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Hematology Case Reactive Leukocytosis effacement

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					Hematology Case 4: Reactive Leukocytosis
The patient is a 55-year-old white male who presents to the emergency room complaining of chest
pain and labored breathing. His temperature is 98.6° F. He is overweight and smokes two packs of
cigarettes a day. His EKG and cardiac enzyme profile reveal changes compatible with an acute
myocardial infarction. The complete blood count (CBC) shows:
Hemoglobin (Hb)                     13 gm/dl (reference range 12 - 15.2 gm/dL)
Total white blood cell              15,500/mm3
Platelets (Plt)                     250,000/mm3
A differential white cell count was included in the CBC report, showing 80% segmented
neutrophils (polymorphonuclear neutrophils), 10% "band" neutrophils, 7% lymphocytes, and 3%
atypical lymphocytes. Based on the reported neutrophilic leukocytosis and "bandemia," an
extensive infectious disease work-up was initiated.
This decision was confusing so you went to the laboratory and performed a 100-cell differential
(white cell) count of the peripheral blood smear. The numbers you obtained basically agree with the
original report except that you counted only 6% "bands" and you identified a myelocyte and a
basophil.




                                                                       1.Peripheral Blood Smear, 4x
                                                                       2.Peripheral Blood Smear,
                                                                10x
                                                                       3.Peripheral Blood Smear,
                                                                100x
                                                                       3a.Segmented Neutrophil
                                                                       3b."Band" Neutrophil




1. Leukocytosis - Peripheral Blood
A low magnification view (4x) of the peripheral blood smear permits you to estimate the total white
blood cell count (TWBC), which in this case was reported to be 15 x10^3/ml. [Note: a rule of
thumb for assessing the TWBC: 15 to 20 WBC/4x field correlates with a TWBC of approximately
15 - 20x10^3/ml]. At a slightly higher magnification (10x), review of the smear now reveals that
most of the leukocytes are granulocytes, specifically neutrophils. A superficial examination of these
neutrophils reveals that some are fully segmented while others are "band" forms. A higher
magnification (100x) reveals a segmented neutrophil demonstrating the typical thin chromatin
strand connecting the lobes (segmented neutrophil -3a). In addition, the smear shows a typical
"band" form with a horseshoe nucleus and no thin chromatin strands ("band" neutrophil -3b).
Although the distinction between these two cells is clear in this picture, such is not always the case.
1. What findings in the CBC report prompted an infectious disease work-up? Neutrophilic
leukocytosis and "bandemia" (i.e., a "left shift").
2. Compare and contrast neutrophilic leukocytosis, leukemoid reaction, chronic myelogenous
leukemia, and acute myeloblastic leukemia.
Neutrophilic leukocytosis refers to an increase in the number of neutrophils in the peripheral
blood. This may be caused by pyogenic infections or by non-microbial stimuli, such as tissue
destruction. The neutrophil increase results primarily from release of mostly mature
neutrophils from the marrow storage pool, and hence neutrophil precursors are rare in the
peripheral blood.
       Leukemoid reaction refers to an extreme form of reactive neutrophilic leukocytosis in
which the peripheral blood contains not only mature neutrophils but also immature
leukocytes, resembling leukemia. However, in leukemoid reactions, there are fewer immature
myeloid cells (myeloblasts are rare), leukocyte alkaline phosphatase is normal or increased,
and cytogenetic abnormalities are absent.
       Acute and chronic myelogenous leukemias are clonal neoplastic disorders that are
discussed later.




                                                                       Basophil (a)
                                                                       Neutrophil (b)
                                                                       Eosinophil (c)




2. Circulating Granulocytes
The three frames in this image (100x) illustrate the different granulocytes that are seen commonly in
the peripheral blood. Granulocytes are leukocytes, or white blood cells, that contain cytoplasmic
granules. Frame (a) demonstrates a basophil with the obvious large basophilic granules typical of
these granulocytes. The nuclear segmentation of these cells is often difficult to appreciate due to the
heavy cytoplasmic granulation. Basophils are the least common of the granulocytes in the
peripheral blood. Basophilia -- or increased number of basophils -- is frequently noted in
myeloproliferative disorders. The most abundant of the circulating granulocytes is the neutrophil
(b). The specific granules of these cells are not readily identified with the light microscope. An
increase in their number (neutrophilia) may be seen in a variety of conditions, including infections
or tissue necrosis (as in myocardial injury). The large red granules of eosinophils (c) are readily
apparent in this picture. Eosinophilia, an increase in eosinophils, will be presented in the next
image.
                                                                     Examples of Eosinophils
                                                                     Lymphocyte




3. Eosinophilia - Peripheral Blood
This slide (100x) demonstrates a leukocytosis composed predominantly of eosinophils, i.e.,
eosinophilia. What are some examples of non-neoplastic eosinophilia? Eosinophilia may be
seen in allergic reactions (including asthma, drug reaction), parasitic infestation, and skin
diseases.




                                                                     Nucleus
                                                                     Cytoplasm




4. Lymphocytes - Peripheral Blood
In the previous image, the lymphocyte manifests the cytologic features of a normal circulating
lymphocyte: small size (about the size of a red cell), high nuclear-to-cytoplasmic ratio (N/C) and
condensed nuclear chromatin. These small lymphocytes constitute about 90% to 95% of circulating
lymphocytes. However, as the cells above demonstrate, larger lymphocytes may also be seen in the
peripheral blood of normal individuals. These larger lymphocytes are given the designation
"atypical lymphocytes" -- to distinguish them from "typical" small circulating lymphocytes -- and
make up less than 5% of lymphocytes in circulation. One "atypical" characteristic is the frequent
presence of nucleoli. Another "atypical" feature is the presence of abundant blue cytoplasm. This
blue hue sometimes is noted only at the periphery of the cytoplasm. Lymphocytosis is found in viral
syndromes, such as infectious mononucleosis, although it is not diagnostic of such infections.
Frequently in these viral syndromes there are an increased number of atypical lymphocytes. The
circulating atypical lymphocytes in these viral syndromes are T cells that have been transformed (or
activated) in response to EBV-infected B cells in infectious mononucleosis. Are these activated
lymphocytes mature or immature (i.e., blasts)? These activated T cells may resemble
immature (primitive) cells, but they are in reality progeny of mature, resting lymphocytes.




                                                                     Atypical Lymphocytes
                                                                     Monocytes




5. Atypical Lymphocytes vs. Monocytes
These pictures (100x) depict the cytologic differences between atypical lymphocytes (a & c) and
monocytes (b). As can be appreciated from these pictures, the differentiation is at times difficult.
However, the cytoplasm of monocytes usually is gray and frequently contains vacuoles (not
demonstrated in this example), while the cytoplasm of atypical lymphocytes reveals the bluish tinge
previously described. Lymphocytes, typical or atypical, can be differentiated by the expression of
surface antigens. Monocytes frequently express CD14, while T cells lack this antigen but express
CD3, CD4, CD5, CD7, CD8. B cells do not express any of these CD antigens; instead, they often
have CD19, CD20, and CD22. What trends can you see regarding CD numbers and
lymphocytes (T and B cells)? CD digits less than CD 10 frequently are associated with T cells.
CD digits around CD 20 frequently are associated with B cells.




                                                                   Leukemic Blasts
                                                              (Lymphoblasts)
                                                                   Atypical Lymphocyte




6. Lymphoblasts vs. Atypical Lymphocytes
The differentiation between atypical lymphocytes and leukemic blasts may, on occasion, be quite
difficult; however, as the two pictures (100x) in this image demonstrate, there are significant
morphologic differences. Blasts often have a very high N/C (nuclear to cytoplasmic) ratio and
stippled chromatin pattern. Atypical lymphocytes, as a rule, have abundant cytoplasm and coarser
chromatin. The clinical and hematologic changes associated with these cases are helpful in this
differentiation as well. Leukemic blasts, as seen in acute leukemia, tend to infiltrate the marrow and
suppress the production of normal hematopoietic elements.




                                                                      Myeloblast
                                                                      Promyelocyte
                                                                      Myelocyte
                                                                      Metamyelocyte
                                                                      Band Neutrophil
                                                                      Segmented Neutrophil




7. Maturation of Neutrophils - Bone Marrow
The different cells seen in the maturation sequence of the neutrophil are identified (100x), from a
myeloblast to a segmented neutrophil. The overall maturation scheme of the neutrophilic line
consists of nuclear shrinkage and segmentation with concurrent condensation of the chromatin. This
process begins with the formation of a functionally immature myeloblast and culminates with the
development of a phagocytic segmented neutrophil. This maturation takes about 15 days and occurs
in the bone marrow. About half of this time is spent in the so-called "mitotic" compartment. The
cells of this compartment -- myeloblast, promyelocyte, myelocyte -- multiply (i.e., are mitotically
active) and undergo maturation. In the last stage of the mitotic compartment, the myelocyte begins
to replace its primary (nonspecific) granules with secondary (specific) granules. The final three
cellular stages of maturation comprise the so-called "storage" compartment. Cells in this pool
consist of metamyelocytes, band neutrophils, and segmented neutrophils or "polys"
(polymorphonuclear neutrophils). When are these cells released into the blood? These cells
remain in the marrow until the segmented forms are preferentially released into the
peripheral blood.

Kidney Case 1: Nephrotic Syndrome:
A previously healthy 35-year-old man, complained of recent fatigue, and swelling of his feet and
ankles. He was also noted to have puffy eyes as well. Physical exam revealed an afebrile man, with
lower extremity edema, extending to the knee, periorbital edema, and a small amount of
ascites. Laboratory features were as follows:
Creatinine           0.8 mg/dL        Urinalysis: protein - 4+
BUN                  18 mg/dL                     glucose - neg
Albumin              2.8 g/dL                     blood - neg
Hematocrit           40%                          bilirubin - neg
                                                  Micro: rare RBCs, no WBCs, many oval fat
Liver function tests normal
                                                  bodies
Triglycerides        400 mg/dL
Cholesterol         375 mg/dL
24 hr urine         11.2 gm/24
protein             hrs

Drug history was negative. ANA, HIV, and hepatitis serology were negative.
The patient developed right flank pain the following day. Ultrasound revealed renal vein
thrombosis. Renal biopsy was performed.




                                                                    Capillary Loops
                                                                    Mesangial Areas




1. Nephrotic Syndrome, Renal Biopsy - Silver Stain
This is a representative glomerulus from the patient's biopsy. The capillary loops show subtle,
uniform thickening. The capillary loops are open (no inflammation) and the mesangial areas and
cellularity are normal. (See image 2, high power.)




                                                                    Capillary Lumen
                                                                    Urinary Space
                                                                    GBM "Spikes"
                                                                    Epithelial Cell
                                                                    Endothelial Cell
                                                                    Mesangial Cells and Matrix




2. Nephrotic Syndrome, Renal Biopsy - Silver Stain
Glomerular basement membranes (GBM's) and spikes stain black, while immune complex deposits
do not stain. Notice that the "spikes" are a part of GBM. The immune complexes are situated in-
between the spikes. Is there any inflammation seen? No inflammation is seen.
What would the IF pattern look like? IF pattern would be granular and peripheral, which
correlates with the immune complex deposits between spikes.
What is the clinical syndrome that this patient presented with? This patient presented with
nephrotic syndrome. He had marked albuminuria, low serum albumin, and consequent
generalized edema. Hyperlipidemia is also often seen in patients with nephrotic syndrome.
This gives rise to lipiduria, seen as oval fat bodies in the urine. Note the absence of red cells.




                                                                     Capillary lumens
                                                                     GBM




3. Nephrotic Syndrome, Renal Biopsy - Immunofluorescence With Anti-IgG
This is a direct immunofluorescence preparation of the patient's biopsy using antisera against IgG.
Note two features: A. Where is the staining? On the GBM. B. What kind of pattern is seen?
Granular. This pattern corresponds to the immune complex deposition. It is the immune
complexes that are stained.




                                                                     Capillary Lumen
                                                                     Urinary Space
                                                                     Dense Deposits
                                                                     GBM "Spikes"
                                                                     GBM
                                                                     Podocytes
                                                                     Schematic of EM




4. Nephrotic Syndrome - Electron Micrograph
This is an electron micrograph of a glomerulus from the patient. Correlate this EM pattern with the
previous IF Pattern. In what area are the deposits located? The deposits are located in the outer
aspect of the basement membrane under the visceral epithelium of the Bowman's capsule.
Thus, they are subepithelial. Are the spikes part of the deposits? No. The spikes are part of
the GBM that lies between the deposits. Based on the results of the light microscopy,
immunofluorescence, and electron microscopy of the renal biopsy, what is the patient's
diagnosis? The biopsy reveals a membranous glomerulopathy, with subepithelial deposits of
electron dense, immune complex type material. By light microscopy, this is illustrated by lack
of increased glomerular cellularity, and uniformly thickened GBMs. A special stain (silver)
shows "spikes" of GBM material projecting between the deposits, a feature better understood
on the EM photo. Immunofluorescence shows the typical granular, peripheral capillary loop
staining with IgG and C3, typical of membranous GN.
What is the purpose of the additional tests the clinician ordered after the diagnosis was made?
After the diagnosis of nephrotic syndrome was made, the clinician did serological tests to rule
out some secondary causes of membranous glomerulonephritis that account for about 40% of
cases. This is important, because they can in many cases be treated. These include SLE,
hepatitis B, malaria, gold and penicillamine for rheumatoid arthritis, Captopril (an
angiotensin-converting enzyme inhibitor), and occult carcinoma.




                                                                  Capillary Lumen
                                                                  Podocyte




5. Nephrotic Syndrome, Minimal Change Disease (MCD) - Silver Stain
The glomerulus shows normal mesangial cellularity, with normal open tufts. The basement
membrane is of uniform thickness. No inflammation is seen. The tubules are of normal shape and
size. The interstitium is also unremarkable. MCD is the most common cause of nephrotic syndrome
in children. Could MCD involve adults? Although the peak incidence is between two and six
years of age, adults may also be affected. Compare this image with membranous GN (Images
1and 2). What does the IF look like in MCD? It is normal.




                                                                  Tubular "Lipoid" Cells
6. Nephrotic Syndrome, Minimal Change Disease (MCD)- Trichrome Stain
Interstitial and tubular foam "lipoid" cells are seen in MCD and other conditions associated with
nephrotic syndrome, hyperlipidemia, and hyperlipiduria. What are the general differences in the
etiology of nephrotic syndrome in children vs. adults? In children primary renal disease
accounts for 95% of the cases, the most common cause being minimal change disease. By
comparison primary renal diseases are responsible for 60% of cases in adults; membranous
glomerulonephritis is the most common primary renal cause.


                                                                    Podocytes
                                                                    Minimal Change Disease,
                                                              Higher Power
                                                                    Basement Membrane
                                                                    Fused Foot Processes
                                                                    Schematic of EM-Minimal
                                                              change
                                                                    Normal EM
                                                                    Podocytes
                                                                    Basement Membrane
                                                                    Endothelial Cell Nucleus
                                                                    Podocyte

7. Minimal Change Disease - Electron Micrograph
In MCD, the EM shows effacement of the foot processes. Is there anything abnormal about the
basement membranes of the capillary loops? No. The only abnormality is foot process
effacement. Are there any immune deposits? No deposits are seen in MCD.
What is the pathogenesis of MCD? This is not fully understood. There is no evidence of anti
GBM antibodies or immune complexes. There seems to be some form of primary injury to
glomerular epithelial cells that effaces foot processes and causes secondary alterations in
GBM.




                                                                    Early Segmental Sclerosis
                                                                    High Power View
                                                                    Intracapillary Foam Cells
                                                                    Epithelial Cell Hyperplasia




8. Focal Segmental Glomerulosclerosis (FSGS), Early Lesion -Trichrome Stain
FSGS is one of the common causes of nephrotic syndrome. Notice the collapse of capillary loops in
a portion of a single glomerulus (segmental capillary collapse), accompanied by intracapillary foam
cells and extracapillary epithelial cell proliferation. In a case of FSGS, what would the IF show in
this segmentally sclerotic focus? IF will be positive in the sclerotic focus, and will stain with
IgM and C3. in a non-sclerotic part? IF is negative in the nonsclerotic part.
Is FSGS the result of immune complex injury? No. The basic lesion in primary FSGS is
disruption of visceral epithelial cells by unknown mechanisms. The sclerosis and entrapment
of plasma protein, including IgM and C3, are non specific. They result from
hyperpermeability at sites of epithelial cell damage.




                                                                      Normal Looking Glomeruli
                                                                      Focal Sclerosis
                                                                      Interstitial Inflammation
                                                                      High Power View
                                                                      Segmental Sclerosis
                                                                      Atrophic Tubules




9. Focal Segmental Glomerulosclerosis (FSGS), Late Lesion -Trichrome Stain
In a later stage of FSGS dense glomerular sclerosis is seen, with more interstitial fibrosis, tubular
atrophy, and chronic inflammation. Is FSGS a descriptive term, a clinicopathologic entity, or
both? FSGS is a descriptive term that implies sclerosis of some, but not all, glomeruli (i.e.,
focal), and in the affected glomerulus only a portion of the capillary tuft is involved (i.e.,
segmental). Such changes may occur in glomerular diseases secondary to HIV infection or
heroin abuse; these may also occur in IgA nephropathy. In addition, idiopathic FSGS is a
distinct clinicopathologic entity. What is the difference in the clinical presentation and
prognosis of minimal change disease vs FSGS in children? Both FSGS and minimal change
disease present with nephrotic syndrome, (the latter is more common). It is essential to
distinguish them because their course and prognosis are different. FSGS is less steroid
responsive and, unlike the benign course in minimal change disease, 50% of cases with FSGS
end up with renal failure in 10 years.
                                                                       Lobular Pattern




10. Membranoproliferative Glomerulonephritis (MPGN) - H&E
MPGN is another relatively common primary renal cause of nephrotic syndrome in both children
and adults. Notice the lobular pattern, the hypercellularity, and the collapse of the capillaries. What
is proliferating in MPGN? Mesangial cells, monocytes, and lymphocytes. What is another
name for MPGN? Mesangiocapillary GN.




                                                                       "Tram-Track" Capillary
                                                                Loops




11. Membranoproliferative Glomerulonephritis (MPGN), Later Stage - Silver Stain
At a later stage of MPGN, many of the capillary loops show a double contour, or "tram-track,"
appearance. What causes the double contour appearance? The double contour appearance is
caused by an apparent "splitting" of the basement membrane. This split appearance results
from the intercalation of mesangial cell processes into the peripheral capillary loops. What do
you expect to see in the IF? IF would show granular and peripheral patterns with/without
mesangial pattern. Are there any spikes in MPGN? (Compare with images 1 and 2.) No
spikes are seen in MPGN.
                                                                    Mesangial Staining
                                                                    Peripheral Staining




12. Membranoproliferative Glomerulonephritis - Immunofluorescence for C3
The pattern here is mesangial and peripheral. Compare with the IF of membranous GN. Could this
pattern be seen in SLE? Yes, because in SLE the immune complexes can deposit in the
mesangium or along the basement membrane. What are the types of MPGN? There are two
types of primary MPGN. Type I, the more common form is an immune complex disease and
shows granular pattern of IgG and C3 deposition by IF. The immune complexes are
subendothelial by EM. Type II, the less common form has C3 but not IgG in a granular-linear
pattern on the basement membrane and as aggregates in the mesangium. The two are
distinguished best by electron microscopy




                                                                    Electron Dense Deposits
                                                                    Endothelial Cell
                                                                    Podocyte
                                                                    Schematic of EM




13. Membranoproliferative Glomerulonephritis - Electron Micrograph
Notice where the deposits are. What cell makes the double track membrane? Extension of the
mesangial cells. Name a systemic disease in which you see MPGN type disease. It can occur in
association with SLE, hepatitis B or C antigenemia, and -1-antitrypsin deficiency.

Hematology Case 2: Hemolytic Anemias
The patient is a 6-year-old male who was seen by his pediatrician because of a low grade fever and
sore throat. Mild yellow coloring of his sclera was noted by his mother two days previously. On
physical examination, the child had mild jaundice. His spleen was moderately enlarged. Selected
laboratory findings are shown below:
Complete Blood Count (CBC)
Hemoglobin                            9.8 g/dL
Reticulocyte count                    14%
Hematocrit                            28%
Leukocyte count                       9,200/mm3
Erythrocyte count                     3.4 x 106/mm3
Platelet count                        375,000/mm3
MCV                                   84 µm3
Serum bilirubin
                                Total 4.5 mg/dL
                               Direct 0.4 mg/dL
MCHC                                  35.0 g/dL
RDW                                   20%
Your microscopic examination of the peripheral blood smear reveals red cell changes as seen in
Image 1. The child had not been known to have anemia previously. There was no history of
bleeding. A paternal uncle had a splenectomy as a child for "low blood." A direct antiglobulin test
(DAT) was negative. Blood was drawn for an osmotic fragility test.
The following day both of the parents and the child returned to the pediatrician's office. A CBC and
blood smear examination were performed on the parents. The father had a normal hemoglobin but
an increased reticulocyte count; spherocytes were observed on his blood smear. His spleen was
palpably enlarged. The mother's studies were normal. The osmotic fragility test on the child showed
increased osmotic sensitivity, which was markedly accentuated after incubating the red cells in
normal saline for 24 hrs.




                                                                       Peripheral Blood Smear,
                                                              Patient
                                                                   Peripheral Blood Smear,
                                                              Normal




1. Peripheral Blood Smear - Unknown
Compare the peripheral blood smear obtained from the case's patient with the normal peripheral
blood smear (Feature 2). Note the size and coloration of red cells (explanation on next image). Is
this patient's anemia due to a production defect or due to hemolysis? Due to hemolysis.
What laboratory tests are used to demonstrate hemolysis? Serum bilirubin, fecal
urobilinogen, serum haptoglobin, chromium-labeling of RBCs (survival), and serum LDH.
What is the cause of jaundice in such patients? Hyperbilirubinemia resulting from increased
destruction of red cells.
How are hemolytic anemias classified? Intrinsic (intracorpuscular) versus extrinsic
(extracorpuscular). What is the diagnosis in this case? Hereditary spherocytosis.
What is the inheritance pattern, red cell defect, mechanism of hemolysis, and treatment in this
disorder? This disease is caused by a genetic defect (most commonly autosomal dominant) in
the red cell membrane cytoskeleton. Abnormal red cells get trapped within the cords of the
splenic red pulp and are phagocytosed by macrophages. Transfusions and/or splenectomy are
sometimes necessary. Why was a DAT ordered? To exclude immune-mediated hemolysis.
Spherocytes can also be seen in immune-hemolytic anemias.




                                                                       Polychromatophilic Red Cell
                                                                       Spherocytes
                                                                       Normal Red Cells
                                                                       Platelets




2. Hereditary Spherocytosis
This image illustrates reticulocytes (largest cells with slightly greyish color), normal mature red
cells (intermediate-sized cells with an area of central pallor), and spherocytes (smallest cells, which
stain darkly and lack central pallor). Spherocytes result from membrane damage due to either a
problem extrinsic to the red cells or an abnormality intrinsic to the red cells. What are the major
categories of intrinsic red blood cell abnormalities? Intrinsic red blood cell abnormalities
include membrane abnormalities, enzyme deficiencies, or hemoglobin abnormalities.
Hereditary spherocytosis, which is the diagnosis in this case, is caused by a genetic defect in the red
cell membrane. An abnormality of membrane skeletal proteins causes progressive loss of
membrane, making the red cells less deformable. The red cells then get trapped within the cords of
the splenic red pulp and are phagocytized by the macrophages. The process is an example of
extravascular hemolysis. Spherocytes are not unique to hereditary spherocytosis and are seen in
other hemolytic states, particularly in immune hemolysis.
                                                                        Polychromatophilic
                                                                  Erythrocytes
                                                                        Reticulocytes




3. Polychromasia/Reticulocytes
The Wright stained slide, at left, demonstrates polychromatophilic red cells in the peripheral blood.
(The term "polychromasia" refers to the bluish tinge of these cells.) These red cells have been
recently released from the bone marrow and still contain significant amounts of RNA, which is the
reason for the bluish tinge (remember, RNA stains blue with the Wright's stain). The right panel is a
reticulocyte stain. In this procedure, RNA is precipitated inside the erythrocytes. Because
precipitated RNA gives the reticulated appearance shown, the cell is called a reticulocyte. For all
practical purposes, reticulocytes and polychromatophilic red cells represent red cells of similar age.
These cells remain as reticulocytes in circulation for about 24 hours. Because these cells have been
recently released from the marrow, it stands to reason that the number of reticulocytes would reflect
the ability of the marrow to respond to the challenges of anemia. In other words, if a person is
anemic and has an elevated reticulocyte count, the likely explanation for his anemia is hemolysis.
That is to say, the red cells are being destroyed outside the bone marrow. What does an anemia
with a low reticulocyte count signify? Anemia with a low reticulocyte response signifies a
production problem within the marrow.




                                                                         Spleen (10x)
                                                                         White Pulp
                                                                         Red Pulp
                                                                         Spleen, Red Pulp (45x)
                                                                         Sinuses
                                                                         Cord




4. Spleen - Hereditary Spherocytosis
The two slides in this image are from a section of spleen removed from a patient with hereditary
spherocytosis. As stated in slide 2, the spleen is the site of destruction of red cells in this disorder.
Why are red blood cells destroyed in this manner? The loss of membrane causes the red cells
to lose their deformability and become unable to negotiate the splenic microvasculature.
This low magnification (10x) picture of the spleen (1) permits review of the general microanatomy
of the spleen. The red and white pulp are noted. It is in the red pulp that red cells are entrapped. A
high magnification of the spleen (45x) (2) shows empty sinuses and trapping of red cells in the
splenic cord. This is the typical finding in hereditary spherocytosis, reflecting the inability of the red
cells in this disease to navigate the unique microvasculature of the spleen.




                                                                         Elliptocytes




5. Hereditary Elliptocytosis
Hereditary elliptocytosis -- like hereditary spherocytosis -- is an example of a hemolytic state
associated with an intrinsic red cell membrane defect. The red cells in this blood smear (100x) have
an elliptical shape, which results from an abnormality in the red cell membrane protein skeleton.
What are clinicopathologic aspects of this disorder? Most patients with this defect have
normal or only a slightly shortened red cell survival time and no significant anemia.




                                                                        1.Blood Smear 100x
                                                                        "Bite" Cell
                                                                        2.Blood Smear, Heinz Body
                                                                  Preparation
                                                                        Heinz Bodies




6. G6PD Deficiency
This hemolytic state is related to a different intrinsic red cell defect: specifically, an enzyme
deficiency. This image (1) shows an abnormal red cell on a blood smear (100x) from a patient with
G6PD deficiency who has been challenged by an oxidant stress. The configuration of this red cell
has the appearance of an apple core and is sometimes referred to as a "bite" cell. Describe the
pathophysiology of G6PD deficiency. Denatured hemoglobin precipitates in the form of Heinz
bodies (Image 2). These precipitates are removed by splenic macrophages causing the configuration
observed on this blood smear. These "pitted" cells are then prone to leak hemoglobin in circulation,
which explains the hemoglobinuria -- a hallmark of intravascular hemolysis -- noted in these
patients. Eventual removal of these red cells from the circulation by macrophages in the spleen --
indicating an extravascular hemolytic component -- contributes to the red cell destruction in these
cases. (2) demonstrates a special Heinz body stain in a patient with a severe form of G6PD
deficiency after an oxidant challenge. The Heinz bodies are the dark inclusions at the periphery of
the red cell. (A special stain is required because the routine Wright stain used on blood smears does
not stain Heinz bodies.)




                                                                       Spherocytes
                                                                       Polychromatophilic Red Cells




7. Immune Hemolysis - Warm Antibody Type
An example of an extrinsic insult causing a hemolytic state is immune-mediated hemolysis. In this
case, the blood smear reveals mainly spherocytes. Spherocytes in this condition are formed due to
partial loss of the red cell membrane caused by attempted phagocytosis of Ig-coated red cells. The
spherocytes are then removed in the spleen (i.e., extravascular hemolysis). So-called warm
antibodies, which may be auto- or alloantibodies, are responsible. (The terms "cold" and "warm"
antibodies relate to the temperature at which the antibody binds most avidly to the red cell antigen.)
What are causes of warm antibody hemolytic anemias? Primary (idiopathic) or secondary
(lymphomas, leukemias, cancers, autoimmune disorders, drugs).
What is the isotype of the antibody in such cases? IgG.




                                                                       Red Cell Agglutination
8. Cold Agglutinin - Hemolytic Anemia
The presence of a cold agglutinin antibody is another example of an extrinsic red cell insult
resulting in an immune-mediated hemolysis. The blood smear in these cases often shows
characteristic clumping of the red cells (as shown here). In contrast to warm antibodies described in
the previous slide, cold agglutinin antibodies are always autoantibodies. The antibody involved is
most often IgM. What is the cause of hemolysis in this process? The in vivo agglutination
property of these antibodies is not the cause of the hemolysis; rather, their ability to fix
complement on the red cell surface at low temperatures results in intravascular hemolysis
when red cells move to warmer areas. In addition, at 37oC, IgM antibody is released from the
cell surface, leaving a coating of C3b. This is an opsonin and such opsonized red cells are
phagocytosed by monocyte-macrophages. Thus, there is extravascular hemolysis.




                                                                        Plasmodium Ring Forms
                                                                        Platelets




Yet another example of an extrinsic insult which can result in a hemolytic anemia is the infestation
of red cells by parasites. This blood smear, taken from a patient with Plasmodium falciparum
malaria, shows two ring-form parasites in the red cell in the center of the field. This red cell will be
destroyed as the parasites mature and are eventually released from the cell. What is the most
common cause of infection-induced hemolytic anemia? Malaria.

Hematology Case 3: Hemolytic Anemias
The patient is a 15-year-old black male who presents to the emergency room (ER) complaining of
severe abdominal pain. His mother informs you that these painful episodes began when he was
about two years old. She also tells you that the patient, his brother, her husband, and she have been
told they have the "sickle gene." However, only her husband has suffered similar painful episodes
("pain crises").
Physical examination demonstrates a soft abdomen and intact bowel sounds. The patient's pulse is
rapid with a blood pressure of 125/90. His temperature is 98.8 F. Complete blood count (CBC)
demonstrates:
Hemoglobin 8 gm/dl (reference range 12 -
                 15.2 gm/dl
WBC count 13,500/mm3
Platelets        350,000/mm3
Your microscopic examination of the peripheral blood smear reveals red cell changes as seen in
Image 1; in addition, you note that there is significant polychromasia. Most of the white cells are
neutrophils (segmented forms and bands).




                                                                        1.Peripheral Blood Smear,
                                                               Patient
                                                                    2.Peripheral Blood Smear,
                                                               Normal




1. Peripheral Blood Smear, Unknown
Compare the peripheral blood smear obtained from the patient (1) with a normal peripheral blood
smear (2). How are the morphologic abnormalities in the red cells seen here related to
polychromasia and this patient's abdominal pain? Red cells in this smear are sickled and are
therefore prone to undergo hemolysis in the spleen; in addition, patients with sickle cell anemia
have episodes of vascular occlusion. The hemolysis causes reticulocytosis and polychromasia. The
vascular occlusions cause painful episodes (crises). What is the pathophysiology of vaso-
occlusive crises in these patients? The pathophysiology of vascular occlusions and resulting
organ damage is not fully understood. It is thought that sickle Hb causes red cell membrane
damage. Such cells, even if not sickled, have increased stickiness to endothelium and tend to
retard blood flow in the microcirculation. In the sluggish blood flow, there is greater
deoxygenation of HbS, and, hence, sickling is facilitated. The abnormal cells thus accumulate
and obstruct blood flow. Why did this patient have no noticeable clinical problems before the
age of two? HbF present in red cells during infancy inhibits polymerization of HbS.
Why don't his mother and brother have abdominal crises but his father does? His mother and
brother have HbAS, i.e., they have sickle cell trait. What is the correlation between the
patient's symptoms and the number of irreversibly sickled cells (ISC)? Why? ISC number
correlates with hemolysis, because irreversibly sickled cells undergo hemolysis in the spleen.
How is the severity of clinical manifestations affected by the red cell concentration of
hemoglobin S and by the presence of other types of hemoglobins? Many factors affect the
severity of sickle cell disease, including intracorpuscular concentration of HbS. Homozygotes
with HbSS have 100% HbS and full blown sickle cell disease; heterozygotes with HbSA have a
milder disease called sickle cell trait. Other Hb's (A, F, C, etc.) inhibit polymerization of HbS.
                                                                       Hgb "C"
                                                                       Crystallized Hgb "C"
                                                                       Target Cells
                                                                       Hgb "S"
                                                                       Irreversibly Sickled Cells




2. Hemoglobinopathies - "C" and "S"
The cases illustrated in these frames are examples of hemolytic states caused by a type of intrinsic
red cell defect: specifically, hemoglobin abnormalities. The peripheral smear (100x) on the left is
from a patient with Hemoglobin C disease. It shows many "target cells" characterized by a
peripheral ring and central deposition of hemoglobin giving them the appearance of a "target" with
a bull's-eye. Another abnormal cell, which stains deep red and has a rectangular shape, is the result
of crystallization of hemoglobin C. Homozygous hemoglobin C or Hemoglobin C disease is usually
associated with mild to moderate chronic anemia. The peripheral smear (100x) on the right is from a
patient with sickle cell anemia. It shows several irreversibly sickled cells (ISC). How are ISC
formed? ISC are the result of permanent membrane damage related to multiple bouts of
polymerization-depolymerization of intracellular hemoglobin S. All the cells shown in this
picture contain hemoglobin S and are prone to sickling; however, only those that have developed
irreversible membrane damage (ISC) appear as sickled cells in the smear.




                                                                       Hb A
                                                                       Hb F
                                                                       Hb S
                                                                       Hb C
                                                                       Sickle Cell Trait
                                                                       Sickle Cell Disease
                                                                       Hemoglobin C Disease




3. Hemoglobin Electrophoresis on Agarose Gel
Hemoglobin electrophoresis is a commonly used laboratory test for qualitative evaluation of
hemoglobinopathies. Hemoglobin molecules in an alkaline solution have a net negative charge and
move toward the anode in an electrophoretic system. Differences in the charge of various types of
hemoglobin results in different mobilities, causing their separation. Lanes 1 and 2 are control lanes.
Lane 1 has adult (HbA) and fetal (HbF) hemoglobins, whereas Lane 2 has HbA, sickle hemoglobin
(HbS) and hemoglobin C (HbC). Lanes 5 and 7 are normal individuals containing only HbA. Lanes
3 and 8 are from patients with sickle cell trait showing both HbA and HbS. Lane 6 is a case of
sickle cell disease with total absence of HbA; HbS is the predominant hemoglobin, with HbF
making up the rest. Lane 4 is an individual with Hemoglobin C disease who has only HbC. What do
you think is the diagnosis of the patient in lane 9? HbSC.




                                                                      Examples of Red Cell
                                                                Precursors




4. Bone Marrow Changes in Hemolysis
The marrow biopsy (left) demonstrates a hypercellular marrow; the marrow aspirate (right) shows
predominantly a normoblastic proliferation, i.e., erythroid hyperplasia. This erythroid hyperplasia is
the expected finding in a hemolytic state. (Note: Although in this example the marrow biopsy
permits identification of the proliferating cells as normoblasts, this is not always the case).

Gastrointestinal Case 3: Inflammatory Bowel Disease
R.N. is a 56-year-old white male with a history of inflammatory bowel disease of 20 years
duration and migratory polyarthritis. Three months prior to admission he experienced an
exacerbation with a 40 pound weight loss and bloody diarrhea. He was admitted to the hospital
with complaints of 20 bloody diarrheal bowel movements per day, diffuse abdominal pain, and
nausea and vomiting. He stated that his flare-ups in the past were resolved with a short course of
Prednisone. He did not report any family history of inflammatory bowel disease.
On physical exam the patient was a cachectic white male in no acute distress. The abdomen
demonstrated positive bowel sounds and was non-distended, soft, and non-tender. There was no
hepatosplenomegaly or masses. The rectal examination demonstrated black, hemoccult-positive
stool but no masses. The remainder of the physical exam was unremarkable.
On admission the following lab results were noted:
                                   TEST RESULT NORMAL VALUE
Hematocrit                         23.3%             40-54%
Albumin                            2g/dl             3.9-4.8 g/dl
CEA (Carcinoembryonic) Antigen 6.4 ng/dl             0-3 ng/ml
The other laboratory values were unremarkable.
The patient was admitted to the hospital and treated with steroid therapy. There was no
symptomatic improvement after eight days of therapy, so the patient was placed NPO (nothing by
mouth) and on TPN (total parenteral nutrition). He was treated with two units of packed red blood
cells. Colonoscopic examination revealed disease in the descending and sigmoid colon and rectum.
Biopsies were taken and the interpretation by the pathologist was more consistent with ulcerative
colitis than with Crohn's disease. There was no evidence of dysplasia. Options were discussed
with the patient, and he decided to have a partial colectomy.




                                                                    Diffusely Inflamed Rectal
                                                               Mucosa
                                                                    Anus




1. Chronic Ulcerative Colitis, Rectum - Gross
Rectum demonstrates a diffusely inflamed mucosal surface. Ulcerative colitis causes chronic
diarrhea with grossly bloody, mucus-filled stools. Since the disease begins in the rectum and
proceeds proximally without interruption (no skip areas), it is usually evident on proctoscopic exam,
where pseudopolyps and ulcers can be seen. Compare the gross and microscopic features of
ulcerative colitis with Crohn's disease. What factors are believed to underlie the pathogenesis of
inflammatory bowel disease (IBD)? The etiology of inflammatory bowel disease (ulcerative
colitis and Crohn's disease) is unknown. Familial aggregation suggests a genetic
predisposition but no definite genetic markers have been identified except for the uniform
presence of HLA-B27 in patients with IBD and ankylosing spondylitis. Current opinion favors
abnormal host immunoreactivity to luminal or mucosal antigens. The occurrence of IBD in
mice lacking selected cytokines (IL-2, IL-10) or disruption of T-cell receptors, favors this
concept. The beneficial effects of immunosuppressive therapy also point to the role of an
aberrant immune response in the pathogenesis of IBD.




                                                                     Diffusely Inflamed Mucosa
                                                                     Appendix
2. Chronic Ulcerative Colitis, Colectomy Specimen - Gross
Large bowel demonstrates a diffusely inflamed mucosal surface extending from the rectum to the
cecum. Note the appendix. The right colon appears to be less involved; however microscopically
the mucosa would demonstrate an active colitis. Is the picture consistent with Crohns' disease?
No, this is not consistent with Crohn's disease because of the uninterrupted involvement of the
mucosa. Although Crohn's disease may involve the colon, the affected areas are sharply
delimited with intervening unaffected areas.




                                                                       Areas of Ulceration
                                                                       Pseudopolyp
                                                                       Submucosa
                                                                       Muscularis Propria




3. Chronic Ulcerative Colitis, Pseudopolyp -Low Power
This image demonstrates a pseudopolyp (bulging mass of inflamed residual mucosa) surrounded by
an extensive area of ulceration. Inflammatory infiltrate is seen in the submucosa. What is the
definition of a polyp? The term polyp is used to describe any nodule or mass that projects
above the level of surrounding mucosa; it may be hyperplastic or neoplastic.
Why are the lesions in ulcerative colitis called pseudopolyps? These lesions are not really the
result of mucosal proliferation, but rather the results of mucosal ulceration. Focal areas that
are unaffected by ulceration appear to project above the denuded mucosa surrounding it.




                                                                       Normal Crypts
                                                                       Distorted Regenerating
                                                               Crypts
                                                                       Ulcer




4. Chronic Ulcerative Colitis - Low Power
Image demonstrates ulcer base covered by fibrin, capillaries, and inflammatory cells. Note the
distorted, regenerating crypts at the edge of the ulcer. Compare the shape of the regenerating crypts
with the normal crypts. How is the extent of these lesions different from that seen in Crohn's
disease? Crohn's disease causes transmural inflammation that ultimately results in fibrosis
and thickening of the wall.




                                                                      Lamina Propria
                                                                      Crypt Abscess




5. Chronic Ulcerative Colitis - High Power
The lamina propria demonstrates an increase in acute and chronic inflammatory cells. A mucosal
crypt is distended by a collection of neutrophils, known as a crypt abscess. Because ulcerative
colitis is a diffuse mucosal disease process, crypt abscesses are common. Crypt abscesses are also
seen in Crohn's disease but are not as common. What histologic feature is seen in Crohn's disease
that is not seen in ulcerative colitis? Granulomas. What are the complications of ulcerative
colitis? The most serious complication is development of carcinoma; with disease affecting the
entire colon, the risk is 10% at 20 years. Other life-threatening complications can include
severe diarrhea and electrolyte disturbances, severe colonic dilatation (toxic megacolon) with
potential for perforation and peritonitis, and massive hemorrhage.




                                                                      Normal Colon
                                                                      Diseased Segments
                                                                      Normal Small Bowel




6. Crohn's Disease, Regional Enteritis - Gross
The specimen is a section of normal ileum, thickened ileum, and right colon. The intestinal wall is
thick, the result of edema, inflammation, fibrosis, and hypertrophy of the muscularis propria. Linear
ulcers are typically present in the diseased segment of bowel. Unlike ulcerative colitis, the diseased
areas are sharply demarcated from adjacent, uninvolved bowel. Note the normal area of colonic
mucosa on the left and normal small bowel on the right. Are any other organs affected in
Crohn's disease? Ulcerative colitis? Both Crohn's disease and ulcerative colitis are systemic
diseases, associated with varied extraintestinal manifestations of immunologic origin. These
include polyarthritis, sacroiliitis, uveitis, sclerosing cholangitis, and erythema nodosum.




                                                                     Gut Lumen
                                                                     Ulceration
                                                                     Submucosa
                                                                     Outer Muscle Layer
                                                                     Lymphoid Aggregate




7. Crohn's Disease - Low Power
The low power view shows focal areas of ulceration, submucosal edema, and transmural
inflammation with lymphoid nodules. Fissures are tracks of necrosis and inflammation that start in
the mucosa and penetrate deeply into the wall. When the entire bowel wall is traversed, a sinus is
formed that may open to the outside skin or adhere to adjacent viscus. What is the risk of colon
carcinoma in Crohn's disease? While the risk is increased, it is less so than with ulcerative
colitis.




                                                                    Distorted Crypts
                                                                    Lymphoid Aggregate




8. Crohn's Disease - Medium Power
Under this magnification, crypt distortion, marked submucosal edema, fibrosis, and lymphoid
aggregates can be seen. In addition, these patients may have non-caseating granulomas in the bowel
wall. They are not seen in every case. What are the complications of Crohn's disease? The
fissures in the mucosa can extend through the wall and form sinus tracts resulting in fistula
formation to other loops of bowel, urinary bladder or vagina; there may be localized
peritonitis and abdominal abscesses; fibrosis of the gut wall may lead to strictures and
obstruction.




                                                                      Mucosa
                                                                      Fissures




9. Crohn's Disease - Low Power
In this view, the marked thickening of the bowel wall can be appreciated. The thickening results
from edema, inflammation, and fibrosis. In addition, two fissures penetrating the wall can also be
seen. These tracks of necrosis and acute inflammation start in the mucosa. Here on cross section, the
mucosal connection of the fissures cannot be seen.




                                                                      Non-caseating Granulomas
                                                                      Mucosa




10. Non-caseating Granulomas - High Power
A non-caseating granuloma is present in the lamina propria of an uninvolved region of colonic
mucosa. Non-caseating granulomas are present in about half of the cases in all tissue layers, both
within areas of active disease and in uninvolved regions of the bowel. Granulomas have been
documented throughout the alimentary tract, from mouth to rectum.

				
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Description: Hematology Case Reactive Leukocytosis effacement