukemia/lymphoma with t(4;11) is known to have very poor prognosis B lymphoblastic leukemia/lymphoma with t(12;21) has excellent prognosis

Precursor B lymphoid neoplasms: B lymphoblastic leukemia/lymphoma, NOS B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities B lymphoblastic leukemia/lymphoma with t(9:22)(q34;q11.2); BCR-ABL1 B lymphoblastic leukemia/lymphoma with t(v;11q23); MLL rearranged B lymphoblastic leukemia/lymphoma with t(12;21)(p13;q22); TEL-AML1 (ETV6-RUNX1) B lymphoblastic leukemia/lymphoma with hyperdiploidy B lymphoblastic leukemia/lymphoma with hypodiploidy (hypodiploid ALL) B lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32); IL3-IGH B lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1) B Lymphoblastic Leukemia/Lymphoma Summary: Most common neoplasm in childhood Classified into B- and T-precursor, and mature B-cell phenotypes Within different immunophenotypic subtypes, cytogenetic abnormalities define subtypes with prognostic significance High cure rate in pediatric cases, as opposed to adult lymphoblastic leukemia and AML Chronic lymphocytic leukemia: Clonal disorder characterized by a proliferation of mature CD5-positive B-lymphocytes Most common type of leukemia in Western world (40% of all leukemias in patients over the age of 65) Majority of patients are >50 years old Asymptomatic; or fatigue, autoimmune hemolytic anemia, infections (neutropenia), organomegaly (infiltration by neoplastic B-cells), bleeding (thrombocytopenia) CLL: Accumulation of small B-cell lymphocytes in peripheral blood, bone marrow and lymphoid organs Chronic lymphocytic leukemia and small lymphocytic lymphoma: Morphologically, immunophenotypically and genotypically identical Differ primarily in the mode of presentation (leukemic vs. lymph node based) In WHO classification known under common name of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) Diagnostic work-up in CLL/SLL: Morphology, Immunophenotyping Immunophenotypic diagnosis of CLL/SLL:

disintegrated small mature lymphocyte

smudge cell
Figure: Peripheral blood in a patient w/CLL. Peripheral blood lymphocytosis composed predominantly of small lymphocytes and smudge cells. Figure: CLL in bone marrow. Bone marrow infiltrate by small lymphocytes (similar proliferation seen in lymph nodes). Loss of heterogeneity. Patient develops cytopenia.

Test q: Increased small lymphocytes w/smudge cells and no blasts are consistent with: CLL.

Lymphocytes

Immunophenotypic diagnosis of CLL/SLL:

Immunophenotypic diagnosis of CLL/SLL: Lymphocyte population (low granularity, bright CD45) Positive for: CD19 CD20 (dim) CD5 CD23 Kappa or lambda light chain (dim)

Above: CD23 is abnormal.

Lambda/kappa reading.

Final diagnosis is based on: Morphology: Lymphocytosis with small lymphocytes and smudge cells (peripheral blood or bone marrow) or diffuse lymph node replacement by small lymphocytes Immunophenotype: CD19+, CD20 dim, CD5+, CD23+, dim kappa or lambda light chain Genetics: Predicting patient prognosis (includes cytogenetics and molecular studies)
Test q: A 75M has a CBC as part of his annual exam. The PBS shows a WBC count of 50,000 w/95% lymphocytes. No blasts as seen. You expect flow cytometry to show: Coexpression of CD5 and CD23. Test q: A 50F presents w/generalized lymphadenopathy and a peripheral WBC count of 60,000. Flow cytometry of an excised lymph node shows CD19+, CD5+, CD23+. Diagnosis? CLL/SLL.

CLL/SLL can develop at two different stages of B-cell differentiation:

Pre-germinal center cells with unmutated immunoglobulin V genes (less mature)

Post-germinal center cells with mutated Ig V genes (more mature)

The unmuted CLL/SLL has worse prognosis and requires more intensive therapy Chronic Lymphocytic Leukemia Summary: Most common leukemia of adults, older individuals Clonal proliferation of mature CD5-positive B-cells involving peripheral blood, bone marrow and lymphoid organs Asymptomatic or symptoms associated with anemia, neutropenia, thrombocytopenia, immune dysfunction Most commonly indolent course, however not curable with current chemotherapy

CLL/SLL :More mature (mutated) version has a better prognosis

Hematopathology Review Tumor Markers: PSA Prostate cancer = prostate specific antigen CEA Colorectal/Pancreatic CA 19-9 Pancreatic Alpha-fetoprotein Hepatic/Yolk sac Beta-HCG Choriocarcinoma/Molar pregnancy CA 125 Ovarian cancer S-100 Melanoma/Astrocytoma Thyroglobulin Thyroid cancer Calcitonin Medullary carcinoma of Thyroid Catecholamines Pheochromocytoma/Neuroblastoma TRAP stain Hairy cell leukemia = tartrate resistant acid phosphatase Alkaline phosphatase Mets to bones, Pagets disease of bone

11/5/10 Translocations: t (9;22) CML (bcr-abl) t (8;14) Burkitts lymphoma (c-myc) t (14;18) Follicular lymphoma (bcl-2) t (15;17) AML M3 (promyelocytic leukemia) t (1;14) T-cell Lymphoblastic lymphoma t (11; 22) Ewings sarcoma t (11; 14) Mantle cell lymphoma (bcl-1) t (11; 18) MALT lymphoma (API-2) t (1; 14) MALT lymphoma (bcl-10)

If tumors metastasize to bone and stimulate osteoblasts, will get increase in alkaline phosphatase. For example, if you have a patient w/prostate cancer and they have an elevation in this enzyme = metastatic disease.
This chart shows bleeding/coagulation disorders. Most problems involve bleeding, some involve hypercoagulation. von Willebrand Disease platelet function problem. Bleeding time is elevated (the amount of time it takes for a laceration to clot). PT is normal. PTT maybe high or normal reason it could be abnormal is because vWF is the carrier for Factor VIII.

or normal

Hemophilia A Factor VIII deficiency the PTT will be elevated. DIC consumptive coagulopathy: bleedingclottingbleeding clottingetc = you use up everything. Bleeding time, PT, and PTT will be high. If you measured fibrinogen, it would be high. If you measured D dimer, it would be high. If you are monitoring coumadin or heparin therapy, PT and PTT will be abnormal. Clnically, following the PT for coumadin therapy works best, and following the PTT for heparin works best.

von Willebrand Disease deficiency of vW factor Glycoprotein I = receptor for vW factor Glycoprotein II = the receptor for fibrinogen

Bernard-Soulier Syndrome deficiency of Glycoprotein I (vW factor receptor) Glanzmann Thrombasthenia deficiency of Glycoprotein II (fibrinogen receptor) May have a quantitative or qualitative problem w/platelets these are 3 of the reasons why they may not work.

There are many factors involved in anti-clotting (must function for survival). If you have a genetic deficiency in Factor V, the anticlotting properites of Prot C and S wont work because they cant work on Factor V (Leiden mutation).

Fibrinolysis:

Above: Patient w/DIC may see MAHA (microangiopathic hemolytic anemia). In the small blood vessels (microangio), will find fibrin dimers and polymers. These, as the RBCs try to pass through, chop them up. Create schistocytes (above right) chopped up RBCs. Could also see schistocytes in a situation w/an artificial heart valve that is chopping RBCs. Goldilocks spot; not too thick and not too thin:

Above (left): Microcytic, hypochromic RBCs. Can tell its microcytic bc the RBCs are smaller than the lymphocyte (top right). Above (middle): Polychromasia reticulocytes (the youngest RBC in the peripheral blood without a nucleus). Normally, 1-2.5% of cells will be reticulocytes. If you have iron deficiency and not making RBCs, will be a lot lower. If you have a hemolytic anemia and you are producing lots of RBCs, will be much higher. Above (right): Special stain for reticulocytes can give a more accurate count.

Above (left): Howell-Jolly bodies indicates the spleen is not working. Spherocytes may see in someone w/hereditary spherocytosis or COULD see in someone w/MAHA (will see spherocytes and schistocytes). Nucleated RBCs indicate that the marrow is pumping out cells faster than normal. Above (middle): examples of schistocytes. Above (right): Iron deficiency anemia. Use lymphocyte to compare size RBCs are smaller = microcytic. Will also see cigar-shaped cells. The size is anisocytosis (RBCs of abnormal size). Unusual shapes poikilocytosis. In this case, we have both anisopoikilocytosis. Megaloblastic anemia. RBCs bigger than lymphocyte.

RR13-15: 23 y.o African American male takes aspirin and Septra for a cold. He later passes red-brown urine and his PBS shows Heinz bodies and bite cells. Dx? A. SS anemia B. SS trait C. Hereditary spherocytosis D. Asplenia E. G-6PD African American descent, aspirin (non-steroidal pain killers) and Septra (sulfa drug) predisposes to a certain type of hemolytic anemia: G-6PD. Will see Heinz bodies and bite cells (looks like bite taken out bc it had iron in it).

Above: Iron stain demonstrating Heinz bodies.

Arrow: bite cell.

Peripheral blood gives you idea of anemia type. Fe deficiency microcytic and hypochromic anemia. Spherocytes think of hereditary spherocytosis. Howell-Jolly bodies means patient does not have a spleen (or spleen is not working) may have sickle cell disease or some other reason they have lost their spleen.
Test q: A 15M has developed a cough and a high fever over the past 4 days. On phys exam, he has a temp of 39.2 degrees C. Diffuse rales are heard 3 3 over all lung fields. Lab studies show Hgb 14.8 g/dL, hematocrit 44.4%, platelet count 496,000/mm , and WBC count 15,600/mm . Exam of the peripheral blood smear shows RBCs w/marked anisocytosis and Howell-Jolly bodies. A sputum culture grows Streptococcus pneumoniae. Which of the following is the most likely diagnosis? Prior splenectomy (Other choices: DiGeorge syndrome, galactosemia, Gaucher disease, or myeloproliferative disorder) Robbins explanation: Splenectomy in childhood reduces immunity to encapsulated bacterial organisms. The spleen recycles old RBCs and removes inclusions such as Howell-Jolly bodies (similar to getting the cherry pits out without damaging the cherry). About one third of all circulating platelets are pooled in the spleen, and granulocytes are marginated in splenic sinusoids, so that when the spleen is absent, the WBC and platelet counts increase. Test q: A 45M farmer is injured by a tractor and undergoes splenectomy. Histology reveals laceration of the capsule but is otherwise unremarkable. He recovers fully. Before leaving the hospital, the patient should: receive pneumorax. (2009, #3 May be a typo, pneumovax is a pneumonia vaccine?)

Case 1. 70 y.o. female with fatigue. No hemoptosis or vaginal bleeding. Normal physical exam except for pallor. Blood smear: see lymphocyte compare RBC size microcytic. Microcytic + abnormal shapes = anisopoikilocytosis. CBC: Hgb 5.9 MCV 56.2 RDW 20.2 WBC 5,900 Plt 383,000 Low Low Elevated

RDW tells you there is a variety of RBC sizes. Would see in iron deficiency, MAHA, also in hereditary spherocytosis. 1a. The RBCs show? A. Macrocytosis B. Hyperchromasia C. Spherocytosis D. Schistocytosis E. Anisopoikilocytosis abnormal in size and shape. Also are hypochromic. All consistent w/Fe deficiency. 1c. Before the patient leaves your office you should? A. Order TSH and T3 B. Order TSH and T4 C. Prescribe multivitamins with iron D. Perform stool exam for occult blood E. Perform stool exam for hookworms and other parasites D. Because its an older patient, MUST perform D. If older individuals have iron deficiency and no obvious chronic bleed, must suspect colon/gastric carcinoma. 1b. What lab result would you expect? A. Elevated serum ferritin B. Elevated serum iron C. High serum iron and low TIBC D. Low serum iron and high TIBC E. Low serum iron and low TIBC D High Total Iron Binding Capacity

Case 2. 34 y.o. female with 2-day history of fever, petichiae and hematuria; also headaches and mental confusion. Have a pentad of problems fever, petichiae (indicates abnormal/low # of platelets), renal disease, headaches (so something is wrong in the CNS), and the only thing missing here is MAHA. Must examine blood smear Blood smear: See lots of schistocytes do not confuse schistocytes w/bite cells. Also see spherocytes here. Hallmark cell of MAHA is schistocyte, but may also see spherocytes. Since its a hemolytic anemia, body tries to respond polychromasia/reticulocytes present. Platelets are missing here.

CBC: Hgb 7.0 MCV 77 RDW 23 WBC 17,000 Plt 14,000

Low High High Very low

Everything fits TTP/MAHA, including RDW. Schistocytes are much different in size than normal RBCs, so RDW is increased. 2a. Describe the RBCs: A. Normochromic B. Microcytic C. Spherocytes and schistocytes D. Drepanocytes and schistocytes E. Howell-Jolly bodies Note: Drepanocytes are aka sickle cells. 2b. Diagnosis? A. Sickle cell anemia B. Hereditary spherocytosis C. Thrombotic thrombocytopenic purpura (TTP) D. Anemia of chronic disease secondary to hypothyroidism E. Folate deficiency 2c. What test result would you expect? A. D dimer elevated Not always, but usually
elevated in TTP. Always elevated in DIC.

B. C. D. E.

Haptoglobin elevated Plasma free hemoglobin decreased BUN normal Urine protein negative

B: haptoglobin is the protein in the blood that combines w/Hgb when RBCs rupture. So if you have a hemolytic anemia, haptoglobin will fall, not increase. C: Plasma free hemoglobin when RBCs break, this goes up (not decreases) D: patient has renal disease, so BUN will not be normal E: Urine protein will not be negative because of renal disease

Case 3. 34 y.o. male in for routine physical has pallor and icterus; splenomegaly on PE Blood smear:See lots of spherocytes but no schistocytes (fragmented cells). See lots of polychromasia/reticulocytes present. CBC: Hgb 11 MCV 84 WBC 6,000 Plt 350,000 Anemic

3a. Describe the RBCs. A. Normochromic B. Schistocytosis C. Microcytic D. Spherocytosis E. Hypochromic

3b. What test should you order to confirm your diagnosis? A. Serum iron B. Osmotic fragility C. Reticulocyte count D. Malaria screen E. D dimer B. If you think the patient has hereditary spherocytosis but no MAHA (because there arent schistocytes), test for osmotic fragility. Spherocytes are easier to lyse than normal RBCs.

Case 4. 37 y.o. female treated lifelong for a seizure disorder complains of fatigue and lightheadedness. Lightheadedness/fatigue anemia. History of seizure disorder patient is probably on anti-seizure meds. If taken for a long time, will produce B12 deficiency. Interferes w/the absorption of B12. Any patient that has been on barbituates or Dilantin may have a macrocytic anemia.

Blood smear: No lymphocyte present to compare size, but we do see a hypersegmented neutrophil. Neutrophils usually have 3 (occasionally 4) segments, but in this disorder, they will have 5-6-7 segments. CBC: Anemic. Hgb 6 MCV 130 WBC 6,000 Plt 220,000 MCV is elevated, consistent w/macrocytic anemia. Low High Normal Normal

4a. All of the following correctly describe the RBCs EXCEPT: A. Normochromic B. Macrocytic C. Anisocytosis D. Ovalocytosis E. Spherocytosis A bone marrow aspirate was performed See multi-segmented neutrophils (bottom right). Also see giant band forms (top left) maybe twice the size of some of the other neutrophils. 4b. What findings on the bone marrow aspirate suggest folate deficiency? A. Hyposegmented neutrophils B. Hypocellularity C. Giant bands and hypersegmented neutrophils D. Miniature bands and hypersegmented neutrophils E. Hypersegmented blasts and promyelocytes The question could say either folate deficiency or B12 deficiency they look the same.

LEUKEMIA: Acute ALL and AML Hi/Low WBC (unpredictable) Rapidly fatal if untreated; anemic and thrombocytopenic Curable
Test q: The current cure rate in the US for children w/ALL is: 80%. (Other choices: 10, 20, 50, 100%)

Chronic CLL and CML Always high WBC (100k or 1million) Slowly progressive- patient lives many years Difficult to cure

Acute Leukemias: can be lymphocytic or granulocytic in origin. 5a. In Acute Leukemias you will almost always find: A. Normal hct, normal platelets B. Normal hct, decreased platelets C. Decreased hct, increased paltelets D. Anemia, thrombocytopenia E. WBC count >100,000 D: Patient is always really sick in acute leukemia. In chronic leukemia most of the time, dont even know patient is sick. 5b. In Chronic Leukemias you will almost always find: A. Anemia, thrombocytopenia B. WBC count >50,000 C. Normal hct, increased platelets D. Anemia, thrombocytosis E. Circulating blasts

Lymphoblasts:

ALL (PAS stain):

PAS stain is + for lymphoblasts, negative for myeloblasts.

Myeloid Precursors:

Myeloid precursors: The one were used to seeing in peripheral blood is the segmented neutrophil. Going backwards in maturation, the closest thing to that is a band. If someone is infected, we usually see segmented PMNs and band PMNs (and occasionally some earlier forms). However, in infection/stress, we usually do not see myeloblasts and promyelocytes.

CML: mixture of forms in peripheral blood. See small # of segmented neutrophils, bands, metamyelocytes, and lots of promyelocytes (top row of forms). The left shift is shifted further in CML. Easiest way to tell if its CML vs. leukemoid reaction is WBC count if over 100,000, its CML. If its 20,000, probably an infection. Problem comes w/a patient who has 40-50,000 could be either. Above: Immature granulocytes (left shift). Lots of bands present, also earlier forms like myelocytes and maybe even a promyelocyte.

LAP stains (Leukocyte alkaline phosphatase) Left: stain positive in leukemoid reaction because they are normal granulocytes (just too many). In CML, none of the cells take the stain because theyre all abnormal cells that do not make leukocyte alkaline phosphatase. LAP score high if benign, LAP score low if malignant.

Above: Karyotype of CML t(9;22). Translocation is diagnostic for CML. Can karyotype or FISH

Above: Abl is green, bcr is red. If translocation, will see yellow/whitish-yellow color. If you did this test for promyelocytic leukemia, would see same type of picture but would be 15;17 translocation.

Above: CML and AML. AML all blasts. CML mixture of blasts (few), maybe occasional promyelocyte, and lots of earlier forms.

Above: If you see an Auer rod, its AML (right). If you see something like on the left, they are BLASTS. Big cells, have nucleoli all over the place = acute leukemia. Cant really tell which one.

Old classification system: 8 different categories of AML, M0-M7. M3 promyelocytic leukemia still retains its name. M0-M7 stages classified based on how the cells look:
Below: Myeloperoxidase (MPO) AMLs are MPO positive.

M3: Promyelocytic leukemia lots of granules, sometimes lots of Auer rods. This is the one that causes MAHA, has specific translocation 15;17, treat w/retinoic acid (vitamin, unique). Most of the AMLs M2-3-4 will stain w/MPO. Will be MPO+, PAS-. PAS is for lymphocytes, MPO is for granulocytic series. Once you get real far to the right (M5-6-7) or the left (M0), will be negative.

Above: Sudan Black stain. If you shift to the R toward monocytes, fat stains are positive

Above: Nonspecific esterase. and non-specific esterase is positive. May indicate that the patient has a monocytic leukemia.

Above: M5 monocytic leukemia. Tend to have extensive involvement in the mouth, gums, buccal mucosa (presenting symptoms ulcers, swollen gingiva).

Above: M6 erythroid (RBC). M7 megakaryocytic version.

Case 6. 25 y.o. male with fever, recurrent URIs and submandibular swelling; also poor wound healing. Blood smear: Circulating blasts and an AUER ROD = AML. CBC: Hgb 9.9 MCV 106.3 WBC 7.9 (70% abnormal cells) Plt 50,000 6a. Diagnosis? A. ALL B. AML C. CLL D. CML E. Hereditary spherocytosis 6b. Which test on a bone marrow aspirate confirms your diagnosis? A. MPO (-) B. MPO (+) C. PAS (+) D. Iron stain (Prussian blue) E. Leukocyte alkaline phosphatase = 20

Case 7. 6 y.o. male with URIs, headache, bone pain and easy bruising; axillary and cervical lymphadenopathy; hepatosplenomegaly. Easy bruising = hints that patient has low platelet count. Blood smear: Again, see blasts. In this case, cant really say for sure what it is. But since its a 6y/o, think ALL acute lymphoblastic leukemia. Blasts have nucleoli, sometimes have unusual cleaves (butt cells). CBC: Hgb 9 MCV 82 WBC 92,000 (86% abnormal) Plt 18,000 thrombocytopenia 7a. Diagnosis? A. ALL B. AML C. CLL D. CML 7b. What test results are expected on a bone marrow aspirate? A. MPO (+), PAS (+) B. MPO (-), PAS (-) C. TdT (+), Precursor B cell (+) D. TdT (-), Precursor T cell (+) E. Sudan black B (+); CD 10 (-)

Case 8. 15 y.o. male with flulike symptoms; axillary and cervical lymphadenopathy; gums are thick and bleeding. Gums think monocytic problem. Blood smear: These blasts are folded up like a boxing glove looks like monocyte nucleus. CBC: Hgb 9 MCV 90 WBC 42,000 (88% abnormal) Plt 22,000

Acute leukemia. 8. A. B. C. D. E. Diagnosis: AML (FAB M1) AML (FAB M3) AML (FAB M5) ALL (L1) Infectious mononucleosis

BM Bx results: o MPO (-) As AMLs shift over toward monocytic side, no longer +. o PAS (-) Negative because they are not lymphoblasts. o Sudan Black B (-) o Non-specific esterase (++) marker for monocytic series. o Serum lysozyme 162 (4-15 normal) product of monocytes elevated. All signs point to FAB M5. Case 9. 39-year-old female has routine workup for fibrocystic disease of breast; splenomegaly present. Blood smear: Way too many cells. All kinds of granulocytic cells band cells, metamyelocytes, myelocytes, more granular forms are probably promyelocytes. Looks like CML. CBC: Hgb 13 MCV 96 WBC 133,000 (seg 56, band 15, meta 13, myelo 4, pro 3); baso 1, eo 1 Plt 130,000

9. What test should you order on a bone marrow aspirate? A. MPO B. PAS C. Sudan black B D. LAP Would be decreased because its CML. If increased, would be benign reaction E. TRAP

9b. What is a better test? A. PAS B. MPO C. Reticulocyte count D. TRAP E. FISH specifically identify the translocation

10. 30-y/o. female; anemia, thrombocytopenia; CBC- blasts w. Auer rods, schistocytes. Diagnosis? A. ALL B. AML, M1 C. AML, M3 AML plus MAHA. Classic picture for M3, aka promyelocytic leukemia. D. AML, M5 E. TTP

Quick Look at Lymphomas: Hodgkin RS Cells Often curable Stage important Few malignant cells (only Hodgkin/RS cells) < cell-mediated Localized radiation Non-Hodgkin Low grade- slowly progressive, rare cure High grade- rapidly progressive; a minority are curable Grade important Mostly lymphoma (All cells are malignant) < humoral Systemic chemotherapy

Dont have to find RS cells to call it Hodgkins disease. If it stains CD15+, CD30+, its enough to call it. No specific translocations. Non-Hodgkin: Grade important. In a low grade lymphoma: will live a long time but not be cured. High grade lymphoma: will die quickly if not treated but chance of cure.

Case 11. 45 y.o. female presents to the OBGYN clinic with diffuse lymphadenopathy. An axillary LN is biopsied.

See lots of follicles, same size, rim of blue cells surrounding. Can also see starry sky tingible-body macrophages in middle of germinal centers. Little blue cells, parafollicular cells, mixture of lymphocytes, macrophages present = BENIGN FOLLICULAR HYPERPLASIA.

11. Diagnosis? A. Follicular hyperplasia B. Follicular lymphoma C. Hodgkin Disease D. SLL E. Tuberculosis

Test q: A 23F presents w/a sore throat and generalized lymphadenopathy. A concerned intern biopsies a cervical lymph node. The cortex shows many follicles, and these areas are CD20+. Tingible-body macrophages are present. Parafollicular areas are CD3+. Diagnosis? Benign lymph node. Test q: An enlarged lymph node is removed from the right neck of a 25y/o male who is free of other symptoms, but fears he may have lymphoma. On histologic exam, the node is follicular. The follicles stain positive for CD20 and the parafollicular areas are positive for CD3. Cells in the follicles stain positive with both anti-Kappa and anti-llambda. Tingible body macrophages in the follicles stain positive for CD68. Diagnosis: Benign reactive lymph node. Test q: A 7M has complained of worsening pain in the right side of his groin region for the past week. Phys exam shows painful, swollen lymph nodes in the right inguinal region. An inguinal lymph node biopsy is performed. Histologically, the node has large, variable sized germinal centers containing tingible-body macrophages. Many neutrophils are present. What is the most likely cause of these histologic changes? Acute lymphadenitis. (Other choices: ALL, sarcoidosis, follicular lymphoma, Cat-scratch fever)

Case 12. A 45-y.o. female presents to OBGYN clinic with diffuse lymphadenopathy. An axillary LN is biopsied

Again, have follicular proliferation. In this case, a lot harder to see exactly where the follicles are. No rim of blue cells, no stars in the middle. Monotonous proliferation of cells, all the same size = FOLLICULAR LYMPHOMA. Can be low-grade or high-grade. If the cells are small, low grade. Big cells, high grade. Follicular lymphoma has a specific translocation t(14;18).

12. Diagnosis? A. Follicular lymphoma B. Follicular hyperplasia C. Hodgkin Disease D. SLL E. Burkitt lymphoma Case 13. 59 y.o. female is seen for fatigue, an abnormal CBC and cervical lymphadenopathy

Lymphocytes present.

All small, round blue lymphocyte cells.

No follicle presentation, just diffuse sheets of little blue cells. Consistent with SLL/CLL.

13. Diagnosis? A. Burkitt lymphoma B. Hodgkin Disease C. Plasmacytoma D. SLL/CLL E. Follicular lymphoma If the disease starts in the lymph nodes, its SLL. If it starts in the peripheral blood or BM, its CLL (chronic lymphocytic leukemia). Same entity, same cells, all malignant cells co-expression of CD5 and CD23. Abnormal because CD5 is Tcell marker and CD23 is B-cell marker.

Case 14 9 y.o. male with abdominal mass. What are the stars A. Lymphoblasts B. Myeloblasts C. Undifferentiated blasts D. Macrophages E. Neutrophils

Above: starry sky. All the sheets of smaller cells are malignant. All the stars are macrophages (benign part).

In Burkitts, you have benign and malignant cells, but the only benign cells are the macrophages which are chewing up all the debris. There is debris present because the malignant cells necrose (turn over so fast that they die; requires a lot of macrophage infiltration to clean up the dead cells).

Case 15: Most of the cells in this hematologic proliferation are A. Reed Sternburg Cells B. Hodgkin cells C. Lymphoblasts D. Myeloblasts E. Benign In Hodgkin Disease, most cells are benign (plasma cells, eosinophils, benign lymphocytes, fibroblasts). Easy to treat HD tumor cell load is much smaller. HD much more important to get low stage, grade not even used. In Non-HLs, the grade is more important than the stage.

Can see ReedSternberg cells of Hodgkin Disease. They have two nuclei, each w/a prominent nucleolus. Immunohistochemi stry would show CD15+ CD30+.

Case 16: An expected lab finding for this patient is A. Acute leukemia on PBS B. Chronic leukemia on PBS C. Hypocalcemia D. Hypercalcemia E. Low total serum protein Multiple myeloma: Myeloma involves the BM and the bones. Breakdown hypercalcemia. Punched out lesions in skull, arm Total serum protein will be elevated because these patients make amyloid in the form of light chains, and they also make increased heavy chains.

BM biopsy see lots of plasma cells.

Again, lots of plasma cells.

Test q: A 65F presents w/severe back pain. She has a history of breast carcinoma treated by radical mastectomy 10 years previously. X-rays show lytic lesions in the vertebrae and ribs. CBC reveals a Hgb of 11.8 g/dL, WBC 4800/uL, and platelets 130,000/uL. Initial chemistry values are in the normal range except for a calcium of 12.8 mg/dL (r.r. 8.9-10.7), a total protein of 10.3 g/dL (r.r. 6.0-8.0), and an albumin of 3.1 g/dL (r.r. 3.5-5.0). Which of the following is the most likely diagnosis? Multiple myeloma. (Other choices: Metastatic breast carcinoma, Osteosarcoma of the spine, Senile osteoporosis, and Waldenstrom macroglobulinemia) Test q: A 71F has had a history of back pain for several months. She recently developed a cough productive of yellowish sputum, and a sputum culture + + grew Streptococcus pneumoniae. On phys exam, she has no lymphadenopathy or hepatosplenomegaly. Lab findings show Na of 141 mmol/L, K 4.2 mmol/L, Cl 104 mm/L, CO2 26 mmol/L, glucose 79 mg/dL, calcium 10.8 mg/dL, phosphorus 3.1 mg/dL, AST 31 U/L, ALT 24 U/L, alkaline phosphatase 291 u/L, total protein 9.3 g/dL, albumin 4.4 g/dL, urea nitrogen 31 mg/dL, and creatinine 3.8 mg/dL. A skull radiograph shows multiple 1-to-4-cm lytic lesions. Which of the following lab findings is most likely to be reported? Bence-Jones proteinuria (Other choices: Increased HbF, Hgb 21.2 g/dL, 3 Positive ANA, and WBC count 450,000/mm ) Robbins: The characteristic punched out bone lesions of multiple myeloma seen on radiographs result from bone destruction mediated by RANKL, a cytokine produced by the myeloma cells that activate osteoclasts. Several cytokines, most notably IL-6, are important growth factors for plasma cells. They are produced by tumor cells and resident marrow stromal cells. High serum levels of IL-6 correlate with active disease and poor prognosis. The monocloncal population of plasma cells often produces a monoclonal serum spike seen in serum or urine protein electrophoresis. Patients can have hypercalcemia and an increased serum alkaline phosphatase level. The neoplastic cells are generally well-differentiated, with features such as a perinuclear hof, similar to normal plasma cells.

For the exam, NEED TO BE ABLE TO IDENTIFY Burkitts, Hodgkins (RS cells, eosinophils, nodular sclerosis), follicular lymphoma (follicles but not normal), follicular hyperplasia (normal follicles), and SLL.

Leftover blood questions:

(I dont think these have been covered yetplease post a reply if any of them have.) Test q: The estimated blood volume of a 70kg man is? 5 liters. Test q: Platelets used for transfusion in the US should be stored w/agitation at: 20 to 24 degrees C. Test q: A 70kg male is injured in an automobile accident w/severe blood loss. You wish to increase the patients hematocrit from 15% to 27%. You should transfuse how many RBC units? 4. Test q: The most common sign of an acute hemolytic transfusion reaction is: fever and chills. Test q: Platelet transfusion is recommended to prevent spontaneous intracranial bleed if the platelet count falls below: 10,000/mm . Test q: The number one cause of hemolytic transfusion in the US is: Clerical error. Test q: Platelets can be used up to __ days after collection. 7. Test q: In blood transfusion, the universal donor and universal recipient are __ respectively: O; AB. Test q: The risk for acquiring HIV-1 from a transfusion in the US is: 1:2,000,000. Test q: Failure due to transfusion is now most commonly associated with: Transfusion related acute lung injury (TRALI) reaction. Test q: Which of the following statements regarding modified blood components is true? Irradiating cellular components decreases the risk of GVHD. Test q: To prevent graft vs. host disease, what type of RBCs are used: Irradiated RBCs.
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