Mod9Week1-PA 3306-Aug 21, 23, 24

M9W1- PA 3306 August 21, 23, 24- Monica Walsh (mwalsh@cmcc.
ca)
Lecture Hour 1: Hematologic Pathology: General

Symptomatology and Bone Marrow Pathology
Note: Not tested on macroscopic and microscopic images; however, we may get X-rays
- Examples of symptoms that can be signs of chronic/ serious illness- long

standing fever, swollen lymph nodes and/ or glands, change in color (jaundice),
vomiting, blood in stool (intestinal bleed), bruise easy (capillary problem, which
can be primary or secondary), chronic fatigue (pathological tirednessà lack of
oxygen carrying capacity aka anemia),
Hematological Symptoms
§ Tiredness, dyspnoea - Reduced oxygen-carrying capacity of blood due to
anaemia
§ Mucosal pallor – Anemiaà asses in mucous membranes like the nose and
mouth
§ Glossitis (sore mouth, smooth tongue, inflammation of tongue) - Mucosal effects
of haematinic deficiency
§ Spoon-shaped nails: Koilonychia – indicative of iron deficiency
M9W1- PA 3306 August 21, 23, 24- Monica Walsh (mwalsh@cmcc.ca)
§ Jaundice - Bilirubin accumulation from haemolysis

§ Abnormal tendency to infections (“weak immune system” or reduced
immunity) – Neutropenia- prone to bacterial infections vs lymphopenia- prone to
viral infection, but very often it is both
§ e.g., in leukaemia or aplastic anaemia or following chemotherapy
§ Hypogammaglobulinemia – low level of gamma globulins- myeloma and
chronic lymphocytic leukemia
§ Lymphopenia - following allogeneic stem cell transplantation
§ Can be due to Immunosuppression due to transplant surgery
§ Splenomegaly
§ Hematological causes: expansion of hemopoiesis in myeloproliferative
disorders, red cell pooling and destruction in hemolytic anemias, infiltration
in leukemias and lymphomas
§ Non-haematological causes: usually due to portal hypertension, storage
disorders, infection
§ Mononucleosisà causes increased size in spleen
§ Note: instruct patient to not do any physical activity or this
can lead to spleen rupture
§ Lymphadenopathy:
§ Non-neoplastic causes, e.g., infectious mononucleosis
§ Infiltration with leukemia or lymphoma
§ Metastatic causes- cancer can spread to lymph nodes (centinal node)
§ Bone pain and fractures - Osteoclast activation in myeloma
§ Purpura, bruising, mucosal or traumatic bleeding -Thrombocytopenia
or platelet dysfunction, hemophilia, Von Willebrand disease
§ Excessive bleeding from a small cut
§ Bruising, muscle and joint bleeding, and traumatic bleeding -
Coagulation factor deficiency
What happens when this system goes out of balance?

- E.g., Overgrowth of lymphocytesà Leukemia
- E.g., Maturation and division of erythrocytic branchà patient becomes anemic
Bone Marrow Pathology

§ Alteration in BM cellularity
o Hyperplastic- lots of cells
o Hypoplastic- reduced number of cells
o Aplastic- empty bone marrow can be caused by chemotherapy, radiation,
other exposure (occupational)
§ Non-neoplastic
o Benign changes: toxicity – arsenic, lead, occupational
§ Lead- common in older houses (pipes, paint), increase octane
number in gas
§ Arsenic- poison
o Infectious/Inflammatory: candida, CMV, Cryptococcus, parvovirus B19
o Systemic disorders: alcohol abuse, bone marrow transplantation,
metabolic defects
§ Certain diets- can have various limitations in nutrients that need to
be supplemented or will lead to deficiency
§ Neoplastic
o Primary bone marrow malignancy
o Secondary (metastatic) bone marrow malignancy
§ Come via blood stream- destroy bone and can suppress things
happening in the bone marrow
Normal Bone Marrow
§ This is the appearance of normal bone marrow at medium 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.
Normal Bone Marrow
§ This is the appearance of normal bone marrow at high magnification. Note the
presence of megakaryocytes (1), erythroid islands (2), and granulocytic
precursors (3). This marrow is taken from the posterior iliac crest in a middle
aged person, so it is about 50% cellular, with steatocytes (4) admixed with the
marrow elements
Normal Bone Marrow
§ This is the appearance of normal bone marrow smear at high magnification. Note
the presence of erythroid precursors (1) and granulocytic precursors (2).
Aplastic Bone Marrow
- Bone marrow became empty- BM exposed to something like radiation/ chemo

- This patient would be defenselessà will not be able to fight off any infections
Clinical Case
§ A 70-year-old man was being evaluated for back pain
§ Two years earlier, he was found to have a monoclonal IgA protein (0.3 g/dL)
§ We don’t know why he was investigated for this immunoglobulin
§ Past history includes the long-standing history of ulcerative colitis with a partial
colectomy and ileostomy 31 years earlier
§ A magnetic resonance imaging scan showed numerous destructive vertebral

lesions
§ This explains his back pain
§ Note: adjusting him could cause spinal cord compression
§ His complete blood count was normal except for a minimal normocytic
normochromic anemia (hemoglobin 12.9 g/dL)
§ Note: normal values will be provided on exams
Bone Marrow Biopsy
Metastatic adenocarcinoma involving the bone marrow
Figure Legend:
A 70-year-old man was being evaluated for back pain. Two years earlier he was found
to have a monoclonal IgA λ protein (0.3 g/dL). Past history included long-standing
history of ulcerative colitis with a partial colectomy and ileostomy 31 years earlier. A
magnetic resonance imaging scan showed numerous destructive vertebral lesions. His
complete blood count was normal except for a minimal normocytic normochromic
anemia (hemoglobin 12.9 g/dL). Peripheral blood smear showed moderate
anisopoikilocytosis without teardrop forms. Bone marrow biopsy and touch prep smears
showed cohesive clusters of tumor cells (panel A). The marrow was replaced by mucin
with occasional cellular groupings of colonic type glandular structures (panel B arrow).
Immunostains were positive for cytokeratins as well as Cdx2, suggesting
gastrointestinal origin, and a diagnosis of metastatic adenocarcinoma was made.
Sigmoidoscopy revealed a large friable mass in the distal rectum and the biopsy
showed invasive adenocarcinoma (cancerous tumor coming from GI glandsà this is a
rare complication from colitis)
The clinical suspicion in this case was of myeloma, based on the radiologic findings and
the previously known monoclonal protein. Colorectal cancer was unexpected, especially
with the previous prophylactic partial colectomy. The bone marrow examination and
endoscopy confirmed the metastatic adenocarcinoma.
Lecture Hour 2: Hematology II- Red Blood Cell Pathology

§ Pathophysiology of Anemia
- Anemiaà name for various diseases with a reduction of RBC and/ or hemoglobin
aka reduction of O2 carrying capacity called hypoxemia
- Tissue hypoxiaà normal blood flow with reduced O2 carrying capacity or blood
has normal O2 capacity, but perfusion is limited
o Two crucial organs: brain (can only live for 5 mins without O2) and
myocardium
o Ischemic organs- claudication, weakness, pallor
o Our organs can adapt to lower oxygen levels (to a certain extent) for
example, changes in the liver- fatty changes, fatty replacement of
functional tissue in heart and kidney; respiratory system will increase
respiratory rate, depth, exertional dyspnea; in CNS we will see dizziness,
fainting, lethargy
- Causes compensatory mechanisms:
o Cardiovascular- Increased heart rate, increased stroke volume, capillary
dilations
o Increased oxygen demands on the heart- increased EPO, which
stimulates bone marrow to produce erythrocytes
§ E.x., athletes use this system to increase o2 carrying capacity to

increase their cardiovascular ability aka blood doping
o Renal- retention of salt and water, increased ECF, and increased renin-
aldosterone response
o Ultimately, cardiovascular compensation to low oxygen supply can cause
cardiac murmurs or high-output cardiac failure.
Erythrocytes Morphology
§ Anisocytosis - abnormal RBC size
§ Poikilocytosis – abnormal shape
§ Elliptocytes – hereditary elliptocytosis
§ Spherocytes – decreased RBC membrane
§ Target Cells- increased RBC membrane: hemoglobinopathies, thalassemia, liver
diseases
§ Acanthocytes - irregular spicules on the surface: uremia, often an artifact
§ Schistocytes – RBC fragments (helmet cells): microangiopathic hemolytic
anemias or traumatic hemolysis
§ Bite cells – “bites” from RBC by splenic macrophages, G6PD deficiency
§ Teardrop cells – dacryocytes – thalassemia, myelofibrosis
§ Sickle cells – drepanocytes – sickle cells anemia
§ Rouleaux – “stack of coins” RBC lining up in a row- multiple myeloma
***Know these terms
Erythrocytic Inclusions
§ Basophilic stippling – cytoplasmic remnants of RNA: reticulocytosis or lead
poisoning
§ Howel-Jolly bodies: remnants of nuclear chromatin: severe anemias, asplenic
patients
o Previously removed spleen
§ Pappenheimer bodies: iron deposition, found in patients after splenectomy
§ Ring sideroblasts: atypical, abnormal nucleated erythroblasts (precursors to
mature red blood cells) with granules of iron accumulated in perinuclear
mitochondria
§ Heinz bodies: denatured Hb: G6PD deficiency
Anemia: General Considerations

§ Anemia is the reduction below normal limits of the total circulation erythrocyte
mass:
o Males Hb<13.5 g/dL, Hct <41%
o Female< 12 g/dL, Hct<36%
§ Females with constant low numbers
§ Laboratory tests used in evaluation of anemia include MCV (mean cell volume),
MCH (mean cell hemoglobin), MCHC (mean cell hemoglobin concentration),
RDW, and reticulocyte count
§ Diagnosis based on reduced levels of: hemoglobin, hematocrit, and RBC count
RBC measures
§ MCV – mean cell volume - average volume of a red blood cell
§ MCH – mean cell hemoglobin - average content (mass) of Hb per RBC
§ MCHC – mean cell hemoglobin concentration - average concentration of Hb in a
given volume of packed RBCs
§ RDW – red cell distribution width – coefficient of variation of RBC volume -
measure of anisocytosis
§ Reticulocytes – anucleate macrocytic precursors of RBCs:
o Maturation into RBC 1 day
o Polychromasia – bluish color due to free ribosomal RNA
o Nucleated Rt – pathological sign
When we have a patient with low hemoglobin, we look into MCV and we have 3
scenarios:
1. Low MCVà T.A.I.L.S
2. High MCVà Megaloblastic or Non-megaloblastic
3. Normal MCV
• High Reticulocyte
o Hemolytic anemia
§ Inherited
§ Acquired
o Bleeding anemia
• Low reticulocyte
o Pancytopeniaà decreased amount/ production of all red and white blood
cells and platelets
o Non-pancytopeniaà all levels of reticulocytes and erythrocytes are low
Anemia
§ Pathophysiology:
o Decreased oxygen transport by blood
o Develops tissue hypoxia
o Note! Patients with anemia have a normal SaO2 and PaO2
§ Clinical symptoms:
o Cardiac symptoms: palpitations, angina (tightness of the chest)
o Vascular symptoms: claudication (in the brain= syncope), pain, skin and
mucous membrane, and sclera's pallor
o Neurological symptoms: headaches, dizziness, syncope
o General symptoms: weakness, lack of energy, fatigue
o Constellation of various non-specific symptoms
§ Pale and tired patient?à send for blood workà if blood work is
normal then we will look somewhere else
Anemia Classification
§ Color:
o Normochromic: normal color, central pallor of about a third of the
diameter of RBC
o Hypochromic – decreased color – increased central pallor of RBC
o Hyperchromic (spherocytosis) – increased color, loss of central pallor of
RBC
Macrocytic normochromicà RBC is bigger than normal

Microcytic, hypochromicà small and no color
Anemia: Classification
§ RBC size: MCV
§ Microcytic – MCV< 80 fl/cell
o Iron Deficiency
o Thalassemia
o Anemia of Chronic Ds
o Sideroblastic anemia
§ Normocytic – 80-100 fl/cell
o Reticulocyte count
§ Low:
• Marrow Failure
• Aplastic Anemia
• Myelofibrosis
• Leukemia/Metastasis
• Renal Failure
• Anemia of Chronic Disease
§ High:
• Sickle Cell Anemia
• G6PD Deficiency
• Hereditary Spherocytosis
• Autoimmune Hemolytic Anemia
• Paroxysmal Hemolytic Anemia
§ Anemia: Classification
o RBC size: MCV
o Macrocytic - >100 fl/cell
§ Megaloblastic Anemia
§ B12 Deficiency
§ Folate Deficiencyà if the patient wants to become pregnant this is

a big deal as this will cause neural tube defects
§ Alcoholic Liver Disease
§ Anemia: Pathogenesis
o Blood loss: acute vs. chronic
§ Patients can have anemia from blood loss- even if it is a small
constant loss of blood, it can still cause problems, especially if the
patient is unaware
o Hemolytic anemia:
§ Hereditary spherocytosis
§ G6PD Deficiency
§ Sickle Cell disease
§ Hemoglobin C disease
§ Thalassemia
§ Paroxysmal nocturnal hemoglobinuria
§ Immunohemolytic anemia
• Autoimmune hemolytic anemia (AIHA)
• Cold AIHA
• Incompatible blood transfusion
• Hemolytic Disease of newborn
§ Anemia: Pathophysiology
o Anemia of diminished erythropoiesis:
§ Megaloblastic anemia: B12 and folate
§ Iron deficiency anemia
§ Anemia of chronic disease
• Renal condition, GI issues, malabsorption
§ Aplastic anemia
§ Myelophthisic anemia- not a chiropractic issue
§ Sideroblastic anemia- not a chiropractic issue
Absorption of vitamin B12, Folic Acid, and Iron
Absorption of vitamin B12 requires initial complexing with intrinsic factor (IF), which is
produced by the parietal cells of the gastric mucosa. Absorption then occurs in the
terminal ileum, where there are receptors for the IF–B12 complex. Dietary folic acid is
conjugated by conjugase enzymes to polyglutamate. Absorption occurs in the jejunum
following deconjugation in the intestinal lumen. Reduction and methylation result in the
generation of methyl tetrahydrofolate, which is then transported by folate-binding
protein. Dietary ferric iron (Fe3+) is reduced to ferrous iron (Fe2+) in the stomach and
absorbed principally in the duodenum. Iron is transported by transferrin in the
circulation.
Iron Deficiency Anemia

§ Iron deficiency anemia develops when body stores of iron drop too low to support
normal red blood cell (RBC) production
§ Causes:
o Inadequate dietary iron
o Iron absorption
o Bleeding
The total body iron in a 70-kg man is about 4 g (contained in the plasma). This is
maintained by a balance between absorption and body losses. Although the body only
absorbs 1 mg daily to maintain equilibrium, the internal requirement for iron is greater
(20-25 mg). An erythrocyte has a lifespan of 120 days so that 0.8% of red blood cells
are destroyed and replaced each day. A man with 5 L of blood volume has 2.5 g of iron
incorporated into the hemoglobin, with a daily turnover of 20 mg for hemoglobin
synthesis and degradation and another 5 mg for other requirements. Most of this iron
passes through the plasma for reutilization. Iron in excess of these requirements is
deposited in body stores (1000 mg) as ferritin or hemosiderin. There is also a significant
amount of iron in myoglobin and respiratory enzymes.
Signs and symptoms

§ Patients with iron deficiency anemia may report the following:
§ Fatigue and diminished capability to perform hard labor

§ Leg cramps on climbing stairs
§ Craving ice (in some cases, cold celery or other cold vegetables) to suck or chew
§ Poor scholastic performance
§ Cold intolerance
§ Reduced resistance to infection
§ Altered behavior (attention deficit disorder)
§ Dysphagia with solid foods (from esophageal webbing)
§ Worsened symptoms of comorbid cardiac or pulmonary disease
Physical examination:
§ Impaired growth in infants
§ Pallor of the mucous membranes (a nonspecific finding)
§ Spoon-shaped nails (koilonychia)
§ A glossy tongue, with atrophy of the lingual papillae
§ Fissures at the corners of the mouth (angular stomatitis)
§ Splenomegaly (in severe, persistent, untreated cases)
§ Pseudotumor cerebri (a rare finding in severe cases)
o Presents as a space occupying lesion in the brain, but there is no lesion
Anemia of chronic disease

§ Anemia of chronic disease (AOCD) is a hypo-proliferative anemia that develops
in response to systemic illness or inflammation
§ Iron entrapment in bone marrow macrophages with subsequent decrease in
utilization of endogenous iron stores
§ Chronic inflammatory conditions increase IL-1 production, which causes
increased lactoferrin, which, in turn, traps iron in bone marrow macrophages
Aplastic anemia
§ Aplastic anemia is a syndrome of
bone marrow failure characterized
by peripheral pancytopenia and
marrow hypoplasia
o Reduced number of cells in
blood stream and bone
marrow
§ Complications - infections and
bleeding
§ Pic: Oral leukoplakia in
dyskeratosis congenita
Myelophthisic anemia
§ Myelophthisis is a form of bone marrow failure that results from the destruction of
bone marrow precursor cells and their stroma, which nurture these cells to
maturation and differentiation
§ Form of fibrosis, secondary to injury by nonhematopoietic cells or pathogens

§ Bone marrow becomes infiltrated by collagen, reticulin, and other forms of
fibrosis, which replace the normal, hematopoietic cells
o Basically, there is less space for bone marrow to produce normal blood
cells
Sideroblastic anemia
§ Sideroblastic anemia is primarily a laboratory diagnosis, made on the basis of
bone-marrow examination with Prussian blue stain (specific for iron)
§ Body has adequate iron stores, but unable to incorporate the iron into Hb
§ May be pyridoxine (vit. B6) responsive and B6 refractory forms
§ Form of myelodysplastic syndrome
Hemoglobin Review
§ Hemoglobin are globular proteins that ferry oxygen (O2) molecules and carbon
dioxide (CO2) molecules throughout the body
§ Each hemoglobin protein structure consists of four polypeptide subunits, which
are held together by ionic bonds, hydrogen bonds, hydrophobic interactions, and
van der Waals forces, as well as four heme pigments, one in each of the subunits
§ These heme groups contain positively-charged iron (Fe2+) molecules which can
reversibly bind to oxygen molecules and transport them to various areas of the
body
§ As the heme groups bind or release their oxygen loads, the overall hemoglobin
undergoes conformational changes which alters their affinity for oxygen
§ Hemoglobin tetramers are comprised of the

four subunits, two α-globin chains and two
β-globin chains all of which take the form of
alpha helices
§ Hemoglobin types:
o HbA - Normal adult hemoglobin (Hb
A) consists of globin containing two
pairs of polypeptide chains, 2 alpha
(α) and 2 beta (β): 2α2β
o HbA2 – minor fraction, contains 2 α-
and 2 delta- (δ-) chains: 2α2δ
o HbF – fetal 2 α-chains, 2 gamma- (γ-)
chains: 2α2γ
o Hb Barts - 4γ chains
o HbH – 4β chains
o HbS - sickle hemoglobin
Thalassemia Syndromes
§ Thalassemic syndromes are genetically determined disorders of hemoglobin
synthesis with decreased production of either alpha or beta polypeptide

chains of hemoglobin molecule
§ Thalassemias are quantitative, not qualitative, abnormalities of Hb
o Aka we have normal amounts of RBCs, but the quality of the RBCs is
different because if the Hemoglobin
§ Alpha-Thalassemia: decreased alpha-globin chains with relative excess of beta-
chains
§ Beta-Thalassemia: decreased beta-globin chains with relative excess of alpha-
chains
Α-Thalassemia
§ Gene deletion affects Hb chains synthesis
§ Normal 4α (αα/αα)
§ Silent carrier: one deletion
o 3α (-α/αα) 75% of α-chains
o Asymptomatic and lab test normal
§ Α-Thal trait: two deletions
o Genotype cis (--/αα) type is seen in Asians
o Genotype trans(-α/-α) type is seen in African Americans
o No development of H disease or hydrops
§ Hb H disease: three deletion
o Number of α genes: 1 (--/-α) – 25% of α-chains
o Increased Hb H β4 – Heinz bodies
§ Hydrops fetalis: four deletions and lethal in utero
o Number of α genes 0 (--/--), 0% α chains
o Increased Barts hemoglobin – (ɣ4)
β-Thalassemia
§ Normal Hb – 2 β-globin chains genes
§ Β-globin expressed postnatally only
§ Postnatal Disease, not Prenatal
§ β-Thal minor
o Asymptomatic
o Increased Hb A2 (8%), and increased Hb F (5%)
§ Β-Thal Intermedia
o Severe anemia, no transfusions needed
§ Β-Thal major (Cooley anemia)
o Patient normal at birth
o Symptoms develop at 6 months as HbF decline
o Severe hemolytic anemia
o Intramedullary destruction – ineffective erythropoiesis
o Hemolysis, jaundice, increased billirubin gallstone formation
o Lifelong transfusions
o Microcytic, hypochromic anemia
Macrocytic Anemia
§ Megaloblastic anemia –heterogeneous group of disorders that share common
morphologic characteristics
o Megaloblast= enlarged RBC
§ Morphological hallmark of megaloblastosis is a megaloblast
§ Megaloblasts are large cells with an increased nuclear/cytoplasmic ratio in which
nuclear maturation is delayed, while cytoplasmic maturation is more advanced
§ Peripheral smears: macrocytic RBCs and occasional megaloblasts are present
§ Megaloblasts are usually abundant in bone marrow aspirates
§ Megaloblastic changes are not limited to RBCs since hypersegmented
neutrophils can be seen on peripheral smears, and pancytopenia occurs in
megaloblastic anemias
§ Etiology: impaired DNA synthesis
§ The most common causes of megaloblastosis are cobalamin (vitamin B-12)
and folate deficiency
§ Vitamin B-12 and folate deficiencies can cause:
o memory loss
o depression,
o personality changes
o psychosis, as well as peripheral neuropathy
Megaloblastic Anemia
§ Vitamin B-12 deficiency can cause subacute combined dorsal and lateral
spinal column degeneration: patients develop ataxia, become weak, and lose
proprioceptive and vibratory senses
§ If not treated, mental and neurological changes can become permanent
§ Megaloblastosis in HIV infection and myelodysplastic disorders is due to a direct
effect on DNA synthesis in hematopoietic and other cells
Cobalamin deficiency
§ The daily requirement cobalamin is about 5-7 µg/day
§ Large amounts of cobalamin are stored in liver and other sites
§ Cobalamin deficiency only develops about 3-4 years after the cessation of
cobalamin uptake
§ Dietary cobalamin deficiency
o strict vegetarians who avoid meat, eggs, and dairy products
o Atrophic gastritis and achlorhydria (reduced HCl production in stomach)
commonly occur in elderly persons
o Impaired release of protein-bound cobalamins interferes with cobalamin
uptake
o Common problem in elderly persons
§ Failure in intrinsic factor (IF) secretion in pernicious anemia due to
autoimmune destruction of gastric parietal cells
o Pernicious anemia is the best-known cause for cobalamin deficiency.
o Cobalamin is not absorbed in the absence of IF
o Pernicious anemia is diagnosed in about 1% of people older than 60
years, and the incidence is slightly higher in women than in men

o H2 antagonists can inhibit IF secretion
§ Pancreatic insufficiency: pancreatic enzymes are not available to facilitate the
release of cobalamins from R-proteins and thus cobalamins are not absorbed. In
Zollinger-Ellison syndrome, the secretion of large amounts of acid inactivates
pancreatic enzymes
§ Disorders of the terminal ileum: terminal ileum is the site of uptake of
cobalamin-IF complexes
o tropical sprue, inflammatory bowel disease, lymphoma, and ileal resection
o Tropical sprue is more severe than nontropical sprue (celiac disease) and
can be associated with both cobalamin and folate deficiencies
o It takes several years for cobalamin deficiency to develop after the onset
of these disorders because of the time required to deplete cobalamin
reserves
§ Blind loop syndrome can result in cobalamin deficiency. Bacterial colonization
can occur in intestines deformed from strictures, surgical blind loops,
scleroderma, inflammatory bowel disease, or amyloidosis. Bacteria then compete
with the host for cobalamin
§ Fish tapeworm Diphyllobothrium latum can compete with the host for ingested
cobalamin. This organism is most often found in Canada, Alaska, and the Baltic
Sea
§ Nitrous oxide exposure can cause megaloblastosis by oxidative inactivation of
cobalamin. Prolonged exposure to nitrous oxide can lead to severe mental and
neurological disorders
§ Medications that can cause cobalamin deficiency includes: purine analogs (6-
mercaptopurine, 6-thioguanine, acyclovir), pyrimidine analogues (5-fluorouracil,
5-azacytidine, zidovudine), ribonucleotide reductase inhibitors (hydroxyurea,
cytarabine arabinoside), and drugs that affect cobalamin metabolism (p-
aminosalicylic acid, phenformin, metformine
Folate deficiency
§ The daily requirement for adults is about 0.4 mg/d. Storage is limited, and
folate deficiency develops about 3-4 weeks after the cessation of folate intake
§ Dietary folate deficiency
§ In the United States, most people obtain sufficient folate from fortified foods
§ Alternate diets may contain little folate. The preparation of foods is a major cause
for folate deficiency, especially in elderly persons. Folates are very thermolabile.
Therefore, excessive heating can lead to inactivation, especially when foods are
diluted in water
§ Failure to increased folate supplementation in response to increased demand
can result in deficiency. There is an increased need for folate in the face of
hemolysis, pregnancy, lactation, rapid growth, hyperalimentation, renal dialysis,
psoriasis, and exfoliative dermatitis
§ Intestinal disorders that impede folate absorption include tropical sprue,
nontropical sprue (celiac disease or gluten sensitivity), amyloidosis, and
inflammatory bowel disease
§ Alcoholism decreases the bioavailability of folate and folate-dependent

biochemical reactions can be impaired
§ Medications that can cause folate deficiency includes phenytoin, metformin,
phenobarbital, dihydrofolate reductase inhibitors (trimethoprim, pyrimethamine),
methotrexate and other antifolates, sulfonamides (competitive inhibitors of 4-
aminobenzoic acid), and valproic acid
Subacute Combined Dorsal And Lateral Spinal Column Degeneration
§ SUBACUTE COMBINED DEGENERATION OF THE CORD is a curious term for

a curious lesion: destruction of the myelin and eventually axons of the posterior
columns of the spinal cord caused by vitamin B12 deficiency
§ Later, the brain and descending pathways are affected
§ This is a tragic disease to miss since it's easy to administer the vitamin. The
neurologic damage can be permanent. There's sensory loss; ask also about
burning pain in the feet
§ Now that food is supplemented with folic acid, there's more of this clinically
§ Like it or not, a vegetarian (not even necessarily a vegan) who does not know
EXACTLY what he/she is doing is likely to end up B12 deficient.
§ By now, the myelopathy in these people is so well known that it's come to be
called "vegetarian's myelopathy”
§ Remember that people who don't take good care of themselves and eventually
can't walk may have subacute combined degeneration of the cord
Polycythemia vera
§ Polycythemia vera (PV) - stem cell disorder characterized as a panhyperplastic,
malignant, and neoplastic marrow disorder
§ Most prominent feature of this disease is an elevated absolute red blood cell
mass because of uncontrolled red blood cell production
§ Increased white blood cell (myeloid)
§ Increased platelet (megakaryocytic) production
§ Due to an abnormal clone of the hematopoietic stem cells with increased
sensitivity to the different growth factors for maturation
§ Lab findings:
o Increased RBC mass
o Absolute leukocytosis
o Thrombocytosis
o Decreased erythropoietin
o Splenomegaly
o Hyper-cellular marrow
o Thrombotic events – increased blood viscosity
o Gout – increased blood cells destruction
§ Secondary polycythemia:
o Increased RBC mass due to compromised ability of blood to supply
oxygen to tissues
§ Causes:
o COPD
o Cyanotic congenital heart disease
o Erythropoietin level is high
o Renal Cell carcinoma - increased production of erythropoietin
§ Relative polycythemia
o Increased RBC count secondary to decreased plasma volume –
dehydration
o RBC mass, erythropoietin level and blood oxygen content normal,
hematocrit increased
Polycythemia vera
Bone marrow film at 100X magnification demonstrating hypercellularity and

increased number of megakaryocytes
Blood film at 400X magnification demonstrating polyglobulia and thrombocytosis

Bone marrow film at 400X magnification demonstrating dominance of erythropoiesis

This blood film at 10,000X magnification shows a giant platelet and an eosinophil.
Erythrocytes show signs of hypochromia because of repeated phlebotomie
Lecture Hour 3&4: Hematologic Pathology- White Blood Cells

Disorders
Reactive Changes in WBC
• Leukocytosis (increased production of leukocytes in response to infection/ stress
on the body)-
o This is a normal reaction to pathology in the body; however, we do react
differently to various conditions/ stressors
o Leukocytes are the fastest reacting blood cells
• Increased neutrophils (neutrophilia) can be due to…
o Increased bone marrow production is seen with acute inflammation
associated with pyogenic bacterial infection or tissue necrosis
§ Tissue necrosis can be due to infection or loss of blood supply for
example, myocardial infarction with neutrophil infiltration of the
necrotic area of myocardium
o Increased release from bone marrow storage pool may be caused by
corticosteroids, stress, or endotoxin
o Increased bands (“left shift”) in peripheral blood is characteristic
§ Increased bands- reaction of the body when inflammatory response

is mounting, and we start releasing newer/ younger neutrophils into
the blood stream indicating significant inflammatory reaction
o Reactive changes include Döhle bodies (aggregates of rough endoplasmic
reticulum [RER] in leukocytes), toxic granulations (prominent granules),
and cytoplasmic vacuoles of neutrophils
• Increased eosinophils (eosinophilia) are seen with:
o Allergies and asthma (type I hypersensitivity reaction)
o Parasites
o Drugs (especially in hospitals)
o Certain skin diseases and cancers (adenocarcinomas, Hodgkin disease)
• Increased monocytes (monocytosis) are seen with:
o Certain chronic diseases, such as some collagen vascular diseases and
inflammatory bowel disease (IBD)
o Certain infections, especially TB
• Increased lymphocytes (lymphocytosis) are seen with:
o Acute (viral) diseases- almost all acute viral diseases
o Chronic inflammatory processes
o Infectious mononucleosis (IM) is an example of a viral disease which is
causing lymphocytosis
o Most common cause is Epstein-Barr virus (EBV) (a herpesvirus) but less
commonly due to other viruses (cytomegalovirus [CMV])
o Note: with lymphocytosis we have an increase in lymphocytes, but some
infections can also cause a change in morphology of lymphocytes
Reactive Changes in WBC: Mononucleosis

• EBV invades B-lymphocytes via CD21 (CR2) receptors
• Cytotoxic (CDB) T-lymphocytes respond against invaded B cells and form
atypical lymphocytes (Downey cells), which are enlarged lymphocytes that have
abundant cytoplasm that is condensed peripherally ("ballerina skirt” appearance);
they are similar in appearance to monocytes - name "mononucleosis"
Mononucleosis
Infectious mononucleosis is a common

viral disease often known simply as mono
or the kissing disease that usually occurs
in individuals between the ages of 15 and
35. The infection generally spreads via
saliva exchange and is thought to be
caused by the Epstein-Barr virus, a
member of the herpesvirus family.
Symptoms of mononucleosis are varied,
but frequently include fever, fatigue, and
sore throat. Swelling of the lymph nodes
is also common and is the origin of
another alternate name for the disease, glandular fever. Less often, a rash, eyelid
swelling, or muscle soreness, as well as the more serious and even less common onset
of pneumonia, meningitis, hepatitis, or peripheral neuritis, may occur. In some cases,
the spleen may become enlarged, and rupture of the organ is the primary cause of the
rare instances of death associated with mononucleosis.
Reactive Changes in WBC: Mononucleosis

• Antibody production: heterophil antibodies (antibodies against other species
such as red cells of sheep and horses) are the basis of the Paul-Bunnell reaction
– monospot test (may be negative first week, so need to repeat test)
• Clinical infectious monocytosis
• Age groups include adolescents and young adults (“kissing disease”)
• Symptoms (classic triad):
o Fever and sore throat (see gray-white membrane on tonsils)- these are
not identifying features of mono
o lymphadenitis (posterior auricular nodes)- specific for mono, but not on
their own
o fourth sign is hepatosplenomegaly- specific for mono
• Mono is an acute, self-limited disease that usually resolves in 4-6 weeks
• Complications include hepatic dysfunction, splenic rupture, and rash if treated
with ampicillin
• Leukopenia – decreased neutrophils are seen with
o Decreased production: aplastic anemia, chemotherapy
o Increased destruction: infections, autoimmune disease (systemic lupus
erythematosus)
o Activation of neutrophil adhesion molecules on endothelium; endotoxins in
septic shock
• Decreased eosinophils are seen with:
o Increased cortisol (causes sequestering of eosinophils in lymph nodes)
§ Cushing syndrome (endogenous corticosteroids)
§ Exogenous corticosteroids
• Decreased lymphocytes are seen with
o Immunodeficiency syndromes: HIV, DiGeorge syndrome (T-cell
deficiency)
o Severe combined immunodeficiency (B- and T-cell deficiency)
o Immune destruction: systemic lupus erythematosus
o Corticosteroids (chronic intake)
o Radiation (lymphocytes are the most sensitive cells to radiation)
§ Atypical lymphocytes are found in the peripheral blood and T-cell
areas of lymph nodes (paracortex)
§ E.g., Patients from Chernobyl who received sub-lethal or lethal
doses of radiation had a very drastic decline off of lymphocytes,
which in turn is a negative prognostic factor for recovery of the
patient.
Lymphadenopathy
• Definition: Lymph node enlargement due to reactive conditions, or neoplasia
o Remember the first lymph node cancer cells invade is called the sentinel
node (aka this is the first node that collects lymph from the area)
o Acute nonspecific lymphadenitis
o Tender enlargement of lymph nodes
§ Focal involvement is seen with bacterial lymphadenitis
o Histology: may see neutrophils within the lymph node
o Note: cat-scratch fever (due to Afipia felis) causes stellate micro
abscesses
o Generalized involvement of lymph nodes is seen with viral infections (see
reactive T-cell immunoblasts in lymph nodes and peripheral blood)
§ Often generalized lymphadenopathy is usually due to generalized
viral infection OR conditions like hematological malignancy
• Chronic nonspecific lymphadenitis
o Nontender enlargement of lymph nodes
o Follicular hyperplasia involves B lymphocytes and may be seen with
rheumatoid arthritis, toxoplasmosis, and early HIV infections
o Paracortical lymphoid hyperplasia involves T cells and may be seen with
viruses, drugs (Dilantin), and systemic lupus erythematosus (SLE)
o Sinus histiocytosis involves macrophages and, in most cases, is
nonspecific
§ An example is lymph nodes draining cancers.
Lymphoid Neoplasms
Lymphadenopathy: caused by a neoplasm

• Neoplasia
• Nontender enlargement of lymph nodes
• Most common tumor to involve lymph nodes is metastatic cancer, initially seen
under lymph node capsule: breast, colon, melanoma
• Malignant lymphoma and infiltration by leukemia another cause for
lymphadenopathy (usually systemic lymphadenopathy)
Bone Marrow Neoplasms - Leukemia

• Leukemia is a neoplastic process which represents a monoclonal proliferation of
one transformed precursor cell
o Most common bone marrow neoplasm
• 80-85% of leukemias and lymphomas are B-cell in origin
• Microscopic classification:
o Lymphoid leukemia
o Myeloid leukemia
• Acute leukemia: composed of immature cells (blasts), sudden onset of a few
months from symptoms to Dx

• Chronic leukemia: composed of mature cells (myelocytes), Dx may be
incidental during lab investigation for other medical reasons
• Signs and Symptoms
o Lymphoma: painless mass
§ Tumor located in organs and tissues
o Advanced lymphoma: metastasis and surrounding organs compression
o Leukemias- Suppression of myelogenesis:
§ Decreased RBC: anemia, fatigue, pallor, dyspnea
§ Decreased mature leukocytes: leukopenia, infections, fever and
sepsis
§ Decreased platelets: thrombocytopenia, bleeding: nose, gums,
subcutaneous hemorrhage, internal organs
o Immune disorders: hypogammaglobulinemia, autoimmune complications
o Bone pain from expansion of marrow
o Lymphadenopathy and splenomegaly: due to infiltration by neoplastic cells
o Hypertrophy of gingivae: leukemic cells infiltration
o Meningisums and cranial nerves deficits due to infiltration of meninges
with leukemic cells
o Blood vessels occlusion: viscosity due to increased number of cells or
proteins: priapism
o CNS symptoms: headache, blurring of vision, strokes
Surface markers
Note: we will not be tested on specific surface markers, but this section is to help our
understanding of blood neoplasms
• B-cell markers: CD10, CD19, CD20
• T-cell markers: CD2, CD3, CD4, CD7, CD8
• Lymphoblasts: TdT (terminal deoxytransferase)
• Myeloid markers: CD13, CD14, CD15, CD64
• Stem cell marker: CD34
• CD - cluster of differentiation - molecule present on certain cells
• Cell surface glycoprotein and functions as a cell-cell adhesion factor
• Used to differentiate cells in diagnostic
Cluster Differentiation
Cytogenetic Analysis
• The malignant cells in many patients with leukemia, lymphoma, or another
malignant hematologic disease have acquired clonal chromosomal abnormalities
• Some specific cytogenetic abnormalities are closely, and sometimes uniquely,
associated with morphologically and clinically distinct subsets of leukemia or
lymphoma, as well as with their prognosis
• The detection of clonal cytogenetic abnormalities can be useful:
o To establish the specific diagnosis, such as the Philadelphia
chromosome in chronic myeloid leukemia
o To distinguish between benign reactive lymphoid or myeloid hyperplasia
and a monoclonal malignant proliferation
o To enhance our understanding of the pathogenesis of the disease and to
identify genes that are critical for the control of cell growth and
leukemogenesis
• The detection of clonal cytogenetic abnormalities can be useful:
o To help in the planning of treatment, since some chromosomal changes
predict for response (or nonresponse) to specific therapies, or to inform
the selection of a targeted therapy
o To estimate prognosis, since the appearance of new karyotypic
abnormalities or the existence of clonal heterogeneity/evolution often
signals a change in the pace of the disease, usually to a more aggressive
course
Chromosomal abnormalities
• Normal cells have 46 chromosomes (22 pairs of autosomes and 2 sex
chromosomes, X or Y)
• The male karyotype is 46,XY and the female is 46,XX
• Trisomy is the gain of an extra chromosome (e.g., trisomy of chromosome 8 is

denoted +8)
• Monosomy is a form of genetic loss (also seen in deletions) in which an entire
chromosome is
• Chromosomal translocation (t):
o the process by which a break occurs in the DNA in at least two different
chromosomes and then genetic material is exchanged between
chromosomes
o This is commonly the driving force behind malignancy
o Reciprocal or balanced translocation refers to an exchange in which
there is no obvious overall loss of chromosomal material
o An example of a reciprocal translocation between chromosome 9
and 22 is the rearrangement giving rise to the Philadelphia
chromosome, t(9;22), seen in chronic myeloid leukemia (CML)
o Note: t= translocation; 9= chromosome 9; 22= chromosome 22 giving us
t(9:22)
o Exam: Philadelphia chromosome gives us chronic myeloid leukemia- this
is the only translocation we need to know
• Chromosomal deletion (del) results in the loss of chromosomal material.
• A loss of genetic material also occurs as a result of loss of an entire chromosome
(monosomy)
• Chromosomal inversion (inv) requires two breaks in the same chromosome
with rotation of the intervening material
• Chromosomal bands are alternating light and dark segments due to various
staining procedures. The banding pattern of each chromosome is unique.
• Recurring abnormality is a numerical or structural abnormality noted in multiple
patients who have a similar disease
• Such abnormalities are characteristic or diagnostic of distinct subtypes of
leukemia or lymphoma that have unique morphological and/or
immunophenotypic features
• Many recurring structural abnormalities have prognostic significance
Chronic Myeloid Leukemia (CML) Karyotype
Myeloid cells of CML are also characterized by the Philadelphia chromosome (Ph1) on
karyotyping. This is a translocation of a portion of the q arm of chromosome 22 to the q
arm of chromosome 9, designated t(9:22).
FISH
• FISH — Fluorescence In Situ Hybridization analysis, labeled probes are
hybridized to either metaphase chromosomes or interphase nuclei, and the
hybridized probes are detected with fluorochromes
• This technique is a rapid and sensitive means of detecting recurring numerical
and structural abnormalities
o Technique is used to discover translocation between chromosomesà we
would see two different colors together in one chromosome indicating
exchange of genetic material
The Philadelphia Chromosome
Schematic presentation of Philadelphia Chromosome
The Philadelphia Chromosome is a well-known DNA translocation that causes CML

(Chronic Myelogenous Leukemia.) Critically short telomeres on chromosomes 9 and 22
may help facilitate this change. The switch (reciprocal translocation) results in the
formation of extra long Ch9 and Philadelphia (Ph) chromosome, mutation carries a new
fused gene bcr-abl)
Derivative chromosome 9 deletions are a significant feature of childhood

Philadelphia chromosome positive acute lymphoblastic leukaemia
Acute Lymphoid Leukemia (ALL)

• Neoplastic monoclonal proliferation of lymphoid cells, predominantly present in
the blood
o Note: this is present in bone marrow as well- but there are so many of
them that they spill into peripheral blood
• Neoplastic proliferation is composed of lymphoblasts.
• ALL is subdivided into several categories, based upon the CD markers present
on the cell.
• Most are precursor B-cell type (CD10 positive)
• Some are early precursor B-cell type (CD10 negative)
• others are pre-B cell type, which is defined by the presence of cytoplasmic Ig
• Peripheral blood has decreased mature forms
• Increased immature (blasts) forms
• Immature forms contain immature chromatin in the nucleus
• Nucleolus has smooth texture
• Bone marrow > 30% blasts – diagnostic criteria
• ALL occurs more commonly in children and represents 70% of all
childhood leukemias
• Children have an increased incidence of ALL if they have Down syndrome
• Most cases of ALL are precursor B-cell type and cells CD19+, CD20+, and
CD10+
• 20% are precursor T-cell type
• Precursor B-cell leukemia presents with leukemia and extensive bone marrow
involvement
• Precursor T-cell leukemia/lymphoma: Male predominance and thymic

involvement
• Good prognostic factors for ALL:
o Hyperdiploid (50 chromosomes per cell)
o Age of patient: 2–10 years
o CD10 positive
o t(12;21) involving TEL1 and AML1
o Low white blood cell count
• Poor prognostic factors for ALL:
o Hypodiploid
o Age younger than 2 years or older than 10 years
o Male gender
o t(9;22), and translocations involving ML1 (chr 11q23)
o High white blood cell count (100,000 cells/μL)
o Important points: ALL, especially during recurrences, can involve the
CNS and testes (sacred sites)
Acute Lymphoid Leukemia (ALL)
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
The WBC's seen here are lymphocytes, but

they are blasts--very immature cells with larger
nuclei that contain nucleoli. Such lymphocytes
are indicative of acute lymphocytic leukemia
(ALL). ALL is more common in children than
adults. Many cases of ALL in children respond
well to treatment, and many are curable.
Also note the RBC- they have increased pallor
in their centers
Chronic Lymphoid Leukemia (CLL)

• Neoplastic monoclonal proliferation of lymphoid cells, predominantly present in
the blood
o Neoplastic proliferation is composed of mature lymphocytes
o Differs from ALL because it deals with immature lymphocytes
• Epidemiology:
o Patients are older than 50 years of age
o male to female ratio is 2:1
o CLL is the most common form of leukemia in the United States
(represents 30% of adult leukemias)
o Associated mutations: del 13q12-14; trisomy 12; deletions 11q and 17p
o Leukemias associated with a mutation have a worse prognosis
• Prognosis of CLL: Overall survival is long (about 10 years)
• Some patients undergo transformation to prolymphocytic leukemia (with 1-year
survival) or to diffuse large B-cell lymphoma, which destroys patients bodies very
quickly
• Complications of CLL
o Hypogammaglobulinemia
o 10–15% of patients develop autoimmune hemolytic anemia
o Immune thrombocytopenia.
o 5% of cases transform to a diffuse large B-cell lymphoma (Richter
transformation)
• Important points regarding CLL
o The features of CLL and small lymphocytic lymphoma (SLL) overlap
o Both conditions are very similar, except that
§ CLL denotes prominent involvement of the blood and
§ SLL denotes prominent involvement of the lymph nodes
§ Close overlap of these two diseases illustrates how leukemia and
lymphoma are not mutually exclusive entities
§ CLL is CD5 positive
§ CD5 is a T-cell marker, but 80% of cases of CLL are B-cell derived;
therefore, expression of CD5 is an aberrant expression of a cell
marker
• Microscopic morphology of CLL
o Cells have a “cracked earth” or “gingersnap” appearance
o Cells are fragile. Cells that break during processing on the slide are
referred to as “smudge cells”
o Exam: smudge cell= CLL
• Clinical presentation of CLL
o Symptoms: Many patients are asymptomatic
o Some patients experience fatigue and weight loss
o Signs:
§ Many patients have a normal physical examination
§ Some patients have splenomegaly
Chronic Lymphoid Leukemia (CLL)
These mature lymphocytes are

increased markedly in number. They are
indicative of chronic lymphocytic
leukemia, a disease most often seen in
older adults. This disease responds
poorly to treatment, but it is indolent
Acute Myeloid (Myelogenous) Leukemia (AML)

• Acute myeloid leukemia (AML, also known as acute myelogenous leukemia and,
less commonly, as acute non-lymphocytic leukemia)
• Group of relatively well-defined hematopoietic neoplasms involving precursor
cells committed to the myeloid line of cellular development (i.e., those giving rise
to granulocytic, monocytic, erythroid, or megakaryocytic elements)
• AML is characterized by a clonal proliferation of myeloid precursors with a
reduced capacity to differentiate into more mature cellular elements
• Accumulation of leukemic blasts or immature forms in the bone marrow,
peripheral blood, and occasionally in other tissues
• Variable reduction in the production of normal red blood cells, platelets, and
mature granulocytes
• Increased production of malignant cells, along with a reduction in these mature
elements, results in a variety of systemic consequences including anemia,
bleeding, and an increased risk of infection
o Patients will present as weak, tired, and will often be fighting off infections
often
• Four main groups of AML recognized in WHO classification system:
o AML with recurrent genetic abnormalities - 11 %
o AML with myelodysplasia-related features - 6%
o Therapy-related AML and MDS - 2 %
o AML, not otherwise specified - 81%
• Epidemiology: Predominantly older patients
• Markers: Neoplastic cells are positive for CD13, CD15, and CD64
• Associated cytogenetic abnormalities
• t(15;17)
• Genes involved: PML and RARA (retinoic acid receptor)
• Consequence of translocation: Cells are fixed at the promyelocyte stage of
differentiation
o The myelocytes never mature they will keep dividing as is
• Treatment with all trans-retinoic acid (ATRA) matures the cells
o ATRA binds to RARA and turns mechanism onà causing cells to mature
• The classic clinical case is a younger patient (median age 40) with symptoms of
leukemia (e.g., bruising, fever) who presents in disseminated intravascular
coagulation (DIC)
Acute Myeloid (Myelogenous) Leukemia (AML)
Leukemias typically fill up the marrow with

abnormal cells, displacing normal
hematopoiesis. The marrow here is essentially
100% cellular but composed almost exclusively
of leukemic cells. Normal hematopoiesis is
reduced via replacement (a "myelophthisic"
process) or by suppressed stem cell division.
Thus, leukemic patients are prone to anemia,
thrombocytopenia, and granulocytopenia and
all of the complications that ensue, particularly
complications of bleeding and infection.
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.
Chronic Myeloid Leukemia (CML)

• Neoplastic monoclonal proliferation of granulocytes
• The granulocytes are more mature precursors of neutrophils (i.e.,
metamyelocytes, myelocytes, and bands)
• CML is responsible for 15–20% of all cases of leukemia
• Although CML is a chronic myeloid leukemia, it also is included in the group of
disorders referred to as chronic myeloproliferative disorders
• Epidemiology: Older patients; median age is 52 years, although a juvenile form
of CML exists but is rare
• Associated cytogenetic abnormality:
o Philadelphia chromosome t(9;22), translocation juxtaposes abl and
bcr
• Complications of CML:
o Progression of CML to AML (two thirds of patients) or to ALL (one third of
patients) - blast crisis, has a poor prognosis

• Splenomegaly
• Microscopic morphology:
o Increased white blood cell count, with a range of myeloid maturation from
myelocytes to band and segmented neutrophils
o The number of basophils is characteristically increased
• Clinical presentation of CML
o Symptoms:
§ Up to 40% of patients are asymptomatic
§ Fatigue
§ Lethargy
§ Shortness of breath
§ Weight loss
§ Early satiety - due to splenomegaly
• Patient might say that they usually eat a lot, but just can’t
any more they are not hungry
o Signs:
§ Markedly elevated white blood cell count (median of 170,000
cells/μL)
§ Low leukocyte alkaline phosphatase (LAP) levels
§ High uric acid levelà likely due to high number of cell death and
when cells die, they release DNA products, thus increasing uric
acid
§ High lactate dehydrogenase (LDH) level
§ Neoplastic cells have an increased amount of DNA and increased
cellular turnover, which causes the high levels of uric acid and LDH
§ Neoplastic cells do not function as normal white blood cells, which
explains the decreased LAP
o Course of CML:
§ Patients are usually diagnosed in the chronic phase (Indolent)
§ Indolent phase lasts 3–5 years and can be asymptomatic
§ Chronic phase leads to an accelerated phase characterized by
fever, weight loss, night sweats, worsening splenomegaly, and
bone pain due to rapid cell turnover
§ Finally, patients progress to a blast crisis
Chronic Myeloid Leukemia (CML)
The CBC here is from a patient with CML. Note

the markedly increased WBC count.
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.
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.
Tumor Lysis Syndrome

• Group of metabolic complications can occur after treatment of neoplastic
disorders
• Caused by massive tumor cell lysis with the release of large amounts of
potassium, phosphate, and nucleic acids into the systemic circulation
o Hyperkalemia
o Hyperphosphatemia
o Hyperuricemia
o Hypocalcemia
o Acute renal failure
• Catabolism of the nucleic acids to uric acid leads to hyperuricemia
• Marked increase in uric acid excretion can result in the precipitation of uric acid in
the renal tubules and can also induce renal vasoconstriction, impaired
autoregulation, decreased renal blood flow, and inflammation, resulting in acute
kidney injury
• Hyperphosphatemia with calcium phosphate deposition in the renal tubules can
also cause acute kidney injury
Hodgkin lymphoma
• Hodgkin lymphoma (HL), formerly called Hodgkin's disease
• Tumor of germinal center or post-germinal center of B cells
• HL has a unique cellular composition, containing a minority of neoplastic cells
(Reed-Sternberg cells and their variants) in an inflammatory background
• It is separated from the other B cell lymphomas based on its unique
clinicopathologic features, and can be divided into two major sub-groups, based
on the appearance and immunophenotype of the tumor cells may respond well to
therapy
• Classical HL
o The tumor cells in this group are also derived from germinal center B cells
o but typically fail to express many of the genes and gene products that
define normal germinal center B cells
o Based on differences in the appearance of the tumor cells and the
composition of the reactive background, classical HL is further divided into
the following subtypes:
§ Classical HL
• Nodular sclerosis classical HL (NSHL)
• Mixed cellularity classical HL (MCHL)
• Lymphocyte rich classical HL (LRHL)
• Lymphocyte depleted classical HL (LDHL)
§ Nodular lymphocyte predominant HL
• tumor cells in this subtype retain the immunophenotypic
features of germinal center B cells
• It is important to determine the stage of the disease and the histologic type of HD
to provide the most appropriate therapy
•
The first four types listed in the table below are "classical HL" and have Reed-
Sternberg cells Immunohistochemically CD15+, CD30+ but negative for CD45-
• Lymphocyte predominance HL has RS cells that are CD20+ but CD15- and
CD30- , more like B cells
Hodgkin Lymphoma – Classification

Hodgkin vs. Non-Hodgkin Lymphoma
Hodgkin Lymphoma
• Hodgkin lymphoma (HL) accounts for approximately 10% of all lymphomas
• Approximately 0.6% of all cancers diagnosed in the developed world annually
• HL has a bimodal age distribution curve
• In the US and other economically advantaged countries, there is one peak in
young adults (approximately age 20 years) and one in older age (approximately
age 65 years)
• Majority of patients are young adults
• There is a slight male predominance, especially in children and in middle and
older adulthood.
• In economically disadvantaged areas, there is an initial peak in childhood for
boys, relatively low rates in young adults, and a late peak in older adults
Bimodal age distribution of Hodgkin lymphoma

Age-specific average annual incidence rates of Hodgkin lymphoma for all races by sex
per 100,000 population, 2004 to 2008, in the SEER program. Two peaks are noted: a
larger peak in young adults, and a second peak in older adults.
• Clinical manifestations of Hodgkin lymphoma:

o Entirely unique, distinctive for the most part
o Hodgkin lymphoma most commonly presents as painless enlargement of
peripheral (superficial) lymph nodes
o Cervical or supraclavicular lymph nodes are enlarged in as many as 80%
of patients at presentation
o Mediastinal involvement is common (10% initially; >50% later)
o In more than 90% of patient’s disease is located above the diaphragm at
the time of initial diagnosis
• Contiguous Spread
o Hodgkin lymphoma appears to spread or
grow by contiguous extension (spreading
from one lymph node to adjacent nodes or
to extra nodal tissue)
o Non-Hodgkin's lymphomas tend to be
less defined in their growth pattern,
commonly being diffuse, without evidence
of contiguous spread
• Clinical symptoms:
o One third of patients with Hodgkin lymphoma have constitutional
symptoms: fever, night sweats, and weight loss (called “B” symptoms)
o Generalized pruritus can occur with nodular sclerosis type.
§ Itchiness
o Painless cervical lymphadenopathy is the most common presenting
symptom of Hodgkin lymphoma

o Pain in the lymph nodes with ingestion of alcohol is classic and
specific, but present in few patients
• B symptoms include:
o Fever greater than 38°C. Pel-Ebstein fever, the classic intermittent fever
associated with Hodgkin disease, occurs at variable intervals of days to
weeks and lasts for 1–2 weeks before resolving. However, fever
associated with lymphoma can follow virtually any pattern.
o Drenching sweats, especially at night.
o Unintentional weight loss of >10% of normal body weight over a period of
6 months or less.
Hodgkin Lymphoma – Staging (do not memorize)
Stage Characteristics
I Only a single lymph node site or extra nodal site is involved
II Two or more lymph node sites on one side of the diaphragm are involved,
or limited contiguous extra nodal site involvement
III Lymph node sites on both sides of the diaphragm are involved, with
splenic or limited contiguous extra nodal site involvement, or both
IV Extensive involvement of extra nodal sites, with or without lymph node
involvement
Lymph Node
Here is a 5 cm lymph node (obviously from a

patient with lymphadenopathy). The node
should normally be soft and pink and less than
1 cm in size. This lymph node is involved with
Hodgkin's disease. This gross appearance
could pass for a non-Hodgkin's lymphoma as
well.
Hodgkin Lymphoma
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 extra nodal
involvement. This picture could probably
suffice for non-Hodgkin's lymphomatous
hepatic disease as well.
Hodgkin Disease: Lymph Node
This is Hodgkin's disease, nodular sclerosis

type. Note the bands of pink collagenous
tissue dividing the field in this lymph node.
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.
In this lymph node, there are numerous scattered

large cells with a surrounding prominent clear space
surrounding the nucleus, an artefact of formalin
fixation.
Reed-Sternberg Cells
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 lymphoma consists of reactive lymphoid
cells. There are four main subtypes of classic
Hodgkin lymphoma with CD15+ Reed-Sternberg
cells and variants: lymphocyte rich, nodular
sclerosis, mixed cellularity, and lymphocyte
depletion. The lymphocyte predominance subtype
with CD15- Reed-Sternberg variant cells acts more
like a low-grade B-cell lymphoma.
Non-Hodgkin's Lymphomas
Note: we quickly went over this info
Type Histological- Immunogenetics Clinical Features
Features
Small Lymphocytic Small and well CD19, 5; Bcl-2 and Seen in older
Lymphoma differentiated B Bcl-6 expression adults, it is
lymphocytes, with essentially the solid
diffuse effacement tissue (lymph node)
of nodal component of
architecture and no chronic lymphocytic
follicles leukemia; disease
tends to be
generalized but
with indolent course
and prolonged
survival; some may
transform to more
aggressive
lymphomas
Follicular Lymphoma Nodal architecture CD19, 20, 79a; Most common type,
(predominantly small is effaced by t(14:18); Bcl-2 seen in adults,
cell) monotonous, expression often involves
crowded follicles multiple lymph
composed of nodes, course is
monomorphous indolent, with
small cleaved B- prolonged survival,
lymphocytes though some may
transform to a large
cell lymphoma
Diffuse Large B-cell Cells are large, with CD19, 20, 79a; Though often
Lymphoma prominent nucleoli some have t(14;18); localized, they tend
and abundant some have Bcl-2 to be aggressive
cytoplasm and and Bcl-6 extranodal masses;
many mitoses. Most expression; linked seen in adults and
are B-cell, but 20% to EBV infection; children, can be
are T-cell negative TdT seen in HIV
phenotype infection
Burkitt Lymphoma Intermediate sized CD10, 19, 20, 79a; Endemic in Africa
B-lymphocytes t(8:14) is with mandibular
(small-noncleaved characteristic; and abdominal
cells) African form linked involvement;
to EBV infection; sporadic elsewhere
negative TdT with abdominal
involvement; affects
mainly children and

young adults
High-grade B-cell Intermediate sized CD19, 20 Sporadic; may be

Lymphoma (small B-lymphocytes seen with HIV
non-cleaved) Burkitt- (small non-cleaved infection
like Lymphoma cells)
Precursor T or B-cell Intermediate sized B-cells are CD19, Seen in children

Lymphoblastic lymphocytes in a 20, sometimes and adolescents; T-
Lymphoma/Leukemia diffuse pattern CD10; T-cells are cell type often in
(Lymphoblastic CD3 and 8; all are mediastinum; very
Lymphoma) TdT positive aggressive and can
progress to acute
lymphocytic
leukemia
Mantle Cell Small to medium CD 19, 20, 43; Seen in adults in

Lymphoma sized B cells t(11;14); Bcl-1 middle age; often
(Cyclin D1) advanced at
expression diagnosis and may
be extranodal,
including multifocal
submucosal
nodules in bowel
Marginal Zone Small to medium CD19, 20, 79a; Seen in middle

Lymphoma sized B cells negative CD5 and aged adults;
10 typically arises in
areas of immune
activation
(Hashimoto
thyroiditis, Sjogren
syndrome,
Helicobacter pylori
gastritis); similar
lesions asociated
with mucosal
lymphoid tissue are
called MALTomas
(mucosa-
associated
lymphoid tissue
tumors); may
transform to diffuse
large B-cell
lymphoma
Note: very quickly went over these pictures
Lymphoma
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).
Small Lymphocytic Lymphoma (SLL)
This pattern of malignant lymphoma is

diffuse and no lymphoid follicles are
identified in this lymph node. Note that the
normal architecture of the lymph node is
obliterated. The lymph node is replaced by
an infiltrate of small (mature-appearing)
neoplastic lymphocytes, and the infiltrate
extends through the capsule of the lymph
node and into the surrounding fat. These
cells will mark as B lymphocytes. The
diagnosis is: small lymphocytic lymphoma
(SLL). This is the tissue equivalent of
chronic lymphocytic leukemia (CLL), and
both often occur together (CLL/SLL). Though both are indolent, they are widespread
and difficult to treat.
non-Hodgkin's lymphoma
Many non-Hodgkin's lymphomas seen in

adults are large cell lymphomas such as the
one here at medium power, but they can be
associated with immunosuppressed states
(such as AIDS), and are typically of B cell
origin. The cells are large, with prominent nucleoli and abundant cytoplasm. This
disease tends to be localized (low stage), but with more rapid enlargement, and a
greater propensity to be extra nodal than the low-grade lymphomas.
B cell lymphoma
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 large B cell
lymphoma (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. Demonstration of CD19 and 20 antigens would classify it as B
cell in origin.
Bone Marrow: malignant lymphoma
A bone marrow biopsy can reveal malignant

lymphoma. Here, there is a peritrabecular
infiltrate of small blue cells which is the
lymphomatous infiltrate.
Post-transplantation lymphoproliferative disorder (PTLD)
Following organ transplantation, particularly

for heart, but also to a lesser extent with
kidney and bone marrow, immunosuppressive
therapy may promote an expansion of
Epstein-Barr virus (EBV) infected T-cells,
seen here with immunoperoxidase staining for
EBV. This is a post-transplantation
lymphoproliferative disorder (PTLD), which
acts like a lymphoma, but will recede when
immunosuppression is diminished, if possible.
Plasma Cell Lesions

• Plasma cell lesions are the neoplastic (monoclonal) proliferation of plasma cells:
o involving single bones (plasmacytoma)
o or multiple bones (multiple myeloma)
• Neoplastic plasma cells secret
o light chains of Ig
o heavy chains of Ig
o complete Ig
o Secretion may include either or all of Ig components
• Free light chains – Bence Jones proteins (urine)
o Exam: Bence Jones Proteins in the urine are free light chains= Dx:
multiple myeloma
• Overproduction of one clone of Ig – M spike in ɣ region of serum electrophoresis
Serum protein electrophoresis
Spike in the gamma globulin during electrophoresis= multiple myeloma
Plasma Cell Lesions

• Plasma cell myeloma (multiple myeloma, myelomatosis, medullary
plasmacytoma) is a bone marrow-based, malignant disorder of postgerminal
center B-cells that is characterized by a clonal proliferation of plasma cells, with
associated serum and/or urine monoclonal proteins
• Malignant plasma cell clone until they start to disrupt the normal marrow
microenvironment leading to immune dysfunction, cytopenias, angiogenesis,
and osteoclastogenesis
Multiple Myeloma
• 50-60 years of age
• Most common tumor arising within a bone
• Most common bones involved: vertebrae, skull, ribs
• Bone pain due to expansion of marrow
• Lytic lesions on bone due to expansion of plasma cells: IL-6 produced by

neoplastic cells, stimulates osteoclastic activity
• Pathological fractures
• Hypercalcemia –due to bone resorption
• ↓ functional Ig – infections: St aureus, Str. Pneumonia, E. coli)
• ↑ light chains – collection in distal convoluted tubules and collecting tubules –
renal insufficiency
• Amyloidosis
• Cytogenetics: t(4;14)
• 99% of MM secrete Ig
• No M spike in non-secretory myelomas
• 60% MM produce IgG
• 20-25% - IgA
• 15-20% light chains only: kappa or lambda
• Bone marrow: > 30% plasma cells
• Accumulation if Igs in plasma cells – Russell bodies
• Exam: Russell bodies= Dx: Multiple Myeloma
Multiple Myeloma
Note: went over these pics very quickly
The skull demonstrates the characteristic rounded "punched out" lesions of multiple
myeloma.
Round lesions filled with a soft reddish

material are indicative of foci of myeloma
in this section of vertebral bone.
At low power, the abnormal plasma cells

of multiple myeloma fill the marrow.
At medium power, the plasma cells of

multiple myeloma here are very similar
to normal plasma cells, but they 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.
Immunoelectrophoresis characterizes the type of
monoclonal immunoglobulin being produced.
Here is a smear of bone marrow aspirate from a

patient with multiple myeloma. Note that there
are numerous plasma cells with eccentric nuclei
and a perinuclear halo of clearer cytoplasm.
MM: Amyloid
A by-product of plasma cells tumors is the

production of amyloid. On routine hematoxylin
and eosin (H&E) sections, amyloid appears as
eosinophilic, amorphous deposits. Special stains
are helpful in identifying these deposits.
Multiple Myeloma
Immunohistochemistry is useful in demonstrating

the clonality of the lesion. Occasionally, the stains
can be hard to interpret, but in general, either the
kappa (pictured here) or the lambda light chain
will show positivity. If both stains are positive, a
tumor diagnosis should be questioned.
Plasmacytoma
• Osseous solitary plasmacytoma of bone is a localized bone tumor of plasma
cells, without evidence of plasma cell myeloma or marrow plasmacytosis
• Extraosseous solitary plasmacytoma of bone is a localized, soft-tissue plasma
cell neoplasm that occurs in a location other than the bone and does not have
evidence of plasma cell myeloma or marrow plasmacytosis
• Most patient progress to multiple myeloma within 10-15 years
• Remember from DI: plasmacytoma is one lesion and multiple myeloma is many
lesionsà plasmacytoma can progress into multiple myeloma
Monoclonal Gammopathy of Undetermined Significance (MGUS)

• Most common of a spectrum of diseases called plasma cell dyscrasias
• Presence of monoclonal immunoglobulin (Ig), also called an M-protein, in the
serum or urine
• M spike without osteolytic lesions, Waldenstrom macroglobulinemia (WM),
amyloidosis (AL) or other lymphoproliferative disorders
• 20% develop multiple myeloma within 10-15 years
• Distinguishing between MM and MGUS is critical
• Patients with MGUS are conservatively treated and do not need chemotherapy
Lymphoplasmacytic Lymphoma (LPL)

• Aka immunocytoma
• Lymphoplasmacytic lymphoma: lymphoma cells have characteristics of both
lymphocytes and plasma cells
• Uncommon type of non-Hodgkin lymphoma (NHL), accounting for about 1–2% of
all lymphomas
• Lymphoplasmacytic lymphoma is usually a slow-growing (indolent) lymphoma
• Sometimes it can transform into an aggressive large cell lymphoma
Waldenstrom macroglobulinemia
• WM is a type of non-Hodgkin lymphoma (NHL) that produces large amounts of
macroglobulin – IgM
• Overproduction of IgM causes slugging of cells in the vessels -hyperviscosity
syndrome
• Visual and neurologic disturbances: dizziness
o headache
o blurring or loss of vision
• Bleeding
• Cryoglobulins: IgM precipitation in cold temperatures – Reynaud phenomenon,
numbness and tingling of the fingers or toes (peripheral neuropathy)
• No Lytic lesions in the bone
• Cytogenetics: t(9;14)
Heavy-Chain Disease
• The heavy chain diseases are rare B-cell proliferative disorders characterized
by the production of a monoclonal (M) protein consisting of a portion of the
immunoglobulin heavy chain without a bound light chain
• Alpha HCD (also called immunoproliferative small intestinal disease (IPSID),
Mediterranean lymphoma, Seligman disease)
• Form of MALT lymphoma, with the same histologic features of MALT type
gastrointestinal lymphomas with marked plasma cell differentiation
• It is the most common form of HCD and occurs in patients from the
Mediterranean region or Middle East, usually young males, and is often
associated with relatively poor sanitation
GAMMA HCD
§ Patients with gamma HCD typically present with systemic symptoms,
lymphadenopathy, splenomegaly, and/or anemia, and occasionally with palatal
and uvular swelling, median age of patients 60 to 70 years
Lecture Hour 5&6: Hematologic Pathology- Pathophysiology

of Blood Coagulation (Hemostasis)
Hemostasis- a chain of various physiological reactions which prevent our body from
losing our own blood. This is a balance of coagulation and anticoagulation.
Coagulation cascade- allows our blood to clot, and we can live with minor injuries (aka
the blood in the circulatory system can still flow without leaving the body)
Anticoagulation process- prevents blood from clotting in our arteries and veins and
allows perfusion of organs and tissues.
Case # 1
§ A 31-year-old woman presents with multiple acute bite wounds on her left hand
caused by her pet monkey.
Case # 2
§ A 6-year-old boy suddenly developed a painful swelling in his left elbow joint, no
apparent trauma reported. His parents noticed frequent bruising, epistaxis (nose
bleed), and spontaneous bleeding since his birth.
Case # 3
§ A 5-year-old child develops a sudden onset of bloody diarrhea, hematuria (dark
urine), and flu-like gastrointestinal illness.
§ Laboratory findings reveal markedly increased level of the blood urea nitrogen
(BUN).
What do the three clinical scenarios have in common?

• The common denominator for all these conditions:
o the involvement of the blood coagulation system (Hemostasis)
• Case 1à Normal blood coagulation in traumatic lacerations.

• Case 2à Hemophilia A or Royal Disease (Factor VIII deficiency).
• Case 3à Hemolytic Uremic Syndrome (HUS) or Hamburger Disease
(contamination with E.coli in the burger).
Learning Objectives:
• After completion of this lecture students will be able to:
• Define physiology of hemostasis:
o Differentiate hemostatic pathways
o Outline the process of blood coagulation
o Explain morphology and function of the platelets
o Classify plasma coagulation factors
• Recognize and Evaluate Bleeding Disorders:
o Inherited platelets disorders
o Acquired platelets disorders
o Inherited Coagulopathies
o Acquired Coagulopathies
Hemostasis
• Etymology: in Ancient Greek heme – blood, and stasis – halting, halting of the
blood.
• Hemostasis is a sequence of events leading to the cessation of bleeding by
a formation of a stable fibrin-platelet plug.
• Hemostasis is one of the major homeostatic mechanisms:
o Keeps balance between thrombosis and bleeding
o Preserves: circulatory volume, cellular and chemical components of the
blood, prevents hypovolemia, hemorrhagic shock and exsanguination
o Maintains circulatory integrity, proper organ perfusion and tissue
oxygenation
Events in Hemostasis
• Blood vessel is cut with a knife.. then what?
1. Vascular Constriction
2. Formation of the Platelet Plug
3. Formation of a Blood Clot
4. Tissue repair
Vascular Wall Injury

• Endothelin-1 is released from endothelial cells
causing vascular spasm
• Vasoconstriction causes blood flow turbulence
and stasis
• Thrombogenic factors release:
o Thromboplastin - Tissue Factor, factor III
o von Willebrand Factor (vWF)
• Coagulation Initiation
• We have endothelial cells with Weibel-Palade Bodies inside and the vascular
wall with collagen inside
• Once there is damage of the endothelium, we have one vWF that catches the
platelets flowing by via the CD42b/ GPIb receptor activating the platelets
• Activated platelets release alpha granules and dense bodies, which are he
integral components of blood coagulation
• Platelets then express other receptors called GPIIb/ GPIIIa, which binds with
fibrinogen in the plasma, which creates a bridging structure with other platelets
• Note that this picture is only showing two platelets, but this process is happening
with a myriad of other platelets and receptors so when the other platelets bind,
and become activated, they release their alpha granules and dense bodies,
which supports aggregation of platelets in all different directions to form a platelet
plug
Platelets
• Blood platelets or thrombocytes: thin, biconvex anucleate disks, 2-4 µm in
diameter
• Production: bone marrow megakaryocyte fragmentation
• Life span ~ 10 days
• Platelets count: 150,000-400,000/µL of blood
• Platelet’s count below 150,000/µL called thrombocytopenia
• Platelet’s count above 400,000/µL named thombocytosis
• The normal platelet count is 150-400k/cc. Thrombocytopenia is a condition in
which there is a fall in platelet count below 150k/cc. Clinically it manifests as a
purpura. Primary or idiopathic purpura usually occurs without identifiable cause.
Platelets Precursors: Megakaryocyte
Bone marrow smear

Platelets in Peripheral Blood
Platelets: Morphology
Blood Coagulation
Prothrombin activator that converts prothrombin into the active form, thrombin.
Thrombin converts soluble fibrinogen into fibrin (insoluble).
Then the Fibrinolytic system is activated.
Intrinsic Pathway Extrinsic Pathway
Surface Contact:
Collage, HMWK (Fitzgerald Factor) Tissue Injury
XII XIIa
Tissue Factor (Thromboplastin)
XI XIa
IX IXa VIIa VII

Ca2+ Ca2+
Tenase Complex
VIII+PF3+IXa+Ca2+
X Xa
Common Pathway Ca2+
Prothrombin Complex: V+Xa+PF3+Ca2+
Prothrombin (II) Thrombin (enzyme)
Fibrinogen (I) Fibrin Monomer + Fibrinopeptides A&B
XIII XIIIa Insoluble cross—linked fibrin
Intrinsic pathway
Surface contact with collagen Fitzgerald Factor converts factor 12 into activated factor
12. Activated factor 12 converts inactive factor 11 to activated factor 11; active factor
11converts inactive 9 to activated factor 9; in the presence of calcium activated factor 9
forms the Tenase Complex, which activates factor 10.
Extrinsic Pathway
Starts with a tissue injury. Tissue factor/ thromboplastin turn inactive form of factor 7
into active form factor 7 with the presence of calcium. Factor 7 activates factor 10.
Common Pathway
activation of prothrombin complex, which cleaves prothrombin, giving us thrombin.
Thrombin creates fibrin monomers and fibrinopeptide A and B from soluble fibrinogen.
Then factor 13 gets activated, which becomes an insoluble cross/ linked fibrin so that
the fibrin can “link” to thrombocytes and begin the formation of the clot.
Fibrinopeptide - peptide released from the amino end of fibrinogen by the action of
thrombin to form fibrin during clotting of the blood.
High-molecular-weight kininogen (HMWK or HK), also known as the Williams-
Fitzgerald-Flaujeac factor or the Fitzgerald factor or the HMWK-kallikrein factor, is
a protein from the blood coagulation system as well as the kinin-kallikrein system.
Fibrinolysis
Intrinsic Pathway Extrinsic Pathway
HMWK
VII
XII
Partial PK
Thromboplastin Prothrombin
XI
Time Time
IX
VIII
Fibrinolytic System
X
Plasminogen
V
Common Pathway PG Activators:
II Tissue-type PA (EC, LU)
FDP
FDP
Urokinase PA (plasma)
s FDP I
s s
Plasmin
FDP FDP
s Fibrin Clot
s
Coagulation Factors (Plasma)

• I. Fibrinogen
• II. Prothrombin
• III. Thromboplastin (Tissue Factor)
• IV. Calcium 2+
• V. Labile Factor, Proaccelerin
• VI. Accelerin (not in use) (factor Va)
• VII. Stable factor (Precacelerin)
• VIII. Anti-hemophilic Factor A
• IX. Anti-hemophilic Factor B (Christmas Factor)
• X. Stuart-Prower Factor
• XI. Plasma thromboplastin
• XII. Hagman factor
• XIII. Fibrin Stabilizing Factor (Laki-Lorand Factor)
• Von Willebrand Factor
• HMWK High Molecular Weight Kininogen (Williams-Fitzgerald-Flaujeac factor)
• Pre-K Prekallikrein (Fletcher Factor)
• Ka Kallikrein
• PPL Platelet Phospholipids
• Test Info: Not testing us on specifics from the clotting pathways, but we do need
to know philosophy and pathophysiological conditions common from intrinsic and
extrinsic and common pathways so we can recognize what part of the
coagulation happens in real life.
Bleeding Disorders
• Clinical manifestation: abnormal bleeding, spontaneous or follow some inciting

event – trauma, surgery, tooth extraction.
• Classification:
o Bleeding disorders caused by Vascular Wall Abnormalities
o Bleeding related to Reduced Platelet Number: Thrombocytopenias
o Bleeding disorders related to Defective Platelet Functions:
Thrombasthenias
o Hemorrhagic diathesises related to Abnormalities in Clotting Factors
Vascular Wall Abnormalities

• Inherited:
o Ehlers-Danlos syndrome (EDS) or Cutis Hyperelastica – are a group of
connective tissue disorders caused by a defect in collagen type I, III and
V. They are autosomal dominant or autosomal recessive inheritance
o Marfan Syndrome – GP Fibrillin, elastic tissue abnormality, autosomal
dominant trait
o Fibrillin is a glycoprotein, which is essential for the formation of elastic
fibers found in connective tissue
• Acquired:
o Scurvy – vitamin C deficiency, defective collagen
o Systemic amyloidosis
o Infectious and hypersensitive vasculidities – rickettsial disease, Henoch–
Schönlein Purpura
• Iatrogenic (caused by medical interventions)
o Chronic glucocorticoid use
Quantitative Platelet Disorders

• Disorders dealing with the amount of platelets
• Increased Production:
o Thrombocytosis > 400,000/µL
o Thrombocytopenias
• Decreased Production < 150,000/µL:
o Aplastic Anemia (drug-induces, viral)
o Bone Marrow malignancies
• Increased Destruction:
o Idiopathic thrombocytopenic Purpura (ITP)
o Thrombotic Thrombocytopenic Purpura (TTP)
o Hemolytic Uremic Syndrome (HUS)
o Disseminated Intravascular Coagulation (DIC)
o Hypersplenism
• Dilutional – multiple transfusions
• Iatrogenic: drug induced, alcohol, thiazides, cytotoxic drugs, heparin
• Infections: measles, HIV
Qualitative Platelet Disorders

• Disorders dealing with the quality of platelets
• Von Willebrand Disease – vWF deficiency
• Bernard-Soulier Syndrome: autosomal
recessive deficiency of GPIb
• Glanzmann's thrombasthenia: deficiency of
GPIIb-IIIa
• Drug-Induced or Drug-Reduced Adhesion
or Aggregation
o Aspirin: irreversible cyclooxygenase
(COX) inhibition – TXA2 synthesis
block
o Clopidogrel (Plavix): - ADP receptors
inhibitor, aggregation
o Abciximab – glycoprotein IIb/IIIa
receptor antagonist
Idiopathic thrombocytopenic Purpura (ITP)

• Aka: primary immune thrombocytopenic
purpura and autoimmune thrombocytopenic
purpura
• Reminder: purpuraà discoloration of the skin
(bluish-violet little bruises)
• Type II Hypersensitivity reaction (IgG-
mediated) against GPIIb-IIIa and GPIb-IX
platelets antigens
• Spleen Produces antibodies against receptors
• IgG bind to GPIIb-IIIa and GPIb-IX antigents
• Macrophages express Fc-receptors and bind to
IgG-coated platelets
• Another mechanism of this is reduced
production of platelets due to reduced number
of megakaryocyte
Thrombotic Thrombocytopenic Purpura (TTP)

• Thrombotic microangiopathy (small vessel
disease)
• Systemic intravascular fibrin-platelet thrombi formation
• Deficiency of ADAMTS13 enzyme (a disintegrin and metalloproteinase with a
thrombospondin type 1 motif, member 13)
• ADAMTS13 – cleaves large multimers of vWF in plasma
• Clinical manifestation:
o Adult women
o Clinical pentad of fever, thrombocytopenia, microangiopathic hemolytic

anemia (intravascular hemolysis), neurologic symptoms, renal condition
Heparin-Induced Thrombocytopenia
• Heparin is an anticoagulant given in hospital setting to patients who are at an
increased risk of blood clots
• Thrombocytopenia due to unfractionated heparin treatment
• 3-5% of patients in 1-2 weeks after therapy starts
• Injected heparin serves as a hapten and elicits immune response by production
of anti-heparin IgGs
• IgG antibodies bind PF4-heparin complex and cause platelets activation and
thrombosis
• Heparin-induced thrombocytopenia and thrombosis (HITT), worsening
thrombosis
• Thrombocytopenia
Hemolytic Uremic Syndrome aka Hamburger Disease

• Often comes from consumption of meat that is undercooked
• Thrombotic microangiopathy, similar to TTP
• Most cases in children and elderly patients
• Gastrenteritis with bloody diarrhea caused by E. coli strain O157:H7
• E. coli O157:H7 produces verotoxin (Shiga-like toxin)
• Endothelial damage followed by platelet activation, aggregation, mcirotrombi

formation
• Acute kidney damage, uremia
• 10% HUS- autoantibodies to factors H, I and CD46, negative regulators of
alternative complement activation
• Deficiency of these factors leads to uncontrolled complement activation after
minor endothelial damage and in massive thrombosis.
• Tx: supportive therapy, dialysis
• Irreversible kidney damage and death in 5-15%
Coagulation Disorders
Thrombotic Disorders
• Virchow triad (Rudolf Virchow (1821-1902)):
o Hypercoagulability
§ Antithrombin III deficiency
§ Protein C and S deficiency
§ Hyperviscosity syndromes
• Cellular
o polycythemia vera
o Hemoblastosis
• Protein-related – IgA, IgG3 hyperproduction
o Waldenström Macroglobulinemia
o Multiple Myeloma
o Hemodynamic alterations:
§ Stasis – prolonged immobilization and congestive cardiac failure
§ Turbulent blood flow
o Endothelial or vessel wall Injury
Remember: thrombotic disorders will have at least one of the above factors, but they
can even have all of them.
Antiphospholipid Antibody Syndrome

• Antibodies against several negatively charged phospholipids
• Autoimmune disorder
• Clinical Features:
o Arterial and venous thrombosis
o Spontaneous abortions
o Immune-mediated thrombocytopenia
• APLAS leading acquired hematologic cause of thrombosis
• Thrombosis in the uteroplacental vasculature – likely the cause of recurrent fetal
loss
Hemophilia A: (Royal Disease, Classic hemophilia)

• X- linked recessive condition
o Only males (XY) can be affected by this disease, and women (XX) will
be asymptomatic carriers of it.
• Deficiency of factor VIII
• Severe prolonged bleeding during surgical procedures
• Spontaneous hemorrhages into muscles and joints (hemarthrosis)
• Easy bruising and hematomas with minor trauma
• Tx: factor VIII transfusion:
o Cryoprecipitate - Cryoprecipitated Antihaemophilic Factor (AHF)
o Recombinant Factor VIII (rFVIII)
• 4,670 British haemophiliacs left infected with Hepatitis C, of whom 1,243
were also infected with HIV. Almost 2,000 have since died as a result, with
scores more needing organ transplants, and dialysis. Some victims
inadvertently infected their partners.
• 5,000 Canadians contracted hepatitis C and HIV through tainted blood in
1985-1990.
European Royal Family Tree

Hemophilia B (Christmas Disease)

• X-linked recessive condition
• Named after the first patient Stephen Christmas
• Deficiency of factor IX
• Clinical manifestation is identical to hemophilia A
Von Willebrand Disease

• Most common inherited bleeding disorder – 1% of US adults
• Autosomal dominant inheritance
• Group of conditions – deficiency or qualitative defect of vWF, more than 20
molecular variants
• Spontaneous bleeding from mucous membrane, menorrhagia, joints –
uncommon
• Defect of platelets function despite of NORMAL plates count
• Abnormalities in platelets adhesion and clot formation
Disseminated Intravascular Coagulation – DIC

• aka Consumption Coagulopathy: Aetiology
• Consumption Coagulopathy is ALWAYS secondary to a preceding disorder.
o Obstetrics: Placental Tissue Factor, amniotic fluid embolism
o Profuse hemorrhage or thromboembolism
o Sepsis (Gram-negative, meningococcal and rickettsia cause TNF release
and coagulation)
o AML type M3 (acute promyelocytic leukemia) granules in promyelocytes
initiate intravascular coagulation
o Adenocarcinomas – mucin activates coagulation cascade
DIC: Pathogenesis
1. Diffuse endothelial Injury.

2. Abnormally increased activation of procoagulant pathways, primary site
microvasculature.
3. Massive intravascular coagulation with formation of microthrombi: acute renal failure
due to glomerular capillaries and renal arterioles occlusion, purpura fulminant (skin),
internal organs necrosis.
4. Radical consumption platelets, and plasma clotting factors and coagulation inhibitors:
profuse, uncontrollable bleeding from mucosal membranes, intracranial bleeding.
DIC: Clinical Presentation

• Fulminant Onset – endotoxic shock, amniotic fluid embolism
• Insidious or chronic – carcinomatosis, or demised fetus retention
• Multiorgan failures, Acute Kidney Injury due to the widespread deposition of
fibrin in the circulation and the consequent compromise of blood supply to
various organs
• Endocrine glands:
o Adrenal cortex hemorrhages due to meningococcemia – Waterhouse-
Friderichsen Syndrome
o Postpartum Pituitary Necrosis or Sheehan Syndrome – form of DIC of
labor and delivery causing postpartum hypopituitarism
Summary
• Hemostasis is a complex biochemical cascade reaction leading to the cessation
of bleeding
• Hemostasis is achieved by the formation of a stable fibrin-platelet hemostatic
plug
• Vascular wall injury triggers transient vasoconstriction, vW factor attachment to
subendothelial collagen, platelet adhesion, and activation of extrinsic and intrinsic
clotting pathways.
• Platelets form aggregates, undergo shape change and degranulation
• Pathology of blood coagulation is manifesting as hemorrhagic syndromes various
severity and similar clinical representation
Lecture Hour 7&8: Fundamentals

This lecture Hour is about the fundamentals of lab testing, which gives us a good
overview of what this course is about. Please note that the practical portion of PA
3306 is all about lab testing and is taught by Dr. Moore.
List of clinical laboratory tests to be understood by chiropractors.

• Urinalysis. • Serum or plasma creatine kinase
• Pregnancy, serum/urine. CK.
• Complete blood count. • Plasma troponin I or T.
• Erythrocyte sedimentation rate. • Serum homocysteine.
• Gram stain and culture. • Serum C reactive protein.
• Serum or plasma glucose. • Serum myoglobin.
• Serum urea. • Serum B Natriuretic peptide.
• Serum creatinine. • Serum thyroid stimulating
• Serum calcium. horrnone, TSH, free thyroxine.
• Serum phosphate. • Serum Protein Specific Antigen,
• Serum parathyroid hormone. free PSA.
• Serum protein. • Serum urate.
• Serum albumin. • Serum rheumatoid factor.
• Serum and urine. Bence Jones • Serum Anti Nuclear Antigen.
protein. • Serum Human Leukocyte
• Protein electrophoresis. Antigen-B27.
• Serum lipids. • Serum sodium.
• Serum alkaline phosphatase. • Serum potassium.
• Serum total bilirubin. • Serum bicarbonate.
• [Serum aspartate amino • Serum iron.
transferase AST]. • Serum ferritin.
• Serum alanine amino transferase • Serum vitamin B12.
ALT. • Erythrocyte folate.
• Prothrombin time. • (Cortisol.

• Faecal occult blood. • Sex hormones).
• Spirometry.
• This list was first defined in 2002 and is currently being petitioned to the Ontario
Health Ministry that Chiropractors can order some or all of these tests.
Laboratory tests that you should know

a) Test for diagnosis
• Laboratory tests in logical groups and what they usually mean clinically.
• Reference ranges are given but these do not have to be remembered. They may
be useful for you to refer to in the 4th year and in clinical practice.
b) Symptoms, organ systems and useful tests

• The signs and symptoms of the common diseases and what laboratory tests are
indicated.
Module 9 route map:
• Topic1. Fundamentals.
• Topic 2. Haematology.
• Topic 3. Heart and Stroke.
• Topic 4. Hypertension.
• Topic 5. Bone.
• Topic 6. Joint.
• Topic 7. Neuromuscular.
Learning objectives:
• Why tests may be ordered.
• Preanalytical errors.
• Analytical errors - precision accuracy, analytical sensitivity, analytical specificity,
interference errors.
• Post analytical errors: what is normal, abnormal, (healthy sick) patients.
• Prevalence and incidence.
• True and false results.
• The “Normal” range.
• Clinical sensitivity and specificity.
• Predictive value of result and likelihood ratio.
• Analytical and biological variance.
• Testing strategy: sensitive tests first to diagnose then specific tests.
• Screening for disease.
• Laboratory organization in Canada.
• Point of care testing.
Clinicians’ use of laboratories in diseases

• Pathology – changes in cell appearance and or function
o e.g. response to antibodies – using labels.
• Chemistry - changes in body cells’ milieu – blood, body fluids.

• Hematology – the cell, inside and out and chemical changes in blood plasma.
• Microbiology – tests for likely infective organisms.
Clinician is expected to use the most efficient, least harmful, cost-effective test
Reasons why laboratory test are ordered:

• Diagnosis:
o to confirm disease.
o to confirm health.
o to “screen” healthy populations for disease.
• Management:
o Prognosis.
o Progression.
o Assess effects of treatment.
• Lab tests should never stand-alone- the clinician should do a history and physical
first to get an idea of what is going on with the patient before ordering a lab test
Other reasons some docs do lab tests (the dark side):

• curiosity.
• to look busy.
• for “science”.
• because of tradition.
• to avoid litigation.
• for profit.
Many tests used in diagnosis fail because:

• Tests were developed in hospitals on very ill patients.
o However, in a hospital setting lab tests give excellent information
• Tests were developed in populations where there is a high prevalence of
disease. Disease is usually of low prevalence.
• Diseases/responses are evolving, e.g. syphilis, bubonic plague (Black Death).
o Disease mutation (e.g., COVID, TB, etc..)
• Intracellular fluid is most of interest but is not available at present except by
scanning e.g. PET.
• Pattern recognition is the most effective diagnostic tool – SYNDROMES. This
means symptoms, signs and laboratory data taken together.
Laboratory tests – whose responsibility?

• Preanalytical: patient and Health Care Provider.
o Patient needs to be honest and describe what’s going on with them well
• Analytical: laboratory staff (with information from the Health Care Provider).
• Post analytical: interpretation by the Health Care Provider.
Some preanalytical factors that may alter laboratory results:

• Diet.
• Drugs.
• Age.
• Sex.
• Hormone cycles.
• Pregnancy.
• Biological rhythms.
• Genotype.
• Posture.
• Exercise.
• Specimen type.
o E.g., Different preservatives
• Specimen storage.
• Sampling error
Mod9 Week1- PA 3306- Aug 21, 23, 24- Monica Walsh (mwalsh@cmcc.ca) 73
The specimen
• What: whole blood, blood plasma or serum, urine, sweat, cerebrospinal fluid,
hair, saliva, orifice swab, erythrocytes, leukocytes
• How: which anticoagulant (for plasma) or none (serum).
o Plasmaà liquid part of the blood in which coagulation has been prevented
via the addition of an anticoagulant
o Serumà liquid that remains after blood has clotted
• Where taken from: arm, leg, biopsy
• Identification.
• How processed: centrifuged, separated, transported, stored.
• Contamination possibilities.
Analytical factors:
• Precision – how close measurements of the same item are to each other
• Accuracy – how close a measurement is to the true or accepted value
• Interferences:
o In vivo – e.g. factors that alter metabolism.
o In vitro – e.g. drugs with similar chemistry to the analyte.
§ E.g., patient taking vit C can alter glucose results
• Analytical sensitivity – what is the least that can be measured?
o 10^-12à a pea in an Olympic sized swimming pool
o Quick and cheap
• Analytical specificity – is the laboratory measuring what it claims to be
measuring?
Analytical factors (repeatability, accuracy, interferences, costs)

• Competent, responsible laboratory staff knows these factors for each test and
should be willing to share them with you.
• Health Care Laboratories (HCL) must be licensed (but insurance and veterinary
laboratories are not).
• HCL are monitored by the State/payer.
• However, still needs user whistle blowers. “Cheating” and/or incompetence is
possible.
Interferences with laboratory tests:

• Most common are
o Lipaemia.
o Haemolysis.
o Icterus (jaundice, bilirubin conjugated).
• Similar chemicals
o Vitamin C, salicylate.
o Metabolites.
o Precursors.
73
Analysis, the dark side

• Malfeasance.
• Financial issues.
• Negligence.
• Conflict of interest.
• Poor science.
• Malpractice.
• Incompetence.
• Overstressed technologists.
• Lack of supervision.
Systeme Internationale, the exceptions:
• The countries in red DO NOT use SI units
Curse of the Babel Tower.

• USA does not use “systeme international” units (SI) for laboratory tests.
• Rest of world does – sort of. Canada is in the “sort of” group.
• PA 3306 uses SI, mostly.
• Some of numbers are same on both systems, most are not.
Post analytical (interpretation)

• Incidence of disease
o How many with disease over a time period.
• Prevalence of disease
o How many with diseases at a defined time.
When you get a laboratory result or are asked to review one

• What do you think it means?
• Why do you have this opinion?
74
• Is this opinion likely to be true based on what else you know about the subject?
• Your judgment is an interpretation, just like any other.
• Use Bayes’ theorem (describes the probability of occurrence of an event related
to any condition)
Post analytical, Bayes’ theorem 2x2 table:
Bayes’ table issues in Clinical Laboratory:

• Natural history of disease– usually not known.
• Non pathological changes that look like disease.
• Definition of disease/non disease – there may be social or cultural biases.
• Prevalence of disease.
• What is/are the decision point(s). Who says so?
• Analytical variance (precision).
• Biological variance in health and in disease.
When to use – the Evidence Based Medicine approach (McMaster U)

• Highly clinically sensitive tests may be good to screen for disease but there
will be many false positives
• Highly clinically specific tests are good for diagnosis but there will be false
negatives. Diseases will be missed but few healthy people will be called sick.
• Which approach to use depends on circumstance.
The real world (according to McMaster Evidence Based Medicine leaders)

• SNOUT - use a highly sensitive test to rule out the disease.
• SPIN - use a highly specific test to rule in the disease.
• Predictive value of positive/negative test results.
• Likelihood ratio (1-sensitivity/specificity).
Likelihood ratio (LR):

• The likelihood that a given test result would be expected in a patient with the
disease compared with the likelihood that the same results would be expected in
a patient without the disease.
75
• Calculation: 1-sensitivity/specificity.
Evidence Based Medicine

• Evidence Based data – hard to come by.
• Most sources of information are not to be trusted – see Cochrane collaboration
for a trustworthy source.
• Likelihood ratios for each test result.
• Need pretest probability to get posttest probability with the expected clinical
sensitivity or specificity of the test
The Evidence Based Medicine method.

• Pretest probability is the prevalence of the disease in the population group.
• Posttest probability is:
o if the test is positive what is the probability that the patient has the
disease?
o if the test is negative, what is the probability that the patient does not have
the disease?
o e.g., we don’t perform pregnancy tests on men, but there are some
circumstances (drugs, cancer, etc..) that can make a man’s pregnancy
test positive
To find the decision points

• Need to study sick people.
• Need to study similar healthy people.
• and (ideally) similar people with similar diseases.
• Apply the appropriate statistical methods.
• Consider the consequences of error for the patient.
Reference range, setting up:

• Define a population.
• Measure with the test.
• Plot the results on a graph.
• Is it Gaussian/Normal/Bell shaped? Then you need at least 30 subjects.
• If not, can you break the results out into sub populations that show Gaussian
curves?
• If not then need > 100 subjects. Delete lowest 2.5% and upper 2.5%
• This means that 1 in 20 normals (healthy) will be called abnormal (sick) or
abnormals called normal.
76
Gaussian curve
The usual set up
77
Decision point, when we cannot do much for the disease e.g. pancreatic cancer.
In a disease that cannot be cured we shift the cutoff point to the right. We do not want to
diagnose someone with pancreatic cancer that does not have pancreatic cancer (aka a
false positive result); however, this could mean not diagnosing someone with pancreatic
cancer who does have pancreatic cancer (aka missing some true positives). Then we
need to think what matters more, giving someone a false diagnosis or not diagnosing
someone with a pathology that cannot be cured anyways?
Decision point, when must get all cases e.g. immigration screening for TB or
COVID.
In this case we want to catch every person who has the disease so we will shift the
cutoff point to the left, which means giving out false positive results. In other words, a
positive COVID test does not mean you have COVID.
Best reference range to use:

• Self when healthy.
• Ontario wide computer file on citizens is being set up.
78
• Part of e-Health – a management disaster behind schedule and well over budget.
• $ 50 million was spent on a diabetes registry in Ontario but not used.
Summary of why laboratory tests may be ambiguous:

• Pre analytical factors: diet, sex etc.
• Analytical factors: analysis variation.
• Post analytical interpretation:
o Incidence.
o Prevalence.
o clinical sensitivity.
o clinical specificity.
o reference ranges.
o biological variation.
CSCC meetings in 2012 and 2022.

• Reports on Patient Safety and Medical Error.
• Preanalytical errors 68% and 26% of these influence patient care.
• Analytical errors 7-13%.
• Post analytical errors 18-47%.
• Under current laws such errors do not have to be revealed but they should.
Primary role of clinician
To increase likelihood of disease in person being tested by increasing the

disease prevalence in the pretest population.
Watch out for unexpected and impossible results.

• 1st. Complain and
o demand a repeat. Laboratories save samples for at least a week.
Pathology samples may be saved “for ever”.
• 2nd. If possible, send sample again.
o Repeat test
• 3rd. Report to authorities.
Case: Plasma Troponin is a test for myocardial infarction. Clinical sensitivity 98%,
specificity 94%. What is the most appropriate place to use this test?
• A) Emergency Department.
• B) Chiropractic Clinic.
• C) Family Medicine Clinic.
• D) On the cardiac unit for diagnosis.
This test is great for ruling in individuals who are going to have a heart attack. You
might use this test on someone who is having chest pain in the ER.
79
Using Troponin for diagnosis of myocardial infarction in an Emergency room.

• Take 100 men with this symptom.
+ -
MI 49 1
Non MI 3 47
• Predictive value of a positive result: 49/52 = 94%.
Serum troponin for MI, the dark side.

• Family practice clinic.
• Take 100 men with chest pain.
• Prevalence of MI in these men is 1% (a guess, most of these men have a muscle
strain, rib injury or pulmonary emboli or infarct).
+ -
MI 1 0
Non MI 6 94
• Predictive value of a positive result 1/7 = 14%.
Troponin as test for MI, summary:

• Clinical sensitivity is 98%
• Clinical specificity is 94%
• If 1% have MI a positive result has a 14% chance of being right
• If 50% then 94% chance of being right.
Improving clinical sensitivity of tests

• Profile – a few or many tests done at once on sample(s) taken at same time.
What is problematic about this?
• Sequential testing. What is the problem?
• Algorithm approach – USA legal system forces this but it leads to test over use
and expense without obvious benefit.
The organization of laboratory services:

• Stat/ urgent – 1 hour.
• Point of Care systems (POCT) – 0-5 minutes.
o Bed side testing
• Routine – 1-8 hours, same day service.
• Special – a week to a month.
• Research – when convenient to the laboratory.
• Goal in modern clinical laboratories is to make all tests Stat.
80
Point of Care testing includes:
A point of care test that you will use.
Typical test questions, from the real world:

• Serum sodium of 300 mmol/L (healthy range is 135-145 mmol/L).
• What is the most likely source of this error?
o Sample is taken from IV line (should be taken from other arm)
81
Case: Serum sodium reported as 300 mmol/L

• Impossible result.
• Healthy range is 135-145.
• What is most likely source of error?
• A) urine not serum. (possible but not likely)
• B) contamination. (very possible).
• C) potassium measured and reported as sodium. (Impossible K=~5mmol/L and
Na=~140mmol/L)
• D) transcription error. (very, very unlikely with modern systems and technologist
training).
Case: serum specimens all K >6 mmol/L

• Healthy is 3.5-4.5 mmol/L. Specimens taken in the Clinic during the afternoon
and evening then sent to laboratory where serum was separated.
• What is a likely explanation for high K? (due to haemolysis)
• A) sample not separated in proper time. (yes, but the sample is stable for an hour
or so).
• B) samples stored at room temperature. (no, but there will be some haemolysis)
• C) samples stored in refrigerator before separation. (yes, cooling shuts down
metabolism and cells leak K so we get a false reading way higher than it should
be. Most likely to be true answer)
• D) potassium reference range is wrong. (No).
Case: plasma cholesterol in shopping mall

• High value found. Checked at lipid clinic at St Michael’s Hospital, which revealed
a normal value.
• Which of the following are likely reasons for this difference?
a) Vendor not subject to Quality Control.
b) Vendor has conflict of interest.
c) St. Michael’s Hospital has conflict of interest.
d) Cholesterol variable.
It turns out the vendor in the shopping mall is a for profit (scam) lipid clinic. This system
was a rip off because the clinic is trying to sell a “cholesterol lowering product”
Case: hair samples sent to two labs.

• Acme Laboratory claims:
o Mineral deficient.
o Vitamin deficient.
• Florida Sun Laboratory claims:
o Zinc at toxic levels.
• How can you best find out which one is telling the truth?
a) Sample split, two names.
b) Use of head and shoulders shampoo.
82
c) Ask about methods used.

d) Ask for External QC data.
C is the most correct answer, but A is reasonable as well.
Case: Laboratory is cheapest, fastest and offers incentives to users

• Results always come back in the middle of the normal range.
• Which of the following is the most likely explanation for this performance?
• A) automation and computer controlled
• B) result is to be expected.
• C) sink tested. (Sample poured away and made up number reported)
• D) better than the rest
Labs run by organized crime to launder money

Samples are poured down the drain and the results are given in the normal range
Laboratory tests fail because:

• Not the right test.
• Not sampled at the right time.
• Patient not prepared properly.
• Transportation and storage issues.
• Laboratory cannot work to the standard that the clinician needs.
• Wrong reference range.
• Disease natural history not known.

• Biological variance not known.
• Prevalence of diseases is low.
• Lack of harmonization of standards between laboratories.
• Deprofessionalisation – point of care system.
Do this case on own time:
Case: 36-year-old man complains of symptoms of upper respiratory tract

infection and loss of performance at long distance running.
• Weight 52 kg.
• Height 170 cm.
• Pulse 48 beats/minute,
• Blood pressure 100/67 mm Hg.
• Many laboratory tests were carried out. Only the abnormal or near abnormal
results are reported next.
83
• Reference ranges were not appropriate because this patient has a metabolic
adaptation to marathon running.
Dr. Moore’s laws, a reminder.

• Laboratory tests are secondary.
• Laboratory tests were designed for really ill patients.
• Every laboratory results contains uncertainty.
• Errors are most likely to be pre analytical.
• Reference range must be appropriate.
• Tests are usually non-specific. The development of specific tests has been
stalled.
84
• Laboratory medicine is badly taught and overused .

Question:
Plasma glucose, healthy people compared to those with diabetes mellitus. Clinical
sensitivity is 60%. Clinical specificity is 90%
• What is the appropriate clinical use for this test?

• A) to screen (many false positives)
• B) Dx with symptoms. (confirms hunch).
• C) Likelihood of diabetes in a healthy population. (many false positives)
• D) No use. (No, see B).
85
References
Teitelbaum, A. Hematological Pathology: General Symptomatology and Bone Marrow

Pathology [Lecture notes as PowerPoint]. PA 3306: Clinical Pathology and Laboratory
Testing, Canadian Memorial Chiropractic College. 2023 August 21 [cited 2023 Aug 29].
Available from https://kiro.cmcc.ca/
Teitelbaum, A. Hematology II: Red Blood Cell Pathology [Lecture notes as PowerPoint].
PA 3306: Clinical Pathology and Laboratory Testing, Canadian Memorial Chiropractic
College. 2023 August 21 [cited 2023 Aug 29]. Available from https://kiro.cmcc.ca/
Teitelbaum, A. Hematological Pathology: White Blood Cells Disorders [Lecture notes as

PowerPoint]. PA 3306: Clinical Pathology and Laboratory Testing, Canadian Memorial
Chiropractic College. 2023 August 23 [cited 2023 Aug 29]. Available from
https://kiro.cmcc.ca/
Teitelbaum, A. Hematological pathology: Pathophysiology of Blood Coagulation

(Hemostasis) and Blood Transfusion Pathology. [Lecture notes as PowerPoint]. PA
3306: Clinical Pathology and Laboratory Testing, Canadian Memorial Chiropractic
College. 2023 August 23 [cited 2023 Aug 29]. Available from https://kiro.cmcc.ca/
Moore, R. Topic 1. Fundamentals v2 [Lecture notes as PowerPoint]. PA 3306: Clinical

Pathology and Laboratory Testing, Canadian Memorial Chiropractic College. 2023
August 24 [cited 2023 Aug 29]. Available from https://kiro.cmcc.ca/
Disclaimer
The material and information within this package is a compendium of information and
course material notes from the DC program. It is to be used by students in the current
academic year of Note Service for only educational purposes and student self-study. It
is not to be reproduced, altered, redistributed in anyway. Materials contained here in are
produced on a cost recovery basis. The CMCC Students’ Council does not profit from
the sale of this material and provides it exclusively as a supplement to the education of
the students of the Canadian Memorial Chiropractic College.
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Lecture Hour 1: Hematologic Pathology: General

- Examples of symptoms that can be signs of chronic/ serious illness- long

§ Jaundice - Bilirubin accumulation from haemolysis

What happens when this system goes out of balance?

Bone Marrow Pathology

Normal Bone Marrow

§ This is the appearance of normal bone marrow at medium magnification. Note

Normal Bone Marrow

Normal Bone Marrow

Aplastic Bone Marrow

- Bone marrow became empty- BM exposed to something like radiation/ chemo

§ A magnetic resonance imaging scan showed numerous destructive vertebral

Bone Marrow Biopsy

Metastatic adenocarcinoma involving the bone marrow

Lecture Hour 2: Hematology II- Red Blood Cell Pathology

§ E.x., athletes use this system to increase o2 carrying capacity to

Anemia: General Considerations

Macrocytic normochromicà RBC is bigger than normal

Microcytic, hypochromicà small and no color

§ Folate Deficiencyà if the patient wants to become pregnant this is

Absorption of vitamin B12, Folic Acid, and Iron

Iron Deficiency Anemia

Signs and symptoms

§ Fatigue and diminished capability to perform hard labor

Anemia of chronic disease

§ Form of fibrosis, secondary to injury by nonhematopoietic cells or pathogens

§ Hemoglobin tetramers are comprised of the

synthesis with decreased production of either alpha or beta polypeptide

years, and the incidence is slightly higher in women than in men

§ Alcoholism decreases the bioavailability of folate and folate-dependent

Subacute Combined Dorsal And Lateral Spinal Column Degeneration

§ SUBACUTE COMBINED DEGENERATION OF THE CORD is a curious term for

Bone marrow film at 100X magnification demonstrating hypercellularity and

Blood film at 400X magnification demonstrating polyglobulia and thrombocytosis

Bone marrow film at 400X magnification demonstrating dominance of erythropoiesis

Lecture Hour 3&4: Hematologic Pathology- White Blood Cells

§ Increased bands- reaction of the body when inflammatory response

Reactive Changes in WBC: Mononucleosis

Infectious mononucleosis is a common

Reactive Changes in WBC: Mononucleosis

Lymphadenopathy: caused by a neoplasm

Bone Marrow Neoplasms - Leukemia

months from symptoms to Dx

• Trisomy is the gain of an extra chromosome (e.g., trisomy of chromosome 8 is

Chronic Myeloid Leukemia (CML) Karyotype

The Philadelphia Chromosome

Schematic presentation of Philadelphia Chromosome

The Philadelphia Chromosome is a well-known DNA translocation that causes CML

Derivative chromosome 9 deletions are a significant feature of childhood

Acute Lymphoid Leukemia (ALL)

• Precursor T-cell leukemia/lymphoma: Male predominance and thymic

Acute Lymphoid Leukemia (ALL)

In contrast to aplastic anemia, leukemia results

The WBC's seen here are lymphocytes, but

Chronic Lymphoid Leukemia (CLL)

Chronic Lymphoid Leukemia (CLL)

These mature lymphocytes are