Heme Lab

Peripheral Smear
-Want to look at RBCs, platelets and nucleated cells in the frame
-RBCs should have 1/3 of cell being pale, darker in periphery
-All should be around same size and shape
-10 platelets per high powered field is normal range (~7 here)
-Nucleated cells - neutrophil is here (3-5 lobes) and band cell

Caption:
The red blood cells here are normal, happy RBC's. They have a zone of central pallor about 1/3 the size of the RBC. The RBC's demonstrate minimal variation in size (anisocytosis) and shape (poikilocytosis). A few small fuzzy blue platelets are seen. In the center of the field are a band neutrophil on the left and a segmented neutrophil on the right.

Caption: (WBC Lab)
A normal mature lymphocyte is seen on the left compared to a segmented PMN on the right. An RBC is seen to be about 2/3 the size of a normal lymphocyte.

Normal smear that happens to have all the cells that could be in the smear
-RBCs are all relatively the same size.
Monocyte (kidney bean shape – fair amount of cytoplasm)
Eosinophils are not common to see in a smear, basophils even less so

Caption:
Click to identify the segmented neutrophil, band neutrophil, lymphocyte, monocyte, eosinophil, basophil, and platelet in the image above:

MCV <70fL!
Possible causes listed
Major signs and symptoms are fatigue and pallor (admittedly vague)
Widened area of pallor visible in the RBC (should only be 1/3 of the cell)
Furlong thinks this could be a transfused patient because there are darker RBCs – seeing a dual population of cells

Caption:
The RBC's here are smaller than normal and have an increased zone of central pallor. This is indicative of a hypochromic (less hemoglobin in each RBC) microcytic (smaller size of each RBC) anemia. There is also increased anisocytosis (variation in size) and poikilocytosis (variation in shape).

Hypochromic cells – most common cause is iron deficiency
Populations most at risk are women in their reproductive years and children

Caption:
The most common cause for a hypochromic microcytic anemia is iron deficiency. The most common nutritional deficiency is lack of dietary iron. Thus, iron deficiency anemia is common. Persons most at risk are children and women in reproductive years (from menstrual blood loss and from pregnancy).

Caption:
This peripheral blood smear demonstrates marked poikilocytosis (abnormally shaped RBC's) as well as some anisocytosis (variation in RBC size), though many are small (microcytes). This patient had beta-thalassemia, a hereditary disorder of beta globin chain synthesis that leads to ineffective erythropoiesis and a microcytic anemia. Some of the RBC's resemble jigsaw puzzle pieces.

Note that we can know this is an RBC and not a WBC because the plasma color is so similar to the RBC – the nucleus just wasn’t ejected yet.
-The notes claim this is lead poisoning that leads to bone marrow injury
-Basophilic stippling is always pathologic and is rRNA bits in the cytoplasm! Other causes are sideroblastic anemia and megaloblastic anemia

Caption:
The nucleated RBC in the center contains basophilic stippling of the cytoplasm. This suggests a toxic injury to the bone marrow, such as with lead poisoning. Such stippling may also appear with severe anemia, such as a megaloblastic anemia.

Macrocytic anemia – MCV >100fL
Associated with B12 and folate deficiencies
Why megaloblastic? Nuclear-cytoplasmic dissynchrony – nuclear stays immature while cytoplasm is maturing! (FINDING IN THE BONE MARROW)
In peripheral blood these are the 2 findings we will see
1. Oval Macrocytes: when you hear OVAL think B12 and folate!
2. Hypersegmented Neutrophils

Caption:
This hypersegmented neutrophil is present along with macro-ovalocytes in a case of pernicious anemia. Compare the size of the RBC's to the lymphocyte at the lower left center.

Always think megaloblastic anemia and B12 or folate deficiencies!!

Caption:
Here is a hypersegmented neutrophil that is present with megaloblastic anemias. There are 8 lobes instead of the usual 3 or 4. Such anemias can be due to folate or to B12 deficiency. The size of the RBC's is also increased (macrocytosis, which is hard to appreciate in a blood smear).

Pyknosis – irreversible condensation of chromatin in a cell undergoing apoptosis or necrosis

SS is homozygous for sickle cell and you get anisocytosis
SC disease behaves like homozygous sickle cell disease
-Both can crystallize if oxygen is low
AS is sickle cell trait and much less likely to enter sickling situation

Caption:
This is sickle cell anemia in sickle cell crisis. The abnormal hemoglobin SS is prone to crystallization when oxygen tension is low, and the RBC's change shape to long, thin sickle forms that sludge in capillaries, further decreasing blood flow and oxygen tension. Persons with sickle cell trait (Hemoglobin AS) are much less likely to have this happen.

Caption:
Here is another example of sickled erythrocytes in a patient with Hgb SS who presented with severe abdominal pain in sickle crisis. The sickled cells are prone to stick together, plugging smaller vessels and leading to decreased blood flow with ischemia.

Caption:
In the center of the field is a rectangular RBC that is indicative of Hemoglobin SC disease. Both hemoglobin S and hemoglobin C are present. The RBC's may sickle, but not as commonly as with Hemoglobin SS disease. The hemoglobin C leads to the formation of "target" cells--RBC's that have a central reddish dot.

Caption:
This peripheral blood smear demonstrates many larger bluish reticulocytes as well as smaller RBC's lacking central pallor--spherocytes. This patient had an autoimmune hemolytic anemia. Antibody coated the RBC's, and portions of the RBC's were removed, decreasing cell size. Many RBC's were removed entirely, resulting in anemia and a bone marrow response with increased erythropoiesis and elevated reticulocyte count (20%). The patient developed an indirect hyperbilirubinemia as well.

MAHA = microangiopathic hemolytic anemia – schistocytes are indicative of this or other intravascular hemolysis (Found with TTP – ADAMST13 deficiency and vWF aggregation)
-TYPICAL FINDING FOR DIC as well!
-This is that crazy situation where you have endothelial damage in your vessels and they basically slice your RBCs up as they go by. Violent imagery but you will never forget it.

Caption:
There are numerous fragmented RBC's seen here. Some of the irregular shapes appear as "helmet" cells. Such fragmented RBC's are known as "schistocytes" and they are indicative of a microangiopathic hemolytic anemia (MAHA) or other cause for intravascular hemolysis. This finding is typical for disseminated intravascular coagulopathy (DIC).

Can get into endothelial cells, can effect the liver
P vivax is shown here
-The smear will help you differentiate species (really don’t want to see P. falciparum!) – she didn’t explain how though

Caption:
Malaria is a parasitic disease caused by the genus Plasmodium, of which there are four species that affect man. Shown here are "ring forms" of Plasmodium vivax in red blood cells. This disorder can produce hemolysis and anemia.

Can see yellow fat cells and different cell lines of the marrow but the little red things are not normal!

Caption:
A myelophthisic process may also result from infections involving the marrow, including fungal, mycobacterial, and parasitic infections. Seen here are multiple amastigotes of Leishmania donovani in a bone marrow smear. The infiltrative process does not have to fill up much of the marrow to produce the peripheral leukoerythroblastic pattern.

Happens with increased serum proteins in the blood i.e. fibrinogen or globulins
-These proteins WILL INCREASE THE SEDIMENTATION RATE!!! Sed rate increases with inflammation and the presence of acute phase proteins – OFTEN IN MULTIPLE MYELOMA (abnormal production of Ig circulating…)
Can think maybe of the linked RBCs as a mustache line of the frenchman named Rouleaux…

Caption:
The RBC's here have stacked together in long chains. This is known as "rouleaux formation" and it happens with increased serum proteins, particularly fibrinogen and globulins. Such long chains of RBC's sediment more readily. This is the mechanism for the sedimentation rate, which increases non-specifically with inflammation and increased "acute phase" serum proteins.

Erythroid precursors are very dark – can’t tell the difference in a bone marrow specimen really unless you do a bone marrow aspirate smear
-ERYTHROID ISLAND
Arrow = Megakaryocytes
Can maybe see neutrophils (PMNs) – predominate in bone marrow
Can say trilinear hematopoeisis is going on – because we see erythroid cells, megakaryocytes and neutrophils
At age 50 – should see 50% fat and 50% cells, varies inversely with age from there (more fat and less cells as you age)

Caption:
This is the appearance of normal bone marrow at high magnification. Note the presence of megakaryocytes, erythroid islands, and granulocytic precursors. This marrow is taken from the posterior iliac crest in a middle aged person, so it is about 50% cellular, with steatocytes admixed with the marrow elements.

Aplastic Anemia
-Reduced hematopoeitic elements – not normal in someone middle-age! Too little cellular component (<25%)
-RBCs, platelets and granulocytes will be markedly decreased – at risk for bleeding
Causes: some hereditary (Fanconi anemia), Infection, drug, toxin or idiopathic

Caption:
Hematopoietic elements in this bone marrow biopsy are markedly reduced. This is a case of aplastic anemia. Of course, besides, RBC's the platelets and granulocytes will often be diminished. Sometimes a drug or toxin is the cause and sometimes infection. When no known cause can be found, it is termed idiopathic aplastic anemia.

May be present in terms of infection (fungal or TB), CT disorders, sarcoidosis, or some types of lymphoma
Cluster of macrophages in center!

Caption:
Here is a granuloma in a bone marrow biopsy, a potential myelophthisic process. Granulomas in marrow tend to be small and poorly formed. This one consists mainly of epithelioid macrophages. Of course, multiple cultures and special stains are done to determine if there is an infectious etiology, such as a mycobacterial or fungal infection. In this case, no organism was demonstrated, and the clinical features fit with sarcoidosis.

Dark blue clusters are replacing the normal hematopoeitic cells
Huge chunks of epithelial cells are here – METS
Patient could be anemic because of this infiltrative process in the marrow

Caption:
The marrow spaces between the trabecular bone are filled with cohesive clusters of dark blue cells and smaller groups of these cells replacing normal hematopoietic cells. This is metastatic carcinoma. The primary site in this case was breast. A bone scan can help to identify metastases. A bone marrow biopsy can confirm the diagnosis.

When looking at bone marrow look first at cellularity (ratio of fat to cells - at age 50 it should be 50:50) – Need Patient’s Age!
-Normal Cellular Components
-Trilinear Hematopoiesis: looking for megakaryocytes, leukoid and erythroid lines
-Heterogeneity here (normal)
*Abnormal Findings: Lymphoid aggregates; metastatic disease; granulomas

Caption:
This is the appearance of normal bone marrow at medium magnification, with cellular marrow and adipose tissue. The large multinucleated cells are megakaryocytes. The erythroid islands and granulocytic precursors form the bulk of the cellular component. This marrow is taken from the posterior iliac crest in a middle aged person, so it is about 50% cellular, with steatocytes admixed with the marrow elements.

Almost 100% cellularity – will never be normal even in an infant
Homogenous cells
Acute Lymphocyte leukemia – B cell more common; clinical presentation are bone marrow failure (anemia, thrombocytopenia, infection) – NOTE that the WBC are high on lab value but that’s because there are blasts circulating; they will not help you fight infection
-Cytogenetic abnormalities: t(12;21), t(1;19) (in B cell acute leukemia); Philadelphia chromosome t(9;22) also in ALL (poor prognosis); t(14,11) associated with T cell
T cell leukemias – slightly order age group because T cells are peaking at a later age; may have thymic mass.

Caption:
In contrast to aplastic anemia, leukemia results in a highly cellular marrow. The marrow between the pink bone trabeculae seen here is nearly 100% cellular, and it consists of leukemic cells of acute lymphocytic leukemia (ALL) that have virtually replaced or suppressed normal hematopoiesis. Thus, though the marrow is quite cellular, there can be peripheral cytopenias. This explains the complications of infection (lack of normal leukocytes), hemorrhage (lack of platelets), and anemia (lack of red blood cells) that often appear with leukemia.

Can decide it’s myeloid instead of lymphoid because myeloid cells make granules and we “see” Auer Rods
We can see here the red things in the cytoplasm (of the non-Furlong slide) that Auer rods (most likely abnormal formation of granules – in NEOPLASTIC CELLS)
CBC in a Patient with AML
-White Cell Count <200 (~80-100)
-Platelets (less than 150)

Caption:
Here are very large, immature myeloblasts with many nucleoli. A distincitve feature of these blasts is a linear red "Auer rod" composed of crystallized granules. These findings are typical for acute myelogenous leukemia (AML) that is most prevalent in young adults.

Myeloproliferative disorders
1. CML – middle-aged adults, clinical presentation is non-specific (happens over months)
2. Polycythemia vera – progresses to burn out phase where there is significant marrow fibrosis (indolent and progressive disorder)
3. Central thrombocytosis - progresses to burn out phase where there is significant marrow fibrosis
4. Primary myelofibrosis (CIM) – 2 stages: Cellular and Fibrotic (See hepato- and splenomegaly
In these disorders you’re getting too many cells being produced, marrow is hypercellular; tend to see HIGH counts with these disorders as well as organomegaly
-Immature myeloid cells and bands are present here
-See MULTIPLE nucleated cells at low mag at various stages! Maturation and differentiation taking place

Caption:
There are numerous granulocytic forms seen here, including immature myeloid cells and bands. This condition is one of the myeloproliferative states and is known as chronic myelogenous leukemia (CML) that is most prevalent in middle-aged adults. A useful test to help distinguish this disease is the leukocyte alkaline phosphatase (LAP) score, which should be low with CML and high with a leukemoid reaction.

Lone megakaryocyte in marrow
These are BLASTS

Caption:
At high power, the bone marrow of a patient with acute myelogenous leukemia is seen here. There is one lone megakaryocyte at the right center. (IS THIS RIGHT CAPTION??0

NOTE: there are not many BLASTS here so you can safely say it is chronic not acute
CML is defined by the Philadelphia chromosome
-Natural progression is to transform to AML (all patients will eventually transform to acute leukemia (usually AML but can be lymphoid)
-New Therapies have prolonged remission and pushes off this progression

Caption:
Here is another view of a peripheral blood smear in a patient with CML. Often, the numbers of basophils and eosinophils, as well as bands and more immature myeloid cells (metamyelocytes and myelocytes) are increased. Unlike AML, there are not many blasts with CML.

Very small lymphocytes (SLL)
Can see bile ducts (arrow); you can say there are lymphoid (dark cells) but that is it!
Need to prove monoclonality to say this is a neoplastic process
Same as SLL (small cell) – if they have lymphocytosis in peripheral smear, will also present with lymphadenopathy
CLL’s immunophenotype is CD5 and CD23 (all B cells have CD19 and CD20 so this isn’t a good marker)
-Can distinguish from Mantle Cell because CLL is CD23+ and Mantle cell is not!
-B-CLL often associated with Trisomy 12 (30% of cases)

Caption:
These infiltrates in the liver are composed of small lymphocytes. The involvement of tissues in cases of chronic lymphocytic leukemia (CLL) is known as small lymphocytic lymphoma (SLL). This disease, CLL/SLL, has an indolent course.

More common in men than women (older patients usually) often present with splenomegaly
-Pancytopenia too since marrow is involved
-Bone marrow involvement is assumed since it is a leukemia; cells are circulating too which give you the splenomegaly

Caption:
This collage of cells on peripheral blood smears depicts abnormal lymphocytes with indistinct cytoplasmic borders and projections, giving the cells a "hairy" appearance. At the lower right can be seen one of these cells with red cytoplasmic staining indicative of tartrate resistant acid phosphatase (TRAP) positivity. This is hairy cell leukemia, an uncommon B cell proliferation seen mostly in older males and marked by marrow involvement, pancytopenia, and splenomegaly.

-Adipose tissue at the top (perinodal) right above the capsule
-Secondary follicles with the mantle zone (dark zone) and germinal center (pale-staining))
-In between follicles is the paracortex with T cells
-Everything else is B cells!
Marginal zone not visible but would be pale staining around the mantle zone

Caption:
Here is the normal appearance of a benign reactive lymph node. At the top is the capsule and just under that a subcapsular sinus where lymphatics enter that drain tissues peripheral to the node. Beneath the capsule is the paracortical zone with lymphoid follicles having a pale germinal center in which the immune responses are often generated. Beneath this are sinusoids extending to the center of the node.

FOLLICULAR PATTERN: follicular lymphoma
Other patterns are not really as clear
NODULAR PATTERN: Mantle Cell Lymphoma and Hodgkin’s Lymphoma
DIFFUSE PATTERN: SLL and diffuse B cell Lymphoma

Caption: Not on website?

They are very large but we can’t really tell it’s lymphoma from the gross picture

Caption:
This is a cross section through the mesentery to reveal multiple enlarged lymph nodes that abut each other. Unlike metastases, nodes with lymphoma tend to have little necrosis and maintain a solid, fleshy tan appearance. High grade non-Hodgkin's lymphoma (NHL) tends to involve a single node, localized group of nodes or extranodal site. Low grade NHL tends to involve multiple lymph nodes.

-Follicular pattern (follicular lymphoma) – low grade
*Other low grade lymphomas: Mantle Cell; lymphoplasmacytic
-Fat infiltrate
-Defined by translocation of chromosome 14 t(14;18), involving Bcl-2 (these regions would be + for bcl-2)
-Positive for CD10; half of these patients will transform into a diffuse large B-cell lymphoma

Caption:
Here is a lymph node involved by lymphoma, a malignant process characterized by the proliferation of neoplastic lymphoid cells. The capsule of the node has been invaded and the lymphomatous cells extend into the surrounding adipose tissue. Note that the follicles are numerous and irregularly sized. This is a malignant lymphoma, small cleaved cell type, follicular (also known as: malignant lymphoma, poorly differentiated lymphocytic type, nodular).

Can occur in all age groups
Cells don’t look like lymphocytes anymore
-Wouldn’t know if this is lymphoid or not lymphoid if you just look at this slide (need the history)
Dispersed open chromatin pattern
Burkitt Lymphoma is the other high grade lymphoma discussed

Caption:
The malignant lymphocytes here are very large with a moderately abundant cytoplasm, and the nuclei are round to ovoid with prominent nucleoli and occasional mitoses. The diagnosis is diffuse malignant lymphoma, large cell type (also known as: immunoblastic lymphoma). The major differential diagnosis in this case would be a metastatic carcinoma. The presence of monoclonal immunoglobulin as demonstrated by immunoperoxidase technique would help to confirm this lesion as a malignant lymphoma.

Often associated with follicular lymphoma
Lymphoid aggregates hugging the bony spicule
Infiltrate is the small blue cells

Caption:
A bone marrow biopsy can reveal malignant lymphoma. Here, there is a peritrabecular infiltrate of small blue cells which is the lymphomatous infiltrate.

Bands of fibrosis are present!
What makes Hodgkin’s different from non-Hodgkin’s?
-Age group is late teens early 20s and then again in 50s
-Occurs typically in SINGLE GROUP OF LYMPH NODES (cervical or axillary often)
-Spreads in systematic fashion to nodes
-Can be associated with night sweats, fever (B symptoms)
-Malignant cell is in the MINORITY
How do we classify Hodgkin’s?
-Classical type (CD15+, CD30+) and nodular lymphocyte predominant (CD20+, EMA+, CD45+)
-Classical subtypes
1. Lymphocyte Depleted
2. Nodular Sclerosis (most common)
3. Mixed Cellularity
4. Lymphocyte rich

Caption:
At medium power, nodular sclerosing Hodgkin's disease has prominent bands of fibrosis. Staging of Hodgkin's disease is important to try and determine therapy and the prognosis. Staging is often done by radiographic means, with CT scans used to determine where lymphadenopathy is located, ultrasonography to determine size and lesions of liver and spleen, and chest radiograph. Histologic diagnosis is typically made from biopsy of an involved lymph node. A bone marrow biopsy is typically performed as well. Staging laparotomy is less commonly used nowadays because the radiographic procedures are excellent.

Nodular pattern
Mononuclear variant – would have CD20 if looking at immunophenotype
Often present with axillary lymphadenopathy
EBV negative usually!
Multilobulated popcorn cell present (variant of the RS cell)

Caption:
This is Hodgkin's disease, lymphocyte predominance type. Many lymphocytes compose this type of HD. A variant Reed-Sternberg cell with a multilobulated nucleus is seen.

Hard to tell this is lymphocytic at all (cells are different)
Not a lot of reactive cells in the background
Often associated with EBV and sometimes HIV
Slightly worse prognosis because patients present at a later stage
**Reed-Sternberg are in Hodgkin’s and often have bilobed nucleus or are multinucleated; much bigger than lymphocytes (giant cells)

Caption:
This is Hodgkin's disease, lymphocyte depletion type. Many Reed-Sternberg cells and variants are present, as seen here at medium and high magnification. This type of HD is not common.

Mixed cellularity in background  macrophages, small lymphocytes, plasma cells, eosinophils
CD15+ on right

Caption:
This is Hodgkin's disease, mixed cellularity type. Note the many different cell types, including small lymphocytes, eosinophils, and macrophages. The immunoperoxidase stain with antibody to CD-15, a marker for Reed-Sternberg cells and variants, is at the right.

Binucleate cells that are indicative of Hogdkin’s

Caption:
Note the large cells with large, pale nuclei containing large purple nucleoli at the arrowheads. These are Reed-Sternberg cells that are indicative of Hodgkin's disease. Most of the cellular content of foci of Hodgkin's disease consists of reactive lymphoid cells. There are four main subtypes of Hodgkin's disease: lymphocyte predominance, nodular sclerosis, mixed cellularity, and lymphocyte depletion.

Caption:
This is a high power view of a Reed-Sternberg cell seen with Hodgkin's disease. Note the large, prominent nucleoli.

If liver is involved it puts the cancer at a higher stage

Caption:
This is a liver that is involved with Hodgkin's disease. The staging of Hodgkin's disease is very important in determining therapy. Thus, it is important to determine whether the patient has only a single lymph node region involved, multiple node regions, or extranodal involvement. This picture could probably suffice for non-Hodgkin's lymphomatous hepatic disease as well.

**Hematopoietic diseases are much more likely to produce splenic masses than mets

Caption:
Thelarge pale nodule and several smaller nodules seen here in this section are the result of splenic involvement by Hodgkin's disease. Hematopoietic diseases are much more likely to produce splenic masses than metastases.

No this is not histology.
Lucent areas in the X-ray are the lesions
Enhanced osteoclastic activity in multiple myeloma
-Protein produced (MIP-1alpha) that activates receptor of a ligand that stimulates osteoclastic activity
Bone pain is common because of the lytic process going on
-As a result they also have hypercalcemia
M spike – Ig produced has many effects
-Toxic to kidney (renal disease is common, #2 cause of death)
-If they’re only making IgM they can’t fight infection well (#1 cause of death)

Caption:
The rounded "punched out" lesions of multiple myeloma appear as lucent areas with this skull radiograph.

Progressive disease

Caption:
In this bone marrow biopsy section at medium power, there are sheets of plasma cells of multiple myeloma that are very similar to normal plasma cells, but the cells may also be poorly differentiated. Usually, the plasma cells are differentiated enough to retain the function of immunoglobulin production. Thus, myelomas can be detected by an immunoglobulin "spike" on protein electrophoresis, or the presence of Bence-Jones proteins (light chains) in the urine. Immunoelectrophroesis characterizes the type of monoclonal immunoglobulin being produced.

Caption:
Here is a smear of bone marrow aspirate from a patient with multiple myeloma. Note that there are numerous well-differentiated plasma cells with eccentric nuclei and a perinuclear halo of clearer cytoplasm. There is also an abnormal plasma cell with a double nucleus.