Medicine

CLINICAL LABORATORY DIAGNOSTICS OF ANEMIAS

CLINICAL LABORATORY DIAGNOSTICS OF ANEMIAS

In its broadest sense, anemia is a functional inability of the blood to supply the tissue with adequate O₂ for proper metabolic function. Anemia is not a disease, but rather the expression of an underlying disorder or disease. Anemia is usually associated with decreased levels of hemoglobin and/or a decreased packed cell volume (hematocrit), and/or a decreased RBC count.

Classification: There are two main ways to classify the anemias:

a. Morphologicall (based on red cell size and hemoglobin content)

(1) Microcytic hypochromic

(MCV < 80 mcm³, diameter of erythrocyte < 6,5 mcm)

(2) Macrocytic hyperchromic

(MCV - mean corpuscular volume >100 mcm³, diameter of erythrocyte > 8 mcm)

(3) Normocytic normochromic

(MCV = 81-99 mcm³,diameter of erythrocyte = 7,2-7,5 mcm)

b. Pathophysiological (based on causative factors)

(1) Blood Loss

(2) Impaired RBC production

(3) Increased RBC destruction

Pathophysiological Classification of Anemias

I. Blood Loss Anemia

A. Acute posthemorrhagic anemia

B. Chronic posthemorrhagic anemia

II. Impaired RBC Production

A. Disturbance of the proliferation and differentiation of stem cells

1. Multipotential stem cells

a. Aplastic anemia

2. Unipotential stem cells

a. Pure red cell aplasia

b. Anemia of renal failure

c. Anemia of endocrine disease

B. Disturbance of the proliferation and maturation of differentiated stem cells

1. Defective DNA synthesis (megaloblastic anemia):

a. Vitamin B12 deficiency

b. Folic acid deficiency

2. Defective hemoglobin synthesis (hypochromic anemias):

a. Iron deficiency anemia (defective heme synthesis)

b. Thalassemia (defective globin synthesis)

3. Unknown or multiple mechanisms

a. Anemia associated with bone marrow infiltration

b. Anemia of chronic disease

c. Lead poisoning

d. Sideroblastic anemia

III. Increased RBC Destruction (Hemolytic Anemias)

A. Intrinsic Disorders

1. Membrane Defects

a. Hereditary spherocytosis

b. Hereditary ovalocytosis

c. Hereditary acanthocytosis (rare)

d. Hereditary stomatocytosis (rare)

e. Paroxysmal nocturnal hemoglobinuria (PNH)

2. Metabolic Defects

a. Glucose-6-phosphate dehydrogenase deficiency

b. Pyruvate kinase deficiency

3. Hemoglobin Defects

a. Hemoglobinopathies

b. Thalassemia

B. Extrinsic Disorders

1. Chemical Agents

2. Vegetable and Animal Poisons

3. Infectious Agents

4. Physical Agents

a. Heat

b. Traumatic hemolysis (Red Cell Fragmentation Syndrome)

5. Immunologically Caused Hemolytic Anemias

Laboratory Investigation of Anemia

Blood count ‑ red blood cell count

hemoglobin

hematocrit

A. Morphology of red blood cell may yield important clues

1. Size

2. Chromia, or color

3. Poikilocytosis ‑ abnormally shaped cells due to either premature aging or abnormal maturation.

a. Crenated cell ‑ artifact of slide preparation. From use of a hypertonic staining fluid ‑draws water out of the cell.

b. Burr cell ‑ cell has blunt horns seen in anemias associated with chromic kidney diseases i.e. uremia

c. Acanthocytes ‑ due to error of lipoprotein metabolism/rare. Associated with mental retardation.

d. Elliptocytosis ‑ cells oval in shape. May be a genetic defect. Homozygous – causes hemolytic anemia. May also be seen in cases of severe burns

e. Sickle cells ‑ contain abnormal Hgb. Upon oxygen deprivation, the cells form a sickle shape.

f. Howell ‑ Jolly bodies ‑ remnant of DNA or RNA. Commonly seen in splenectomy patients.

g. Cabot rings ‑ similar to Howell Jolly bodies

h. Basophilic stippling ‑ due to poisoning by heavy metals, especially lead. Results from precipitation of ribosomal material within the red cell.

i. Pappenheimer bodies ‑ also called Siderocytes. Can get them in small numbers in all anemias except Fe deficiency. Must be specially stained. Maturation defect.

j. Schistocytes ‑ irregularly shaped fragments of red cells which may be seen in hemolytic anemias.

Polychromasia ‑ (Reticulocytosis)

Polychromatophilic erythrocytes are newly released red cells, lilac in color and contain residual RNA which can be stained with a supravital stain such as New Methylene Blue. Levels of 0. 5‑1.5 % are usually considered normal. A lot of polychromasia indicates increased cell production, usually hemolysis. Lots of new cells have to be produced to keep up with the loss.

Blood Loss Anemia

Posthemorrhagic anemia is an anemia which develops as a result of hemorrhage. There are two types of anemias of this group according to the character of hemorrhage: 1) acute posthemorrhagic and 2) chronic posthemorrhagic anemia.

Acute posthemorrhagic anemia arises after fast massive hemorrhage as a result wounding of vessels or their damage by pathological process.

Chronic posthemorrhagic anemia develops after repeated hemorrhages, caused by injury of blood vessels during number of diseases (dysmenorrhea, ulcer of stomach, hemorrhoids etc.)

The picture of blood of acute posthemorrhagic anemias depends on time which has passed after hemorrhage. Depending on it it is possible pick out three periods, each of them is characterized by the certain picture of peripheral blood.

1. The first several hours after acute hemorrhage. At this period of time the total amount of blood, and also total number of erythrocytes in an organism decreases. However in unit of blood volume the contents of erythrocytes and concentration of hemoglobin is normal.

2. The period of time from several hours untill several days after acute hemorrhage. Dillution of blood takes place as a result of transition of liquid from interstitial spaces into blood vessels. As a result of it the quantity of erythrocytes and hemoglobin in unit of volume of blood decreases, as well as hematocrit. A color index stays without changes (normochromic anemia). Qualitative changes of erythrocytes in blood smear are not found yet.

3. The period of time from several days untill 1-2 weeks after acute hemorrhage. The most typical feature of picture of blood in this period is occurrence of plenty regenerative forms of erythrocytes, due to amplification of erythropoiesis in red bone marrow. Because young unripe erythrocytes contain less hemoglobin in comparison with mature cells, the color index decreases also and anemia becomes hypochromic.

During chronic posthemorrhagic anemia after the loss of iron hematologic attributesof irondeficiency anemia develop: concentration of hemoglobin and color index decrease, in blood smear there are degenerate forms of erythrocytes (micro- and poikilocytosis, hypochromy). Quantity of erythrocytes and hematocrit may remain without changes.

Iron-deficiency anemia

Iron has a pivotal role in many metabolic processes, and the average adult contains 3–5 g of iron, of which two-thirds is in the oxygen carrying molecule haemoglobin.

A normal Western diet provides about 15·mg of iron daily, of which 5–10% is absorbed (~·1·mg), principally in the duodenum and upper jejunum, where the acidic conditions help the absorption of iron in the ferrous form. Absorption is helped by the presence of other reducing substances, such as hydrochloric acid and ascorbic acid. The body has the capacity to increase its iron absorption in the face of increased demand, for example, in pregnancy, lactation, growth spurts

and iron deficiency. Once absorbed from the bowel, iron is transported across the mucosal cell to the blood, where it is carried by the protein transferrin to developing red cells in the bone marrow. Iron stores comprise ferritin, a labile and readily accessible source of iron and haemosiderin, an insoluble form found predominantly in macrophages. About 1·mg of iron a day is shed from the body in urine, faeces, sweat and cells shed from the skin and gastrointestinal tract. Menstrual losses of an additional 20·mg per month and the increased requirements of pregnancy (500–1000·mg) contribute to the higher incidence of iron deficiency in women of reproductive age.

Causes of iron deficiency anaemia

Reproductive system

• Menorrhagia

Gastrointestinal tract

Bleeding

• Oesophagitis

• Oesophageal varices

• Hiatus hernia (ulcerated)

• Peptic ulcer

• Inflammatory bowel disease

• Haemorrhoids (rarely)

• Carcinoma: stomach, colorectal

• Angiodysplasia

• Hereditary haemorrhagic telangiectasia (rare)

Malabsorption

• Coeliac disease

• Atrophic gastritis (also may result from iron deficiency)

Physiological

• Growth spurts (especially in premature infants)

• Pregnancy

Dietary

• Vegans

• Elderly

• Other

• Patients with chronic renal failure undergoing haemodialysis and receiving erythropoietin

Laboratory investigation of iron-deficiency anemia

1. Total blood count.

RBC

Reticulocytes

Platelets

RBC parameters:

· MCV (mean corpuscular volume – N 75-95mcm³)

· MCH (mean corpuscular hemoglobin – N 24-33 picograms),

· MCHC (mean corpuscular hemoglobin concentration – N 30-38 %),

· RDW (red cell distribution width – N 11,5-14 %).

At the onset of disease amount of red blood cells is not reduced, but they are reduced in size (microcytes) and not saturated enough with hemoglobin (hypochromiya). Color index is low (0,7-0,5) and MCHC also decreases. In blood smears small hypochromic erythrocytes are dominated with unequal size and shape (anizocytosis, poykilocytosis). In severe anemia erythroblasts may appear. The number of reticulocytes does not change. But if the anemia is caused by acute hemorrhage, immediately after it, the level of reticulocytes is increased, which is an important sign of bleeding. Osmotic resistance of erythrocytes is not changed or is slightly increased. The level of platelets does not change, only in case of hemorrhage is slightly increased.

2. Biochemical profile.

Ferritin, serum

Ferritin is a high molecular weight protein that consists of approximately 20 % iron. It is found in all cells, but especially in hepatocytes and reticuloendothelial cells, where it serves as an iron reserve. A small amount is present in plasma and serum and reflects iron stores in normal individuals. Iron is released from ferritin and binds to transferrin for transport to developing red blood cells in the bone marrow. Inadequate iron stores may result in iron deficient erythropoiesis.

Ferritin is also a useful screening test to distinguish iron deficiency from thalassemia minor in patients with anemia and erythrocyte microcytosis; ferritin is decreased in iron deficiency and normal or increased in thalassemia.

Interpretation

Reference Range: 45-340 ng/dL

· Values less than 100 ng/dL usually indicate depleted iron stores

· Ferritin is decreased in patients with an iron-depletion state

· Ferritin may be normal to mildly increased in functional iron deficiency

(anemia of inflammation) and in anemia secondary to thalassemia minor

· Ferritin is increased in inflammation

· Ferritin may be markedly increased in hereditary hemochromatosis and

other iron overload states, acute hepatitis, and many malignancies and in Gaucher’s disease

Iron, serum

Ingested iron is absorbed primarily from the intestinal tract, temporarily stored as ferritin in mucosal cells, and then released into the blood as Fe³⁺ - transferrin in equilibrium with a very small amount of free Fe³⁺. Serum iron can be used as one test to evaluate patients for iron deficiency, especially in combination with iron binding capacity (transferrin and transferrin saturation). Serum iron alone is unreliable due to considerable physiologic variation in the results.

Many normal subjects demonstrate a predictable diurnal variation with highest values in the morning and lowest values in the evening. Values in an individual may vary 10-40 % within a single day or day-to-day due to changes in iron absorption, marrow iron uptake, or storage iron outflow. Therefore, serum iron results should always be interpreted in the context of other studies.

Interpretation

Reference Range: 50-150 ug/dL

· Useful for diagnosis of iron depletion states especially when used in combination with transferrin and transferrin saturation

· Can be used for evaluation of chronic iron overload states

· Subject to physiologic variation including diurnal variation, and variation in response to iron therapy

Iron binding capacity, total (total serum transferrin) N- 1,7-4,7 mg/l, or 30,6 -84,6 micromole/l

Soluble transferrin receptor (sTfR), serum

Transferrin receptors are present on the external surface of the plasma membrane. In order for iron to be internalized into cells, the iron-transferrin complex binds to these receptors. It is then internalized through endosomes and the iron released into the cytoplasm. Proteolytic cleavage of the transferrin receptor releases a truncated version of the transferrin receptor as soluble transferrin receptor circulating in the blood. Membrane expression of transferrin receptors (TfR) are regulated by iron status. There is increased expression of TfR in iron deficiency states and this results in an increase in soluble TfR as well. In iron repletion states, there is a decrease in membrane and soluble TfR. TfR is not an acute phase reactant. While ferritin, which is an acute phase reactant, increases in response to inflammatory states, malignancy, infection, and chronic disease, soluble TfR is not affected by these confounding pathologies and may help determine the status of iron stores in patients with inflammation. TfR should not be used routinely for evaluation of iron status as it is referral test for most hospital laboratories with a higher cost and slower turn around time than ferritin, transferrin and transferrin saturation.

Interpretation

Reference Range: 1.8 – 4.6 mg/L

· Useful for evaluating iron status in patients with inflammation

· sTfR concentration is inversely related to iron status; sTfR elevates in

response to iron deficiency and decreases in response to iron repletion

· Patients with hemolysis or recent blood loss may have falsely elevated

sTfR levels

· sTfR is elevated in patients with thalassemia and sickle cell disease.

Transferrin, serum

Transferrin is the principle plasma protein for transport of iron. Its concentration

correlates with the total iron-binding capacity of serum. For diagnosis of iron depletion states, transferrin and iron-binding capacity may be used interchangeably. Transferrin is synthesized primarily in the liver. In otherwise healthy individuals with iron depletion states, transferrin levels in serum increase due to an increase in synthesis. High levels can be seen in pregnancy and during estrogen administration. Decreased transferrin may be seen in chronic liver disease, malnutrition, and protein loss. It is important to note that transferrin is decreased in malignancy and in both acute and chronic inflammation.

Interpretation

Reference Range: 200-400 mg/dL

· Transferrin is significantly increased in iron depletion states

· Transferrin is decreased in inflammatory states including anemia of chronic inflammation (functional iron deficiency)

· May be decreased in malnutrition, chronic liver disease, malignancy, and protein loss

· High levels of transferrin may be seen in pregnancy (and may be an indicator of iron depletion) and during estrogen administration.

Transferrin Saturation

Transferrin, the principal plasma protein for transport of iron, binds iron strongly at

physiologic pH. Transferrin is generally 20-45% saturated with iron. The additional amount of iron that can be bound is the unsaturated iron-binding capacity (UIBC). The sum of the serum iron and UBIC represents the total iron-binding capacity (TIBC). TIBC is an indirect measure of transferrin concentration and the two terms are often used interchangeably. The transferrin saturation (TSat) is usually reported as percent saturation (100 x serum iron/TIBC or transferrin).

Interpretation

Reference Range: 20-45 %

·Transferrin saturation less than 20 % is indicative of an iron deficiency state, either, latent iron deficiency, functional iron deficiency (usually associated with a decrease in transferrin but a disproportionately larger decrease in iron resulting in a transferrin saturation < 20 %) or true iron deficiency where a decrease in serum iron is associated with an increase in transferrin

· Transferrin saturation > 45 % may suggest hereditary hemochromatosis

· Transferrin saturation > 45 % may indicate an iron overload state (hemosiderosis) due to multiple transfusions or iron poisoning

· A transient increase in transferrin saturation is seen after intravenous iron infusion. The duration is dependent on the type and dose of the iron infusion.

3. Investigation of bone marrow.

Diagnostic bone marrow sampling is seldom performed in simple iron defi ciency, but, if the diagnosis is in doubt, a marrow aspirate may be carried out to demonstrate absent bone marrow stores.

4. Investigation of free protoporphyrin in erythrocytes.

Porphyrins are widely distributed in living cells throughout nature and play essential roles in various metabolic processes such as photosynthesis, transportation of oxygen and cellular respiration. In man the most important of the porphyrins is protoporphyrin 9, type III, which in combination with iron and specific proteins forms such compounds as hemoglobin, myoglobin, cytochrome,

peroxidase and catalase. In addition to its occurrence in these compounds, protoporphyrin has been found in apparently uncombined or free form in erythrocytes, hence the term "free erythrocyte protoporphyrin"

Adults: 16-36 mcg/dL red cells (0.28-0.64 micromol/L red cells)

The sequence of events (left to right) that occur with gradual depletion of body stores of iron. Serum ferritin and stainable iron in tissue stores are the most sensitive laboratory indicators of mild iron deficiency and are particularly useful in differentiating iron deficiency from the anemia of chronic disorders. The percentage saturation of transferrin with iron and free erythrocyte protoporphyrin values do not become abnormal until tissue stores are depleted of iron. Subsequently, a decrease in the hemoglobin concentration occurs because iron is unavailable for heme synthesis. Red blood cell indices do not become abnormal for several months after tissue stores are depleted of iron.

Описание: Image

Normal blood smear: RBC, platelets, lymphocyte

Hemolytic anemias

Red cells being enucleated, their survival within the blood circulation depends on the stability of their various components that cannot be replaced through protein synthesis. The normal life span of the red cell is 120 days. The normal red blood cell production compensates for the destruction and loss of red cells. Thus one in every 120 red cells (or 0.8%) is replaced every day by a newly formed red cell. These newly formed red cells, called "reticulocytes", can be recognized with

special stains because of the transient persistence of ribosomes in their cytoplasm.

Hemolysis is defined as a shortening of the red cell survival below the normal of 120 days.

MECHANISMS OF HEMOLYSIS

1. Fragmentation:

Mechanical destruction of the red cells. Examples include red cells trapped by malfunctioning artificial heart valves or fragmentation of red cells that are going through fibrin clots in the microcirculation (e.g. disseminated intra-vascular coagulation).

2. Osmotic Lysis:

The concentration of protein in the red cell is very high (32-36 gm% of hemoglobin). This creates a high osmotic force that tends to let water and sodium "leak" into the cell. This water needs to be pumped out by a membrane pump that uses ATP as a power source. If not enough ATP can be generated (as in pyruvate kinase deficiency) then the pump does not function properly and the red cell swells with water and may ultimately explode.

3. Erythrophagocytosis:

In autoimmune hemolytic anemia, red cell coated with antibodies are taken up and destroyed by macrophages, which have receptors for the Fc portion of the antibody (immunoglobulin).

4. Complement – Mediated Hemolysis:

The "complement" system is made up of a series of serum proteins which are sequentially activated, ultimately creating an active enzyme that punches a hole in the membrane of the red cell. This results in water rushing into the red cell, thus causing osmotic lysis. The complement system may be activated by bacteria, viruses, and certain antibodies.

LABORATORY FINDINGS IN HEMOLYSIS

1. The blood smear should be examined for potential clues about the cause of hemolysis. The reticulocyte count is elevated, indicating the increased production of red cells in an attempt to compensate for abnormal red cell losses.

2. Biochemical changes indicate the increased turnover of red cells:

· increased serum bilirubin (unconjugated form, or "indirect")

· increased serum lactate dehydrogenase (L.D.H.), an enzyme released from red cells

· increased serum iron (due to its release from the heme moiety of the hemoglobin)

In intravascular hemolysis:

· Decreased serum haptoglobin

· Increased free serum hemoglobin

· Hemosiderinuria

3. The bone marrow is sometimes examined to clarify the diagnosis. It will then show an expansion (hyperplasia) of the erythroid precursors (decreased marrow fat and lower myeloid/erythroid ratio as the erythroid cells increase in number to compensate for the hemolysis).

Rarely, the red cell survival is directly evaluated using autologous red cells labeled with radioactive chromium-51 (51Cr).

Hemoglobinopathy: A genetic defect that results in abnormal structure of one of the globin chains of the hemoglobin molecule.

Thalassemia: A genetic defect that results in production of an abnormally low quantity of a given hemoglobin chain or chains. The defect may affect the a, b, g, or d chain, or may affect some combination of the b, g, and d chain in the same patient (but never the a and b chain together). The result is an imbalance in production of globin chains and the production of an inadequate number of red cells.

The Hb S gene is found primarily in populations of native tropical African origin (which include most African-Americans). The incidence of the gene in some African populations is as high as 40 %; in African-Americans the incidence is 8 %. Unfortunately, homozygous expression produces sickle cell disease, which is a chronic hemolytic anemia and vasoocclusive condition that usually takes the life of the patient.

Sickle cell anemia is a particularly bad disease in that not only is it a hemolytic anemia, but also a vasoocclusive condition. The clinical findings can then be divided into one of these two groups:

a. Effects of chronic hemolysis

1. Anemia. Pretty much self-explanatory

2. Jaundice, due to rapid heme turnover and subsequent generation of bilirubin

3. Cholelithiasis. It has been classically taught that sickle cell patients are prone to the formation of calcium bilirubinate gallstones due to excess bilirubin secretion into the hepatobiliary tree.

4. Aplastic crisis. Many of us have brief episodes of marrow aplasia as a result of common viral infections. With a normal erythrocyte life span of 120 days, no anemia results from an unnoticed marrow shut-down of a few days. However, the sickle cell patients, with their markedly abbreviated RBC life span, can have a precipitous fall in hematocrit (and retic count) under such conditions. This may be life-threatening.

5. Hemolytic crisis. Most sickle cell patients establish a stable, tonic level of hemolysis. Rarely, for obscure reasons, they experience a catastrophic fall in hematocrit, increasing intensity of jaundice, and increasing reticulocyte count. This is called a “hemolytic crisis.”

b. Effects of vaso-occlusion

1. Dactylitis. Resulting presumably from infarction or ischemia of the bones of the hands and feet, this is often the presenting manifestation of sickle cell disease in a six-months-old infant. The hands and feet are swollen and painful.

2. Autosplenectomy. In childhood, the spleen is enlarged due to excess activity in destruction of the sickled erythrocytes. Gradually, the spleen infarcts itself down to a fibrous nubbin.

3. Priapism. This refers to a painful and sustained penile erection, apparently due to sludging of sickled cells in the corpora cavernosa. Sometimes the penis has to be surgically decompressed. Repeated episodes of priapism cause the spongy erectile tissues to be replaced by fibrous tissue, with impotence being the end result.

4. Renal papillary necrosis. The physiologic function of the loops of Henle make the renal medulla an eldritch, unbodylike area of high hematocrit, high osmolarity, low pH, hemodynamic stasis, and low pO₂. All of these conditions predispose to sickling and infarctive loss of the papillae of the pyramids. The result is inability to concentrate and dilute urine. Even sickle cell trait individuals may experience episodes of hematuria, presumably due to this mechanism.

5. Infarctive (painful) crisis. Increased sickling activity may be brought about by any general stress on the body, especially infection. Almost any organ may suffer acute infarction (includinmg the heart), and pain is the chief symptom.

6. Sequestration crisis. This occurs mostly in infants and young children and is characterized by sudden pooling of sickled erythrocytes in the RES and vascular compartment. This produces a sudden fall in hematocrit. Sequestration crisis may be the most common cause of death in sickle cell patients in the youngest age group.

7. Leg ulcers. After all of the disasters mentioned above, this seems trivial. However, the deep, nonhealing ulcers of skin and tela subcutanea (classically around the medial malleolus) may be the only clinical manifestation of sickle cell disease in an otherwise well-compensated patient. These may be the only bugaboo standing between the patient and a productive, financially solvent life.

Megaloblastic anemia

Megaloblastic anemia is an anemia (of macrocytic classification) that results from inhibition of DNA synthesis in red blood cell production. When DNA synthesis is impaired, the cell cycle cannot progress from the growth stage to the mitosis stage. This leads to continuing cell growth without division, which presents as macrocytosis.

Causes:

1.Vitamin B12 deficiency leading to folate deficiency:

§ Achlorhydria-induced malabsorption

§ Deficient intake

§ Deficient intrinsic factor (pernicious anemia or gastrectomy

)

§ Celiac disease

§ Biological competition for Vitamin B12 by diverticulosis, fistula, intestinal anastomosis, and infection by the marine parasite Diphyllobothrium latum (fish tapeworm)

§ Selective Vitamin B12 malabsorption (congenital—juvenile megaloblastic anemia 1—and drug-induced)

§ Chronic pancreatitis

§ Ileal resection and bypass

§ Nitrous oxide anesthesia (usually requires repeated instances).

2. Folate deficiency:

§ Alcoholism

§ Deficient intake

§ Increased needs: pregnancy, infant, rapid cellular proliferation, and cirrhosis

§ Malabsorption (congenital and drug-induced)

§ Intestinal and jejunal resection

§ (indirect) Deficient thiamine and factors (e.g., enzymes) responsible for folate metabolism.

3. Combined Deficiency: Vitamin B12 & folate.

§ Inherited Pyrimidine Synthesis Disorders: Orotic aciduria

§ Inherited DNA Synthesis Disorders

4. Toxins and Drugs:

a. Folic acid antagonists (methotrexate)

b. Purine synthesis antagonists (6-mercaptopurine)

c. Pyrimidine antagonists (cytosine arabinoside)

d. Phenytoin

e. Nitrous Oxide

Hematological findings

The blood film can point towards vitamin deficiency:

§ Decreased red blood cell (RBC) count and hemoglobin levels

§ Increased mean corpuscular volume (MCV, >95 fl) and mean corpuscular hemoglobin (MCH)

§ Normal mean corpuscular hemoglobin concentration (MCHC, 32–36 g/dL)

§ The reticulocyte count is decreased due to destruction of fragile and abnormal megaloblastic erythroid precursor.

§ The platelet count may be reduced.

§ Neutrophil granulocytes may show multisegmented nuclei ("senile neutrophil"). This is thought to be due to decreased production and a compensatory prolonged lifespan for circulating neutrophils, which increase numbers of nuclear segments with age.

§ Anisocytosis (increased variation in RBC size) and poikilocytosis (abnormally shaped RBCs).

§ Macrocytes (larger than normal RBCs) are present.

§ Ovalocytes (oval-shaped RBCs) are present.

§ Howell-Jolly bodies (chromosomal remnant) also present.

Blood chemistries will also show:

§ In increased lactic acid dehydrogenase (LDH) level. The isozyme is LDH-2 which is typical of the serum and hematopoetic cells.

§ Increased homocysteine and methylmalonic acid in Vitamin B₁₂ deficiency

§ Increased homocysteine in folate deficiency

Bone marrow shows megaloblastic hyperplasia.

Vitamin B₁₂ deficiency

The total amount of vitamin B₁₂ stored in the body is between two and five mg in adults. Approximately 50 % is stored in the liver, but approximately 0.1 % is lost each day, due to secretions into the gut — not all of the vitamin in the gut is reabsorbed. While bile is the main vehicle for B₁₂ excretion, most of the B₁₂ secreted in bile is recycled via enterohepatic circulation.

Vitamin B₁₂ can be found in large quantities in animal products, including meat, poultry, fish, seafood, eggs, and dairy products.

Causes:

§ Inadequate dietary intake of vitamin B₁₂. Vitamin B₁₂occurs in animal products (eggs, meat, milk) as the animal absorbs it by grazing in soil (or consuming unwashed crops or consuming other animals or feed composed of other animals that have done so, or fortified feed, etc.); significant portions of the world population have vitamin B₁₂deficiency, and it is theorized to be a problem of absorption as opposed to consumption. Vegan populations may have characteristic deficiency, more notably Western countries, unless one uses supplements or eats enriched food, though B₁₂ deficiency is observed in omnivorous, vegan and vegetarian populations frequently and everyone should be tested regularly. Children are at primary risk for dietary deficiency, since they have fewer vitamin stores and a relatively larger vitamin need per calorie of intake.

§ Selective impaired absorption of vitamin B₁₂ due to intrinsic factor deficiency. This may be caused by the loss of gastric parietal cells in chronic atrophic gastritis (in which case, the resulting megaloblastic anemia takes the name of "pernicious anemia"), or may result from wide surgical resection of stomach (for any reason), or from rare hereditary causes of impaired synthesis of intrinsic factor.

§ Impaired absorption of vitamin B₁₂ in the setting of a more generalized malabsorption or maldigestion syndrome. This includes any form of structural damage or wide surgical resection of the terminal ileum (the principal site of vitamin B₁₂ absorption).

§ Forms of achlorhydria (including that artificially induced by drugs such as proton pump inhibitors) can cause B₁₂ malabsorption from foods, since acid is needed to split B₁₂from food proteins and salivary binding proteins. This process is thought to be the most common cause of low B₁₂ in the elderly, who often have some degree of achlorhydria without being formally low in intrinsic factor. This process does not affect absorption of small amounts of B₁₂ in supplements such as multivitamins, since it is not bound to proteins, as is the B₁₂ in foods.

§ Surgical removal of the small bowel (for example in Crohn's disease) such that the patient presents with short bowel syndrome and is unable to absorb vitamin B₁₂. This can be treated with regular injections of vitamin B₁₂.

§ Long-term use of ranitidine hydrochloride may contribute to deficiency of vitamin B₁₂.

§ Coeliac disease may also cause impaired absorption of this vitamin, though this is due not to loss of intrinsic factor, but rather damage to the small bowel.

§ Some bariatric surgical procedures, especially those that involve removal of part of the stomach, such as Roux-en-Y gastric bypass surgery. (Procedures such as theadjustable gastric band type do not appear to affect B₁₂ metabolism significantly).

§ Bacterial overgrowth in parts of the small bowel are thought to be able to absorb B₁₂. An example occurs in so-called blind loop syndrome.

§ The diabetes medication metformin may interfere with B₁₂ dietary absorption.

§ Hereditary causes such as severe MTHFR deficiency, homocystinuria, and transcobalamin deficiency.

§ Some studies have shown that giardiasis, or similar parasitic infections may be a cause of vitamin B₁₂ deficiency.

§ Malnutrition of alcoholism.

§ Nitrous oxide abuse.

Clinical symptoms: The main syndrome of vitamin B₁₂ deficiency is Biermer's disease (pernicious anemia). It is characterized by a triad of symptoms:

1. Anemia with bone marrow promegaloblastosis (megaloblastic anemia). This is due to the inhibition of DNA synthesis (specifically purines and thymidine)

2. Gastrointestinal symptoms : These are thought to be due to defective DNA synthesis inhibiting replication in a site with a high turnover of cells. This may also be due to the autoimmune attack on the parietal cells of the stomach in pernicious anemia. There is an association with GAVE syndrome (commonly called watermelon stomach) and pernicious anemia.

3. Neurological symptoms: Sensory or motor deficiencies (absent reflexes, diminished vibration or soft touch sensation), subacute combined degeneration of spinal cord, or even symptoms of dementia and or other psychiatric symptoms may be present. The presence of peripheral sensory-motor symptoms or subacute combined degeneration of spinal cord strongly suggests the presence of a B₁₂ deficiency instead of folate deficiency.

Each of those symptoms can occur either alone or along with others. The neurological complex, defined as myelosis funicularis, consists of the following symptoms:

1. Impaired perception of deep touch, pressure and vibration, abolishment of sense of touch, very annoying and persistent paresthesias

2. Ataxia of dorsal cord type

3. Decrease or abolishment of deep muscle-tendon reflexes

4. Pathological reflexes — Babinski, Rossolimo and others, also severe paresis

The Schilling test is a medical investigation used for patients with vitamin B₁₂ deficiency. The purpose of the test is to determine whether the patient has pernicious anemia.

In the first part of the test, the patient is given radiolabeled vitamin B₁₂ to drink or eat. The most commonly used radiolabels are ⁵⁷Co and ⁵⁸Co. An intramuscular injection of unlabeled vitamin B₁₂ is given an hour later. This is not enough to replete or saturate body stores of B₁₂ (this requires about 10 B₁₂ injections over some length of time). The purpose of the single injection is to temporarily saturate B₁₂ receptors in the liver with enough normal vitamin B₁₂ to prevent radioactive vitamin B₁₂ binding in body tissues (especially in the liver), so that if absorbed from the G.I. tract, it will pass into the urine. The patient's urine is then collected over the next 24 hours to assess the absorption.

Normally, the ingested radiolabeled vitamin B₁₂ will be absorbed into the body. Since the body already has liver receptors for transcobalamin/vitamin B₁₂ saturated by the injection, much of the ingested vitamin B₁₂ will be excreted in the urine.

§ A normal result shows at least 10 % of the radiolabeled vitamin B₁₂ in the urine over the first 24 hours.

§ In patients with pernicious anemia or with deficiency due to impaired absorption, less than 10 % of the radiolabeled vitamin B₁₂ is detected.

The normal test will result in a higher amount of the radiolabeled cobalamin in the urine because it will have been absorbed by the intestinal epithelium, but passed into the urine because all hepatic B₁₂ receptors were occupied. An abnormal result will cause less of the labeled cobalamin to appear in the urine because it will remain in the intestine and be passed into the feces.

If an abnormality is found, i.e. the B₁₂ in the urine is only present in low levels, the test is repeated, this time with additional oral intrinsic factor.

§ If this second urine collection is normal, this shows a lack of intrinsic factor production, or pernicious anemia.

§ A low result on the second test implies abnormal intestinal absorption (malabsorption), which could be caused by coeliac disease, biliary disease, Whipple's disease, fish tapeworm infestation (Diphyllobothrium latum), or liver disease. Malabsorption of B₁₂ can be caused by intestinal dysfunction from a low vitamin level in-and-of-itself, causing test result confusion if repletion has not been done for some days previously.

Myelodysplastic syndrome (MDS)

MDS are a morphologically and clinically heterogeneous group of acquired stem cell disorders characterized by ineffective and dysplastic haematopoiesis in one or more cell lines that affect predominantly older subjects. The patients usually present with anaemia and dysplastic morphologic abnormalities in cells of more than one lineage (neutropenia, thrombocytopenia, monocytosis, or a combination of types of cytopenias). The diagnosis is essentially based on the morphological examination of a blood smear and a bone marrow biopsy specimen. The French-American-British classification divides the MDS into five subtypes based on the number of immature blast cells and morphological abnormalities in blood and bone marrow and the presence of ringed sideroblasts.

The median age at diagnosis of a MDS is between 60 and 75 years; a few patients are younger than 50; MDS diagnoses are rare in children. Males are slightly more commonly affected than females. Signs and symptoms are nonspecific and generally related to the blood cytopenias:

§ Anemia (low RBC count or reduced hemoglobin) —chronic tiredness, shortness of breath, chilled sensation, sometimes chest pain

§ Neutropenia (low neutrophil count) —increased susceptibility to infection

§ Thrombocytopenia (low platelet count) —increased susceptibility to bleeding and ecchymosis (bruising), as well as subcutaneoushemorrhaging resulting in purpura or petechia

Many individuals are asymptomatic, and blood cytopenia or other problems are identified as a part of a routine blood count:

§ neutropenia, anemia and thrombocytopenia (low cell counts of white and red blood cells, and platelets, respectively);

§ splenomegaly or rarely hepatomegaly;

§ abnormal granules in cells, abnormal nuclear shape and size; and/or

§ chromosomal abnormalities, including chromosomal translocations and abnormal chromosome number.

Blood smear from an adult female with a myelodysplastic syndrome related to radiotherapy and chemotherapy for Hodgkin disease. A hypogranular neutrophil with a pseudo-Pelger-Huet nucleus is shown. The red blood cells show marked poikilocytosis, in part related to post-splenectomy status. (Wright-Giemsa stain).

IIron deficiency anemia. a and b Erythrocytemorphology in iron deficiency anemia: ring-shaped erythrocytes (1), microcytes (2) faintly visible target cells (3), and a lymphocyte (4) for size comparison. Normal-sized erythrocytes (5) after transfusion. c Bone marrow cytology in iron deficiency anemia shows only increased hematopoiesis and left shift to basophilic erythroblasts (1). d Absence of iron deposits after iron staining (Prussian blue reaction). Megakaryocyte (1).

The erythrocytes vary greatly in size (anisocytosis) and shape (1) (poikilocytosis), and show basophilic stippling (2). Burr cell (3), which has no specific diagnostic significance.

Thalassemia. a Thalassemia minor: often no target cells, but an increase in the number of small erythrocytes (shown here in comparison with a lymphocyte), so that sometimes there is no anemia. b More advanced thalassemia minor: strong anisocytosis and poikilocytosis (1), basophilic stippling (2), and sporadic target cells (3). c Thalassemia major: erythroblasts (1), target cell (2), polychromatic erythrocytes (3), and Howell–Jolly bodies (4) (in a case of functional asplenia). Lymphocyte (5) and granulocyte (6).

Native sickle cells (1) are found only in homozygous sickle cell anemia, otherwise only target cells (2) are present. d Sickle cell test under reduced oxygen tension: almost all erythrocytes appear as sickle cells in the homozygous case presented here.

Hemolytic anemia. a and b Newly formed erythrocytes appear as large, polychromatic erythrocytes (1) after Pappenheim staining (a); supravital staining (b) reveals spot-like precipitates (reticulocyte = 2). Thrombocyte (3). c Bone marrow cells in hemolytic anemia at lowmagnification: increased hematopoiesis with cell clusters. Orthochromatic erythroblasts predominate. A basophilic erythroblast shows loosened nuclear structure (arrow), a sign of secondary folic acid deficiency.

Hyperchromic anemia. a Marked anisocytosis. In addition to normalsized erythrocytes (1), macrocytes (2) and large ovoid megalocytes are seen (3). Hypersegmented granulocyte (4). b In hyperchromic anemia, red cell precursors may be released into the peripheral blood: here, a polychromatic erythroblast. c and d Bone marrow in megaloblastic anemia: slight (1) or marked (2) loosening up of the nuclear structure, in some cases with binuclearity (3). Giant forms of band granulocytes and metamyelocytes (4) are often present.

Myelodysplastic syndrome (MDS) as differential diagnosis in hyperchromic anemia. a Strongly basophilic stippling in the cytoplasm of a macrocyte (in myelodysplasia). b Myeloblast with hyperchromic erythrocyte as an example of a myelodysplastic blood sample in the differential diagnosis versus hyperchromic anemia. c A high proportion of reticulocytes speaks against megaloblastic anemia and for hemolysis (in this case with an absence of pyruvate kinase activity). d Bone marrow in myelodysplasia (type RAEB), with clinical hyperchromic

anemia.

Polychromatic erythrocyte with fine, dense basophilic stippling. Erythrocyte with Howell–Jolly bodies (arrow) in addition to a lymphocyte (after splenectomy)

Erythrocyte with a delicate Cabot ring (arrow) (here in a case of osteomyelosclerosis)

Fixation and staining artifacts.

CLINICAL LABORATORY DIAGNOSTICS OF HEMOBLASTOSIS

Leukemia appears to be a clonal disease resulting from the abnormal uncontrolled proliferation of a single stem cell from which a new clone of cells develops. For some unknown reason, these abnormal cells have a selective growth advantage over normal cells. Present data indicate that these cells are functionally different and biochemically abnormal.

Etiology is Unknown: Possible Causes:

A. Viruses are the proven etiological agent in some animal leukemias, and may well be the causative agent in some human leukemias as well.

B. Marrow damage due to irradiation increases the frequency of some leukemias, but not others.

C. A variety of chemicals and drugs have been implicated as possible etiological agents of leukemia, especially benzene.

D. Possible genetic factors have been implicated, especially in Chronic Lymphocytic Leukemia.

Leukemia Classification

Leukemias are classified into 2 major groups. Chronic in which the onset is insidious, the disease is usually less aggressive, and the cells involved are usually more mature cells. Acute in which the onset is usually rapid, the disease is very aggressive, and the cells involved are usually poorly differentiated with many blasts.

Both acute and chronic leukemias are further classified according to the prominent cell line involved in the expansion:

1. If the prominent cell line is of the myeloid series it is a myelocytic leukemia (sometimes also called granulocytic).

2. If the prominent cell line is of the lymphoid series it is a lymphocytic leukemia

Therefore, there are four basic types of leukemia.

I. Acute myelocytic leukemia – AML - (includes myeloblastic, promyelocytic, monocytic, myelomonocytic, erythrocytic, and megakaryocytic)

II. Acute lymphocytic leukemia – ALL - (includes T cell, B cell, and Null cell)

III. Chronic myelocytic leukemia – CML - (includes myelocytic and myelomonocytic)

IV. Chronic lymphocytic leukemia – CLL - (includes plasmocytic multiple myeloma, Hairy cell, prolymphocytic, large granular cell lymphocytic, Sezary’s syndrome, and circulating lymphoma)

Acute

Chronic

Acute leukemias can occur in all age groups

n ALL is more common in children

n AML is more common in adults

Chronic leukemias are usually a disease of adults

n CLL is extremely rare in children and unusual before the age of 40

n CML has a peak age of 30-50

Age Distribution: optimum ages for development:

1. ALL: 3‑4 years old

2. AML: 15‑20 years old

3. CLL: 50 years old

4. CML: 20‑50 years old

5. Monocytic: middle age (rarely before 30)

Comparison of acute and chronic leukemias:

Acute Chronic

Age all ages usually adults

Clinical onset sudden insidious

Course (untreated) 6 mo. or less 2-6 years

Leukemic cells immature >30% blasts more mature cells

Anemia prominent mild

Thrombocytopenia prominent mild

WBC count variable increased

Lymphadenopathy mild present; often prominent

Splenomegaly mild present; often prominent

Acute Leukemia

Is a result of:

Malignant transformation of a stem cell leading to unregulated proliferation and

arrest in maturation at the primitive blast stage. Remember that a blast is the most immature cell that can be recognized as committed to a particular cell line.

Clinical features

Leukemic proliferation, accumulation, and invasion of normal tissues, including the liver, spleen, lymph nodes, central nervous system, and skin, cause lesions ranging from rashes to tumors. Failure of the bone marrow and normal hematopoiesis may result in pancytopenia with death from hemorrhaging and infections.

Pathophysiology of the clinical manifestations of acute leukemias

1. Marrow failure due to infiltration

-anemia -fatigue, pallor,

-thrombocytopenia -bleeding, spontaneous bruising

-neutropenia-infections, sepsis

2. Infiltration of other organs

-liver, spleen, lymph nodes (particularly in ALL)

-lymphadenopathy

-hepatosplenomegaly

-mediastinal masses(T-ALL)

-gums –gum hypertrophy (monocytic subtype of AML)

-bone pain, esp. in children with ALL

-any organ or tissue

-skin-leukemia cutis

-soft tissue -chloromas

-testis

-CNS

-solid organs

3. Leukostasis (only seen with WBC >>50x10⁹ /L)

-CNS -strokes

-lungs -pulmonary infiltrates, hypoxemia

4. Constitutional symptoms

-fevers, sweats are common

-weight loss uncommon

other

-exposure of substances that can initiate coagulation can cause DIC

Lab evaluation

The lab diagnosis is based on two things:

1. Finding a significant increase in the number of immature cells in the bone marrow including blasts, promyelocytes, promonocytes (> 30 % blasts is diagnostic)

2. Identification of the cell lineage of the leukemic cells

Peripheral blood:

Anemia (normochromic, normocytic)

Decreased platlets

Variable WBC count:

The degree of peripheral blood involvement determines classification:

n Leukemic – increased WBCs due to blasts

n Subleukemic – blasts without increased WBCs

n Aleukemic – decreased WBCs with no blasts

Classification of the immature cells involved may be done by:

Morphology – an experienced morphologist can look at the size of the blast, the amount of cytoplasm, the nuclear chromatin pattern, the presence of nucleoli and the presence of auer rods (are a pink staining, splinter shaped inclusion due to a rod shaped alignment of primary granules found only in myeloproliferative processes) to identify the blast type:

n AML – the myeloblast is a large blast with a moderate amount of cytoplasm, fine lacey chromatin, and prominent nucleoli. 10-40% of myeloblasts contain auer rods.

n ALL – in contrast to the myeloblast, the lymphoblast is a small blast with scant cytoplasm, dense chromatin, indistinct nucleoli, and no auer rods.

myeloblastsjpg lymphoblast

Myeloblast with auer rods Lymphoblasts

Auer rods in AML

Cytochemistry

Cytochemistry – help to classify the lineage of a leukemic cell (myeloid versus lymphoid). Myeloperoxidase – is found in the primary granules of granulocytic cells starting at the late blast stage. Monocytes may be weakly positive.

myeloperoxidase

Sudan black stains phospholipids, neutral fats and sterols found in primary and secondary granules of granulocytic cells and to a lesser extent in monocytic lysosomes.

sudan black

Rare positives occur in lymphoid cells. Nonspecific esterase – is used to identify monocytic cells which are diffusely positive. T lymphocytes may have focal staining.

esterase

Acid phosphatase may be found in myeloblasts and lymphoblasts. T lymphocytes have a high level of acid phosphatase and this can be used to help make a diagnosis of acute T-lymphocytic leukemia.

adid ptase

Leukocyte alkaline phosphatase – is located in the tertiary granules of segmented neutrophils, bands and metamyelocytes. The LAP score is determined by counting 100 mature neutrophils and bands. Each cell is graded from 0 to 5. The total LAP score is calculated by adding up the scores for each cell.

alk ptase

Chronic Lymphocytic Leukemia

Neoplastic proliferation of small mature-appearing lymphocytes

>99 % are B-cell derived

Older patients (> 40)

Disease may be discovered incidentally

Fatigue, weakness, weight loss, anorexia, and/or recurrent infections may occur

Variable splenomegaly and non tender lymphadenopathy

Leukemic counterpart of small lymphocytic

Lymphoma

Lab diagnostics

Absolute lymphocytosis (> 4000/mm³)

Smudge cells

Variable anemia, neutropenia, and thrombocytopenia

Hypercellular bone marrow with lymphocytic infiltrates

Hypogammaglobulinemia

Elevated serum LDH

Bone Marrow in CLL

Chronic Myelocytic Leukemia (CML)

15-20 % of all leukemias

Young or middle-aged patients

Asymptomatic, fatigue, abdominal fullness, early satiety, weight loss, anorexia

Splenomegaly, bone pain, bone tenderness

Proliferation of myeloid cells in blood and bone marrow (mostly myelocytes polys)

Slow progression (3 yr survival without Rx)

Ph 1 chromosome in > 95 %

Peripheral Blood in CML

Bone Marrow in CML

Hypercellular bone marrow

High Myeloid : Erythroid Ratio

Myeloid hyperplasia

Relatively few blast cells

Mostly mature neutrophils

Increased basophils and eosinophils

Increased megakaryocytes

Bone Marrow in CML

Multiple Myeloma

Multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a cancer of plasma cells, a type of white blood cell normally responsible for producing antibodies. In multiple myeloma, collections of abnormal plasma cells accumulate in the bone marrow, where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein — an abnormal antibody which can cause kidney problems. Bone lesions and hypercalcemia (high calcium levels) are also often encountered.

Myeloma is diagnosed with blood tests (serum protein electrophoresis, serum free kappa/lambda light chain assay), bone marrow examination, urine protein electrophoresis, and X-rays of commonly involved bones.

The presence of unexplained anemia, kidney dysfunction, a high erythrocyte sedimentation rate (ESR), lytic bone lesions, elevated beta-2 microglobulin, and/or a high serum protein (especially raised globulins or immunoglobulin) may prompt further testing. The globulin level may be normal in established disease. A doctor will request protein electrophoresis of the blood and urine, which might show the presence of a paraprotein (monoclonal protein, or M protein) band, with or without reduction of the other (normal) immunoglobulins. One type of paraprotein is the Bence Jones protein which is a urinary paraprotein composed of free light chains. Quantitative measurements of the paraprotein are necessary to establish a diagnosis and to monitor the disease. The paraprotein is an abnormal immunoglobulin produced by the tumor clone. Very rarely, the myeloma is nonsecretory (not producing immunoglobulins).

In theory, multiple myeloma can produce all classes of immunoglobulin, but IgG paraproteins are most common, followed by IgA and IgM. IgD and IgE myeloma are very rare. In addition, light and or heavy chains (the building blocks of antibodies) may be secreted in isolation: κ- or λ-light chains or any of the five types of heavy chains (α-, γ-, δ-, ε- or μ-heavy chains).

Additional findings include: a raised calcium (when osteoclasts are breaking down bone, releasing calcium into the bloodstream), raised serum creatinine due to reduced renal function, which is mainly due to casts of paraprotein deposition in the kidney, although the cast may also contain complete immunoglobulins and albumin.

Cancer of plasma cells

Disease of older men and women (> 60 years)

Produce abundant useless monoclonal Ig (paraprotein, M-protein)

Decreased normal Ig, infections occur

Lytic bone lesions, bone pain, pathologic fractures, hypercalcemia

Tumorous masses of plasma cells (spine, skull, ribs, pelvis)

Renal failure may develop

Peripheral Blood in MM

Bone Marrow in Multple Myeloma

Lymphogranulomatosis

Lymphogranulomatosis – is a malignant tumor of lymphoid tissue with lesion of lymph nodes and organs, characterized by growth of the giant cells called Reed-Sternberg- Beresovsky cells, large and small atypical cells (Hodgkin,s cells) and inflammatory infiltration.

Hodgkin disease: a giant mononuclear cell with a large nucleolus (arrow) and wide cytoplasmic

layer (Hodgkin cell), surrounded by small and medium-sized lymphocytes.

Hodgkin disease: giant binuclear cell (Reed–Sternberg giant cell).

Hodgkin's lymphoma, also known as Hodgkin lymphoma and previously known as Hodgkin's disease, is a type of lymphoma, which is a cancer originating from white blood cells called lymphocytes.

Patients with Hodgkin's lymphoma may present with the following symptoms:

§ Lymph nodes: the most common symptom of Hodgkin's is the painless enlargement of one or more lymph nodes, or lymphadenopathy. The nodes may also feel rubbery and swollen when examined. The nodes of the neck and shoulders (cervical and supraclavicular) are most frequently involved (80–90 % of the time, on average). The lymph nodes of the chest are often affected, and these may be noticed on a chest radiograph.

§ Itchy skin

§ Night sweats

§ Unexplained weight loss

§ Splenomegaly: enlargement of the spleen occurs in about 30 % of people with Hodgkin's lymphoma. The enlargement, however, is seldom massive and the size of the spleen may fluctuate during the course of treatment.

§ Hepatomegaly: enlargement of the liver, due to liver involvement, is present in about 5 % of cases.

§ Hepatosplenomegaly: the enlargement of both the liver and spleen caused by the same disease.

§ Red-coloured patches on the skin, easy bleeding and petechiae

due to low platelet count (as a result of bone marrow infiltration, increased trapping in the spleen etc. – i.e. decreased production, increased removal)

§ Systemic symptoms: about one-third of patients with Hodgkin's disease may also present with systemic symptoms, including low-grade fever; night sweats; unexplained weight loss of at least 10 % of the patient's total body mass in six months or less, itchy skin (pruritus) due to increased levels of eosinophils in the bloodstream; or fatigue (lassitude).