Hematology Course1

Dear 5th year student Re: Hematology course The hematology course has been designed to be interactive and clinically oriented. Your hematology course consists of didactic lectures, multidisplinary seminars, case scenarios and practical. Please, read the course objectives prior to starting the course to exactly know what is required from each of you at the end of the course. Case scenarios supplemented in this course are meant to enhance your abilities in problem solving: understanding the patient’s complaints, looking for proper signs of any hematological problem, understand the sequence of events, formulate a plan, order the proper tests and be able to initiate a therapy and follow outcome of treatment. To gain the maximum benefit, you are requested to read these case scenarios upfront write your answer in a separate sheet of paper and be ready to give it to your tutor upon request. Active participation and discussion is required from every body in class, your tutor is only a facilitator and not information provider during these sessions. You will be divided into groups (seminar rooms) and be assigned different tutor each module. Please, send me your comments any time. This is the only way to improve our teaching abilities. Sincerely 5th year hematology course Coordinator

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INDEX Sr. No. 1 2 3 4 5 6

Particulars AIM & OBJECTIVES COURSE TIMETABLE APPROACH TO ANEMIA HEMATOLOGY LABORATORY – NORMAL VALUES PRINCIPLES OF HEMATOLOGY TESTS SLIDES

Page No 5 7 10 13 14 17

MODULE I – DISORDERS OF RED BLOOD CELLS 4 5 6 7 8 9 10 11

NUTRITIONAL ANEMIA MEGALOBLASTIC ANEMIA FOLIC ACID DEFICIENCY ALCOHOL AND HEMATOPOIESIS APLASTIC ANEMIA THE HEMOLYTIC ANEMIAS BLOOD TRANSFUSION PRACTICE PROBLEMS FOR TUTORIALS

20 22 23 24 24 25 28 31

MODULE II- WBC DISORDERS 12 13 14 15 16 17 18 19

LYMPHADENOPATHY AND SPLENOMEGALY BENIGN LEUKOCYTE DISORDERS HEMATOPATHOLOGY TESTING HODGKIN’S AND NON-HODGKIN’S LYMPHOMAS MYELOMA LEUKEMIAS PROBLEMS FOR TUTORIALS MYELOPROLIFERATIVE DISORDERS

37 38 40 42 46 48 51 54

MODULE III & IV - HEMOSTASIS AND THROMBOSIS 20 21 22 23

BLEEDING DISORDERS COAGULATION DISORDERS ANTITHROMBOTIC AGENTS PROBLEMS FOR TUTORIALS

57 61 66 68

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PROBLEMS FOR TUTORIALS Sr. No.

Particulars

Page No

MODULE I – DISORDERS OF RED BLOOD CELLS 1 2 3 4 5 6 7 8 9 10

ANEMIA AND CONFUSION ANEMIA AND CONFUSION IN ALCOHOLIC ANEMIA AND RETICULOCYTOSIS ANEMIA AND ARTHRITIS ANEMIA AND HEART DISEASE ANEMIA, WEAKNESS AND JOINT PAIN ANEMIA AND MACROCYTOSIS FEVER, ANEMIA AND RETICULOCYTOSIS CHRONIC FATIGUE AND ANEMIA A YOUNG PATIENT WITH RECURRENT BONY ACHES

31 31 32 32 32 33 33 33 34 35

MODULE II- WBC DISORDERS 1 2 3 4 5 6 7 8

ADULT MAN WITH LYMPHOCYTOSIS A YOUNG FEMALE WITH NECK LUMP POLYCYTHEMIA ATYPICAL LYMPHOCYTOSIS LEUKOCYTOSIS AND SPLENOMEGALY PANCYTOPENIA LEUKOCYTOSIS LYMPHADENOPATHY

51 51 51 51 52 52 52 52

MODULE III & IV - HEMOSTASIS AND THROMBOSIS 1 2 3 4 5 6 7 8 9 10 11

A YOUNG BOY WITH SWOLLEN KNEE A BLEEDING YOUNG WOMAN FEVER AND COAGULOPATHY A BLEEDER BABY BOY BLEEDING GUMS AND BRUISING A YOUNG PATIENT WITH SUDDEN SHORTNESS OF BREATH OFTEN MISSED BLEEDING PROBLEM POST OPERATIVE DVT ALCOHOLIC AND COAGULOPATHY FEVER POST SPLENECTOMY BLEEDING WITH SEVER THROMBOCYTOPENIA

68 68 69 69 69 70 70 71 71 71 72

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COURSE IN CLINICAL HAEMATOLOGY Aim Extensive knowledge of common disorders as encountered in the practice of clinical hematology; to enable the students to acquire requisite clinical skills for their diagnosis, and for planning and carrying out appropriate interventions for their prevention and management; to emphasize the emotional response and to understand the consequences of patient’s sickness.

Objectives At the completion of the course in clinical hematology, the student shall demonstrate: ­ Knowledge of cardinal manifestations of hematological diseases ­ Comprehension of etiology, pathophysiology pathogenesis, diagnosis, and principles of management of hematological diseases. ­ Understanding of the basic principles of laboratory investigations of hematological diseases, and the ability to interpret relevant data to arrive at appropriate diagnosis of a hematological disorder. ­ Ability to correlate relevant aspects of basic sciences and molecular biology with the clinical manifestations of hematological diseases. ­ Recognition of the impact of disease on patient's social, emotional and professional life.

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Module II : White Blood Disorders At the completion of the study of disorders affecting white blood cells, the students shall be able to: -

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Specific Learning Objectives The course in clinical hematology consists of 4 units with specified learning objectives for each unit.

Module I: Disorders of Red Blood Cells At the completion of the study of the disorders of red blood cells, the students shall be able to: -

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describe causes and pathophysiology of common types of nutritional anemias. enumerate the common causes of haemolytic anemias. discuss the pathophysiologic and molecular mechanisms that cause hemolysis, and to differentiate between intravascular and extravascular cause of hemolysis describe the clinical presentation, plan of

investigations, and be able to interpret, integrate and correlate salient points in clinical history, physical findings and laboratory data with a view to making a diagnosis of the type and cause of anemia in a patient plan and prescribe a rational schedule of management, based on a sound knowledge of the mechanism of action, dose, duration and side effects of appropriate drug therapy identify patients with anemia requiring blood component therapy, and to plan a management schedule including the type, amount, rate and the frequency of such administration advise the patient and the family regarding measures to be adopted for prevention or recurrence of anemia

differentiate benign from malignant causes of leukocytosis describe the clinical presentation, plan of investigations, and be able to interpret relevant laboratory data, so as to reach likely diagnosis and prognosis in a patient who may present with leukocytosis and/or lymphadenopathy explain basic molecular abnormalities underlying malignant transformation of haematopojetic cells, and resulting in hematological malignancies plan and prescribe a rational schedule of management, based on a sound knowledge of the mechanism of action, dose, duration and side effects of appropriate drug therapy

Module III: Coagulation Disorders At the completion of the study of coagulation disorders, the students shall be able to: -

-

discuss common causes of bleeding disorders and their pathogenesis describe the clinical presentation, plan of investigations and interpret, integrate and correlate salient points in clinical history, physical findings and laboratory data so as to reach a correct diagnosis in a patient presenting with a bleeding disorder describe the common mode of clinical presentation and demonstrate ability to

5

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interpret relevant laboratory data in patients with following bleeding disorders Haemophilia von Willebrand's disease Acquired coagulation deficiency secondary to vitamin K deficiency, liver disease, chronic renal failure, and DIC. discuss common causes of thrombocytopenia. plan and interpret salient points in clinical history, physical findings and relevant laboratory data to establish diagnosis of the type and cause of thrombocytopenia in a patient. Plan and administer rational therapy to a patient with thrombocytopenia. Describe the indications, schedule of administration and side effects of the use of fresh frozen plasma, cryoprecipitate and platelets concentrates in the management of bleeding disorders

Module IV: Thrombotic Disorders At the completion of this unit of study, the students shall be able to: -

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discuss pathophysiologic mechanisms of thrombosis and describe molecular basis of inherited thrombophilia enumerate common causes of acquired and inherited thrombotic disorders demonstrate ability to correlate salient points in clinical history and physical findings and to plan and interpret laboratory investigations so as to make dignosis of a thromboembilic disorder describe mechanism of action, dose, frequency, mode of administration, and adverse effects of heparin and coumadin anticoagulants

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HAEMATOLOGY COURSE 5 YEAR STUDENTS 2005-2006 TH

FIRST WEEK Date

Time P.M.

Saturday 17/12/05

2 – 2.50

Lecture: Approach to Anemia

3 – 3.50

Case Study: Microcytic Anemia in a young woman

2 – 2.50

Case Study: Macrocytic Anemia and confusion

Medicine Pathology

3 – 3.50

Lecture: Hemolytic Anemia (Immune & Hereditary)

Medicine

2 – 2.50

Case Study: Normocytic Anemia and Reticulocytosis

Medicine Pathology

3 – 3.50

Lecture: Hemoglobinopathy (Thalassemia/SCA)

Pathology

2 – 2.50

Communication Skills

Community Med.

3 – 3.50

Case Study: A young man with recurrent bony aches & jaundice

Medicine Pathology

4 – 4.50

Case Study: Unexplained Microcytic Anemia in a Kuwaiti patient

Medicine Pathology

2 – 3.50

Practical (Anemia)

Pathology

Sunday 18/12/05

Monday 19/12/05

Tuesday 20/12/05

Wednesday 21/12/05

Topic

Lecturer/Department Pathology Medicine Pathology

SECOND WEEK Page 7 of 72

Saturday 24/12/05

2 – 2.50

Lecture: Acute Leukemia

3 – 3.50

Lecture: Myeloproliferative Disorders (CML/ET/PRV/MF)

Medicine Medicine

Date

Time P.M.

Sunday 25/12/05

2 – 2.50

Case Study: Polycythemia in a smoker

Medicine Pathology

3 – 3.50

Case Study: Leukocytosis and Splenomegally

Medicine Pathology

2 – 2.50

Lecture: Lymphoproliferative Disorders

Medicine

Monday 26/12/05

Topic

3 – 3.50 Case Study: A young girl with neck lump Tuesday 27/12/05

Lecturer/Department

Medicine Pathology

2 – 2.50 Communication Skills

Community Medicine

3 – 3.50 Lecture: Blood Component Therapy

Pathology

Wednesday 2 – 3.50 28/12/05

Practical (WBC disorder)

Pathology

THIRD WEEK Saturday

2 – 2.50 Lecture: Approach to patient with

Medicine

Page 8 of 72

31/12/05

Sunday 01/01/06

bleeding tendency 3 – 3.50 Lecture: Thrombocytopenia/Hemophilia

Medicine

2 – 2.50

Case Study: Woman with recurrent bleeding

Medicine Pathology

3 – 3.50

Case Study: A bleeder child

Medicine Pathology

Monday 02/01/06

2 – 3.50 MDS: Thrombosis

Medicine Pathology Radiology

Tuesday 03/01/06

2 – 2.50 Case Study: Elderly man with unexplained thrombocytosis

Medicine Pathology

3 – 3.50 A boy with painful knee

Medicine Pathology

Wednesday 04/01/06

2 – 3.50 Pre-exam Revision

FOURTH WEEK Saturday 07/01/06 Sunday 08/01/06 Monday 09/01/06

HOLIDAY

9.30 – 11.00 AM

HAEMATOLOGY ASSESSMENT

Medicine Pathology

HOLIDAY

Tuesday 10/01/06

HOLIDAY

Wednesday 11/01/06

HOLIDAY

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Approach to Anemia Classification of Anemia The classification of anemias is based on the size of the red blood cell and thus, divided into Microcytic (hypochromic), Macrocytic and Normocytic (normochromic). Normograms for the anemias can be used to order the appropriate tests for each group as follows:

Microcytic Anemia F e r r it in R educed

N o rm a l

I r o n d e f ic ie n c y

Further investigations to find the cause are necessary

H B e l e c t r o p h o r e s is

In c re a s e d

A n e m i a o f C h r o n i c D is e a s e

B o n e m a rro w R i n g e d s id e r o b l a s t s

If HB electrophoresis is normal then do alpha gene mapping

Hypochromia Microcytic Note: Anisocyosis: RDW poikilocytes

Thalassemia Trait

Variables

IDA

RBC count

< 5 x1012/L

> 5 x1012/L

MCV to HB level

Proportional

Disproportional

Mesner criteria MCV/RBC count

> 13

< 13

Red cell distribution width (RDW)

Elevated

Normal

Morphology

Anisocytosis Poikilocytosis / Elliptocytes

Target cells

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Normocytic Anemia It is important to have a retics count done in normocytic anemia. As a high retics count indicates a healthy proliferating bone marrow. And this usually indicates hemolytic process or bleeding episode. It is important to calculate the absolute retics count or retics production index. Please, look at tests listed above for the investigations of Normocytic anemia.

R e t ic u lo c y t e s In c re a s e d

H e m o ly s is I m m u n e v s . n o n - im m u n e LDH/Bili/haptoglobin / Urine hemosiderin Coomb's test / Cold agglutinins G-6PD / HAMS test / Osmotic fragility

N o rm a l D e c re a s e d P o s t H e m o r r h a g ic

B o n e M a rro w E x a m A b n o rm a l

N o rm a l

H y p o b l a s t ic I n f ilt ra t io n D y s e r y t h r o p o i e t ic

RFT LFT E n d o c r in e F e r r it in

Polychromasia: expressed as high reitcs count.

Fragmented RBC: schistocyte

Page 11 of 72

Macrocytic anemia Here we need to differentiate between megaloblastic and non-megaloblastic anemias. In cases of megaloblastic anemia we need to request schilling test in case of Vit.B12 deficiency. In case of Folate deficiency we need a detailed dietary history, as poor dietary intake is usually responsible. Please look at the investigations listed below for Macrocytic anemia.

B lo o d a n d M a r ro w M o r p h o lo g y M e g a l o b l a s t ic

N o n - M e g a l o b l a s t ic

C lin ic a l D a ta S e r u m V it a m in e s

L iv e r d is e a s e M yxed e m a M D S

N o d e fic ie n c y

F o la t e D e f ic ie n c y

B 1 2 d e f ic ie n c y

C o n g e n it a l D ru g s

D ie t

S c h ilin g t e s t

Dimorphic anemia If things do not “compute’ i.e. the hematological picture dose not fit with scheme then consider dimorphic blood pictures (two different combined types of anemia).

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HEMATOLOGY LABORATORY – NORMAL VALUES (Expressed in S.I. Units)

GENERAL HEMATOLOGY

Thrombin Clotting Time

9 – 11 sec

Bleeding Time (IVY)

2 - 10 min

Test WBC (x109/L)

Male 4.0 – 10.0

Female 4.0 – 10.0

FDP Assay

0 – 10 ug/L

RBC (x 109/L)

4.5 – 6.5

4.0 – 5.6

Fibrinogen Level

1.7 – 3.4 g/L

Hb (g/L)

135-180

115-160

Factor Assays

Het (L/L)

0.40 – 0.5

0.3 - 0.4

Antithrombin III

0.8 – 1.2 u/mL

MCV (fL)

79.0 – 97.0

79.0 – 97.0

D-dimer

0 – 0.5

MCH (pg)

27.0 – 32.0

27.0 – 32.0

MCHC (g/L)

320 – 360

320 – 360

MPV (fL)

7.5 – 11.1

7.5 – 11.1

RDW (um)

8.6 – 13.0

8.6 – 13.0

PLT (x109/L)

150 – 450

150 – 450

RETIC (x109/L)

10.0 – 75.0

10.0 – 75.0

ESR (mm/h) (Wintrobe)

0.0 – 7.0

0.0 – 15.0

WBC Differential

%

0.50 = 1.5 u/Ml

Absolute (x 109/L)

NEUT

0 .40 - 0.75

2.5 – 7.5

Lymph

0.20 - 0.40

1.5 – 3.0

MONO

0.02 - 0.10

0.2 – 0.8

EOS

0.01 – 0.06

0.04 – 0.4

BASO

< 0.01

0.00 – 0.1

COAGULATION Prothrombin Time (P.T.)

9 – 13 sec

International Normalized Ratio (INR)

< 13

Partial Thromboplastin Time (P.T.T)

26 – 36 sec

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PRINCIPLES OF HEMATOLOGY TESTS HEMOGLOBINOMETRY, CELL COUNTS AND INDICES

SIZE

Accurate electronic cell counting has largely replaced previous counting chamber methods in most laboratories. These procedures may, or may not, be automated but either way the parameters shown below may be measured or calculated.

SHAPE

WBC RBC Hb Hct MCV MCH MCHC RDW

9

x 10 /L x 1012/L g/L L/L fL pg g/L um

DIAGNOSTIC PATTERNS OF RBC INDICES 1. Normal MCV, MCHC • Normocytic, normachromic. Consider aplastic anemia, chronic disease, malignancies, renal failure, endocrine hypofunction, hemolysis, or acute blood loss. Iron deficiency may also be normocytic and normochromic initially. 2. Low MCV ± low MCHC • Microcytic ± hypochromic. Consider iron deficiency, chronic disease, thalassemia and rarely sideroblastic anemia and lead poisoning. 3. Increased MCV • Macrocytic. Consider Vitamin B-12 or folate deficiency, reticulocytosis, alcholism, liver disease and myelodysplasia

RED CELL MORPHOLOGY Careful examination of a peripheral blood film is an integral part of the initial laboratory investigation confirming abnormalities detected by the Coulter Counter and seeking further clues to a specific diagnosis. At the same time, leukocytes and platelets should be assessed. Morphologic assessement includes:

Normocytic Microcytic Macrocytic Anisocytosis

- normal size (7.5u diameter) - 6u diameter - 9u diameter - variation in size

Normal • Biconcave disc Poikilocytosis • Variation in size Oval (or elliptical cells) • Large numbers suggest hereditary elliptocytosis, but oval cells are also seen in many anemias (common in iron deficiency and thalassemia). Oval macrocytes suggest megaloblastic anemia Spherocytes • Due to membrane loss, producing round spheres which are densely staining, and appear small. Large numbers suggest hereditary spherocytosis or immune hemolytic anemia (IgG type) Target cells • Due to membrane excess. Suggest liver disease, thalassemia, hemoglobinopathy or postsplenectomy state. Fragmented cells • Many forms (schistocytes, helmet cells). Suggest fragmentation hemolysis with prosthetic heart valves, micro-angiopathic process (DIC, TTP, HUS) Sickle cells • In homozygous form, mainly Acanthocytes (spine cells) • Liver disease, post splenectomy state Spurr cells (echinocytes) • Uremia, often artifact STAINING Normachromic • Central pallor occupies one-third of RBC Hypochromic • Decreased Hgb concentration (increased central pallor). Polychromasia • Bluish-gray staining (indicates reticulocytes).

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INCLUSIONS Basophilic stippling •

Ribosome precipitation; seen in many anemias, but helpful in diagnosis of thalassemia and lead poisoning. Howell-Jolly bodies – nuclear remnants, usually seen in post-splenectomy. Nucleated RBC's • Suggest extramedullary hematopoiesis (myeloid metaplasia), active erythropoiesis (bleeding, hemolysis) or metastatic cancer in bone marrow. Heinz Bodies • Require special supravital stain; represent denatured Hgb and seen with oxidative hemolysis, some enzymopathies and hemoglobinopathies. Parasites • Eg. Malaria

DISTRIBUTION Rouleaux • Suggest immunoproliferative disease if extensive, but lesser amounts common in many acute and chronic diseases. Autoagglutination • Aggregates of RBC's due to cold agglutinations.

RETICULOCYTE COUNT Detects young erythrocytes (by precipitation of residual RNA with special stain) and usually provides a reliable source of erythropoiesis. Reticulocytes last upto 48 hours in circulation and 1% erythrocytes are made each day; therefore, up to 2% reticulocytosis is considered normal (absolute 10 – 75 x 109/L). Reticulocytopenia • Suggests bone marrow depression, infiltration (eg leukemia) or lack of erythropoietin, iron, etc. Reticulocytosis • Suggests hemolysis, blood loss, or response to treatment. If the reticulocyte count is reported as a percentage and the patient is anemic, it should be converted to an absolute count to assess effectiveness of reticulocyte response.

•

Qualitative – eg. Normoblastic vs megaloblastic erythropoiesis. Presence or absence of iron stores. Ring sideroblasts 2. Assessment of marrow infiltrates • Leukemia, myeloma, metastatic carcinoma Closed bone marrow biopsy (Jamshidi needle) provides a better sample for assessment of cellularity and is essential if the bone marrow examination is being performed to detect marrow fibrosis, or neoplastic infiltration (carcinoma, lymphoma, Hodgkin's disease)

SEDIMENTATION RATE The popular "Sed rate" is one of the simplest, yet poorly utilized test in laboratory medicine. The usual method used involves placing a known volume of anticoagulated blood in a special tube of specified length (100 mm Wintrobe, or 200 mm Westergren) and allowing the red cells to sediment for one hour. In normal persons the red cells sediment only 7 – 15 mm (Wintrobe) during this hour but this rate increases with many illness due to an increase in globulins eg increased gammaglobulins (in myeloma, liver disease or chronic infection) or acute phase reactants such as haptoglobin and fibrinogen (in any traumatic, infectious, inflammatory or neoplastic illness) The Sedimentation Rate has been used to differentiate organic (elevated) from functional (normal) disease and to diagnose or to follow progress of inflammatory and infectious processes. A normal Sedimentation Rate does not rule out serious illness, and an elevated Sedimentation Rate is a non-specific finding.

OTHER LABORATORY PROCEDURES A careful evaluation of some of all of the previous basic laboratory tests may be sufficient for a diagnosis but often they serve, in conjunction with clinical assessment, to direct specific investigations. These investigations are described elsewhere. From this point, careful selection of laboratory tests is essential. Widespread ordering of many expensive and unnecessary investigations without regard to basic test results is wasteful and unrewarding.

SPECIAL CONSIDERATIONS FOR PEDIATRIC PATIENTS BONE MARROW EXAMINATION Most common indications for marrow aspirations include 1. Assessement of cytopenias (anemia, neutropenia, thrombocytopenia) • Quantitative – eg. Hypocellular vs hypercellular • Presence of absence of precursor cells (eg. Megakaryocytes)

The following differences between adult and pediatric patients require emphasis: 1. Level of Hemoglobin: At birth, the Hb is very high but shortly after birth, there is decrease in Hb (as well as Hematocrit and RBC count). The lowest point is reached between 3 and 6 months with Hb as low as 100g/L being normal. By one year of age infants are often physiologically slightly iron deficient. This is more marked in

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premature infants who require supplemental iron to avoid iron deficiency anemia.

2. White Blood Count and Differential: Initially in neonates there is neutrophilic leukocytosis, but within a week a lymphocyte predominance develops and lasts until about five years of age when the percentage of lymphocytes and granulocytes is equal. Later the child develops the usual adult picture of neutrophil predominance. Pertusis causes a striking lymphocytosis with white counts up to 100 x 109/L. The lymphocytes

are small and not atypical as seen in viral infections.

3. Lymph nodes: There is a marked lymphoid hyperplasia of the lymphoid system in children reaching a peak between 3 and 5 years. Tonsillar enlargement is a common finding and enlargement of the cervical axillary and inguinal lymph nodes is frequently found on routing examination.

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Basophilic stippling

Burr Cells

Elliptocytes

Heinz Bodies

Helmet cells

Hemoglobin C crystals

Howell-Jolly bodies

Hypochromia

Macroovalocytes

Malaria-Plasmodium Falcipuram

Malaria- Plasmodium Vivax

Megakaryoblast and promegakaryocyte

Megalocytes-1

Megalocytes-2

Microcytosis

Pappenheimer bodies- iron stain

Pappenheimer bodiesWright stain

Poikilocytes

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Polychromatophils

Rouleaux

Sickle Cell

Spherocytes

Target Cells

Teardrop cells

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MODULE I DISORDERS OF RED BLOOD CELLS OBJECTIVES •

Describe causes and pathophysiology of common types of nutritional anemias.

•

Enumerate the common causes of haemolytic anemias.

•

Discuss the pathophysiologic and molecular mechanisms that cause hemolysis, and to differentiate between intravascular and extravascular cause of hemolysis.

•

Describe the clinical presentation, plan ofinvestigations, and be able to interpret, integrate and correlate salient points in clinical history, physical findings and laboratory data with a view to making a diagnosis of the type and cause of anemia in a patient.

•

Plan and prescribe a rational schedule of management, based on a sound knowledge of the mechanism of action, dose, duration and side effects of appropriate drug therapy.

•

Identify patients with anemia requiring blood component therapy, and to plan a management schedule including the type, amount, rate and the frequency of such administration.

•

Advise the patient and the family regarding measures to be adopted for prevention or recurrence of anemia.

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NUTRITIONAL ANEMIA: IRON ABSORPTION AND METABOLISM Iron is absorbed largely in the duodenum and upper jejunum. The absorption of ferric iron, but not ferrous or hemoglobin iron, is facilitated by hydrochloric acid, and impaired after gastrectomy. The rate of iron absorption is regulated by physiological needs, and is increased in iron deficiency and other anemias. Human control their total body iron by absorption and not iron excretion. The average North American diet contains about 10 - 15 mg of iron daily. Total daily iron loss in men is about 1 mg (0.5 mg in feces, 0.1 mg in sweat). The average daily iron loss in women during reproductive life is about 2 mg.

IRON DEFICIENCY Iron depletion without anemia is very common; some degree of iron deficiency is present in approximately 10% of most populations. Mild anemia, due to iron deficiency, is frequently not hypochromia and there are other causes of hypochromic anemia other than iron deficiency; hence hypochromic anemia and iron deficiency anemia are not synonymous.

ETIOLOGY OF IRON DEFICIENCY SPECIFIC CAUSES 1. Menstruation The following points are important to assess the severity of menstrual bleeding: A. The number of pads used. Greater than 12 pads is considered abnormal. B. If double pads are used, does blood soak through? C. Are there large clots? D. How many days do the periods last; more than five days being suggestive of increased bleeding. E. Pregnancy and Lactation:

2. Impaired Absorption of Iron This is rarely a cause of iron deficiency except in the presence of sprue or as the result gastrointestinal surgery. Most patients who have had total gastrectomy and up to 50% of patients with sub-total gastrectomy develop iron deficiency, although this may require several years after surgery.

3. Gastrointestinal Bleeding Determining the source of gastrointestinal bleeding is a challenge. Radiologic examinations and endoscopy are the chief aids to the diagnosis. With respect to gastric bleeding, corrosive injury from several drugs (e.g. Aspirin, phenylbutazone, Indomethacin) occurs with considerable frequency.

4. Intravascular Hemolysis Paroxysmal nocturnal hemoglobinuria (an exceedingly rare disease) and much more commonly, intravascular hemolysis due to erythrocyte trauma from a cardiac valvular prosthesis may both result in an iron deficiency Intravascular hemolysis leads to trapping of hemoglobin in renal tubular cells followed by formation of hemosiderin which is lost in desquamated cells. Hence, hemosiderinuria occurs with a loss of as much as 15 mg of iron per day.

CLINICAL FEATURES OF IRON DEFICIENCY The iron deficient patient may be completely a symptomatic, even in the presence of marked anemia if this has developed gradually. There is good evidence that the anemia itself may not be responsible for the symptoms, muscle, etc. Symptoms frequently reported include: fatigue, tinnitus, palpations, weakness and lightheadedness. Sore tongue or sore mouth sometimes occurs and there may rarely be severe stomatitis. There may be some loss of papillae from the tongue and fissures at the corners of the mouth. Mild splenomegaly occurs rarely. Koilonychias (spoon nails), a condition in which nails are flattened or even concave and are brittle, occurs uncommonly but is of considerable diagnostic help when it occurs.

LABORATORY INVESTIGATION 1. Examination of the Stained Blood Film and Red Cell Studies. The classical appearance of red cells in iron deficiency is that of hypochromia and microcytosis. These features are often helpful in making a diagnosis but two problems prevent complete reliance on it. One is that there are causes such as "anemia of chronic disease", thalassemia and sideroblastic anemia for erythrocyte hypochromia other than iron deficiency. The second problem is that the anemia of iron deficiency may be normochromic and normocytic until quite severe.

2. Serum Iron and Iron Binding Capacity Determination (these studies may be limited values) Normal Range Total Iron Binding Capacity 45 - 81 umol/L (TIBC) Unsaturated Iron Binding Capacity 27 - 54 umol/L (UIBC)

Serum Iron *Percent saturation = serum iron x 100% Total iron binding capacity *Values of less than 15% are suggestive of Page 20 of 72

iron deficiency. 3. Serum Ferritin The level of ferritin in the plasma appears to be a reliable indicator of the size of body iron stores in most situations. The normal range is approximately (adult male: 15-350 ug/L; adult female: 15-200 ug/L). Values below the normal range indicate iron deficiency and correlate well with absent marrow iron. With chronic inflammatory or malignant disease, ferritin tends to be elevated or at least normal, even when iron stores are absent. Therefore, a low serum ferritin is diagnostic of iron deficiency, whereas a normal level in a patient with chronic inflammation, may not actually reflect the iron stores.

4. Bone Marrow Iron stores are usually evaluated following Prussian blue staining of the bone marrow smear. This is a highly reliable method of assessing iron deficiency, since this store is exhausted before anemia occurs.

5. Examination of Stools for Blood Occult bleeding causing iron deficiency is usually of too small a volume to cause blackening of the stool. Testing for occult bleeding may be done with the use of benzidine, Guaiac or orthotolidine.

TREATMENT OF IRON DEFICIENCY 1. Principles of Treatment: (Determine the cause)

c. An iron-deficient patient with normal gastrointestinal absorptive function will respond to any oral iron preparation give in an adequate dosage for an adequate period of time. d. Patient tolerance is largely related to dosage. e. Ferrous sulphate is much cheaper than any other iron preparation.

2. Oral Iron Preparations In a daily dose of 0.9 grams given as 300 mg t.i.d., p.o., ferrous sulphate results in rapid correction of iron deficiency anemia after a latent period of approximately 14 days. Three hundred (300) mg of ferrous sulphate t.i.d. provides 180 mg of elemental iron per day, of which about 10% is absorbed by the normal individual and up to 25 or even 40% in the iron-deficient individual. Replenishment of iron stores requires at least four months of treatment after the return of the hemoglobin concentration to normal. Thus, in the absence of continuing blood loss, oral iron therapy should be continued for at least six months. Continued blood loss requires more chronic therapy.

3. Parenteral Iron Therapy. The indications for parenteral iron therapy are very limited since oral therapy is Generally well tolerated, effective and inexpensive.

a. Iron deficiency is the only disorder that responds to iron administration. b. A search for chronic blood loss often must accompany treatment.

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MEGALOBLASTIC ANEMIA Megaloblastic anemia is a term used for an anemia with a macrocytic blood film and megaloblastic erythroid maturation in the bone marrow. These changes are due to defective DNA synthesis, most often secondary to deficiency of vitamin B12, or folic acid. Other organs, such as G.I. tract and CNS may be affected. The common findings are:

PERIPHERAL BLOOD Pancyopenia: A moderate reduction in leukocytes and platelets is usual, and anemia may vary from mild to severe. Blood film: The erythrocytes vary greatly in size and shape, but macrocytes predominate and oval macrocytes may be seen. The neutrophils have hypersegmented nuclei (6 or more lobes). Polychromasia is not conspicuous (and reticulocyte count is not increased).

VITAMIN B12 DEFICIENCY NUTRITIONAL REQUIREMENTS Vitamin B12 is required in small amounts (1 - 2 ug) daily and since adequate B12 is found in all foods of animal origin it is also impossible for anyone other than a strict vegetarian to become B12 deficient on a dietary basis. Vitamin B12 is not present in vegetables and fruit. Body stores are 2 - 3 mg enough to last 3 - 4 years.

ABSORPTION Normal B12 is absorbed selectively in the ileum by the intrinsic factor mechanism. Intrinsic factor is a glycoprotein, secreted by gastric parietal cells. It quickly binds with vitamin B12 released from food in the stomach, and transports the B12 to specific sites of attachment on the brush border of the ileal mucosa. The B12 is absorbed, after several hours delay in the ileal mucosa, and is carried in the blood stream attached to proteins (transcoballamins).

CAUSES OF VITAMIN B12 DEFICIENCY In comparison to the lymph, many RBC are large and oval

Hypersegmented Neutrophil

Bone Marrow: erythroid hyperplasia, and

Nutritional - vegans MALABSORPTION Gastric causes - pernicious anemia (P.A.) ­ gastrectomy Intestinal causes - Blind loop syndrome ­ sprue, ileal resection ­ Crohn's disease ­ Fish tape

megaloblastic maturation as shown below

.

Giant metamyelocyte

Dysmegakaryopoiesis Erythroblast

OTHER LABORATORY TESTS Due to the breakdown of erythroid cells, mainly in the bone marrow (intramedullary hemolysis) the following abnormalities may be seen, although there is no need for them to be ordered: 1. Serum bilirubin - mild increase, mainly indirect fraction 2. Lactic dehydrogenase (LDH) - may be greatly increase; LDH1 predominates 3. Decreased haptoglobin, and increased methemalbumin.

PERNICIOUS ANEMIA Adult P.A. occurs in both men and women, usually over the age of forty, and is characterized in the typical case by triad of: a) Megaloblastic anemia: b) Glossitis: recurrent sore tongue and mouth, with progressive atrophy of papillae, leading to a smooth, red tongue. c) CNS changes: peripheral neuritis is common, and subacute combined degeneration is unique to B12 deficiency (it does not occur with folate deficiency).

AUTOIMMUNE ASPECTS: It has been suggested that P.A. is an "autoimmune disease". The B12 malabsorption is secondary to lack of intrinsic factor production, due to gastric atrophy. The gastric mucosa is invaded by lymphocytes and plasma cells, and there is a high incidence of parietal cell antibody (95%) and intrinsic factors antibody (30 60%).

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LABORATORY INVESTIGATION The specific laboratory tests are: 1. Serum B12 Vitamin B12 is measured in the serum by radioimmunoassay. Serum B12 should be measured usually along with folate, before any treatment is given, or a Schilling test performed. A low serum B12 and normal serum folate is virtually diagnostic of B12 deficiency; further tests are necessary to determine the cause. A slight decrease in serum B12 may occur with severe folate deficiency. Serum B12 may be reduced in patients on birth control pills.

2. Gastric Juice All adult patients with P.A. have achlorhydria but is not common practice to measure this.

3. Defective B12 Absorption The Schilling test may be performed if doubt exists as to B12 absorption.

TREATMENT In pernicious anemia, 1000 ugm cyano-cobalamin intramuscularly each month will sustain a normal blood picture.

FOLIC ACID DEFICIENCY REQUIREMENTS AND DIETARY SOURCE Man is unable to synthesize his estimated daily requirement of about 50 ug per day (infants 25-50 ug per day). Meats, particularly liver, and green vegetables are good dietary sources of folic acid. Boiling vegetables in excess water will efficiently extract much of the folate content. Requirements are increased in pregnancy, hemolytic anemia, and hyperthyroidism.

ABSORPTION AND FUNCTION OF FOLIC ACID Folic acid is absorbed in the duodenum and jejunum by an active transport mechanism. Folic acid metabolites act as coenzymes in the metabolism of both DNA and RNA. There is a complex interrelationship between vitamin B12 and folic acid metabolism and function such that DNA synthesis is impaired with a deficiency of either vitamin and pharmacological doses of the opposite vitamin will partly correct the impaired DNA synthesis, megaloblastosis and anemia.

CAUSES OF FOLIC ACID DEFICIENCY 1. Dietary Deficiency Requires approximately four months to produce anemia in the healthy subject.

2. Intestinal Disease Especially gluten enteropathy (coeliac disease) and tropical sprue.

3. Drugs Commonly dilantin, primidone, and barbiturates. Oral contraceptive agents also appear occasionally to cause malabsorption. The folic acid antagonist methotrexate inhibits dihydrofolate reductase

activity and therefore conversion to active coenzyme forms.

4. Alcohol The folate deficiency of alcoholics is complex and probably involves dietary deficiency as well as impaired absorption and perhaps also decreased hepatic storage and altered utilization.

5. Pregnancy Megaloblastic anemia of pregnancy usually responds to folic acid and usually laboratory evidence of folate depletion is obtained. Requirements for the pregnant woman are not well established, but are increased and pregnant women should receive supplemental folic acid (as well as iron) to prevent deficiencies.

LABORATORY INVESTIGAIONS OF FOLATE DEFICIENCY Normal Values Serum Folate Red Cell Folate

6.8 - 18.4 nmol/L 363 nmol/L

Low serum and red cell Folate with normal or slightly reduced B12 levels is diagnostic of Folate deficiency. As with B12 deficiency, the megaloblastosis in the marrow is rapidly corrected, and reticulocytosis is prompt (3 - 7 days).

TREATMENT OF FOLIC ACID DEFICIENCY In addition to removing causative factors where possible, the administration of folic acid orally is usually required.

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ALCOHOL AND HEMATOPOIESIS Chronic and excessive alcohol ingestion causes many hematological complications by direct toxic effects, by contributing to nutritional deficiency or by complicating liver dysfunction as follows: 1. Macrocytic Anemia Usually due to folic acid deficiency on a nutritional basis (megaloblastic anemia) but even in the absence of folate deficiency, alcohol and liver disease can cause macrocytic anemia. 2. Iron Deficiency Anemia Usually due to chronic G.I. bleeding from esophageal varices or gastritis 3. Hemolytic Anemia Usually a complication of severe alcoholism with liver disease and often hyperlipidemia. Spur cells

are the morphological clue to this type of hemolysis. 4. Sideroblastic Anemia Direct toxic effect of alcohol on hemesynthesis causes ring sideroblasts, ineffective erythropoiesis, and a hypochromic anemia. 5. Hypersplenism Due to cirrhosis and portal hypertension causing congestive splenomegaly. Pancytopenia is usually present. Thrombocytopenia, often severe, may be caused by a direct toxic effect of alcohol, even in the absence of folate deficiency.

APLASTIC ANEMIA Aplastic anemia is an uncommon, but often serious, blood disorder characterized by pancytopenia due to the decreased functional capacity of a hypoplastic (fatty) bone marrow. Even in severe cases of aplastic anemia there may be small residual islands of functioning bone marrow which may cause confusion in diagnosis, and which lead to a concept that the micro-environment (the soil) rather than the stem cell (the seed) might be abnormal in these patients. However, the success of bone marrow transplantation strongly supports the concept that deficient or defective stem cells are responsible.

CLASSIFICATION IDIOPATHIC: anemia) SECONDARY

2. Anti-inflammatory drugs – phenylbutazone, indomethacin, gold, etc 3. Diuretics – thiazides 4. Anticonvulsants – dilantin Chloramphenicol rarely causes a severe irreversible aplastic anemia (1 in 30,000 cases treated) which cannot be predicted, but frequently causes a mild reversible pancytopenia (Idiosyncratic reaction). Phenylbutazone is now the commonest cause, since the use to chloramphenicol has decreased. The aplastic anemia associated with viral hepatitis is severe and often fatal. Immunologic causes of some cases of aplastic anemia have been postulated but not proven

- Heriditary (Fanconi's - Acquired - Chemical and Drugs - Radiation - Infection (viral hepatitis) - Immunologic (?autoimmune)

The cause of about 50% cases cannot be determined and these are termed idiopathic. Fanconi's anemia is an extremely rare hereditary aplastic anemia, often associated with short stature, skeletal abnormalities, hypoplastic kidneys, etc. Exposure to chemicals containing benzene is particularly hazadarous. Drugs which most frequently are associated include: 1. Antibiotics – chloraphenicol, sulfas, streptomycin

CLINICAL AND LABORATORY FEATURES Pallor, bleeding and bruising are common and infections are frequent. Lymphadenopathy and hepatosplenomegaly are characteristically absent. The anemia may be severe, usually normocytic but sometimes slightly macrocytic. Granulocytopenia is regularly seen, and lymphopenia is variable. Thrombocytopenia is usually marked, and megathrombocytes are absent.

MANAGEMENT The role of red cell, granulocyte, and platelets transfusions are discussed with the management of leukemias. When aplastic anemia is severe (granulocyte count 0.5 x 10 9/L, platelet count 20 x 10 9/L) and not reversible, the prognosis is very grave (85% mortality in one year, with median survival of 3

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months). If an HLA-compatible sibling is available as a bone marrow donor, then prompt bone marrow transplantation is the treatment of choice (not that an HLA-compatible sibling is only available in 1/4 to 1/3 of cases. For milder cases, or those unsuitable for marrow transplantation Antithymocyte globulin can cause a significant improvement in a number of cases.

high dose androgen therapy may cause improvement in anemia but not leucopenia nor thrombocytopenia. Steroids are often given a trial of 2 -4 weeks but should then be discontinued if not obviously helpful. Splenectomy has no definite role to play in these cases. The status of immunosuppressive treatment (Cyclosporin) is investigational but helpful in some cases.

THE HEMOLYTIC ANEMIAS Increased RBC destruction is the common factor in all hemolytic anemias. Hemolysis is detected by: 1. Evidence of erythrocyte destruction. 2. Bone marrow compensation 3. Decreased erythrocyte survival EVIDENCE OF ERYTHROCYTE DESTRUCTION a) Hemoglobin, hematocrit – both decreased unless hemolysis is "compensated" (normal bone marrow can increase RBC production at least 6 to 8 times) b) Hyperbilirubinemia – increased serum bilirubin, mainly indirect (unconjugated type), and increased urobilinogen in urine. c) * Increased plasma hemoglobin * Decreased serum haptoglobin * Hemoglobinuria * Hemosideriuria * Mainly intravascular hemolysis BONE MARROW COMPENSATION a) Reticulocytosis – seen as polychromasia on routine blood films, but can be specifically demonstrated on reticulocyte count. b) Erythroid hyperplasia of bone marrow DECREASED ERYTHROCYTE SURVIVAL Seldom necessary Cr51 most often used as radioactive label for patient's own erythrocytes. Requires 10 to 14 days.

CLASSIFICATION I INTRINSIC DEFECTS (INTRACORPUSCULAR) A. HEREDITARY 1. Membrane defect – HS, HE 2. Hemoglobinopathies a. Quantitative – Thalassemias b. Qualitative – Hb S, C, S-C etc 3. Enzymopathies – G6PD, PK, etc B. ACQUIRED 1. Paroxymal Nocturnal Hemoglobinuria (PNH)

II EXTRINSIC DEFECTS (EXTRACORPUSCULAR) 1. Autoimmune hemolytic anemia (Coombs positive) a. Warm antibody (IgG)- idiopathic, secondary (lupus, lymphoma, Aldomet, etc) b. Cold antibody (IgM) – idiopathic (cold hemagglutinin disease), secondary (mycoplasma, infectious mononucleosis) 2. Alloimmune hemolytic anemias – Hemolytic disease of newborn, post transfusion 3. Drug induced hemolytic anemia a. Chemical effect in absence of G6PD deficiency, or with G6PD deficiency 4. Mechanical hemolytic anemia a. Micro-angiopathic – hemolytic – uremic syndrome, TTP, DIC, etc b. Cardiac fragmentation hemolysis 5. Secondary hemolytic anemia – infectious, liver or renal disease, etc.

I INTRINSIC DEFECTS A. HEREDITARY HEMOLYTIC ANEMIAS 1. MEMBRANE DEFECTS: Hereditary Spherocytosis is an uncommon form of hemolytic anemia (incidence of 1 in 5,000 population) with an autosomal dominant inheritance. Spherocytes are usually evident on examination a routine blood film. If not obvious, then an osmotic fragility test should be ordered to detect the osmotically fragile pre-spherocyte cells. Reticulocytosis and splenomegaly are usually prominent. Splenectomy is the definitive treatment after which hemolysis ceases, although spherocytes persist in the blood. Hereditary Elliptocytosis is also called hereditary ovalocytosis and is relatively common (1 in 2,000 population) with an autosomal dominant inheritance. Most cases have little or no hemolysis but 10% or less have significant hemolytic anemia requiring splenectomy.

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2. HEMOGLOBINOPATHIES: Thalassemia is a complex group of anemias usually classified with the hemolytic anemias because hemolysis is marked in the severe homozygous forms, but the primary defect is a quantitative reduction in synthesis of globin chains. Beta chain synthesis is decreased in beta thalassemia which is mainly seen in individuals of Mediterranean background. The heterozygous form (beta thalassemia minor) is fairly common is characterized by extreme microcytosis, and hypochromasia but little or no anemia. Diagnosis is confirmed by demonstrating an increased Hb A2, and no treatment is required (although laboratory findings resemble iron deficiency, iron stores are usually normal or increased and iron therapy is contradicted) The homozygous form (beta thalassemia major) causes severe hemolysis in childhood with splenomegaly, and iron overload usually leading to death in adolescence unless vigorous transfusion and ironchelation therapy is employed. Alpha chain synthesis is decreased in alpha thalassemia which is commoner in Orientals; the inheritance is more variable resulting in clinical disorders ranging from a very mild anemia, through moderate hemolytic anemia (Hb H disease), to a very severe form causing death in utero. Sickle cell Anemia- Sickle cell trait is common (10% of American Blacks) but is usually asymptomatic and detected only by the presence of abnormal Hb electrophoresis (SA) or a positive sickle cell preparation. If the heterozygous sickle cell trait is combined with the gene for Hb C, then a more severe disorder (Hb S-C disease) occurs with hemolysis, splenomegaly, and hemolytic crises. Sickle cell anemia (or disease) is the homozygous form (Hb SS) characterized by a severe hemolytic anemia beginning in early childhood, causing splenic infarcts (autosplenectomy), bone infarts, hemolytic or aplastic crises and often death in young adult life. Treatment is unsatisfactory (supportive care with transfusions, fluid therapy and analgesia). 3. ENZYMOPATHIES: Hereditary deficiencies of G6PD or Pyruvate kinase (PK) are commonest. The gene for G6PD deficiency is sex-linked and many isoenzyme types exist. Most lead to acute hemolysis on exposure to oxidant drugs, or possible FAVA beans, while others cause chronic hemolysis. Blacks and Mediterranean races are most commonly affected. Management involves avoidances of oxidant drugs. PK deficiency causes chronic hemolysis and splenomegaly; splenectomy is usually helpful.

Diagnosis of enzymopathies requires demonstration of the specific enzyme deficiency by complex laboratory procedures. B. ACQUIRED HEMOLYTIC ANEMIAS 1. PAROXYSMAL NOCTURNAL HEMOGLOBINURA (PNH); Very rare hemolytic disorder. Acquired increased sensitivity of the RBC membrane to complement, causing intravascular hemolysis and hemoglobinura. Diagnostic test is Ham's acidified serum test. PNH may be associated with aplastic anemia or myelofibrosis. Treatment: supportive blood transfusions.

II EXTRINSIC DEFECTS (EXTRA CORPUSCULAR) 1. AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA): Requires the presence of: a. Hemolytic anemia b. Antibody directed against an intrinsic antigen on the patient's own erythrocytes; find antibody or complement on the surface of the patient's erythrocytes (direct antiglobulin or Coombs test), and often antibodies in the patient's serum (indirect antiglobulin or Coombs test) IgG antibodies lead to destruction of RBC's in the R-E system of spleen and liver mainly. IgM antibodies usually fix complement and produce intravascular hemolysis.

a. WARM ANTIBODY TYPE (IGG): This is the commonest type, usually chronic, and often (50%) secondary to other diseases (lupus, lymphoma, chronic lymphocytic leukemia). Splenomegaly and lymphadenopathy suggest secondary type. Drug-induced hemolytic anemias occur by these mechanisms: i. Innocent bystander type- quinidine ii. Hapten type – penicillin (massive doses) iii. Autoimmune type – Almodet (alpha methyl DOPA); a positive direct antiglobulin (Coombs) test occurs in one third of patients on drug for several months, but hemolysis is very rare. Diagnosis is suggested by hemolysis, spherocytosis and polychromasia, and confirmed by a positive direct antiglobulin (Coombs) test. Treatment includes: 1. Steroids – up to 60 – 80 mg Prednisone daily with gradual tapering depending upon response.

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2. Splenectomy – 50 % response; reserve for cases unresponsive to steroids, or requiring large maintenance steroid dosage. 3. Blood transfusion – difficult to crossmatch; use least incompatible blood and transfused only if absolutely necessary. 4. Immunosuppressive therapy (Imuran, Cyclophosphamide) – for cases unresponsive to steroids and splenectomy. 5. Folic Acid (5 mg daily) – this is given to all chronic hemolytic anemias as prophylaxis for folic acid deficiency due to increased folate utilization b. COLD ANTIBODY TYPE (IGM): These are uncommon and characterized by RBC agglutination and hemolysis when exposed to cold temperature. They may be: i. Idiopathic – chronic cold agglutinin disease (CAD); relatively benign hemolytic anemia in older persons, with Raynaud's syndrome and hemoglobinuria ii. Secondary – with lymphoma, or infectious (mycoplasma, infectious mononucleosis) Diagnosis is suggested by autoagglutination on blood film and hemolysis; it is confirmed by demonstrating increased cold agglutinatinins (anti-I or anti-i) Treatment involves avoiding cold temperature and possible blood transfusion. Chronic cases may need chemotherapy (Chlorambucil). Steroids and splenectomy are not indicated.

ii.

thrombotic thrombocytopenic purpura (TTP) septicemia, DIC and carcinomatosis may cause RBC fragmentation with or without thrombocytopenia Cardiac Hemolytic Anemia – Due to prosthetic aortic valve usually; occasionally with Teflon patch, or calcified valve. Iron deficiency is commonly associated because the chronic intravascular hemolysis leads to chronic urinary iron (hemosiderin) loss.

5. SECONDARY HEMOLYTIC ANEMIA: A variety of infections (eg. Clostridia, malaria) may cause erythrocyte damage and hemolysis. Hepatic and renal disease result in mild chronic hemolysis usually, but defective erythropoiesis from chronic disease, iron deficiency may also play a role

2. ALLOIMMUNE HEMOLYTIC ANEMIAS: These are due to passage of maternal antibodies across placenta to damage fetal RBC's (hemolytic disease of new born) or to blood transfusion with antibodies present acting against foreign RBC's. Delayed hemolytic transfusion reactions of varying severity may occur. 3. DRUG-INDUCED HEMOLYTIC ANEMIA: Chemical Effect – Many oxidant drugs (Phenacetin, Salazopyrine, etc) in high dosage cause hemolysis in normals, but G6PD deficient patients are particular susceptible to many drugs in small amounts. The end result is methemoglobin, sulfhemoglobin and Heinz body formation with resultant RBC fragmentation. 4. MECHANICAL HEMOLYTIC ANEMIA: The trauma to RBC membrane produces fragmentation (schistocytes, helmet cells, burr cells, spherocytes, etc). May be i. Micro-angiopathic Hemolytic Anemia. – Toxemia, abruption placenta, malignant hypertension, hemolytic-uremic syndrome,

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BLOOD TRANSFUSION PRACTICE DONOR AND PATIENT SELECTION Donors should be volunteers, and are carefully screened for history of infectious disease and medications. However, no transfusion can be completely free of risk because: 1. Present screening procedures do not eliminate hepatitis transmission (especially non-A, non-B hepatitis). HIV screening of all blood products occurs now. 2. Some patients may react to non-RBC components – e.g. allergic and febrile reactions. 3. Auto-antibodies produced to RBC antigens (other than ABO and Rh) may limit future transfusion therapy. In spite of this, most transfusions (95%) produce no adverse effects, and serious reactions are rare. Patient selection depends on clinical judgement but the following guidelines might be used:

1. Degree and chronicity of anemia: Patients with chronic anemia of moderate degree (Hb 60 – 80 g/L) often maintain reasonable activity, and the need for transfusion should be correlated with symptomatology (eg severe fatigue, aggravation of angina, heart failure etc) 2. Active or potential bleeding: It is generally accepted that Hb should be kept about 100 g/L in bleeding patients and in patients who undergo surgery. 3. Possible response of hemantinics. Usually better to investigate anemia, and treat specifically (eg iron, B12) if possible.

PRINCIPLES OF BLOOD TRANSFUSION 1. TYPE OF CROSSMATCH a. Group and Reserve Serum – no crossmatch; consists of blood group and antibody screen; serum is reserved for cross-matching later, if needed. Some institutions do not crossmatch if the antibody screen is negative b. Routine Crossmatch- complete; takes atleast one hour. c. Urgent or Emergency Crossmatch – shorter incubation (about 30 minutes) to provide blood for urgent clinical situations. d. Uncrossmatched Blood – give ABO and Rh type same as patient (rather than Rh negative). Only given in most extreme emergencies, unless patient has been previously tested (group and reserve serum) in which the case the need for crossmatching is much less

Each hospital will have guidelines for appropriate procedure (cross match or group and reserve) for each type of operation. 2. BLOOD COMPONENT THERAPY a. Whole Blood – stored up to 5 weeks in CPD anticoagulant. Used for treatment of acute blood loss and replacement of red cell mass and volume) rarely available; blood components are used to provide the equivalent of whole blood in those situations b. Packed Red Cells – storage and cell survival same as whole blood. Preferred for most anemias requiring transfusion. c. Plasma Products –fresh frozen plasma, stored plasma, albumin, cryoprecipitate and factor concentrates. d. Platelet Concentrate e. Gamma Globulin and Specific Antisera – Intravenous and intramascular gamma globulin preparations are available, as are specific antisera (anti-hepatitis B, CMV, Zoster) 3. TRANSFUSION PROCEDURE a. Rate of Transfusion. Depends on clinical situation. One unit of blood may be infused in 15 minutes in rapidly bleeding patients. Packed cell transfusions for anemia usually take about 2 hours (longer than 4 hours should be avoided due to risk of contamination) b. Amount Transfused. Depends on clinical situation. Single unit transfusions are generally avoided. The patient's Hb should increase approximately 10 g/L per unit of packed cells transfused, in average size adult. c. Diluents. Normal saline is the recommended diluent for blood products. A small amount (50 mL) will correct the increased viscosity of packed red cells (about 0.70). Plasma and 5% albumin are possible alternatives. Glucose solutions cause RBC agglutination and hemolysis so must not be used. Ringer's solution contains calcium which can overcome the citrate anticoagulant and cause clots to form in the tubing; so red cells must not be diluted with Ringer's solution. ADVERSE EFFECTS OF BLOOD TRANSFUSIONS When any significant reaction, which cannot be explained or treated, occurs during a transfusion, it is advisable to terminate the transfusion and investigate for hemolytic transfusion reaction. However, most reactions are not hemolytic. Types of reactions include:

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1. HEMOLYTIC REACTIONS: a. Immediate. Chills, fever, dyspnea, bone pain, hypotension, red urine, renal failure and bleeding (D.I.C) may all occur. Terminate transfusion and investigate by sending unused blood, venous blood specimen and urine specimen to Blood Bank (for repeat crossmatch, direct Coomb's test and test for hemoglobinura) May need supportive treatment for shock, bleeding or renal failure. b. Delayed. Fever, jaundice, or recurrent anemia occurring 3 days or more after transfusion. Investigate for hemolysis, including Coomb's test. This may reflect an amnestic antibody response to previous antigen exposure. 2. FEBRILE REACTIONS: Chills, fever and occasionally hypoxia usually in multi-transfused or multiparous patients with WBC antibodies. Treat with antipyretic, and try to prevent in future with leukocyte poor blood or washed packed cells. There are several methods to produce leukocyte poor blood, leukocyte centrifugation in line filters, washed packed cells, frozen blood, but line filters are usually used. 3. ALLERGIC REACTIONS: Usually urticaria. Common (up to 3% of transfusions). Treat with antihistamina – may finish transfusion if reaction not severe, responds to therapy, and does not worsen when transfusion is restarted carefully. If recurrent, prescribe and antihistamine before future transfusions. Rarely anaphylactic reactions occur and there are often in patients with IgA deficiency. Such patients should be given washed blood in the future. 4. VASCULAR OVERLOAD: Congestive heart failure may be precipitated, particularly in elderly patients with chronic anemia, given whole blood, or too rapid transfusion of packed cells and blood components. 5. OTHER: Chills – rapid transfusion of refrigerated blood; contaminated blood – very rare. Disease transmission – hepatitis, cytomegalovirus, malaria, AIDS Iron overload – with large numbers of transfusions Hyperkalemia – in renal failure Citrate toxicity – rarely, with massive transfusions and liver failure, or prematurity.

TRANSFUSION OF PLASMA AND PLATELET COMPONENTS Blood and blood component therapy is influenced by problems of product availability and storage far more than most branches of medicine. A thorough knowledge of these limitations, and continuing communication with Blood Bank staff will ensure that

these products are utilized to their best possible advantage. The following guidelines are currently employed: 1. FRESH FROZEN PLASMA (FFP): Storage: 20oC for 1 year Contents: Must be frozen within 12 hours of blood donation so that it will contain all coagulation factors. No platelets are present, and it contains allo-antibodies as in stored plasma. Use: Replacement of coagulation factors which cannot be provided by other products (i.e. for factor VIII alone, in hemophilia, factor VIII concentrate would be used) Its main use is for treatment of multiple coagulation deficiencies occurring after massive transfusion (10 or more units) or with D.I.C. Time is required for thawing (up to 30 minutes) so stored plasma 5% albumin is preferred for immediate replacement in severe hemorrhage. It is evident that excessive use of this component will mean less cryoprecipitate, or factor VIII concentrate, can be made, thereby limiting the treatment of hemophiliacs. 2. CRYOPRECIPITATE, FACTOR VIII CONCENTRATE: Storage: -20oC for 1 year (cryoprecipitate); o 4 C (Factor VIII) or room temperature for several months Contents: Both are made from plasma frozen within 12 hours of blood donation Cryoprecipitate is a partly purified factor VIII preparation containing about 50% of the factor VIII in the original blood donation, in small volume (10 mL). Each unit of cryoprecipitate (one unit is made from one blood donation) contains between 70 – 100 units of factor VIII ( 1 unit of factor VIII equals the amount present in 1 mL of plasma). Factor VIII concentrate is a more highly purified preparation of factor VIII, requiring more extensive fractionation procedures; it contains a standardized amount of Factor VIII, per vial and is more easily administered so is the preferred product for factor VIII replacement in some situations (particularly for home care programs for severe hemophilia). Recently a recombinant factor VIII product has become available and has replaced Standard Factor VIII concentrate. 3. ALBUMIN: Supply: 25% albumin solution (100 mL), salt poor, stored at 4oC Use: Hypoalbuminemia (usually with complicated edema) where improvement in the patient can be reasonable expected, i.e. do not just treat a laboratory abnormality without regard to patient's condition, or underlying disease – albumin is usually not given for cirrhosis and nephritic syndromes. It is commonly used for supportive

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care in burn patients, and during hyperalimentation. Supply: 5% albumin solution (250 mL) Use: This product provides a hepatitis-free colloid substitute for stored plasma where volume replacement is needed (eg. Hemorrhage with shock, plasma exchanged) but where coagulation factors are not. It is much more expensive than crystalloid solutions (eg. Normal saline, Ringer's solution) so should only be used when colloid (protein) effect is really needed. NOTE: Albumin and immune globulin are the only blood products which are free of risk of hepatitis transmission due to methods of production. All other products carry this risk, albeit small, despite extensive donor screening and viral testing; fibrinogen had a greatly increased risk because it was pooled, as opposed to single donor product, and is no longer issued (cryoprecipitate may be used instead to treat hypofibrinogenemia). The factor concentrates (VIII and IX) are now specially treated ; this should prevent the viral transmissions which were previously high with these products.

4. PLATELET CONCENTRATE: Supply: Made by differential centrifugation of blood donation less than 2 hours old. Stored for 5 days at room temperature. Contents: One unit of platelet concentrate contains about 50 to 75% of platelets present in the original blood donation in a volume of about 50 ml plasma. It also contains allo-antibodies (anti-A or B). In general 6 units of platelet concentrate are given at a time, and one would expect an increase in platelet count in the patient of approximately 10

x 10 9/L for each unit transfused, 1 hour after transfusion in a patient of average size (eg, increase platelet count by 60 x 10 9/L if 6 units given). Use: Platelet concentrate should be infused promptly (1/2 an hour) through a regular blood transfusion set, not previously used for blood transfusion. Platelet response is limited by such factors as fever, splenomegaly and presence of auto-antiobodies or allo-antibodies. In general, the presence of these antibodies makes platelet transfusion almost worthless, although occasionally a therapeutic response may be seen in a bleeding thrombocytopenic patient of this type. Best results are obtained in patients who have not been previously transfused, or pregnant, and where thrombocytopenia is due to decreased production, rather than increased destruction or consumption of platelets. Prophylactic platelet transfusion may be given to patients with severe thrombocytopenia (platelet count < 10 x 10 9/L) due to a disease of limited duration (eg. Leukemia during chemotherapy, drug toxicity, some aplastic anemias) and will be then required 2 to 3 times weekly. Sensitization to platelet and HLA antigens often occurs over 1 to 2 months and this limits further transfusions of random donor platelets. Platelets obtained by platelet-pheresis from donors who are selected for HLA compatibility may be helpful when patients become refractory to random-donor platelet transfusions. 5. PENTASTARCH: 10% hexastarch used for the plasma volume expansion instead of 5% albumin. Used in hypovolemia, secondary to sepsis, blood loss

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PROBLEMS FOR TUTORIALS 1. ANEMIA AND CONFUSION A 62-year-old woman was admitted to hospital after a progressive illness of approximately 6 months duration, characterized by progressive pallor, fatigue, and general weakness. More recently, she had developed shortness of breath on exertion and required 2-3 pillows at night for orthopnea. She had fallen several times at night getting up to the bathroom, and friends found her to be unsteady on her feet. Despite her frequent complaints of a sore mouth she had been eating well and had no G.I. complaints. Her past health has been good, and she had no operations. On physical examination, the intern found her to be pale, slightly jaundiced and in no respiratory distress with the head of the bed elevated. Her tongue was red and smooth. There was slight jugular venous distension, hepatomegaly, and pitting edema in the legs. Laboratory Investigations: Hemoglobin 50 g/L WBC 3.0 x 109/L Platelets count 60 x 109/L MCV 120 fl MCHC 350 g/L Reticulocytes count 3.0% (36 x 109/L) Normal (10-75 x 109/L)

Peripheral Blood

Questions: 1. List the clinical problems. 2. What other physical findings related to her unsteadiness not mentioned but which you would expect to be present? 3. What do you expect the blood film to show? Describe BM features seen below? 4. What other laboratory tests you would order? 5. Given the diagnosis, discuss the management, including: a. Where should she be treated (hospital or home)? b. Approximately when you would expect improvement to occur with regards to: i. Hemoglobin ii. Reticulocytes count iii. Pallor iv. Unsteadiness

Bone marrow: Identify the labeled structures

2. ANEMIA AND CONFUSION IN ALCOHOLIC A 54-year-old woman was brought to the Emergency department by her daughter who found her at the home, weak and confused. Since the patient's husband had died five years ago, she was known to have increased her alcohol consumption and frequently failed to eat properly. She denied any G.I. Symptoms, except chronic constipation. On physical examination she was a febrile, pale and not jaundiced. Her breath smelt of alcohol and she was slightly confused. There were no focal neurological findings. Her tongue was smooth. On abdominal examination there was demonstrable ascites, the liver was not enlarged, and the spleen was palpable 4 cm below the left costal margin. Numerous spider nevi were present on her upper chest and shoulders, and several bruises were noted on her legs. Laboratory Investigations: Hemoglobin 50 g/L MCV 105 fL WBC 3.6 x 109/L Platelets count 86 x 109/L Reticulocytes count 40 x 109/L Questions: 1. List the likeliest causes for: a. Macrocytosis b. Pancytopenia c. Low Reticulocytes count d. Confusion Three days after admission, the patient was no longer confused and was eating well. Further investigation had been delayed by the weekend and on the 4th hospital day further blood tests, as well as a bone marrow aspirate, were performed. Later that day she suddenly vomited a large amount of blood and became confused. Her heart rate increased to 130/minute and blood pressure fell to 70 mm Hg systolic. A STAT Hb. was 42 G/L., prothrombin

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time was 24 seconds (INR: 2.5), and PTT was 60 seconds.

2. On the 4th hospital day, what changes would you expect to see if the following tests were performed that day a. Reticulocytes count b. Serum and RBC folate c. Serum B12 d. Bone marrow 3. You are called that evening to manage this patient after she vomited blood: a. Explain why the hemoglobin hasn't fallen. b. What would your first order for a laboratory test be? c. What type of I.V. solution would you order immediately? d. What further I.V. therapy would you recommend?

3. ANEMIA AND RETICULOCYTOSIS A 22-year-old woman was admitted to hospital with the chief complaints of progressive weakness, and pallor of one-month duration. She had also noticed some joint discomfort over recent months, but denied any other significant complaints. Past history and family history were non-contributory. On physical examination, she appeared pale and slightly jaundiced. A faint skin rash was noted over her cheeks. There was no lymphadenopathy or hepatomegaly. Spleen was palpable 2 cm. below the left costal margin. No joint abnormalities were detected. Laboratory Investigations: Hemoglobin 60 g/L MCV 90 fL MCHC 360 g/L Reticulocytes count 16% (300 x 109/L) WBC 4.0 x 109/L Platelets count 130 x 109/L Direct Antiglobulin (Coombs') - Positive (+++) Questions: 1. List the clinical problems. 2. What would you expect to see on the blood film? 3. Explain why the MCV is normal. 4. List 2 reasons for the thrombocytopenia. 5. In considering management of this patient: a. What would your first choice of treatment be? b. Would you recommend a blood transfusion? c. If the splenectomy is considered later, what precautions and advice to the patient should be provided?

4. ANEMIA AND ARTHRITIS

A 46-year-old woman attended her doctor's office complaining of weakness and joint pains. The weakness had been present for several years, during which time she had noticed progressive joint discomfort and stiffness mainly in her hands, elbows and knees. She had been taking up to 8 aspirin tablets daily for the past year and was on no other medications. Her diet was good and she had no G.I. symptoms except indigestion related to the aspirin. She had no past history of serious illness and was 4 years postmenopausal. On examination, she was slightly pale and not jaundiced. There was no lymphadenopathy or hepatosplenomegaly. She had obvious rheumatoid arthritis with joint deformity of both hands. Laboratory Investigations: Hemoglobin 94 g/L MCV 74 fL MCHC 310 g/L WBC 6.3 x 109/L Platelets count 230 x 109/L

Questions: 1. Classify the anemia. 2. List 2 possible causes for the anemia. 3. List 3 most appropriate laboratory tests. 4. Assume that you conclude the patient requires iron therapy: a. What type and amount of iron treatment would you prescribe? b. How long would you advise the patient to continue iron treatment? c. The patient wants to get better as quickly as possible and asks for either a blood transfusion or iron shots. Do you agree with either of these treatments? If not, what advice would you give to the patient to support your recommendations? d. With iron, at what rate would you expect the Hb to rise?

5. ANEMIA AND HEART DISEASE A 42-year-old man regularly visited his physician for routine cardiac studies following open-heart surgery three years ago. At that time an aortic valve prosthesis was inserted for rheumatic aortic stenosis, and he had been anticoagulated under careful supervision since then. He had felt well until recent weeks when he began to complain of dyspnea and fatigue. He noticed a reddish discoloration to his urine often during the day.

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On the examination, he appeared pale, but was not jaundiced. There was a tachycardia (100/minute), and a moderate (grade III/VI) early diastolic murmur maximal at the left sternal border. There was no evidence of cardiac failure. Liver and spleen were not enlarged. Laboratory Investigations: Hemoglobin 80 g/L MCV 75 fL MCHC 300 g/L Reticulocytes count 2% (75 x 109/L) WBC 7.5 x 109/L Platelets count 300 x 109/L Blood film RBC fragmentation, hypochromia

Mean corpuscular volume (MCV) 70u3 Mean corpuscular hemoglobin (MCH) 17.5 uugm Mean corpuscular hemoglobin concentration (MCHC) 25% White blood cells (WBC) 10/mm3 with normal differential Sedimentation rate: 62mm/hr CXR / ECG: normal Questions: 1. List the clinical problems? 2. After reviewing CBC results, what are the next important simple tests you would order? 3. Classify the anemia and what further tests would you order at this stage 4. How would you manage this patient?

7. ANEMIA AND MACROCYTOSIS Questions: 1. List the clinical problems. 2. The Reticulocytes count is not appropriate to the degree of anemia. Explain. 3. Classify the anemia. 4. Further tests indicate iron deficiency, why do you think this occurred? 5. Outline your management of this patient.

6. ANEMIA, WEAKNESS AND JOINT PAIN A 46-year old woman comes to your office complaining of increasing fatigability and weakness for the past 4 months. She has had rheumatoid arthritis for 8 years and 12 to 16 tablets of aspirin per day have controlled her joint symptoms. The patient says she had no sever pain, tenderness, redness, swelling, or heat in her joint for 2 years while on regular aspirin. However, she is less energetic and she finds she must stop and rest during her household work, and by the end of the day she is exhausted. She denies crying spells and has had no anorexia, weight loss or insomnia. She denies any history of abdominal pain, heartburn, jaundice, and black or tarry stools. She dose like to chew on ice cubes. There is no history suggestive of diabetes. Past history reveals no additional pertinent information. Family history reveals that she is of Italian origin and one sister was mildly anemic and was told she had Mediterranean anemia. On physical examination, the patient is slightly obese, vital signs are normal. Slight pallor is noted. Cardiac, pulmonary and abdominal examination was normal. There is no stool in the rectum for guaiac (occult blood) testing. No signs of active rheumatoid arthritis. Laboratory tests: Hemoglobin 70 gm/l, hematocrit (Hct.) 28%

42-years-old clerk presented to his G.P complaining of inability to do his daily jogging and exercise and reported an incident of dizziness without loss of consciousness. He has been feeling gradual deterioration during the past few months. He did not have any illness and was not on any medication. There was no blood loss from any site and he had normal eating habits. Apart from undergoing tonsillectomy at the age of 5 years, there was no significant past history. On examination, the patient was fully alert, pale with a tinge of jaundice. The majority of his hair was gray and upon further questioning, the patient admitted to having gray hair since the age of 20 years. There was no lymphadenopathy. Examination of the heart revealed an ejection systolic murmur over the apex. Examination of the abdomen was normal Preliminary blood tests revealed the following: WBC: 3.5 x 109 /L neutrophils 70% Hb.: 83 g / L ( N: M: 140-180 F: 120-160) MCV 115 fl (N 80 - 95) Platelets: 100 x 109 / L (N 150 – 400) s. Iron: 30 μmol / L (males N 14 – 31) s. Transferrin 2.4 g / L (2 - 4) Blood film showed the presence of macro-ovalocytes and hypersegmented neutrophils Questions: 1. What are the hematological abnormalities elicited in the full blood count result? 2. What may be the cause of this abnormality? 3. What is the most likely diagnosis? 4. How would you confirm this diagnosis?

8. FEVER, ANEMIA AND RETICULOCYTOSIS A 24 –year old Kuwaiti man is admitted to the hospital because of increasing fatigue following a respiratory

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infection. He felt well until 5 days ago when he developed nasal stuffiness, sore throat, and dry cough. Three days later his cough became productive of thick yellow sputum, the temperature rose to 39 o C, and he began to note sharp pain in the left posterior thorax with deep inspiration. His physician made clinical diagnosis of pneumonia and prescribed Septrin and 3 – 4 aspirin tablets daily for fever. Cough and fever abated, but he felt progressively weaker and was therefore hospitalized. Past history reveals that the patient had been healthy all his life. He had been told that he had” yellow jaundice” as a new born but that he had not required transfusion or other specific therapy. The review of systems is entirely negative except for the fact that he has noted his urine to be dark brown for the past 1 to 2 days. Family history is unobtainable. His parents are dead and his only sibling, a sister is in good health. On physical examination he appears acutely ill. There is pallor, the sclera is slightly icteric. T 37.4o C, P 95/min, BP 120/60, RR 16/min. Respiratory movements of the left thorax are asymmetrical with splinting on the left. There is dullness to percussion, breath sounds are high pitched bronchial, and many fine to medium rales are heard over the left lung posteriorly. The heart exam reveals a grade 1/6 systolic murmur heard best along the left sternal border. Abdominal exam revealed no organomegally. Laboratory tests HB 75 mg/l , hematocrit 25% (notice: the plasma appears pink), retics: 15% WBC: 14.5/mm3 with 81% neutrophils, 12% bands, 2% metamyelocytes and 5% lymphocytes. Platelets count is 250. Blood film: reveals generally normocytic normochromic red cells, but there is moderate poikilocytosis with occasional fragmented cells and microspherocytes. No true sickled cells are seen. There are many large polychromatophilic red cells and rare nucleated red cells. Urine: is clear, brown-colored, protein +, specific gravity 1.025, and glucose, ketons and urobilinogen are negative. Dipstick test for blood is positive. No cells or casts are seen on spun sediment. CXR: a homogenous density involves much of the left lower lung field. No pleural effusion seen. Renal and liver function is normal. Serum bilirubin is 50 mmol/l (normal< 25), 35 unconjugated, 15 conjugated. Questions 1. What does the history of jaundice as a newborn indicates? 2. What are the causes of dark urine? 3. Why do we look for icterus under the upper eyelid? What dose the Combination of icterus and

dark urine implies? What gives urine this color in this case? 4. What do you make out of the rticulocytes count? Why the plasma is pink-colored? 5. What is you differential diagnosis? What other tests would you like to order?

9. CHRONIC FATIGUE AND ANEMIA A 28-year-old Kuwaiti lady has been complaining of chronic fatigue and known to have anemia for a long time on iron supplement but without any appreciable response. The first time she was told to have anemia at the age of 10 years in the range of 90 – 100 mg/L. She has been prescribed different types of iron supplements but with out any actual change in HB level. She is single and here menstrual period is regular and scanty in amount. She has a brother who was told to have a similar degree of anemia but she dose not know the exact nature of his problem. On examination, the patient is healthy and vital signs are normal. Slight pallor is noted. Cardiac, pulmonary, and abdominal examination is normal. Laboratory tests: White blood cells (WBC) 10/mm3 with normal differential Hemoglobin 110 gm/L, Hematocrit 37% Mean corpuscular volume (MCV) 58 u3 Mean corpuscular hemoglobin (MCH) 17.5 uumg Mean corpuscular hemoglobin concentration (MCHC) 25% Red cell distribution width (RDW) 15 Peripheral blood film: microcytic, hypochromic, target cells Questions: 1. How do you assess the severity of menstrual period? 2. Comment on the hematological picture and what is your differential diagnosis? 3. Comment on the HB electrophoresis? 4. If the HB electrophoresis was normal, what else should you consider? 5. What would you council this patient? Cellulose Acetate Method (pH 8.4) Cathode -

Hemolyse d blood applicatio n

C E A2

Sample 4.5%

S D

F

+ Anode

A

0.5% 95%

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10. A YOUNG PATIENT WITH RECURRENT BONY ACHES A 17-year-old male high school student presented to the emergency department complaining of a sudden onset right-sided chest pain. He is known to have repeated pain attacks of similar nature that involving different sites of his body since childhood, requiring frequent hospitalizations. Family history revealed his sister to have similar problem. Physical examination revealed a pulse rate of 110/ minute, Bp 110/70, and temperature of 38 °C. He was pale and slightly icteric. Tonsils were congested. Chest examination revealed the presence of decreased air entry on the right side. No organomegally. Lab Investigations CBC WBC HB Hematocrit MCV MCH MCHC RDW Reticulocytes Count s-Total Bilirubin s-Indirect Bilirubin s-Direct Bilirubin

Reference Range 15.0 (4 – 10 x 109/L) 87 (140 – 180 g/L) 26 (42 – 50) 88 (80 – 94) 30 (27 – 31 pg) 340 (330 – 360 g/L) 17 (11.5 – 14.5 %) 150 (60 – 120 x 109/L) 50 45 5

Differential WBC Count Range Neutrophils 11.5 (1.5 – 6.0 x 109/L) Lymphocytes 2.5 (1.0 – 3.0 x 109/L) Monocytes 0.7 (0.2 – 0.6 x 109/L) Eosinophils 0.2 (0.1 – 0.5 x 109/L) Basophils 0.1 ( 0.1 – 0.3 x 109/L)

Cathode -

Hemolys ed blood applicati on

Peripheral blood smear: Polychromasia / Occasional nucleated RBC seen Hemoglobin electrophoresis provided Questions: 1. Comment on the peripheral blood findings. What are the Howell-Jolly bodies and what is their significance? 2. Comment on the hemoglobin electrophoresis findings. 3. What is the diagnosis? And what do describe this attack of pain? 4. What is the cause of the hyperbilirubinemia in this case? 5. How do you manage this case? 6. What is the role of exchange transfusion in this disease? 7. What are the complications of this disease?

+ Anode

C E A2

S D

70%

A F

10%

20%

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MODULE II WHITE BLOOD DISORDERS OBJECTIVES •

Differentiate benign from malignant causes of leukocytosis.

•

Describe the clinical presentation, plan of investigations, and be able to interpret relevant laboratory data, so as to reach likely diagnosis and prognosis in a patient who may present with leukocytosis and/or lymphadenopathy.

•

Explain basic molecular abnormalities underlying malignant transformation of haematopojetic cells, and resulting in hematological malignancies.

•

Plan and prescribe a rational schedule of management, based on a sound knowledge of the mechanism of action, dose, duration and side effects of appropriate drug therapy

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LYMPHADENOPATHY AND SPLENOMEGALY GENERAL OBJECTIVES The student should have a general understanding of the spectrum of disorders presenting with enlargement of lymph nodes and/or spleen, including methods of presentation, investigations required to diagnose these disorders, urgency with which diagnosis should be made, and treatment commence and finally the prognosis and complications which might occur during the treatment program. SPECIFIC OBJECTIVES At the end of this section the student should be able to: 1. Describe diagnostic possibilities, given a series of white cell counts and differentials, and/or clinical presentations of patients with enlarged lymph nodes or spleen. 2. List the diagnostic tests required, if any, to confirm the suspected diagnosis. 3. Describe the major clinical and laboratory features of the common or important disease in this category: Infectious mononucleosis Acute and chronic leukemias Lymphomas and Hodgkin's disease Myeloma 4. Differentiate between benign and malignant causes of leukocytosis and polycythemia. 5. Describe the expected outlook for patients once any of the above diseases are diagnosed 6. List the possible treatment options, when available, for theses diseases, and describe the risks / benefits expected with these treatments. 7. describe the management of the common complications which might arise during the course of these disorders; these complications include bone marrow suppression, local effects of enlarged lymph nodes and spleen, pain and hyperviscosity. 8. Describe the role of blood component therapy in the management of these patients, including type of component, rate of administration, and expected responses CLINICAL ASSESSMENT Particular points in history include a search for sites of local infection, or if the lymphadenopathy is generalized and/ or splenomegaly is present, particular attention must be paid to systemic symptoms (fever, night sweats, pruritus, weight loss, anemia and jaundice), medications, travel history, and presence of other diseases (eg. Rheumatoid arthiritis, lupus, etc). Physical examination should include a careful documentation of the size and consistency of enlarged

lymph nodes in cervical, axillary and inguinal areas (occasionally epitrochlear and popliteal as well), extent of hepatosplenomegaly, presence of tonsillar enlargement, skin lesions and bone tenderness LABORATORY ASSESSMENT The qualitative assessment of white blood cells (leukocytes) is almost performed by automated cell counters (Coulter Counter) rather than older inaccurate and labor-intensive counting chambers. The normal WBC count is 4.0 – 10.0 x 109/L The qualitative assessment of white blood cells is most commonly performed by manual microscope techniques, with reporting of a WBC Differential (percentage of cells assigned to each category) after viewing 100 or more cells. If the WBC is too low (1.5 x 109/L) then a buffy coat preparation may be prepared to increase the number of cells which can be viewed microscopically in a reasonable period of time. Many of the automated cell counters provide partial differential counts. Most commonly granulocyte and lymphocyte counts. Such differentials have the advantages of precision since many more cells are assessed than by manual methods, and less technologist time is involved. However, most do not give full differentials and do not give a printout when abnormal cells are present or the differential counts are significantly abnormal. Fully automated differential counters require a large volume of tests to be cost effective and are not as helpful in settings where a large percentage of the differential are abnormal. The bone marrow may provide information about granulocyte production, and presence of infiltrative disorders such as leukemia, lymphoma and myeloma. The normal sequence of granulocyte development is as follows: Myeloblast → promyelocyte → myelocyte → metamyelocyte → band → neutrophil Normally there are 3 – 4 times as many granulocyte precursors, than erythroid precursors, in the bone marrow (M/E ratio 3/1). This reflects the presence of a large granulocyte pool or reserve, and also the short life span of granulocytes compared with erythrocytes. The maturation of granulocytes in the marrow is sequential. If there are increased numbers of less mature forms, then this is termed "shift-to-the-left". It could represent a reactive process (granulocytic hyperplasia) or a malignant process (leukemia and the morphological distinction may be difficult.

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BENIGN LEUKOCYTE DISORDERS GRANULOCYTOPENIA This term refers to neutropenia, or reduction in the absolute neutrophil count; decreased in the eosinophils and basophils cannot be accurately measured, and are of no practical importance. The severity of the neutropenia is usually graded as follows: Normal neutrophil count 2.5 – 7.5 x 109/L Mild neutropenia 1.0 – 2.5 x 109/L Moderate neutropenia 0.5 – 1.0 x 109/L Severe neutropenia < 0.5 x 10 9/L A serious risk of spontaneous infection is usually not present until severe neutropenia develops. CLASSIFICATION OF NEUTROPENIA 1. Decreased granulopoiesis – marrow infiltration / fibrosis, drugs, radiation 2. Ineffective granulopoiesis – megaloblastic anemia 3. Decreased granulocyte survival – hypersplenism, severe infection, immune DECREASED GRANULOPOIESIS This may be associated with decrease in other marrow elements causing pancytopenia (see aplastic anemia, leukemia, myelofibrosis, etc). When granulocytopenia occurs selectively due to decreased granulopoiesis, it is most frequently caused by drugs. Even then, many of these may be due to drug-related granulocyte antibodies which are not detectable by current techniques. Some drugs, such as chemotherapeutic agents, will predict cause neutropenia, but usually also thrombocytopenia and anemia. These are usually slow in onset and dose-dependent. Other drugs may unexpectedly cause neutropenia, often severe and sometimes fatal, in a previously sensitized patient, or by unknown mechanisms (idiosyncratic reaction). Many drugs have been implicated, similar to those which cause aplastic anemia. The major groups include anti-inflammatory agents, anticonvulsants, antibiotics, antimal arials, antithyroid drugs and phenothiazines. The neutropenia may last for days / weeks. INEFFECTIVE GRANULOPOIESIS This is seen most often in megaloblastic anemias, characterized by a pancytopenia but a hypercellular megaloblastic bone marrow; the precursors of all cells lines dies in the marrow cavity before being released. Thus, ineffective hematopoiesis is corrected by appropriate therapy such as B12 or folate vitamin treatment or in the case of alcohol excess cessation of alcohol consumption. DECREASED GRANULOCYTE SURVIVAL In severe bacterial infections such as lobar pneumonia, and various septicemias, neutropenia can

occur due to granulocyte consumption or complement activation, and depletion of the marrow granulocyte pool. It is often an ominous sign. In hypersplenism, there is usually a pancytopenia due to splenic pooling of hematopoietic cells and hypercellular marrow, with correction of blood counts after splenectomy. Severe neutropenia may occur with Rheumatoid arthritis (Fety's syndrome) usually with splenomegaly as well. This may be due to autoantibodies against granulocytes, which also likely occur in some patients with lupus, but laboratory techniques are not presently available to detect these antibodies. The management of neutropenia involves discontinuing any drugs which may be associated, correcting any vitamin deficiency or nutritional problems, and if possible, controlling any underlying disease. If hypersplenism cannot be reversed by other means, and if the cytopenias are severe enough to warrant the risks involved, then a splenectomy may be indicated. LYMPHOPENIA May also occur (absolute lymphocyte count < 1.5 x 109/L) due to drugs (steroids), hereditary immuno-deficiency disorders or associated with some other diseases, such as Hodgkin's disease & HIV infection. It may be present, usually to a lesser degree than neutropenia, in association with the pancytopenia of aplastic anemia, hypersplenism, etc. GRANULOCYTOSIS When the numbers of granulocytes are increased in comparison to the numbers of lymphocytes, but in absolute terms, then this is called "relative granulocytosis". It is usually seen with the lymphopenias described previously. When the actual number of granulocytes is increased (and this is calculated from the percentage of granulocytes and the total WBC count) this is termed "absolute granulocytosis" and these conditions are now considered further, depending on which granulocyte is increased. a. Neutrophilia (neutrophil granulocytosis) An absolute neutrophil count greater than 7.5 x 109/L is called neutrophilia; the causes which would be considered are: i. Controlled proliferation This is a normal response to infections (mainly bacterial), trauma, inflammatory diseases an d even neoplasm. Leukocytosis up to 50 x 109/L is common and elevations as high as 100 x 109/L can occur. Above this level, however, a leukemic process will almost always be present. There will often be left-shift with

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reactive neutrophilia but this will seldom extend as far back as the myeloblast and promyelocyte stages. When the reactive leukocytosis is so extreme and/or left-shifted as to resemble a leukemia, it is termed "leukemoid" reation. ii. Uncontrolled proliferation This represents a malignant myelproliferative process such as leukemia, myeloid metaplasia or polycythemia, rather than reactive or secondary, neutrophilia. iii. Storage pool activation Roughly 1/3 of the total granulocyte mass is present as a storage pool in the marrow. This reserve may be rapidly mobilized in response to many acute traumatic and inflammatory processes resulting in a prompt neutrophilia in peripheral blood. The mature granulocytes are depleted in the marrow for several days until granulocyte hyperplasia occurs. iv. Marginal pool activation 2/3 of the total granulocyte mass is present outside of the marrow, and this is divided equally between a central pool (1/3 of total mass) and a marginal pool (the last 1/3). The number of marginating neutrophils may be changed acutely due to catecholamines (any stressful situation) or by migration inbibition with steroids, causing the neutrophils to demarginate and be counted in the central pool. The result is an absolute neutrophilia on WBC counting, even though the number of circulating granulocytes hasnot actually changed. b. Eosinophilia Absolute eosionophilia (0.4 x 109/L) is most commonly associated with the following disorders: • Allergies – allergic rhinitis, asthma, etc • Parasitic infections – amebiasis, nematodes, trematodes, etc • Dermatitis – dermatis herpetiformis, eczema, etc. • Hypereosinophilic syndromes – Loefler's syndrome, polyarteritis, etc • Neoplasms – various carcinomas, Hodgkin's disease,etc. It is extremely rare for eosinophilia to occur as a primary myeloproliferative disorder (eg. Leukemia) c. Basophilia Increase in basophils is rare, and virtually always part of other leukocyte abnormalities in myeloproliferative diseases (CGL, myeloid metaplasia, polycythemia) d. Monocytosis (0.8 x 109/L) Elevation of monocytes is uncommon. If may occur with some chronic infections, preleukemia or monocytic leukemia. e. Lymphocytosis

As noted previously, children have a large proportion of lymphocytes on WBC differential than adults, and frequently these lymphocytes appear atypical, or reactive, in response to the immunological challenges and frequent viral infections during childhood. Absolute lymphocytosis in adults requires a lymphocyte count greater than 3.5 x 10 9/L. This will usually be due to some underlying disease, usually a viral infection, but if it is persistent, without underlying cause, then it may represent an early stage of chronic lymphocytic leukemia – the distinction cannot be made on morphological grounds alone. Up to 5% of lymphocytes in adults and 10% in children may appear slightly atypical or reactive; these lymphocytes are larger with less mature nucleus, and the cytoplasm is often basophilic, and in large amount so that the cell membrane appears compressed by surrounding erythrocytes on the blood film. Causes for lymphocytosis of moderate to marked degree, include infectious mononucleosis, cytomegalovirus, infectious lymphocytosis, Bordetella pertussis (whooping cough), toxoplasmosis, HIV and hypersensitivity reactions. The infectious lymphocytosis of whooping cough is distinctive because the lymphocytes are all small lymphocytes, not atypical, and the lymphocyte count may reach extreme degrees (50 -100 x 109/L) resembling chronic lymphocytic leukemia, which is not seen in children. For practical purposes, major degrees of atypical lymphocytosis (20 – 40% of WBC's) in adults will be due to infectious mononucleosis (EB virus) or cytomegalovirus; the latter may cause an identical clinical picture but with a negative mono test, and is much less commonly diagnosed. Infectious mononucleosis characteristically presents with fever, sore throat and cervical lymphadenopathy; jaundice is uncommon (5%) but laboratory evidence of hepatic dysfunction is common (elevated transaminase in over 80%). Skin rash may occur, particularly if these patients are treated with ampicillin. Occasionally hemolytic anemia (cold agglutinins) and thrombocytopenia may be prominent features. Splenomegaly is present in half of the cases but must be carefully examined because of the risk of splenic rupture. The diagnosis is confirmed by a positive monospot test in 95% of cases, and the treatment is usually only supportive (bed rest, analgesics, or antipyretics) Steroids are indicated only for serious complications (CNS involvement, pharyngeal obstruction, sever thrombocytopenia) Sometimes the lymphocytes are fragile, and are ruptured during the preparation of the blood film; they are then referred to as "smudge cells". These are most common in chronic lymphocytic leukemia but can also be seen with reactive lymphocytosis (eg. Viral infection) so are not diagnostic of C.L.L

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HEMATOPATHOLOGY TESTING DIAGNOSIS OF ACUTE LEUKEMIA Classification of acute leukemias according to FAB criteria requires morphologic examination of a bone marrow aspirate smear. This is required to obtain a blast count, as well as to determine the blast morphology, the number and types of maturing cells, and whether dysplastic features are present. Cytochemical Stains It is often impossible to determine whether the blasts in an acute leukemia are of myeloid or lymphoid origin from morphology alone. For this reason, cytochemical staining is performed. Stain Myeloperoxidase

Cells stained Myeloid

Non-specific esterase

Monocytes, blasts in monocytic leukemia

Periodic acid-Schiff

Normal granulocytes, blasts in lymphocytic Leukemia ("string of pearls"), abnormal Erythroblasts in erythroleukemia ("chunky")

Tartrate-resistant acid phosphatase (TRAP) Leukocyte alkaline phosphatase

that is involved in the rearrangement of immunoglobulin and T cell receptor genes. If a leukemia is atypical (i.e. of T cell origin) additional antibodies can be used to further characterized the leukemic cells. If the leukemia cells. If the leukemia has a mature B cell phenotype (i.e. expresses CD19 and/or CD10 but not TdT, as in Burkitt’s leukemia), antibodies recognizing κ and λ immunoglobulin light chains are added to the panel to establish clonality. If the leukemia is of plasma cell origin (i.e. multiple myeloma), staining for cytoplasmic immunoglobulin is performed by immunofluorescent microscopy or flow cytometry. This is done since plasma cells generally lose cell surface expression of the useful markers, including κ and λ.

Hairy cell leukemia cells High in leukemoid reactions , low in CML

Flow Cytometry Flow cytometry is used to determine the cell type of leukemic blasts. Stem Cell

CD34

Myeloid

CD13, CD33

B cell

CD10 (CALLA), CD19

T cell

CD2, CD5, CD7

Megakaryocyte

CD61

In addition to the above markers, the "leukemic panel" includes immunofluorescent staining for terminal deoxynucleotidyl Tranferase (TdT). This is an enzyme present in the nuclei of immature lymphocytes

DIAGNOSIS OF LYMPHOMA AND LYMPHOPROLIFERATIVE DISORDERS Most lymphomas are characterized by immunophenotyping cells from the lymph node or other tissues in suspension or frozen tissue sections. Flow Cytometry Flow cytometry is the method of choice for characterizing lymphomas involving the peripheral blood or bone marrow. Since most lymphomas are of mature B cell origin, finding a B cell population that expresses exclusively κ or λ light chains on the cell surface is sufficient for the diagnosis of malignancy. Actual classification requires morphologic examination, as well as determining the expression of other cell surface markers. The following table lists the antibodies used for the characterization of lymphomas: ___________________________________________ _ Cell Surface Marker C ells Identified CD5

Pan-T cell marker; also expressed on some B cell lymphomas (small lymphocytic lymphoma/CLL, mantle cell lymphoma)

CD19, CD20

Pan-B cell markers (CD20 is low or absent in SLL/CLL)

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κ, λ

Immunoglobulin light chains, used to determine clonality in B cell lymphomas

CD2

Pan-T cell marker, also expressed on NK cells

CD4, CD8

T cell subsets

CD1

mature T cells, Langerhans cells

CD3, CD7

Pan-T cell markers

CD16/56, CD57

NK cell markers

CD25

IL2 receptor; present on activated T cells, some T cell malignancies, and hairy cell leukemia

MOLECULAR STUDIES Immunoglobulin and T Cell Receptor Gene Rearrangement As a part of normal development, lymphocytes rearrange their immunoglobulin (B cells) or T cll receptor genes (T cells). This results in alarge repertoire of receptors for antigen recognition. It also allows determination of B or T cell clonality, since each lymphocyte has a different DNA structure. In a mixed population of lymphocytes, no one lymphocyte clone is of sufficient number to be detected by DNA blot analysis, however when one clone predominates, its receptor gene rearrangement can be detected. When a single clone is detectable, this is generally equivalent to lymphoid malignancy, although there are a number of specific conditions where clonality does not necessarily mean malignancy.

chromosome 22. The resulting fusion protein, BCRABL is thought to cause neoplastic transformation. This chromosomal translocation can be detected by DNA blot analysis using a probe for the BCR gene. This particular probe easily detects the translocation if as few as 1% of the cells present contain the Philadelphia chromosome. This probe detects the translocation in greater than 90% of CML. Because the breakpoint is different, the probe only detects about 33% of Philadelphia chromosome-positive ALL. This test can be used to make the differential diagnosis of CML vs. other reason for leukocytosis or thrombocytosis. It can also be used to detect minimal residual disease following bone marrow transplantation, although it is not as sensitive as PCR. In addition to increased sensitivity, this method will allow detection of both types of t(9;22) breakpoints (i.e. CML vs. ALL types). Detection of t(14;18) In up to 90% of follicular lymphomas, a translocation between chromosomes 14 and 18 can be found. This translocation results in the insertion of the Bcl-2 gene from chromosome 18 into the immunoglobulin locus on chromosome 14, Bcl-2 is a mitochonderial protein that regulates apoptosis or programmed cell death; the function of Bcl-2 appears to be the prevention of cell death by apoptosis. The t(14; 18) results in over expression of Bcl-2 allows cells to survive longer, and thereby accumulate additional mutations which lead to neoplastic cell transformation. The t (14; 18) can be detected by PCR. The main use of this test is to determine whether peripheral blood, bone marrow, or various stem cell transplantation products are contaminated by lymphoma cells. The test can also be used to make a diagnosis of follicular lymphoma, particularly on needle biopsy specimens where follicular architecture may not be apparent.

The technique used for detection of gene rearrangements is DNA blot analysis. Genomic DNA is isolated from a tissue biopsy, peripheral blood, or bone marrow specimen. Recently the polymerase chain reaction (PCR) has been used to detect immunoglobulin heavy chain gene rearrangement. BCR Gene Rearrangement Chronic myelogenous leukemia (CML) is caused by a translocation between chromosomes 9 and 22 (the Philadelphia chromosome). The result of the translocation is the juxtaposition of the c-abl protooncogene on chromosome 9 with the BCR gene on

SKY allows for direct visualization of a specific chromosomal abnormality (numeric and translocation

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Result of FISH

Normal cell

MCL – lymphoma cell

Microarray technology allows for a large number of genetic abnormalities to be screened on a single chip that is then scanned and analyzed by a computer • • •

FISH allows for direct visualization of a specific chromosomal abnormality. FISH studies are less sensitive than PCR-based methods, but can detect abnormalities, such as monosomies and trisomies, that cannot be studied by PCR analysis.

HODGKIN’S AND NON-HODGKIN’S LYMPHOMAS HODGKIN'S DISEASE

Diagnostic Reed-Sternburg (RS) cell

Thomas Hodgkin’s Immune stain:

Hodgkin's disease is not common (annual incidence about 2 per 100,000) but it has generalized intense interest because of its predilection to affect young people, and its potential for cure with either radiation or chemotherapy. It is generally considered to be a type of lymphoma but clearly differentiated from the other "non-Hodgkin's" lymphoma to be discussed later. Common clinical features include fever, night sweats, pruritus, weight loss, lymphadenopathy and splenomegaly. Tissue diagnosis is mandatory, and achieved by lymph node biopsy or biopsy of another involved organ (eg. Liver, lung) Pathological classification: The W.H.O. classification will be used for classification of Hodgkin's disease - Hodgkin's Disease, Lymphocyte predominance (LPHD) - Classical Hodgkin's disease Nodular Sclerosis (NSHD) Mixed cellularity (MCHD) Lymphocyte Depletion (LDHD) Lymphocyte-Rich (LRHD)

CD15 & CD30 positive in classical HD LPHD:

abundant lymphocytes

Nodular Sclerosis: nodularity / RS cells abundant

LDHD: few lymphocytes seen

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MCHD: pleomorphism , PET Scan

mixed lymph, eosinophils, plasma cell. The clinical stage or extent of the disease however is more important in planning therapy and influencing prognosis. The following simplified stages are currently used: Stage I – disease in one lymph node region Stage II – disease in two or more lymph nodes but confined to one side of diaphragm Stage III – disease confined to lymph nodes or spleen and present on both sides of diaphragm Stage IV – disease outside confines of lymph nodes or spleen. (i.e. involves organs) The presence or absence of systemic symptoms are also important; these include: A– absence of fever, night sweats or weight loss (10% of body weight) B– presence of any of the above. The procedures required to stage patients with Hodgkin's disease vary somewhat from centre to centre, but generally include the following: 1. Routine hematology and biochemical tests including liver function 2. Chest X-ray – PA and lateral; CT chest or tomogram if indicated 3. CT Scan of abdomen

4. Nuclear scan: Gallium scan: shows active uptake in the lymphoid tumor. More recently PET scan is more utilized as it is more sensitive and more specific Gallium uptake –Anterior mediastinal mass

FDG PET scan in a newly diagnosed lymphoma. Widespread nodal disease is shown above and below the diaphragm. Spleen (arrowheads) is diffusely hypermetabolic, suggesting lymphomatous involvement.

5. Bone marrow aspirate and biopsy are done as part of staging work up to exclude bone marrow involvement. This is more likely if there are 'B' symptoms and hematological abnormalities. The principles of management of HD include the following: a. LPHD : local radiotherapy b. Classical HD stages I, IIA include 4 courses of chemotherapy and involved field radiotherapy c. Advanced stages (IIB, III, IV) includes systemic chemotherapy of 6 – 8 cycles. Local radiotherapy may be given for initial bulk (tumors > 10 cm) The potential for cure exists for all stages and all therapies but is much higher for earlier stages and asymptomatic disease. It is important to optimize treatment to minimize side effects. Chemotherapy, induces considerable morbidity; nausea and vomiting, anorexia, hair loss and long term can lead to sterility. However, the most commonly used protocol is known as ABVD (Adriamycin, Bleomycin, Vinblastin , DTIC) has less toxicity and is the most commonly used.. It has low infertility rate and less leukomogenic effect. Currently cure rates are high > 70 % for patients with good prognosis. However, the relapse rate is over 40% in patients with bad prognosis (advanced stages). For those who relapses a high dose chemotherapy and autologous peripheral stem cell transplant is offered. The progression free survival is 50 % at 5 years. LYMPHOMAS The “non-Hodgkin's" lymphomas are a somewhat variable group of malignant neoplasms primarily involving lymph nodes and/or spleen, although almost any organ can be involved. The clinical presentation is virtually identical to those of patients with Hodgkin's disease. Lymphomas are more likely to affect extra nodal sites e.g. gastrointestinal tract, than Hodgkin's disease in which this presentation is uncommon.

Classification of Non-Hodgkin’s Lymphoma –

B Cell Neoplasms • Precursor B-cell neoplasms

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• Mature B cell neoplasms T and NK Cell Neoplasms • Precursor T cell neoplasms • Mature T and NK cell neoplasms Examples of these cases: • Diffuse large B cell lymphoma (DLBCL) • Follicular lymphoma • Mucosa associated lymphoid tissue (MALT) • Burkitt lymphoma • CLL/SLL • Cutaneous T-cell lymphomas MF/Sezary –

CLL

CLL

Follicular NHL

Diffuse large B cell Lymphoma express CD 20 antigen CD20 + Large B- cells

Burkitt lymphoma with starry sky appearance

Mucosa associated lymphoid tissue MALT) lymphoma of the stomach

The NHLs can be divided into two prognostic groups: the indolent lymphomas and the aggressive lymphomas. Indolent NHL types have a relatively good prognosis, with median survival as long as 10 years, but they usually are not curable in advanced clinical stages. Early stage (I & II) indolent NHL can be effectively treated with radiation therapy along. Most of the indolent types are nodular (or follicular) in morphology. The aggressive type of NHL has a shorter natural history, but a significant number of these patients can be cured with intensive combination chemotherapy regimens) In general the well differentiated histological types and follicular (nodular) pattern have an indolent course. At present these cannot be cured but have a relapsing course and survive 7-10 years. Some live considerably longer. The high grade lymphomas have a more aggressive course. Previously such lymphomas had a bad reputation with early death. Combination chemotherapy with regime that usually contain cyclophosphamide, adriamycin (Doxorubicin), as well as other drugs have changed this outlook. The moast common chemotherapy protocol used is CHOP (Cyclophosphamide, Hydrodunarubicin, Onconvin, Presnisone). Now approximately 50% of patients with diffuse large cell lymphomas may have long term remissions and probable cure. Recently it has been shown the use of monoclonal antibody therapy increases the chances of response and cure rate. The most widely effective and popular monoclonal antibody is an anti CD20 monoclonal antibody, known as Mabthera or Rituxan. Currently, the use of anti CD20 monoclonal antibody in addition to chemotherapy is the standard of care for the most common aggressive lymphoma (diffuse large B cell lymphoma).Once relapse occurs, patients usually undergo a high dose chemotherapy and autologous stem cell transplant.. Results are better too in the treatment of two very aggressive high grade lymphomas with increasing evidence for possible cures in this group, namely Lymphoblastic lymphoma and Burkitt’s lymphoma. The current therapy for these two lymphomas include intensive systemic chemotherapy and CNS directed prophylaxis. Most of the indolent lymphomas, particularly the follicular and small lymphocytic lymphoma, have reached a widespread stage at the time of presentation. Bone marrow and liver are frequently involved in addition to generalized lymphadenopathy and splenomegaly. Follicular (nodular) lymphomas of insidious onset without significant symptomatology do not need treatment and many years may pass until treatment becomes indicated. When treatment is required, gentle chemotherapy with Chlorambucil will usually suffice for elderly patient. However, for younger patients a

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systemic chemotherapy and monoclonal antibody therapy may produce better results and long remissions.. For localized treatment radiotherapy may be indicated but this is not common as most cases have generalized disease or are of more aggressive type such that chemotherapy is the treatment of choice.

Radiptherapy may also be used in combination with a chemotherapy programme to treat bulky disease. It often plays an important role in treatment of relapsed disease when chemotherapy has failed or to treat a palliative symptomatic disease when cure is not possible.

Common chromosome translocations in nonHodgkin lymphoma Chromosome abberation

Lymphoma

Genes involved

t(14;18)(q32;q21)

Follicular lymphoma Diffuse large B-cell lymphoma

BCL-2, IgH

t(8;14)(q24;q32)

Burkitt lymphoma

C-MYC, IgH

t(8;22)(q24;q11)

Burkitt lymphoma

C-MYC, IgL

t(2;8)(p11;q24)

Burkitt lymphoma

C-MYC, IgK

t(11;14)(q13;q32)

Mantle cell lymphoma

CCND1, IgH

t(11;18)(q21;q21)

Marginal zone/extranodal MALT

API2, MALT1

t(14;18)(q32;q21)

Marginal zone/extranodal MALT

MALT1, IgH

t(1;14)(p22;q21)

Marginal zone/extranodal MALT

BCL-10, IgH

t(1;2)(p22;p12)

Marginal zone/extranodal MALT

BCL-10,IgK

t(3;14)(p13;q32)

”de novo” Diffuse large B cell

BCL-6, IgH

t(3;22)q27;q11)

”de novo” Diffuse large B cell

BCL-6, IgL

t(2;3)(p12;q27)

”de novo” Diffuse large B cell

BCL-6, IgK

t(2;5)(q23;q35)

Anaplastic large cell T/null

ALK, NPM

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MYELOMA Myeloma is a disease caused by malignant proliferation of plasma cells. It usually presents as a bone disease, with bone pain, but multiple other presentations are possible due to many complications which may occur. Most cases of myeloma are due to clones of plasma cells producing immunoglobin G (IgG), but IgA myeloma is also common – IgD and IgE myeloma are rare. Myeloma must be differentiated from: 1. Benign Monoclonal Gammopathy – long-standing non-progressive monoclonal increase in immunoglobulin without marrow plasmacytosis or bone lesions. Usually the Ig elevation is mild (25 g/L) and urinary light chain proteinuria is present. 2. Waldenstrom’s macroglobulinemia – this uncommon disorder presents with monoclonal elevation of IgM; in contrast to myeloma, bone disease is usually absent, whereas lymphadenopathy and splenomegaly may occur. The cellular infiltrate is heterogeneous (lymphocytes, plasma cells and plasmacytoid lymphocytes) Hyperviscosity symptoms may be prominent. The disease is usually slowly progressive; if therapy is needed then Chlorambucil will usually suffice. Myeloma – the following presentations are worth noting. CLINICAL As noted above, bone pain due to osteolytic bone lesions with or without pathological fractures or vertebral compression fractures from osteoporosis or osteolytic lesions may all occur. Symptoms of anemia (fatigue, pallor), leucopenia (infections) or thrombocytopenia (bruising or bleeding) may all be present due to myeloma replacement of bone marrow. Renal failure may result from myeloma kidney (light chain, or Bence-Jones protein, precipitation), amyloidosis, hypercalcemia, hyperuricemia or infection. Amyloidosis may also cause tongue enlargement, arthropathy, hepatosplenomegaly, purpura, carpal tunnel syndrome, etc. Hypercalcemia may induce polyuria, polydipsia, vomiting and confusion. LABORATORY Bone marrow Infiltrated with plasma cells

a. Blood picture - anemia, leucopenia and thrombocytopenia

b. Bone marrow - 10 – 20% plasma cells usually required for diagnosis of myeloma. Multinucleated and immature forms are common. c. Biochemical tests for - total protein (increased) and albumin (decreased) - hypercalcemia - hyperuricemia - renal function - β-2 microglobulin - LDH - C- reactive protein - Cytogenetic studies - serum protein electrophoresis – detects and quantitates the monoclonal protein; about 15% however, have hypogammaglobulinemia.

-

Urine protein (24 hour) and protein electrophoresis – quantities the amount of light chain proteinuria (routine urinary dipstick test for protein does not detect light chains). d. Immunology tests - Ig quantitation Immunisation electropheris – confirms Ig type (IgG, IgA, etc) and light chain (kappa or lambda) - Urinary light chains – selective kappa or lambda light chain proteinuria e. X-rays - skeletal survey to detect osteolytic lesions or diffuse osteoporosis. X-rays of skull, spine and pelvis are particularly important, but ribs and long bones can be involved and should be xrayed if clinically indicated. Note that alkaline phosphatase is usually not elevated, and bone scans are usually normal because the bone lesions are almost totally osteolytic in nature. MANAGEMENT 1. CHEMOTHERAPY Melphalan and Prednisone given for 4 days each month are helpful in palliative treatment, causing control or regression of myeloma in most, but not all, patients. Morbidity from chemotherapy is minimal. It may be continued as long as patient lives, or stopped

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after improvement is complete, or stable, to be restarted at the time of relapse. Cyclophosphamide may be substituted for melphalan if it is no longer effective. There is definite increase of leukemic transformation in patient receiving alkylating agent chemotherapy but the improved quality of life and survival warrant this risk. Treatment of multiple myeloma in symptomatic patient is dictated by age and presence of co-morbidities. For patients aged below 70 years initial induction by combination chemotherapy of Vincristine, Doxarubicin, Dexemethasone (VAD) 3-4 cycles followed by high dose chemotherapy and autologous stem cell transplantation is the treatment of choice and results in longer progression free survival and overall survival compared to conventional chemotherapy. 2. RADIOTHERAPY Excellent treatment for symptomatic isolated bone involvement, unresponsive to chemotherapy. Radiotherapy is used in patients with cord compression and in prevention of impending pathological fractures. 3. SURGERY For pathological fractures 4. SUPPORTIVE CARE 1. Biophosphonates: Bone involvement in multiple myeloma is a major reason for morbidity of the disease. Biophosphanates inhibit bone resorption. Pamidronate or the newer more potent Zoledronate are usually used to prevent skeletal events. Ideally such treatments should be combined with calcium and hormone replacement in patients with no contradiction to their use. 2. Erythropoetin: Anemia is frequent in patients with multiple myeloma. Recombiant human erythropoietin decreases transfusion requirement, increases means hemoglobin, improves quality of life and performance status in aneamic myeloma patients. Erythropoetin may be used in weekly or three times weekly schedules 3. Vertebroplasty and Kyphoplasty: Painful vertebral compression is a major cause of morbidity in multiple myeloma patients. Vertebroplasty and Kyphoplasty helps in relieving pain in most patients

4. Treatment of infection: Multiple myeloma and its treatment including chemotherapy and transplantation render patients extremely vulnerable to infection. Prompt treatment of infection and in selected cases prophylactic antiinfective therapy reduces morbidity and morbidity. 5. SPECIAL SITUATIONS • Amyloidoisis is usually, but not always, unresponsive to any treatment. • Hypercalcemia – must be promptly and aggressively treated with large volumes of intravenous saline, steroids, and chemotherapy or biphosphonates. • Allogeneic bone marrow transplantation can cure multiple myeloma, however, high rates of treatment related mortality and poor overall survival precludes its routine use. Low intensity allogeneic bone marrow transplantation following cytoreductive chemotherapy, autologous transplantation may however be used in high risk cases. Patients not eligible for high dose therapy should be treated with combination of Melphalan & Prednisolone or Dexamethasone with Thalidomide. Thalidomide and its analogoue lenalidomide has antiangiogenic, antimyeloma and immunnomodulatory effect and has been found to be effective in newly diagnosed myeloma as well as in relapsed patients. Bortezomib (valcade), a proteosome inhibitor has been found to be effective in relapsed multiple myeloma and is under trial in patients with newly diagnosed multiple myeloma. • Renal failure – treatment of hypercalcemia, hyperuricemia and the myeloma are all important to reverse as much of the renal failure as possible. If renal failure is established and severe, then careful medical management of the azotemia, and electrolytes, will be needed; dialysis is sometimes employed if the life expectancy and general condition of the patient warrants this. The treatment of myeloma improves the quality of life (decreases bone pain, improves blood counts, etc) and prolongs life (median survival is now 3 – 4 years compared to 1 year untreated). Current treatment does not cure the disease.

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LEUKEMIAS ACUTE LEUKEMIA The acute leukemias are not common (annual incidence rate of 3.5 / 100,000) but a major cause of morbidity and mortality particularly in young people (acute lymphoblastic leukemia is the commonest cancer in children). Although the incidence is not increasing, the prevalence is, since many cases who previously would have died quickly are now achieving prolonged remissions with chemotherapy and supportive care. The hallmark of the acute leukemias is the characteristic proliferation of immature (blast) cells in the bone marrow, and often in the peripheral blood where WBC counts can be increased up to 100 x 109/L or more. If the diagnosis is obvious from the WBC differential (large number of blast cells) then a bone marrow is not essential, but if there is any doubt when a bone marrow will be required to demonstrate the involvement, or often complete replacement by rapidly proliferating blast cells. The blast cells fail to differentiate into more mature forms (i.e. granulocytes, lymphocytes or monocytes) and inhibit maturation of normal clones of marrow cells. The acute leukemias are divided into the following categories. 5. Acute myeloblastic leukemia (AML) – variants include acute myelomonocytic leukemia (AMML), promyelocytic leukemia and erythroleukemia 6. Acute lymphoblastic leukemia (ALL) – variants include acute prolymphocytic leukemia, undifferentiated leukemia 7. Acute monocytic leukemia Morphological criteria (plus cytochemistry) used to be the prime method of differentiating the various types of leukemia. Now with the use of monoclonal antibodies, cell surface antigens can be identified, enable a more accurate differentiation of acute leukemias. This is particularly important in determining the type of acute lymphoblastic leukemias. Cell surface marker analysis of ALL now determines the type of treatment used and probable prognosis. Morphological criteria are still important and both techniques are necessary for diagnosis. In AML the use of cell surface marker analysis has not yet altered treatment approaches. AML is the commonest acute leukemia in adults (90%) whereas ALL is commonest in children (90%), 70% are CALLA positive. Presentation of all forms of leukemia is distressingly similar, a previously healthy person develops symptoms from reduction in circulating blood cells (anemia, thrombocytopenia, fever or lymphadenopathy / splenomegaly, with the duration of illness usually being very short (a few days to a few months). ALL may cause enlarged lymph

nodes (including mediastinal) and splenomegaly, whereas these features are usually absent in the commoner AML in adults. Acute monocytic leukemia presents typically with gingival enlargement from monoblast infiltration. MANAGEMENT CHEMOTHERAPY The initial goal of chemotherapy is remission induction ( a complete remission has occurred when there is no evidence of leukemia in the blood and < 5% blast cells in the bone marrow) This is readily achieved in children with ALL (95%) but only in about 60% adults with AML. Combination chemotherapy is required with various protocols (drugs such as vincristine, prednisone, L-asparaginase, methotrexate, 6-Mercaptopurine, adriamycin, cytosine, arabinoside, and 6-thioguanine) are used in cyclic fashion to bone marrow patient tolerance. In the treatment of AML this aggressive chemotherapy is continued for two or more courses after complete remission is achieved for consolidation. Maintenance treatment is not usually used as it has not been proven to prolong survival in AML. In treatment of ALL following induction of remission, consolidation therapy is given which is followed by maintenance treatment. The total time treatment undertaken is 2 – 3 years. Most maintenance treatments are given on an outpatient basis and patients return to regular routines of daily living. In childhood ALL, the risk of CNS involvement or meningeal leukemia is so high ( and not counteracted by systemic chemotherapy) that these patients receive prophylactic intrathecal chemotherapy and is a subset cranial radiation to prevent CNS relapse. The outlook for childhood ALL is much improved with modern chemotherapy protocols with the prospect of cure in up to 70% of cases. In adults results are not so encouraging. A median survival of 18 months for AML responders and 30% long term survivors (5 years) can be achieved. For ALL recently 25-40% long term survival has been achieved. Morbidity during aggressive induction chemotherapy is high (nausea and vomiting, alopecia, bleeding / bruising, infections, anxiety and depression). Maintenance chemotherapy used in ALL is generally well tolerated. Once relapse is occurred second remissions are much more difficult to achieve particularly in AML. If the patient has an HLAidentical sibling then bone marrow transplantation may be a viable alternative for AML (first remission) or ALL (second remission)

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SUPPORTIVE CARE At presentation these patients are often anemic, infected and bleeding; during remission induction these potential complications are very common, and the following guidelines generally apply to their management: a. Anemia - packed red cell transfusions are given as needed for symptomatic anemia b. Thrombocytopenia - during remission induction, spontaneous hemorrhage is common if the thrombocytopenia is severe (platelet count 10 x 109/L) so many centers provide prophylactic platelet transfusions during this period; others are more selective, attempting to monitor the seriousness of the thrombocytopenia by clinical assessment (amount of purpura, epistaxis, mucosal bleeding, etc). The goal is to prevent intracerebral hemorrhage which is rapidly fatal if it occurs. Any drugs which cause platelet dysfunction must be omitted e.g. Aspirin, and intramuscular injections are prohibited. c. Granulocytopenia - although reverse isolation procedures do not alter the frequency of serious infections in these patients, most acute leukemias are provided with a private room, whenver possible, and visitors with infections are discouraged. Masks may help if attending staff have a potentially transmittable upper respiratory infection and hands should be washed before examining the patient. If a neuropenic patient develops a chill and/or fever, the patient should be promptly assessed for site of infection, cultured immediately (Throat swab, urine C & S, blood cultures etc) and if a septicemia is likely, should be placed on broad spectrum (eg. Cephalosporin plus aminoglycoside) intravenous antibiotics as soon as cultures have been obtained. Granulocyte transfusions have not been proven to be of help in this situations. d. Emotional support - These patients require regular visits, frank discussions and frequent reassurance from compassionate and knowledgeable medical, nursing and other personnel to cope with the multiple physical and emotional problems which routinely are encountered. CHRONIC GRANULOCYTIC LEUKEMIA Chronic granulocytic leukemia (CGL) accounts for 20% of all cases of leukemias, and mainly onset is in middle age although cases in both adolescents and elderly patients are seen. The onset is insidious over several months, or more, and patients often present with significant splenomegaly, possibly symptomatic and hypermetabolic symptoms (fever, night sweats, etc) Diagnosis is usually obvious after clinical examination and assessment of the WBC differential. Leukocytosis is present with left shift in

granulocytic series, often back to myeloblast state (but the "hiatus" between blast cells and mature cells as seen in acute leukemia is absent in CGL) Anemia is usually mild, and a wide range of platelet counts may be seen (to 1000 x 109/L) The bone marrow also demonstrates the increased and left-shifted granulopoiesis, but is generally not required for diagnosis (there are no diagnostic morphologic features which differentiate CGL from reactive granulocyte hyperplasia) It may, however, provide a sample for karyotyping for Philadelphia chromosome if needed. The Philadelphia chromosome (loss of short arm of chromosome number 22 due to 22:9 translocation) is present in 8590% of CGL patients; if absent, in a patient who appears to have CGL by other tests, the prognosis is worsened. The Philadelphia chromosome is only rarely present in other myeloproliferative diseases. Another test which might help differentiate between CGL and leukemoid reaction (granulocytic hyperplasia) is leukocyte alkaline phosphatase (LAP); this is a histochemical test for alkaline phosphatase in leukocyte (neutrophil) granules – it is characteristically decreased – absent in CGL but normal – increased in leukemoid reactions. It is performed on peripheral blood, rather than bone marrow. MANAGEMENT OF CML 1. Oral chemotherapy: During chronic phase, hematological remission is produced in more than 70% of patients treated with Hydroxyurea or Busulphan. Hydroxyurea is preferred over Busulphan because of better toxicity profile, progression free survival and overall survival. However, no significant cytogenetic remission occurs with Hydroxyurea or Busulphan and over all pace of progression to blast crisis is unchanged. These treatments are therefore considered to be palliative. 2. Allogeneic bone marrow transplantation: Allogeneic hematopoietic cell transplantation is the only known curative treatment for CML. However only minority of patients have HLA matched siblings. Treatment related morbidity and mortality is significant and increase with increasing age of the patient. Therefore currently this modality is offered to patients less than 40 years of age. In younger patients HLA matched unrelated donor transplantation is feasible. 3. Interferon Alpha Before the use of Imatnib mesylate, interferon alpha was the standard therapy for CML in patients not eligible for allogenic bone marrow transplantation. Interferon alpha alone or in combination with Cytosinarabinoside produced hematological remission in most patients and cytogenetic responses in 20% patients. There is

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significant toxicity and interferon has even superseded by Imatnib 4. Imatinib Mesylate Imatinib (Gleevec) is a tyrosine kinase inhibitor. It inhibits PDGF, Abl & C-kit tyrosine kinases by interfering with the ATP binding site. It produces hematological responses in 94% patients and major cytogenetic responses in 83% patients. The drug has excellent toxicity profile with common side effects being mild nausea, myalgia, edema and diarrhea. However, it is not clear at present whether it is a curative modality for CML. More potent tyrosine kinase inhibitors are currently undergoing clinical trials. CHRONIC LYMPHOCYTIC LEUKEMIA Chronic lymphocytic leukemia (CLL) is the commonest form of leukemia, appearing mainly in old age, often symptomless and requiring no treatment. Hypermetabolic symptoms such as fever, night sweats, fatigue and weight loss may occur. Lymph nodes and spleen are regularly enlarged often for many years, without causing symptoms, but can achieve such enlargement as to require treatment for pressure or cosmetic symptoms. The laboratory diagnosis is usually simple. There is lymphocytosis which may reach extreme degrees (500 x 109/L or more) without other obvious causes (eg. Viral infection) The lymphocytes seen are usually small lymphocytes and appear morphologically normal. Frequently they are fragile so that they rupture during preparation of the slide; they are then called "smudge cells". Although not diagnostic of C.L.L they are so frequently seen in this type of leukemia that they are quite typical. The bone marrow contains a lymphocytic infiltration but often is not needed to make the diagnosis of CLL. Flow cytometry reveals a

characteristic antigenic profile in CLL lymphocytes. The lymphocytes are positive for CD45, 19,20,22,23, HLADR and characteristically show co-expression with T cell antigen CD5 and light chain restriction. The immunophenotyping helps to distinguish it from other lymphoproliferative disorders. Anemia and /or thrombocytopenia may result from marrow involvement, hypersplenism, or autoantibodies (immune hemolytic anemia of IgG type of secondary I.T.P) Hypogammaglobulinemia is common. MANAGEMENT OF CLL Patients with early stage of CLL do not require treatment. In young patients, Fludarabine + Cyclophosphamide is currently the treatment of choice. It produces higher incidence of remission (complete and overall) compared to Chlorambucil or combination chemotherapy. The drug may be associated with cumulative myelotoxicity and increased risk of opportunistic infection. In older patients, intermittent Chlorambucil is an appropriate therapy, because of it ease of administration and relatively favourable toxicity profile. Moncolonal antibody, anti CD20 (Rituximab) is effective when used in combination with chemotherapy and anti CD52- Alemtuzumab (Campath) have significant activity in CLL. These agents are however used for patients who have failed Fludarabine therapy. Coomb's positive autoimmune hemolytic anemia and immune thrombocytopnenic purpura are treated with Prednisolone. Radiotherapy is reserved for symptomatic lymph node enlargement not responding to chemotherapy.

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PROBLEMS FOR TUTORIALS 1. ADULT MAN WITH LYMPHOCYTOSIS A 65-years-old man was brought to the emergency unit by his son because he has fainted at home. According to his son, the man was gradually feeling week with inability to do his daily routine. He had loss of appetite and lost 5 Kg. over the last 6 months. He had no previous history of cardiac disease or hypertension but had diabetes for which he was on oral hypoglycemic drugs for the past 5 years. On examination, the man was conscious, oriented, a febrile, pulse: 90 b. / min. B.P: 130 / 80, he was not jaundiced but had pale conjunctiva. Examination of the neck revealed bilateral cervical and supraclavicular lymphadenopathy. There were also two axillaries and one inguinal lymph node. Examination of the heart and chest was normal. Abdominal examination revealed a splenomegaly of 10 cm. below the left costal margin (LCM) Investigations showed: Hb. 95 g/L N M: 140-160 F: 120-160 MCV: 105 fl (N 80- 95) WBC count: 50 x 109/L Lymphocytes 80% Platelets 90 x 109 / L Blood Sugar: 8.5 mmol / L Na: 140 mmol/L K: 4.2 mmol/L Questions: 1. What are the important findings in the history and clinical examination of this patient? 2. What is the differential diagnosis 3. What is the most likely diagnosis? 4. What do you think is the cause of the anemia?

2. A YOUNG

FEMALE WITH NECK LUMP

A 17-year-old Kuwaiti girl sought medical at tension because she noticed lumps in the left side of her neck. She denied any B-Symptoms. Her past medical history is unremarkable. On examination, there is a 3x3-cm firm, non-tender, freely movable nodule in the left neck just above the sternoclavicular joint, and several smaller nodes to the left along the clavicle. All vital signs are normal. There is no fever or icterus. The thyroid gland is normal. The chest, heart, breasts are normal. The spleen is palpable 2 cm below the left costal margin on deep inspiration.

Questions: 1. What are the B-symptoms and how do you define them? 2. What is your differential diagnosis? 3. What initial tests would you like to order? 4. How do reach a final diagnosis? What do the cells shown below imply?

3. POLYCYTHEMIA Problem: A 60 year old man with history of heavy smoking and chronic bronchitis was admitted to hospital with the onset of severe dyspnea. This man's chronic chest disease has been present for twenty years. At the time of admission to hospital, he was dyspneic and plethoric with both central and peripheral cyanosis. There was no clubbing. Examination of the chest revealed diffuse wheezing. Laboratory Investigations: Hemoglobin 210 g/L Hematocrit 0.69 WBC 19 x 109/L Platelet Count 950 x 109/L Chest X-ray showed over inflation of the lungs bilaterally Required questions: 1. Is this likely to be a primary or secondary erythrocytosis (polycythemia)? Why? 2. What additional investigations would you perform? 3. Discuss treatment in terms of Polycythemia

4. ATYPICAL LYMPHOCYTOSIS Problem: This 18 year old woman saw her physician for increasing tiredness over the past two weeks associated with a sore throat and fever. Physical examination revealed a young woman who had bilateral slightly tender anterior cervical nodes 2.5 cm in diameter. The spleen was palpable 3 cm below the left costal margin Laboratory Investigations: Hemoglobin 132 g/L WBC 12.4 x 109/L .01 bands .46 segmented neutrophils .26 lymphocytes .25 atypical lymphocytes .02 monocytes Platelet count 110 x 109/L Required questions: 1. List 2 causes of atypical lymphocytosis 2. What is the most likely cause in this patient 3. The patient worries about having cancer. List 3 reasons why you think it is unlikely 4. The patient requests an antibiotic for new sore throat. You do not agree with this. Why not, and what would you do instead

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5. What are the appropriate confirmatory tests? 5. LEUKOCYTOSIS AND SPLENOMEGALY Problem: This 51 year old woman was referred to hospital. One month prior to admission she had sharp pains in the left upper quadrant of her abdomen. She palpated a large mass herself, and consulted her private physician who thought the mass was spleen. He noted that the leukocyte count was elevated. Laboratory investigations Hemoglobin 100 g/L Platelet count 650 x109/L WBC 70 x 109/L .02 blasts .03 promyelocytes .18 myelocytes .17 metamyelocytes .25 bands .20 neutrophils .04 lymphocytes .02 monocytes .05 eosinophils Required Questions: 1. What is the most likely diagnosis? 2. What confirmatory tests might you order 3. The patient asks if her spleen should be removed since it bothers her. Do you agreed and why? 4. The patient has just started a clothing business and wants to make plans for the future. What advise would you provide regarding: a. Possibility of bone marrow transplantation b. Side effects of the treatment you would prescribe c. Complication which is likely to occur and when. 5. New treatment. 6. PANCYTOPENIA Problem: This 59 year old woman was referred to hospital because of a low WBC count. The patient denied malaise of fatigue. Two weeks before admission, she had an acute respiratory tract infection treated with penicillin. Except for pallor and few bruises, there were no other physical findings. Laboratory Investigations Hemoglobin 80 g/L MCV 82 fL MCHC 350 g/L WBC 3.8 x 109/L Platelet count 15 x 109/L Required Questions 1. List any further laboratory tests you would request 2. List 2 possible diagnosis for the blood changes 3. The day after admission, her temperature spiked to 39oC and she had a chill. She also developed many bruises, recurring nosebleeds

and some gum bleeding. By this time it was known that her neutrophil count was very low (less than 0.5 x 109/L) Describe your management with regard to: a. Tests to investigate the fever b. Any treatment for the febrile illness. What should she avoid? c. Treatment of the thrombocytopenia? i. What blood products should be ordered? ii. How much? iii. Expected response 7. LEUKOCYTOSIS Problem: This 78 year old man visited his family physician for an annual "checkup" He was in good health except for mild symptoms of congestive heart failure being treated with digoxin and hydrochlorothiazide daily. On examination, there was no tonsillar enlargement, lymphadenopathy or hepatosplenomegaly Laboratory Investigations: Hemoglobin 148 g/L MCV 88 fL MCHC 34- g/L WBC 58.0 x 109/L .15 neutrophils .65 lymphocytes .20 smudge cells Platelet count 175 x 109/L Required Questions: 1. What is the most likely diagnosis to explain the blood findings? 2. Are any further tests required 3. What advise would you give this patient regarding: a. Need for treatment now? b. Indications for treatment in the future c. Side effects of treatment if required? d. Overall disease course 8. LYMPHADENOPATHY Problem: A 30-year old female who was previously well until 4 weeks ago when she noted a lump in her left axilla. During the past 2 weeks she has complained of fatigue. Physical examination revealed a 3 cm, non-tender soft mass in the left axilla. There were no abnormalities detected on breast exam and there as no evidence of splenomegaly or lymphadenopathy elsewhere. Laboratory Investigations: Hemoglobin 115 Hematocrit .3 White Blood Count 4.5 Platelet count 200 Chest X-ray small mediastinal mass Required Questions:

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1. List 3 additional pieces of information from

4. What other investigations are necessary if this

the history you would require 2. What test would you arrange to make the diagnosis? 3. What are the most likely diagnoses?

is either Non-Hodgkin's Lymphoma or Hodgkin's Disease? 5. Assuming the diagnosis of Hodgkin's Disease, what additional investigations may be done? 6. What is the importance of the information gained in the above questions?

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MYELOPROLIFERATIVE DISORDERS The designation "myeloproliferative disorder" is a general term given to wide range of malignant proliferation of myeloid tissue, which can be classified as follows: MYELOPROLIFERATIVE DISORDERS 1. Polycythemia vera 2. Myelofibrosis (myelosclerosis) 3. Myeloid metaplasia (with or without myelofibrosis) 4. Chronic granulocytic leukemia 5. Thrombocythemia 6. Acute myeloblastic leukemia We have included CGL and AML under leukemias, and will restrict further discussions to polycythemia, myelofibrosis (myeloid metaplasia) and thrombocythemia. POLYCYTHEMIA VERA Before deciding whether a patient has polycythemia vera, the physician must first usually exclude two other possibilities: b. Spurious (stress, or relative) erythrocytosis – patients with this disorder are detected by the isolated findings of elevated Hb and Hct, often in association with hypertension (Gias-bock's syndrome) Blood volume studies (Cr51 labelled RBC's) demonstrate a normal red cell mass, however, and the "apparent" erythrocytosis is due to a reduction in plasma volume. This condition requires recognition, to differentiate it from true polycythemia, but no treatment. c. Secondary polycythemia (erythrocytosis)- this represents a response to tissue hypoxia, or autologous erythropoietin production and the following should be considered: i. Chronic lung disease – particularly chronic bronchitis ii. Congenital heart disease – cyanotic, with right to left shunt iii. Renal disease – renal cell carcinoma, cysts, renal artery stenosis iv. Liver disease – hepatoma v. High altitude vi. Hemoglobinopathy – high affinity hemoglobin (rare) vii. Cerebellar hemangioblastoma (rare) Most of the above can be ruled out on clinical assessment. The one diagnosis which must not be missed is the renal cell carcinoma which may still be curable if detected at a localized stage; if any doubt exists as to a cause for erythrocytosis, appropriate investigations of the urinary tract should be considered.

In both of the above possible categories the abnormality is usually limited to increased Hb, Hct, and RBC count, without an elevation of WBC count or platelet count. The red cell mass, as assessed by Cr51 blood volume, is increased in both secondary polycythemia, and primary polycythemia (P. vera) Polycythemia vera is a malignant proliferation of myeloid cells usually involving all marrow cell lines leading to erythrocytosis, leukocytosis (neutrophilia) and thrombocytosis. Splenomegaly is present in 75% of patients at diagnosis) Common symptoms include plethora (ruddy cyanosis of face and extremities), hyperviscosity symptoms (transient bleeding, TIA's, angina, claudication etc) bleeding and pruritus (which is common, often sever, and aggravated by bathing "bath pruritus") Management includes control of red cell mass (hyperviscosity) and prevention of bleeding complications. If erythrocytosis is marked but not leukocytosis or thrombocytosis, then a therapeutic phlebotomy program (removal of 500 ml blood usually weekly) may be instituted; usually this must be continued till the patient develops iron deficiency, limiting erythropoiesis and this may take several months. If there is complicating thrombocytosis, symptomatic splenomegaly or hypermetabolic symptoms, then radioactive phosphorus (P32) will provide good long term control of P.vera, and should be considered particularly in older patients. Chemotherapy has also been used, but there is a definite leukemogenia potential with either chemotherapy (chlorambucil, myleran) or P32 so this must be carefully assessed for each patient. Younger patients should receive hydroxyurea, as chemotherapy, due to its lower leukemogenic risk. Recently Aspirin has been shown to decrease the risk of thrombosis. Uric acid is usually increased and most patients are treated with allopurinol to prevent gout or other complications of hyperuricemia. MYELOFIBROSIS, MYELOID METAPLASIA These two conditions often exist together, although myeloid metaplasia can occur without fibrosis in the bone marrow (called "agnogenia" myeloid metaplasia). The marrow fibrosis may result in bone proliferation as well (myelosclerosis) and be visible on x-ray but usually it doesn't. It does result in decreased hematopoiesis, and the concomitant myeloid metaplasia present mainly in the liver and spleen is rather inefficient. Accordingly, the patients with these disorders usually have anemia and thrombocytopenia, both of which may be severe, whereas the leukocytes are often increased and left shifted.

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The peripheral blood film is usually characteristic, with a "leukoerythroblastic" blood picture – nucleated RBC's and left-shifted granulocyte series. Other features include teardrop poikilocytes, and megathrombocytes – all of these abnormalities result from abnormal hematopoiesis taking place outside the bone marrow. Marrow biopsy is required to demonstrate the marrow fibrosis. Patients usually present with symptoms of anemia and/or thrombocytopenia, with systemic complains of fever, night sweats, and weight loss. Splenomegaly is usually prominent and may be massive producing major pressure symptoms. Splenic infarcts are common. Treatment is unsatisfactory – Supportive care with red cell transfusions for symptomatic anemia and platelet transfusions for thrombocytopenia bleeding complications may be required. Androgens may provide mild benefit for the anemia and folic acid may

be helpful if deficiency is documented. Splenectomy may be required to control symptoms of massive splenomegaly; this operation is risky, however, and platelet function should be studied prior to surgery since many patients have abnormally functioning platelets in addition to the thrombocytopenia. ESSENTIAL THROMBOCYTHEMIA If the thrombocytosis is extreme (1,000 x 109/L) it should probably be controlled by chemotherapy Hydroxyurea (myleran) particularly if any surgery is planned and is elective enough to provide time for this treatment to be effective (1-2 months) Emergency treatment with plateletphoresis is rarely needed. These patients tend to develop other myeloproliferative disorders (polycythemia, leukemia) but usually many years pass before this occurs. Baby Aspirin may reduce the risk of arterial thrombosis.

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MODULE III & IV HAEMOSTASIS & THROMBOSIS OBJECTIVES •

Discuss common causes of bleeding disorders and their pathogenesis.

•

Describe the clinical presentation, plan of investigations and interpret, integrate and correlate salient points in clinical history, physical findings and laboratory data so as to reach a correct diagnosis in a patient presenting with a bleeding disorder.

•

Describe the common mode of clinical presentation and demonstrate ability to interpret relevant laboratory data in patients with following bleeding disorders.

•

Haemophilia

•

von Willebrand's disease

•

Acquired coagulation deficiency secondary to vitamin K deficiency, liver disease, chronic renal failure, and DIC.

•

Discuss common causes of thrombocytopenia.

•

Plan and interpret salient points in clinical history, physical findings and relevant laboratory data to establish diagnosis of the type and cause of thrombocytopenia in a patient.

•

Plan and administer rational therapy to a patient with thrombocytopenia.

•

Describe the indications, schedule of administration and side effects of the use of fresh frozen plasma, cryoprecipitate and platelets concentrates in the management of bleeding disorders.

•

Discuss pathophysiologic mechanisms of thrombosis and describe molecular basis of inherited thrombophilia.

•

Enumerate common causes of acquired and inherited thrombotic disorders.

•

Demonstrate ability to correlate salient points in clinical history and physical findings and to plan and interpret laboratory investigations so as to make dignosis of a thromboembilic disorder.

•

Describe mechanism of action, dose, frequency, mode of administration, and adverse effects of heparin and coumadin anticoagulants

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BLEEDING DISORDERS GENERAL OBJECTIVES The student should have a general understanding of hemostasis, the types of common or important bleeding problems which occur with hemostatic defects, and the principles of management for these problems. SPECIFIC OBJECTIVES At the end of this section, the student should be able to: 1. list possible causes of bleeding problem given either a clinical presentation of bleeding, abnormal tests of hemostasis or both 2. List any additional tests required, if any to define the precise cause of a bleeding problem. 3. Describe the clinical presentation and expected laboratory abnormalities for common or important bleeding problems; these include Hemophilia, von Willebrand's disease, Acquired coagulation deficiencies, secondary to vitamin K deficiency, liver disease and DIC, thrombocytopenia secondary to ITP, consumption or decreased production. 4. Demonstrate an understanding of the use of heparin and coumarin anticoagulants including types of drugs, dosage, frequency and route of administration for both prophylaxis and therapy of thromboembolic disease 5. Describe the mode of inheritance for hereditary coagulation disorders 6. Describe the use of Fresh Frozen Plasma, Cryoprecipitate and Platelet concentrates in the management of bleeding disorders, including indications and contradictions for its use, expected response and possible complications. PLATELET / VASCULAR DISORDERS OF HEMOSTASIS CLASSIFICATION OF PURPURA (BRUISING) THROMBOCYTOPENIC PURPURA 1. Decreased Production a. Hereditary – very rare b. Marrow suppression – medications, radiation, alcohol, chemotherapy c. Marrow infiltration – leukemia, metastatic tumors d. Marrow ineffective – megaloblastic, dysmegakaryopoiesis 2. Decreased Survival a. Immune – I.T.P, medications, sepsis, neonatal 3. Dilutional a. Massive transfusion

1. Defective platelet function a. Hereditary – von Willebrand's Disease (VWD), defective aggregation b. Acquired – medications, anemia, myeloproliferative diseases. 2. Vascular Disorders a. Allergic (Henoch-Schonlein purpura) b. Non-allergic – mechanical, senile, steroid, dysproteinemia, connective tissue disorders. APPROACH TO PATIENTS WITH BRUISING (PURPURA) In general, patients with thrombocytopenia, defective platelet function, or vascular disorders tend to bruise either with minimal trauma or even spontaneously, depending on the severity of the problem. The various factors tend to be additive ( i.e. one would expect more bruising in an elderly patient with defective connective tissues, who is thrombocytopenia and taking Aspirin. Also the combination of platelet/vascular disorders discussed in this section with coagulation disorder discussed in subsequent sections usually leads to more severe bruising and bleeding problems than either would by itself. The commonest manifestations of these disorders are bruises and petechiae involving the muco-cutaneous surfaces. Petechiae appear as small (1 – 3 mm diameter) red spots, which do not blanch; they may occur on any cutaneous or mucosal surface but tend to be more evident on dependent areas, such as legs in ambulatory patients. Widespread and/or mucosal lesions indicate a serious problem. In addition to these manifestations patients with several platelet and vascular disorders may also present with abnormal bleeding at many sites (e.g. epistaxis, GI hemorrhage, cerebral hemorrhage, intramascular hematomas, etc). Excessive bleeding often occurs with surgery and trauma CLINICAL ASSESSMENT Special attention must be paid during the interview to elicit information about current bleeding and bruising problems as well as previous history, including surgery, dental extractions and childbirth. A meticulous enquiry into the use to drugs, medications and alcohol is mandatory and the review of the family history must be thorough to detect hereditary problems. 70% of all hereditary bleeding diseases will have an evident family pedigree Physical examination should document the extent and location of petechiae and bruises as well as the presence of absence of other vascular malformations e.g. telangiectasia, hematoma, confluent hemorrhagic stomatitis. It is also important to rule out

NON-THROMBOCYTOPENIC PURPURA

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other underlying diseases such as liver disease or hematologic disorders. LABORATORY ASSESSMENT 1. CBC, Differential, Platelet count: Platelet must be either estimated or counted in a bleeding or bruising patient to document thrombocytopenia. If the estimate made on observation of the blood film is normal, this is sufficient to rule out thrombocytopenia. If platelets appear reduced, a platelet count is usually performed to quantitate platlet number accurately. Mordern cell-counter equipment provide accurate counts rapidly, often alont with other measurements (Hb, WBC, indices, etc); if this equipment is not available then manual platelet counts must be performed – these are more time consuming and less accurate. When thrombocytopenia is sever (<109/L) most laboratories report it as such rather than providing a figure since counts below that level cannot be accurately measured. All Abnormal platelet counts must be confirmed by microscopic examination of a blood film. The CBC and Differential are required to determine whether a thrombocytopenia is selective (usually indicating peripheral destruction, often immune in origin) or associated with decrease in other cellular elements (anemia, leucopenia) and/or an abnormal differential (eg leukemia). Morphology of the platelets is important to assess eg. Abnormal shape or cytology 2. Coagulation Screening Tests: If abnormal in a thrombocytopenic patient this suggest involvement with coagulation system. Certain diagnosis, such as D.I.C, liver disease / hypersplenism and lupus require exclusion. 3. Bleeding Time: This test is usually performed by making 2 short standard incisions on the patient's forearm, often using a template to regulate the length and depth of the incisions. The time taken for the bleeding from the incisions to cease is a good measure of the in vivo significance of thrombocytopenia or platelet dysfunction. It is usually performed in a bruising patient with normal numbers of platelets to detect platelet function abnormalities. If it is normal, and platelet number is normal then no further platelet tests (eg aggregation) are indicated. A prolonged bleeding time in the presence of normal platelet number implies a platelet function defect and further studies may be indicated unless the cause is evident (eg. Uremia) One should exclude the recent ingestion of drugs which alter platelet function (note that ASA may prolong the bleeding time for up to 1 week) prior to ordering the test.

In thrombocytopenic patients a bleeding time is usually contraindicated unless the result would have a direct influence on management. Limitation: This test is not precise and does not have predictive value. 4. Bone marrow: A bone marrow aspirate and preferably a biopsy will quickly differentiate between thrombocytopenia due to decreased production (decreased megakaryocytes) versus that due to increased destruction (normal to increased megakaryocytes) it is usually performed when the cause for thrombocytopenia is not obvious by other non-invasive tests, or clinical examination. SIGNIFICANCE OF THROMBOCYTOPENIA AND ASSOCIATED CLINICAL FINDINGS Severe thrombocytopenia (platelet count < 20 x 109/L and particularly < 10 x109/L) – risk of spontaneous bruising and bleeding, which may be life threatening (eg. Intracranial hemorrhage) Moderate thrombocytopenia (platelet count 20 – 80 x 109/L – may bruise with mild trauma but usually not spontaneously; excessive bleeding is likely to occur at surgery. Mild thrombocytopenia (platelet count 80 – 150 x 109/L) – no problem expected with or without trauma or surgery. NOTE: 1. Ingestion of drugs which affect platelet function will make any degree of thrombocytopenia more likely to cause bleeding/bruising and should be avoided. 2. Intramuscular injections should be avoided in all patients with platelet /vascular and coagulation disorders to avoid development of intramuscular hematomas. 3. Both the cause and the degree of thrombocytopenia must be considered when assessing the clinical significance of thrombocytopenia (patients who are compensating for increased platelet destruction such as I.T.P are much likely to bleed than those suffering from decreased platelet production such as patients with leukemia and aplastic anemia.) The role of platelet transfusions in the management of patients with thrombocytopenia or platelet dysfunction is outlined in a previous section (Blood Transfusion Practice). COMMON AND/OR IMPORTANT DISORDERS Generally speaking the most common causes of bleeding under the heading of “Platelet and Vascular

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Disorders" are thrombocytopenias. The most common thrombocytopenias are probably: 1. Those seen with general marrow suppression or aplasia produced whether accidentally (i.e. Chloramphenicol, Phenylbutazone) or therapeutically (i.e. Myelosuppressive drugs) 2. Those secondary to marrow infiltrations such as leukemia and secondary tumor. 3. Hypersplenism 4. In association with D.I.C 5. The specific immune thrombocytopenias (ITP or following certain drugs such as Quinine and Quinidine, Heparin) 6. Association with infections, especially HIV Other than the thrombocytopenias the platelet function defect in association with Von Willebrand's disease and with aspirin are important to be aware of as are several of the vascular disorders such as the nonallergic, non-thrombocytopenic simple purpuras seen commonly in elderly and in patients on steroids, and allergic (Henoch-Schonlein) purpura seen not uncommonly in children. The following are some comments regarding some of the above subjects which are not covered elsewhere: IMMUNE THROMBOCYTOPENIAS Acute I.T.P This disease occurs predominantly in children of either sex consisting usually of a severe abruptly onsetting thrombocytopenic purpura usually 2-21 days following viral infection. Most recover spontaneously in 1-2 months. It is important to rule out bacterial sepsis as a cause. Treatment with Prednisone 1-2 mg./kg or intravenous gamma globulin 1g/kg is often given if significant mucocutaneous hemorrhage is present. 80% will recover regardless of treatment. 50% will have normal platelets in 6 weeks and 80% are well in 6 months. Approximately 20% become chronic lasting 6 months or more and some remain severe enough to require steroids or other immunosuppressive therapy for 6-12 months with splenectomy only being performed if no other therapies prevent significant bleeding. Chronic I.T.P: This is a disease of adults occurring in 3:1 in females, usually onsetting insidiously and rarely resolve spontaneously. Usual treatment is Prednisone 1 – 2 mg/kg with a response in the platelet count usually occurring in 3-4 days to 2 weeks. Splenectomy is considered if no response occurs after several weeks of large doses or in patients continuing to require Prednisone for longer than 6 months or having major side effects and continuing bruising and bleeding occurs. Because of the extremely short life span of transfused platelets, platelet transfusions are used only in life-threatening hemorrhage.

PLATELET FUNCTION DEFECTS VWD is one of the most common of the hereditary hemostatic disorders. Other hereditary platelet function disorders are rare. The acquired platelet defect seen with ASA is important as it is so common, and may aggravate other hemostatic problems. The platelet defects seen in other diseases are usually mild, but may cause serious problems at surgery. Treatment of vWD requires the injection of DDAVP (arginine vasopressin) to release vWF from the individual's endothelium or viral attenuated products that contain vWF (eg. Humate-P) VASCULAR DISORDERS Allergic (Henoch-Scholein Purpura): This disease is seen not uncommonly in children, especially boys with peak incidence at 3- 7 years and usually occurring 1-3 weeks following an upper respiratory infection. Onset is sudden with malaise, headache, fever, urticarial rash, and 50% have joint or abdominal pain. There are no specific lab findings. Treatment with corticosteroids is usually not effective but commonly used in the sicker child with joint and abdominal pain. The problem usually last 2-4 weeks but relapses are not uncommon. Non-Allergic: Non-thrombocytopenic purpuras such as simple purpura (seen in 50% of healthy women) senile purpura and steroid purpura are common but usually very mild and require no treatment. THROMBOCYTOSIS Thrombocytosis applies to any patient with a platelet count greater than normal (450 x 109/L). Mild degrees are fairly common and usually not clinically significant. Severe degrees (1,000 x 109/L) may result in serious problems (eg CVA) particularly in patients with underlying vascular disease or stasis. Therefore, the management of these patients requires knowledge of the platelet count, the cause of the increased platelets, and the general medical/surgical condition of the patient. CLASSIFICATION 1. Primary: a. Myeloproliferative disorders b. Essential thrombocytosis (thrombocythemia) c. Associated with other myeloproliferative disorders, eg. Myeloid metaplasia / myelofibrosis, polycythemia, rubra vera, chronic myelogenous leukemia 2. Secondary: a. Post-splenectomy b. Response to chronic bleeding c. Iron deficiency

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d. Chronic Inflammation / infection e. Underlying malignancy The diagnosis and management of patients with primary thrombocytosis is discussed in the myeloproliferative section of the Lymphadenopathy and Splenomegaly Section. In patients with secondary thrombocytosis the cause may be obvious (post-splenectomy, iron deficiency) but in others it may be very obscure (e.g. underlying malignancy) and necessitate a thorough

search to detect what is sometimes a small primary neoplasm. Patients with secondary thrombocytosis rarely require treatment for the thrombocytosis itself since it is usually mild (< 1000 x10 9/L), uncomplicated and transient or reversible with treatment of the underlying disease. Drugs that alter platelet function (e.g ASA) may be used if thrombocytosis is more sever, or occurs in a patient at high risk for vascular occlusions but this is seldom required.

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COAGULATION DISORDERS Coagulation disorders comprise a wide variety of both hereditary and acquired hemostatic problems. The hereditary disorders vary from uncommon to very rare, and only the more common types will be discussed in some detail; the general principles, however, apply to them all. The acquired disorders are more common, usually being secondary to other conditions (infections, pregnancy, liver failure), medications (Coumadin) or poor nutrition. APPROACH TO PATIENTS WITH BLEEDING PROBLEMS Whereas patients with platelet / vascular disorders predominantly with excessive bleeding involving the mucocutaneous surfaces, the coagulopathies present mainly with bleeding of potential spaces eg. Joints or soft tissue eg muscles as opposed to bruising, although there is some overlap. With minor surgery, mild trauma, and dental extractions, bleeding is usually prompt with platelet disorders but may be delayed with coagulation defects since initial hemostasis is provided by platelets (platelet plug) and backed up later by a fibrin thrombus. In coagulation disorders bleeding may occur from any orifice and in almost any organ. Typically, in hereditary coagulation factor deficiencies, hemarthrosis are most frequent and disabling. During the clinical examination of patients with bleeding problems careful attention must be paid to past history and family history. The pattern of inheritance will be helpful (e.g. males affected and females as carriers of sex linked recessive for hemophilia, but equal distribution in males and females for VWD) LABORATORY TESTS FOR COAGULATION DISORDERS The end result of most coagulation test is the formation of a fibrin clot which is usually detected by a variety of automated equipment. The main tests are: 1. International Normalized Ration (INR) (Prothrombin Time (P.T.)) This test measures the extrinsic clotting system by adding extrinsic thromboplastin to citrated plasma. Prolongation greater than 1.2 is considered abnormal. An isolated deficiency of factors VII, X, V, II or I will prolong the INR (note that deficiencies of the common pathway usually prolong both INR and PTT; isolated factor VII deficiency will only prolong the INR) 2. Partial Thromboplastin Time (P.T.T): This is also known as activated P.T.T since current tests incorporate some form of activator

increasing the reliability and reproductivity. The normal range varies from one laboratory to another, but usually the upper limit is less than 40 seconds; it will be prolonged when the amount of any single factor decreases to less than approximately 30% ( 0.30 u/ml) The test measures the intrinsic clotting system as mentioned previously, defects in the common pathway (Factors X, V, II and I) prolong the P.T.T. as well as the INR; isolated deficiencies in factors XII, XI, IX, and VIII result in prolongation of P.T.T. only) 3. Thrombin Time (T.T) or Thrombin Clotting Time (T.C.T) The thrombin time measures the brief time (about 9 seconds) required to convert fibrinogen to fibrin after the addition of thrombin to citrated plasma. It is most often prolonged when there is a deficiency of fibrinogen, a defect in fibrinogen (dysfibrinogenemia) or if heparin is present in the samples. 4. Fibrinogen (Factor I) The fibrinogen level can be measured in plasma; since it is an “acute phase reactant” it is frequently elevated in many infections, inflammatory or traumatic illnesses. A decreased fibrinogen level may be isolated as a rare hereditary deficiency, but more commonly is reduced along with other factors in liver disease, D.I.C or fibrinolysis. 5. Fibrin-Fibrinogen Degradation Products (FDP) FDP are the result of fibrin and fibrinogen proteolysis, indicating excess plasmin activation. They do not differentiate between primary and secondary fibrinolysis and mild to moderate elevations are commonly seen in seriously ill patients or physiologically stressed individuals. Marked elevations, particularly when associated with other coagulation deficiencies and thrombocytopenia, require that D.I.C be ruled out. Elevated FDP are also seen in severe liver disease, since plasmin is not metabolized properly in the damaged liver. Since other coagulation deficiencies and thrombocytopenia may be seen in liver disease, as well as D.I.C, it is extremely difficult to differentiate between these two problems. A test for fibrin monomers (protamine sulphate test) may help since it should not be positive if intravascular thrombin activation has occurred e.g. deposition of fibrin monomer and polymer. More recently, a test called D. Dimer has been developed. These tests measures degradation products of cross linked fibrin and thus not

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fibrinogen breakdown products. This test is replacing FDP measurement in many laboratories as it does not require special collection tubes. HEREDITARY COAGULATION DEFICIENCIES Hereditary deficiencies of each clotting factor have been reported. Most of these deficiencies are very rare; only deficiencies of Factor VIII (Hemophilia, and VWD), Factor IX (Christmas Disease) and Factor XII (Hageman deficiency) warrant more discussion. In screening for all hereditary deficiencies, it is important to remember what the INR, PTT tests measure at their various levels of sensitivities. Deficiencies of Factors XII, XI, IX, VIII → Prolonged PTT, Normal INR Deficiency of Factor VII → Prolonged INR, Normal PTT Deficiency of Factors X, V, II, I → Prolonged INR and PTT CLASSICAL HEMOPHILIA (HEMOPHILIA A) (Factor VIII Deficiency) This is the commonest hereditary factor deficiency. It is inherited as a sex-linked recessive disorder, being manifest almost entirely in males, with females as carriers. 25% of patients have no family history. The clinical problems vary with severity of the deficiency. Mild hemophiliacs (5-30%) will only have excessive bleeding with surgery or trauma, whereas at the other end of the spectrum the severe hemophiliacs (< 1% Factor VIII) are constantly threatened with spontaneous hemorrhagic problems (mainly hemarthrosis, intramuscular hematomas, and hematuria) All hemophiliacs must avoid injury, and refrain from any medications (eg. ASA) which would alter platelet function and enhance the bleeding problem. Patients with mild hemophilia or who are carriers may only require DDAVP to temporarily boost the individual’s endogenous Factor VIII for minor surgical procedures and then it should be given prior to and post operatively until wound healing has occurred. Moderate and severe hemophiliacs on the other hand, will require regular infusions of Factor VIII concentrate at onset of joint pain, or on a prophylactic basis, often given by themselves on a home care program monitored by a Hemophilia Centre; the predictability, efficiency and ease of administration outweigh the possible risks of disease transmission such as hepatitis for these patients so commercial factor VIII concentrates are favored. HIV infection is not considered a significant contaminant since the introduction of improved pasteurized concentrates in 1988. Further the recombinant factor IV is generated through DNA technology. This makes it save from infection point of view. However, formation of antibodies directed to factor VIII is still a problem.

HEMOPHILIA B DISEASE (FACTOR IX DEFICIENCY) This deficiency is less common than Hemophilia A but has the same inheritance and clinical presentations depending on the severity of the deficiency. It can only be differentiated by coagulation factor assays which are only reliably performed in laboratories experienced with these tests. The same principle apply to the management of factor IX deficiency as for factor VIII deficiency regarding the need for surgical prophylaxis, and adjusting type and frequency of coagulation factor support depending on severity of deficiency and the clinical circumstances. If the deficiency is mild to moderate it should be corrected with plasma ( usually FFP although stored plasma would suffice), but if severe then adequate replacement would require infusion of a commercial Factor IX concentrate (again a pasteurized pooled lyophilized product with decreased risk of viral borne disease transmission). von WILLEBRAND’S DISEASE (VWD) vWD is inherited as an autosomal dominant disorder affected both males and females equally. In addition to Factor VIII deficiency, which is usually mild, the lack of von Willebrand factor also causes a platelet dysfunction. Measurement of Factor VIII coagulant and VWF antigen, VWF Ristocetin cofactor are necessary for making a diagnosis. However they carry a risk of thrombotic complications. Pure factor IX also available. The preferred treatment for such cases is DDAVP. A cryoprecipitate is rich in vWF and is generally administered in a dose of one bag per 10 kg body weight. Certain virus-attenuated concentrates (Humate-P) contain substantial amounts of vWF and are preferred to cryoprecipitate if available. HAGEMAN FACTOR DEFICIENCY (FACTOR XII) This deficiency is mentioned only to point out that it may cause a prolonged P.T.T. and therefore must be differentiated from other factor deficiencies (VIII, IX, and XI) by appropriate Factor Assays. However, it does not result in clinical bleeding problems since it is a "contract factor" and therefore causes only an "invitro" abnormality. ACQUIRED COAGULATION DEFICIENCIES VITAMIN K DEFICIENCY The Vitamin K-dependent coagulation factors are Factors II, VII, IX and X. If the factor deficiency is mild only the INR is elevated, but if more severe then both INR and P.T.T are elevated since coagulation factors of both intrinsic and extrinsic systems are

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involved. The thrombin time and fibrinogen levels are normal thereby differentiating Vitamin K deficiency from either heparin effect (prolonged thrombin time but normal fibrinogen) or liver disease (low fibrinogen, also). Causes of Vitamin K deficiency include: 1. Dietary deficiency – this is most commonly seen in ill patients on intravenous for 1-2 weeks particularly if also on antibiotics which kill the bacterial flora in the intestine. 2. New born (Hemorrhagic Disease of Newborn) – prevented by routine vitamin K 3. Malabsorption 4. Chronic billiary tract obstruction 5. Oral anticoagulants – vitamin K antagonists Patients who are at risk of developing Vitamin K deficiency should receive prophylactic vitamin K, usually by parenteral route. Patients who have established vitamin K deficiency should receive parenteral vitamin K (5-10 mg I.V) but this requires up to 8 hours to correct the deficiency and up to 24 hours for this to be complete. If the patient is bleeding and one cannot wait for complete correction of the INR by the vitamin K then plasma infusions can provide immediate but temporary correction; 2-4 or more units, of FFP, are usually required to correct the prolonged INR/PTT. LIVER DISEASE All coagulation factors except for factor VIII are produced in liver, so severe liver disease is associated with a decrease in most factors. Often platelets are also reduced due to hypersplenism and since fibrinolysis is also increased the multiple abnormalities (prolonged INR, P.T.T, T.T., decreased fibrinogen, platelets and increased FDP) mimic those seen with D.I.C Except in uncomplicated obstructive liver disease the coagulation defects are not corrected by parenteral vitamin K. For temporary correction, during times of significant bleeding, infusions of FFP may be required (again usually 4 or more units are needed to completely correct the coagulopathy). If thrombocytopenia is moderate to severe then platelet transfusions may also be used. DISSEMINATED INTRAVASCULAR COAGULATION (D.I.C) This occurs when there is a generalized activation of the coagulation factors with the final elaboration of thrombin turning fibrinogen to fibrin. It results in fragmentation of RBC's, consumption of coagulation factors and platelets, deposition of fibrin and stimulation of secondary fibrinolysis. As a result there is anemia, hemolysis, thrombocytopenia, coagulation deficiencies (prolonged INR, P.T.T. , T.T., decreased fibrinogen), and increased FDP. CAUSES 1. Pregnancy-related - abruptio placenta, retained placenta

- septic abortion - amniotic fluid embolism - eclampsia 2. Infections - especially gram negative septicemia 3. Surgery - especially prostate surgery, and surgery on cancer patients 4. Immediate Hemolytic Transfusion Reaction - Eg. ABO incompatibility 5. Malignancies - mainly prostate Ca., promylelocytic leukemia As mentioned previously, the multiple hempostatic abnormalities of liver disease closely mimic D.I.C and diagnosis is very difficult in jaundiced patients. There are also other conditions which present with fragmentation hemolysis and thrombocytopenia (eg. Thrombotic thrombocytopenic purpura or T.T.P., and Hemolytic-Uremic Syndrome, or HUS) where fibrin formation (thrombin activation) has not occurred. MANAGEMENT OF D.I.C INVOLVES: 1. Aggressive treatment of underlying cause - evacuation of uterus for obstetrical complications - broad spectrum I.V. antibiotics for septicemia 2. Replacement of deficient (consumed) factors and platelets - FFP or possibly cryoprecipitate for coagulation defects - Platelet concentrates for thrombocytopenia - Some clinicians also advocate heparin to stop the activation of coagulation, but most do not since these patients frequently bleed much more when heparinized. It is more likely to be ordered for patients who have subacute or chronic D.I.C. often when the underlying cause cannot be quickly controlled (eg promyelocytic leukemia, prostate cancer). DILUTONAL COAGULOPATHY During the management of serious bleeding problems with massive transfusions (e.g. Greater than 50% of blood volume exchange in 24 hours) there is a decline in coagulation factors since the replacement products (packed red cells, crystalloid or albumin solutions) do not contain coagulation factors. If stored plasma is also used it will lack labile factors (V and VIII) Therefore it is appropriate to monitor INR and P.T.T in massively transfused patients, or preferably to supply FFP judiciously (e.g. 1 unit FFP for each 1-2 equivalent units of stored plasma, 5% albumin or crystalloid) after a total blood volume exchange has occurred in a short period to prevent the development of a coagulopathy. Platelets are also not present, or functionless, in most blood components, but significant thrombocytopenia from dilution during massive transfusion usually takes much longer to develop than

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does the coagulopathy. Platelets are also not present, or functionless, in most blood components, but significant thrombocytopenia from dilution during massive transfusion usually takes much longer to develop than does the coagulopathy. Platelets are also not present, or functionless, in most blood components, but significant thrombocytopenia from dilution during massive transfusion usually takes much longer to develop than does the coagulopathy. The final amounts of replacement platelet and coagulation factors will be determined by the clinical status of the patient eg. Age, underlying conditions etc.

­ ­

­

Types of thrombophilia: 1. Hereditary b. Natural anticoagulant deficiencies: ­ Activated protein C resistance (Factor V leiden) 25 – 40 % ­ Protein C deficiency 5% ­ Protein S deficiency 5% ­ AntithrombinIIIdeficiency 5 %

Venous thrombo-embolic disorders Deep venous thrombosis (DVT) affects mainly the veins in the lower leg and the thigh. It involves the formation of a clot (thrombus ) in the larger veins of the area. This clot may interfere with circulation, and it may break off and travel through the blood stream (embolize). A resulting embolus can lodge in the lungs, heart, or other area, causing severe damage to that organ. A pulmonary embolism (thromboembolism) occurs when a blood clot, generally a venous thrombus, becomes dislodged from its site of formation and embolizes to the arterial blood supply of one of the lungs.

Pathogenesis: Thrombosis is most often due to combinations of Virchow’s triad: • • •

Problems with the vessel wall (i.e. atherosclerosis) Problems with the blood flow (i.e. sluggish or turbulant flow) Problems with the blood contents (i.e. low natural anticoagulant levels)

Venous thrombosis is more predominantly a fibrindependent process involving the pro-coagulant cascade of proteins. In contrast, arterial thrombosis is more predominantly a platelet-dependent process. These different pathophysiologies influence the efficacy of treatment strategies.

THROMBOPHILIA [HYPER COAGULABLE STATE] Who should be investigated? ­ ­ ­ ­

Venous thrombosis < 40 years Arterila thrombosis < 30 years Recurrent thrombosis (more than one episode) Thrombosis at unusual sites (Buddchiari, Portal vein, Sagital sinus)

Unexplained neonatal thrombosis Family history of thrombosis Recurrent fetal loss

c. Dysfibrinolytic disorder: ­ Dysplasminogenemia ­ tPA deficiency ­ PAI dysregulation ­ alpha-2 microglobulin deficiency d. Others: ­ G20210A prothrombin gene mutation ­ Dysfibrinogenemia ­ Homocysteinemia

2. Acquired a. Antiphospholipid antibody syndrome b. Malignancy ­ Hematological : . PNH . Acute promyelocytic leukemia . PNH

-

Oncologic:

.

Pancreatic . Gastric . Mucin secreting c. Nephrotic syndrome d. Surgery and immobolization Normal ranges: Protein C 70 – 140 % Protein S 60 – 140 % AT-III 80 – 125 % APTT/APC < 2.2 males APCR APTT <1.9 females Secondary causes of Protein C deficiency: ­ Warfarin therapy ­ Liver disease ­ DIC Secondary causes of Protein S deficiency: ­ Liver disease ­ DIC ­ Nephrotic syndrome

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­

Pregnancy

Secondary causes of AT-III deficiency: ­ Heparin therapy ­ Nephrotic syndrome ­ DIC ­ Liver disease

Symptoms and Investigations: DVT: leg pain, tenderness, swelling (edema), increased warmth, and change in skin color (redness) The presence of deep venous thrombosis may be detected by: • venography of the leg • doppler ultrasound • plethysmography • positive D-dimer test PE Signs of PE are sudden-onset dyspnea, tacypnea, chest pain of "pleuritic" nature, cough hemoptysis, and in severe cases, hypotension, shock, loss of consciousness, and death. Although most cases have no clinical evidence of deep venous thrombosis in the legs, findings that indicate this may aid in the diagnosis. ECG may occasionally show right heart strain (the "S1Q3T3 pattern). The Gold standard for diagnosing pulmonary embolism (PE) is still pulmonary angiography. In most of the cases, however, when PE is suspected on the basis of shortness of breath and chest pain, the following scans may confirm the presence of an embolus: "Ventilation-perfusion scan" (or V/Q scan), which shows that some areas of the lung are being ventilated but not perfused with blood (due to obstruction by a clot). Increasingly, the V/Q scan is being replaced with

computed tomography (spiral CT). Advantages are clinical equivalence, better access for patients and the possibility of picking up other lung disorders from the differential diagnosis in case there is no pulmonary embolism

Treatment of deep vein thrombosis: ­ ­

Unfractionated Heparin : intravenous bolus dose 80 u/kg followed by continuous infusion at a rate of 18 u/kg/hr., then adjust dose according to APTT. ­ Alternatively, start LMW heparin, dose per body weight and no need for laboratory monitoring. - Start coumadin within 24 hour of starting heparin, monitor INR that need to be maintained at 2 – 3. Once this range achieved for at least 2 days you may discontinue heparin. Warfarin therapy often requires frequent dose adjustment and monitoring of the INR. In proximal DVT and PE, INR between 2.0 and 3.0 are generally considered ideal. If another episode of DVT or PE occurs under warfarin treatment, the INR window is often increased to 3.0-4.0 (unless there are contraindications). ­ Continue with warfarin therapy for at least 6 months. or "lifelong" if there have been previous DVTs or PEs.

Treatment outcome: - DVT recurrence: during heparin therapy< 5 % during warfarin therapy < 2 % during the first year 2–5% ­ Most recurrence have hereditary or acquired thrombophilia ­ 20 % of thrombophilic patients have recurrence in 1 year ­ > 50 % of thrombophilic patients have recurrence in 5 years

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ANTITHROMBOTIC AGENTS 2. IV.Bolus: 5,000 – 10,000 u Q 4th P.T.T just prior to

HEPARIN Structure – A potent organic acid which is a mixture of sulphate containing mucopolysacchiarides with molecular weights between 3,000 – 30,000 strong negative charge, prepared from animal tissues. Mechanism of Action – combines readily with plasma proteins, notably Antithrombin III (heparin co-factor). Heparin markedly accelerated the inhibition action of AT III on thrombin and Factors XII, XI, IX and X. Heparin also activates lipoprotein lipase. It does not cross the placenta. Administration – continuous intravenous infusion or intermittent bolus and subcutaneous (NOT INTRAMUSCULAR) Onset – I.V. – immediate maximum effect; s.c. – gradual effect and maximum 2-3 hours Duration of Effect – mean half life is 60 minutes in normal; shortened with extensive thrombosis and pulmonary embolism; prolonged slightly in renal failure. Degradation and Excretion – Degraded in the liver in large doses, some is taken up by R.E.S and a small amount may remain unbound in the plasma. Antidote – Protamine sulphate – I.V. – slowly; effective immediately 10 mg for 1,000 units Heparin; 100 mg. maximum at one time; decrease dose according to time elapsed since Heparin given to allow for half life decline in the infused heparin. Major Uses – thromboplebitis; pulmonary embolism; artificial pumps and filters (i.e renal dialysis and bypass pumps) Contraindications – active bleeding sites – i.e. GI tract; known hemostatic defect i.e. factor VIII deficiency; recent CNS surgery; thrombocytopenia; severe hypertension. Problems – Bleeding; uncommon, rarely fatal; occasionally produces thrombocytopenia; antiplatelet drugs enhance bleeding and should not be used (i.e. Aspirin) Monitoring – Heparin prolongs various clotting test times (anticoagulant effect) in direct proportion to the amount of heparin in the sample tested. P.T.T (activated partial throboplastin time); therapeutic range is 60-80 seconds; dependent on laboratory method and should be approximately 1½ - 2 times control range. Dosage and Control: 1. Continous I.V: 5,000 u bolus I.V, then 280 u/hours in 5% d/W or saline, use hourly mini drip chamber (syringe pump better if available); check P.T.T 2 x daily and adjust accordingly; if too low, give 5,000 u bolus and increase drip rate.

next dose should be therapeutic

3. Prophylaxis: 5,000 u q.8h or q.12 h., sc for surgical cases start on admission, or 2h. preoperative and continue q. 8-12 h. postoperative; peak P.T.T 2-3 hours after injection should be below the therapeutic range. Note: The P.T.T is also prolonged by warfarin derivates. Perform a CBC count every three days while on heparin to avoid heparin induced anemia or thrombocytopenia.

Low-Molecular-Weight Heparin Low-molecular-weight heparin is a relatively new class of anticoagulants that are derived from standard heparin through either chemical or enzymatic depolymerization. Whereas standard heparin has a molecular weight of 5,000 to 30,000 daltons, lowmolecular-weight heparin ranges from 1,000 to 10,000 daltons, resulting in properties that are distinct from those of traditional heparin.

Mechanism of Action: -Binding of LMW heparins to antithrombin accelerates the inhibition of coagulation factor Xa and thrombin about 1000-fold -Unlike unfractionated heparin, which blocks thrombin and factor Xa equally well, LMW heparins primarily block coagulation factor Xa

Advantages of Low-Molecular-Weight Heparin The clinical advantages of low-molecular-weight heparin include predictability, dose-dependent plasma levels, a long half-life and less bleeding for a given antithrombotic effect. Furthermore, immune-mediated thrombocytopenia is not associated with short-term use of low-molecular-weight heparin, and the risk of heparin-induced osteoporosis may be lower than the risk with the use of standard heparin. Low-molecularweight heparin is administered according to body weight once or twice daily, both during the high-risk period when prophylaxis for DVT is recommended and also when waiting for oral anticoagulation to take effect in the treatment of DVT. The activated partial thromboplastin time (aPTT) does not need to be monitored, and the dosage does not need to be

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adjusted. Since low-molecular-weight heparin is given subcutaneously, outpatient administration by the patient, with or without the assistance of a visiting nurse or family member, is both possible and costeffective.

Preparation (trade name): • • • •

Dalteparin sodium (Fragmin) Enoxaparin sodium (Clexane) Nadroparin calcium (Fraxiparin) Tinzaparin sodium (Innohep)

WARFARIN DERIVATES: (COUMADIN) Structure – A water-soluble derivative of Coumeric Acid; Mechanism of Action – not clear; bound to albumin; acts in the liver to block the synthesis of the Vitamin K dependent Factors (II, VII, IX, X), and, therefore, interfere with both the intrinsic (II, IX and X) and extrinsic (II, VII, X) system – may block an enzyme regulating Vitamin K metabolism or may be a competitive inhibitor of Vitamin K for specific sites on regular protein. Administration – by mouth; Onset and Duration of Effect – does not destroy circulating factors but prevent production; therefore dependent on half-life of the above factors which range from 3 or 4 hours to 72 hours, prothrombin time may be therapeutic in 3-4 days but full therapeutic effect requires 3-5 days. Degradation and Excretion – not absolutely clear, probably the liver; degradation products excreted in the urine. Antidote – Vitamin K1 (p.o. or I.V. ; takes a minimum of 8-12 hours for effect

a) mild INR increase – 1-2 mg Vit K po or SC b) For severe hypoprothrombinemia alone – 5-10 mg Vitamin K1 c) If accompanied by bleeding – 25 mg. Vitamin K1 + plasma Major uses – Prophylaxis + treatment of – thrombophlebitis, pulmonary embolism, embolic heart disease i.e rheumatic mitral valve disease Treatment – follow Heparin therapy for the above. Contraindication – active bleeding sites, specially GI; known hemostatic defet – i.e. Factor VIII deficiency; recent C.N.S surgery, unreliable patient or no laboratory control; liver and kidney disease (relative); pregnancy; thrombocytopenia. Problems 1. Enhance the Effect of Coumadin – decreased Vitamin K absorption, i.e. malabsorption, pancreatic disease; decreased gut production of vitamin K, i.e. broad spectrum antibiotics; displacement of albumin binding site, i.e. phenylbutazone. 2. Decrease the Effect of Coumadin – (require increased dosage of Coumadin) liver microenzyme inducer, i.e. barbiturates. Dosage and Control – 10 mg p.o. for 2-3 days according to INR, and then a maintenance dose according to INR (usually about4 mg daily). The warfarin dose should be administered at a consistent time of the day and the INR blood sample should be drawn at a consistent time of day. INR, therapeutic range: a. Simple Venous Thromboembolism INR of 2.0 – 3.0 b. Arterial and Valvular Prosthesis INR of 3.5 – 4.5 Frequency should be daily initially, lengthening to one weekly or at the most every other week once wellcontrolled.

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PROBLEMS FOR TUTORIALS HEMOSTASIS / THROMBOSIS 1. A YOUNG BOY WITH SWOLLEN KNEE A 1-year old infant was noted to ooze intermittently from a circumcision, and his thigh had become swollen and tense following a vitamin K injection. He was born at term to a 26-year old gravida IV para III woman following an uneventful pregnancy. Delivery was vaginal over a small midline episiotomy, and he cried vigorously. The mother's health had always been excellent, and she denied any abnormal bleeding or bruising. Her family history was likewise unrevealing. Before the infant was transferred to the neonatal special unit, the following studies returned: Hemoglobin 167g/L Hematocrit 52% Platelet count 339 x 109/L White blood cell count 19.4 x 109/L Prothrombin time (PT) 30 sec (nl 10-16 sec) Activated partial thromb- 115sec (nl 31-55 sec) oplastin time (aPTT) On examination, the infant was pink, easily aroused, and had good muscle tone. Bright red blood discolored the circumsion dressing. The left thigh was swollen, its circumference measurably larger than the right. A repeat hematocrit several hours later was 37%, the aPTT was 105 sec and the PT was 16 sec. Coagulation studies were as follows: factor XII: 60% (nl 13-93%); factor XI: 45% (nl 10-66%); factor IX: 39% (nl 2591%); factor VIII: less than 1%(nl 40-180%); factor XIII screen: normal; fibrinogen: 2.55 g/L (nl 1.67-4.0) Questions: 1. What is your interpretation of the Lab findings? 2. How the presentation is different in those with Platelet dysfunction? 3. How would you manage this patient? 4. What would be your advice for the parents ? Two months later this mother brought her baby to the emergency room because he had cried inconsolably all night. By that age he had attained the expected milestones and was generally an active, happy baby. The mother denied that he had any fever but noted that he did not use one arm at all. Any attempt to move it caused him to cry. On examination, none of his joints or muscles was swollen or tender; but trying to bend one elbow made him cry. Within an hour of infusing factor VIII concentrate, all symptoms subsided. At age 6 years, the patient was seen at an annual multidisciplinary clinic for routine evaluation. At age 5, his parents began home care, injecting him with

factor concentrates after thorough training by the hemophilia center staff. Now he is bright, very active boy who seems to seek out precisely those games and stunts from which his parents have tried to discourage his participation. His infusions are at times difficult because he squirms. To avoid them he does not consistently inform his parents until pain is severe, or he begins to limp noticeably. A re-view of a year's infusion records reveals he had 68 bleeding events requiring 78 infusions, mostly involving knees and ankles. A disproportionate number involved his right ankle and were repeated the next day because of persistent pain and swelling. For the most part, pain began in school and his parents infused him at home, often hours later. In the clinic he appeared to walk normally, but his right ankle was swollen, nontender, but boggy to palpation. Its range of motion was slightly decreased. An X-ray revealed moderate demineralization of the adjacent bones but no narrowing of the joint spaces, subchondral cyst formation, or bony spurs. Pertinent laboratory studies showed only mildly elevated ALT: 65 U; AST: 72U (normal levels less than 40 U/mL) 1. What do you make of this follow up information and what would you recommend to this patient 2. What further tests would you order

2. A BLEEDING YOUNG WOMAN Brenda, a 14-year-old girl, came to the emergency room with 18 days of profuse vaginal bleeding and with pallor and weakness. This was her second menstrual period, and because it was painful, she had taken several aspirins. She denied any recent sexual activity and said she had always been in perfect health. A age 12, however, she had bled so much after a tooth extraction that her dentist had to pact and suture the bleeding site. Like one of her younger brothers, she bruised easily. Her father had once required a blood transfusion after a tonsillectomy but had an uneventful postoperation course following a herniorrhaphy. Her blood pressure was 110/60 recumbent and 90/50 sitting up; resting pulse was 100. She was dizzy and anxious when she was upright. Her abdomen was soft and nontender; and no masses were felt. On bimanual pelvic examination, the uterus was midline, anteverted and normal in size; the adnexae were soft and nontender. There was much blood flowing from an intact cervical os to the vagina. The laboratory findings were as follows: Hemoglobin 60g/L MCV 85 fL WBC 16.4 x 109/L, with normal differential

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aPTT 39 sec (nl 33-27 sec) PT 14 sec Pregnancy test negative

Thrombin Time (TT): 20 (control 14 s) Fibrinogen: 1.7 g/L (N2 – 4) D- dimer 4000 (N < 500)

She was admitted to the hospital and received one unit of packed red blood cells and two 25-mg doses of conjugated equine estrogen, with some improvement. She later received a progestational agent. She was discharged on medroxyprogesterone for 10 days. On next visit, a bleeding time was 12 min (nl less than 9 min), and the following clotting studies were obtained:

Questions: 1. Comment on the results of the tests? Comment on the peripheral blood film below. 2. What is your presumptive diagnosis? 3. How would you manage this patient?

aPTT PT Factor XII Factor XI Factor IX Factor VIII:C Factor VIII:Ag Ristocetin cofactor

38.5 sec (nl 27-37 sec) 14 sec (nl 11-14 sec) 60% (nl 52-164%) 72% (nl 67-127%) 73% (nl 55-163%) 38% (nl 40-149%) 35% (nl 40-149%) 45% (nl 50-150%)

Questions: 1. What is the clinical presentation suggests 2. How do you interpret the lab data 3. How would you manage this patient 4. Comment on inheritance mode

3. FEVER AND COAGULOPATHY 3 years old boy was brought to the emergency unit by his mother. The child has been unwell for the past three weeks with fever and loss of appetite. He has been diagnosed to have an upper respiratory tract infection by the family doctor and was given Amoxill but without any improvement. Two days before presentation, the mother noticed the appearance of pinpoint reddish spots on his shins and arms. The boy has been generally well, received all his vaccinations and does not have any known illness or allergies. On examination, the boy was fully conscious but irritable. Temp: 39ºC. There was a big bruise on his forehead and petechial hemorrhages on his legs, arms and abdomen. Examination of the neck revealed the presence of two small lymph nodes in the cervical region. Examination of the chest and heart was normal. Examination of the abdomen was normal apart from feeling a splenic tip. Investigations showed: WBC: 40 x 109 /L Hb.: 105 g/L Normal Male: 140-180, Female: 120140 Platelets: 25 x 109 /L N 150 - 400 Prothrombin time (PT): 20 sec (control 13 s) / INR:2 APTT: 60 (control 38 s)

4. A BLEEDER BABY BOY A 1-year-old boy was referred from a dentist to the emergency unit after failing to control a gum bleed, which took place at a new teething site. The mother mentioned that the child also bled profusely following a circumcision, which was done at the age of two months. Apart from these two incidents, the child has been generally well and growing normally. The boy was the second child in the family, has a 5 years old sister who has no medical problem. The parents were non- consanguineous but the mother mentioned that she has a brother who is a “bleeder”. On examination, the child was healthy, a febrile, oozing blood from a teething site. He also had few bruises on his shins. Examination of the chest, abdomen was normal Investigations revealed: WBC: 6.9 x 109 /L Hb. 120 g /L Platelets: 345 x 109 /L PT : 15 s (N 10 – 14s) APTT : 55 s (N 25 - 35 s) Fibrinogen 3.2 g/L D-dimer: 200 μg/ml (N <500) Bleeding time 5 min (N 2-7) Question: 1. What is your presumptive diagnosis? 2. How would you prove this diagnosis? 3. How would you manage this patient?

5. BLEEDING GUMS AND BRUISING A 23- year woman came to the emergency room complaining of bruises over the body and bleeding gums for the preceding 36 hours. The patient never has never had easy bruisability, epistaxis, gum bleeding, or bleeding into the joints. Menarche was at age 13 and

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menses have been normal. The last menstrual period was 16 days prior to admission. And was normal in amount and duration. The patient had several dental extractions at age 17 and oozed for 12 hours thereafter. There has not been excessive bleeding with minor trauma. She has not had surgery or pregnancies. Family history is negative for bleeding disorders.

Examination reveals a respiratory rate of 26/min, HR 110/min, BP 110/60. The right calf was plethoric, swollen and tender. The reminder of the examination, including the chest examination, was normal

On physical examination the patient is a welldeveloped young woman without pallor or jaundice. Vital signs are normal. Fundoscopic examination is unremarkable. There is oozing of the gingivae with several small hemorrhagic bullae on the buccal mucosa. There is an ecchymoses and oozing in the right antecubital area secondary to venipuncture and there are several ecchymoses and multiple petechiae on both lower extremities. There is no palpable adenopathy or splenomegaly. The remainder of the examination is normal.

Investigations: CBC HB 121 g/L RBC Count 4 x 1012/L Hct 0.41 MCV 85 fl MCH 31 pg/L MCHC 340 pg/L RDW 13 WBC 12 x 109/L Platelets Count 280 x 109/L

Laboratory Data

Coagulation Screen APTT 26 seconds PT 11.4 seconds INR 1.0 Fibrinogen 2.5 g/L

PT 13 / 12 –second control INR: 1 APTT / 33 –second control 30 second WBC 6x109/L – normal differential HB 125 mg/L Platelets 8,000/mm3 Questions: 1. What dose the combination of bruising and oral mucosal bleeding implies? What dose the negative clues in the history suggests? 2. Based on the history what is your differential diagnosis? 3. Looking at the CBC results, what is the next immediate test you would ask for? What dose the normal INR and APTT results imply? 4. Considering the low platelets count, what relevant piece of history you would ask the patient? And what are the other relevant tests you would ask for? 5. What is your differential diagnosis of thrombocytopenia in this patient? Given the most likely diagnosis how would you manage this patient? 6. A YOUNG PATIENT WITH SUDDEN SHORTNESS OF BREATH A 24-year-old female university student presented to the emergency room complaining of a sudden onset left sided pleuritic chest pain and Shortness of breath of one-hour duration. She admitted to having a painful swollen right calf of several days duration. She has recently returned from a summer holiday in Florida. She had been taking the oral contraceptive pills for 6 months. She has a younger brother with a history of deep venous thrombosis. Her father has taken warfarin for many years. Her mother and 3 siblings are well.

A Doppler ultrasound of the right leg revealed thrombus in the popliteal and superficial femoral vein.

Specific Tests Test

Results

Normal

ATIII Protein S (free) Protein S (Total) Protein C Activity Protein C Antigen APCR Ratio

0.9 0.75 1.10 0.12 0.15 2.8

0.75 – 1.15 0.50 – 1.50 0.75 – 1.20 0.50 – 1.75 0.65 – 1.50 >2

Questions 1. Discuss the investigations of suspected deep venous thrombosis and pulmonary embolism

2. What conditions or situations may have predisposed to this event? 3. What are the causes of hypercoagulable state? 4. Discuss the management of this patient under the following headings: ­ Anticoagulant therapy ­ Monitoring therapy ­ Family study 7. OFTEN MISSED BLEEDING PROBLEM A 34-year old woman was seen by a gynecologist for tubal ligation. During the interview, she described a lifelong problem with easy bruising with minimal trauma. She also described the frequent nosebleeds as a child and heavy menstrual periods, particularly in the last two years.

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Physical examination was entirely normal, except for a few small bruises on her legs. There were no petechiae. Laboratory Investigations Hemoglobin 130 g/L WBC 7.9 x 10 9/L Platelet count 280 x 109/L P.T.T 47 seconds Thrombin Time 9/9 seconds Bleeding time 12 minutes INR 1.0 Questions: 1. List 2 additional points in the history which you would require 2. What is the most likely diagnosis? 3. How is this disorder inherited? 4. The patient is booked for tubal ligation a. What precautions would you advise her about daily lifestyle? b. Which of the following would be most appropriate before the operation or post-operatively i. FFP ii. Cryoprecipitate iii. Factor VIII concentrate iv. None of the above 8. POST OPERATIVE DVT Four days after a left nephrectomy, a 45 year old man developed a deep vein thrombosis in his left leg. He had no respiratory symptoms. The foot of the bed was elevated and he was given IV injection of Heparin, 5000 units, followed by a continuous IV heparin infusion at 1000 units per hour. Later on his anticoagulation was changed to coumadin orally. Questions: 1. List 3 techniques which might have helped prevent the DVT 2. The intern orders "Coumadin 10 mg daily", what is wrong with this? 3. The intern orders "INR daily", what is wrong with this order? After 10 days of coumadin therapy, he complained of nausea, and faintness. He had a large melena stool. He was pale, clammy and perspired profusely. Pulse was 110/min and regular. Blood pressure was 95/96 with significant postural drop. Hemoglobin was 110 g/L., (down from his last known Hb of 130 g/L. three days earlier) INR = 6.1 4. Describe your management under the following headings: a. Amount of coumadin you would prescribe? b. What medication would you order, how much and by what route?

c. If the bleeding progressed is there anything you could prescribe to correct the coagulation problem more quickly? 9. ALCOHOLIC AND COAGULOPATHY A 53 year old formed alcoholic and previously been investigated during a G.I. bleed and was found to have cirrhosis and esophageal varices. She was brought to the Emergency Room by friends who had found her lying on the floor of her apartment. In the Emergency Room she vomited "coffee-ground" material, and had a melena stool. On examination, her pulse was 100/minute and blood pressure was 106/70 supine. She had mild scleral icterus, spider nevi on her face and chest, moderate splenomegaly (4 cm below left costal margin) and mild ascites. The liver was palpable. Laboratory Investigations: Hemoglobin 84 g/L MCV 100 fL MCHC 320 g/L WBC 3.6 x 109/L Platelet count 70 x 109/L INR 4.0 seconds P.T.T 54 seconds Questions: 1. List 3 possible causes for the anemia? 2. List 3 possible causes for the thrombocytopenia. 3. Explain the elevated INR and P.T.T. 4. The intern orders: a. bleeding time – is this a reasonable order 5. Discuss your management regarding a. Initial I.V. order b. The anemia c. The thrombocytopenia d. The coagulation defect (2 points) 10. FEVER POST SPLENECTOMY A 60 year old man was admitted to hospital with a three day history of fever, rigors, cough and yellow sputum and pleuritic left lower chest pain. He had been previously been healthy except for a previous splenectomy 12 years before following a traumatic rupture of his spleen in a farm accident. On examination he was very ill with a high fever and signs of pulmonary consolidation in the lower lobe of his left lung. Pulse was 140/minute and blood pressure 130/60. He had many petechiae and bruises on his trunk and extremities. Laboratory Investigations: Hemoglobin 116 g/L WBC 15 .5 x 109/L .68 neutrophils .16 bands .02 metamyelocytes .01 myelocytes

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RBC fragmentation Howell Jolly bodies Platelet count Reticulocyte count INR P.T.T Thrombin Time Fibrinogen

.13 lymphocytes ++ + 26 x 109/L 60 x 109/L 3.5 seconds 80 seconds 18/9 seconds 0.5 g/L

5. After three weeks there has been little response to treatment, and splenectomy is advised; what chance is there of complete remission of the thrombocytopenia with splenectomy? – 50%, 80%, 100% 6. What is the main long-term risk of splenectomy? What could you do in an attempt to avoid this complication? What is the role of IVIgG in this disease?

Questions: 1. Give a brief explanation for: a. abnormal WBC differential b. RBC fragmentation c. Howell-Jolly bodies d. Thrombocytopenia e. Normal reticulocyte count f. Coagulation defects 2. Discuss your management under the following headings: a. Thrombocytopenia – type, amount and rate of blood products; expected response b. Coagulation defect 11. BLEEDING WITH SEVERE

THROMBOCYTOPENIA A 25-year old woman with no previous history of abnormal bleeding or bruising visited her doctor after 3 week history of increasing spontaneous bruising. Her last menstrual period, two weeks before was normal, but she did have a prolonged nosebleed two days ago. Her only medications included multivitamin tablet, occasional diazepam for sleeping, and aspirin tablets for headaches (her last aspirin was three days ago). Her doctor noticed some crusted blood in both nostrils, a few hemorrhaging bullae on her tongue and buccal mucosa, and numerous petechaie and bruises on her extremities. She was afebrile, and appeared otherwise healthy. Laboratory Investigations Hemoglobin 130 g/L MCV 88 fl MCHC 340 g/L WBC 6.5 x 109/L Differential normal Platelet count 10 x 109/L Required Questions: 1. What is the most likely diagnosis? 2. Should the patient be treated at home or in hospital? 3. What additional test or procedure would usually be done? 4. What medication would you initially prescribe?

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