cr hematology. rbcs disorders anemias&others wbcs disorders benign & malignant hemostatic...

CR Hematology

RBCs DisordersAnemias

&Others

WBCs DisordersBenign & Malignant

Hemostatic Disorders

Hematological Disorders

Introduction

• Blood consists of 55% plasma and 45% formed elements.

• Formed elements include erythrocytes, leukocytes, and thrombocytes.

Erythrocytes• Normal range 4.0-5.0

million per mm3 in adults.

• Biconcave shape.• Diameter 7 microns.• Cells for transport

of O2 and CO2.

• Life span 120 days.

Leukocytes

• Normal range 4 - 11 thousand per mm3

in adults.• Five types.• Size 8-20 microns.• Involved in fighting

infection, combating allergic reactions, and immune responses.

Thrombocytes

• Smallest cells in the blood.

• Normal range 150,000-400,000.

• Active role in coagulation and hemostasis.

RBC Disorders

Anemia• Defined by measurement of hemoglobin

concentration.• Manifestations (symptoms) are related to

duration and severity of anemia– Body has physiologic responses to chronic

anemia such that many patients are asymptomatic until hg < 8 g/dl .

– Fatigue, pallor, dyspnea, dizziness and dyspnea on exertion

Signs

• Pallor of mucous membranes (conjunctiva, tongue, palm of the hands).

• Nails are delicate and break easily.• Hair is thin.• Rough skin.

Classifications of Anemias

Microcytic, HypochromicMicrocytosis – small cells (MCV <80)

– Iron deficiency– Sideroblastic– Anemia of chronic disease– Lead poisoning– Thalassemia trait

Microcytic, Hypochromic

• Many RBCs smaller than

nucleus of normal

lymphocytes, increased central pallor.

Classifications of AnemiasNormochromic

– Hereditary Spherocytosis– PNH– G6PD deficiency– Aplastic anemia– Acute blood loss

Classifications of Anemias

Macrocytic– Vitamin B12 deficiency– Folate deficiency– Liver disease– Drugs– MPD

Macrocytic RBCs

• Most RBCs larger than nucleus of normal

lymphocytes.

• increased MCV.

Macrocytic Anemia

Macrocytosis – large cells (MCV >100)

Check vitamin B12, RBC folate (why?), fasting homocysteine, and methylmalonic acid (MMA)

*HC and MMA are elevated in subclinical B12 and folate deficiency

IRON DEFICIENCY ANAEMIA.

Iron deficiency is the most common cause of anemia in every common country of the world, and it is the most important cause of microcytic hypochromic anaemia.

Nutritional and metabolic aspects of the iron:

Iron in the body is about 2.5-3 g. Iron in the Haemoglobin of the RBC

represents a greatest percent of body constitutes.

Iron presents in the body in two forms:

- Ferrittin. - Haemosiderin.

Causes of iron deficiency anaemia:

1. Chronic blood loss, especially gastrointestinal tract.

2. Increased demands, during pregnancy, infancy, growth, lactation and menstruated women.

3. Malabsorption especially in the cases of gastroectomy and peptic ulcer.

4. Poor diet.

Clinical features:1. When IDA is developing, the RE stores

(hemosiderin and ferritin) become completely depleted before anemia occurs.

2. At an early stage, no clinical abnormalities.

3. Later, patient may develops general symptoms and signs of anemia.

4. Spoon or ridged nails in severe case of IDA.

5. Dysphagia.

Laboratory findings:•Red cell indices:

Low Hb conc.MCV, MCH, MCHC*

•Blood film:Hypochromic microcytic Picture.Occasional Target cells.Pencil shaped poikilocytes.Normal reticulocyte count.

•Bone marrow iron:Normal to hypercellular.RBC precursors are increased in number.Iron stain negative.

•Chemical testing on serum:Serum iron Decreased Transferrin/TIBC Normal to HighSerum ferritin Decreased (Very low)

DD. Of microcytic anemia

Sideroblastic anemias

Characterized by

Increase in total body iron

Presence of ringed sideroblasts in bone marrow

Hypochromic anemia.

Classification

• Hereditary form• Acquired form (more common)• Idiopathic – Refractory anemia with ringed

sideroblasts(RARS)• secondery

Pathophysiology

• Disturbances of enzymes regulating heme synthesis• Ringed sideroblasts form when non-

ferritin iron accumulated in the mitochondria that circle the normoblast nucleus

Hereditary Sideroblastic Anemia

• Most common form is sex-linked and due to an abnormal aminolevulinate

synthetase enzyme (ALAS)• Decreased heme synthesis due to block in iron utilization perceived by body as

increased need for iron associated with increased iron absorption results in iron overload

Acquired Sideroblastic Anemia

Idiopathic RARS– acquired stem cell disorder Secondary• Lead poisoning (plumbism) Inhibits cellular enzymes involved in heme

synthesis• Malignancy

Laboratory findings in SA –Peripheral Blood

• Moderate to severe anemia• Target cells• Basophilic stippling • ↑Fe, N to ↓TIBC, ↑% saturation, ↑ ferritin

• Bone marrow: -Erythroid hyperplasia -Ringed sideroblasts in more than 15% of

normoblasts.**Lower number of ringed sideroblast in variety of

hematological disorders.

Macrocytic anemia

Macrocytic anemia

• Other causes include:– Drug toxicity– Hypothyroidism– Liver disease– Myelodysplasia– MPO

Megaloblastic macrocytosis

• The smear in a patient with macrocytic anemia is helpful in identification of megaloblastic changes – macrocytes and hypersegmented neutrophils (>5 lobes)

• DD: B12 deficiency, folate deficiency, drugs that cause abn.DNA synthesis or folate metabolism, liver disease and myelodysplastic syndromes

• Non-megaloblastic macrocytosis, on smear patients may have large target cells and acanthocytes.

Folate deficiency Found in: Fruits (e.g. citrus, melon, bananas),

leafy green vegetables. Causes include:

Malabsorption MedicationsMalignancy HemodialysisDiseases/conditions associated with rapid cell

turnover such as pregnancy, infancy,….

Vitamin B12 deficiency Found in : (meat, fish) The body stores large amounts of B12 therefore

decreased dietary intake rarely lead to deficiency Medications to decrease stomach acid can also

contribute to B12 deficiency (antacids) Vegetarians can also contribute to B12 deficiency In addition to causing anemia, B12 deficiency can lead

to a metabolic peripheral=neuropathy and gastrointestinal disorders.

Diagnosis

1. Blood cell count:

• macrocytic anemia ( MCV>100fl )• thrombocytopenia• leucopenia (granulocytopenia)• low reticulocyte count 2. Blood smear: • macrocytosis , anisocytosis.• hypersegmentation of granulocytes

Diagnosis

3. Laboratory features hyperbilirubinemia elevation of lactate dehrogenase (LDH) serum iron concentration- normal or increased 4. Bone marrow smear hypercellular erythroid cell changes (megaloblasts, an abnormally large

cell with nuclear- cytoplasmic asynchrony) myeloid cell changes (hypertsegmentation) megakaryocytes are decreased and show abnormal

morphology

Hemolytic AnemiaNormocytic

Normochromic

Hemolytic Anemia•Congenital

–Membrane defects•Hereditary spherocytosis•Hereditary elliptocytosis

–Enzyme defects•G6PD deficiency, PKD.…,

Hemolytic Anemia

• Acquired– Classified according to site of RBC destruction and/or whether

mediated by immune system:• Intravascular• Extravascular• Immune• Non-immune

– Causes: –• Transfusion of incompatible blood• Autoimmune

– Warm (IgG-mediated) ; most common– Cold (IgM-mediated)

• Prosthetic valves• TTP/HUS• DIC• Cancer• Drugs

Haematological findings in HS

• Anaemia is usual.[Increased mean corpuscular hemoglobin concentration (MCHC)]

• Reticulocytosis 5-20%• Microspherocytes are seen in the blood film.

(densely staining with smaller diameters than normal red cells).

Other investigations

• The classic finding is that the osmotic fragility is increased.

• Autohaemolysis is increased and corrected by glucose.

• Direct antiglobulin test is normal

G6PD deficiency

• G6PD functions to reduce nicotinamide adenine dinucleotide phosphate (NADP) while oxidizing glucose-6-phosphate.

• NADPH is needed for the production of reduced glutathione (GSH) which is important to defend the red cells against oxidant stress.

Clinical features

• G6PD deficiency is usually asymptomatic.• Neonatal jaundice.• Acute haemolytic anaemia in response to

oxidant stress: drugs, fava beans or infections.

Laboratory diagnosis

• Between crises blood count is normal.• The enzyme deficiency is detected by

– One of a number of screening tests or– By direct enzyme assay on red cells.

• During the crisis, the blood film may show contracted and fragmented cells, bite, blister cells, ………

• Enzyme assay may give a false normal level in the phase of acute haemolysis.

• The blood film shows irregularly contracted cells [deep red arrows]

and sometimes hemighosts [deep blue arrow] in which all the hemoglobin appears to have retracted to one side of the erythrocyte

Polycythemia / Erythrocytosis

• Abnormal elevation of hemoglobin– Rule out “relative” polcythemia caused by contraction of plasma volume,

e.g. dehydration– Primary

• Polycythemia Vera– RBC production independent of EPO

» EPO level is low / positive JAK-2 is diagnostic– Uncommon– May be associated with leukocytosis, thrombocytosis, splenomegaly– Hyperviscosity

» Headache, vertigo, visual changes, mental confusion– Risk of transformation into acute leukemia– Treatment??

– Secondary• RBC production in response to increased EPO production

– EPO level is usually high• Very common• Usual etiology is chronic hypoxia (COPD)**……………. (250-500 ml) to maintain hct 45-50% and treat underlying problem

Reticulocytes• Immature RBCs.• Contain residual

ribosomal RNA.• Reticulum stains

blue using a supravital stain (new methylene blue).

• Counted and expressed as % of total red cells.

Reticulocyte Count

Retic % = # retics per 100 RBCs

Corrected retic= % retics x pt. HCT

45

Haemoglobinopathies

• A group of inherited disorders characterized by structural variations of the Hb molecule. They are Disorders of globin synthesis rather than heme synthesis.

• These may result from : 1. Synthesis of abnormal Hb2. Reduced rate of synthesis of NORMAL α or β

globin chains• Genetic defects of Hb are the most common

genetic disorders worldwide.

What are hemoglobinopathies?

SICKLE CELL ANAEMIA

Sickle cell disease is a group of haemoglobin disorders, in which there is inherence globin abnormality, caused by substitution of valine for glutamic acid in position 6 in the ß chain.

• Hb S is insoluble and forms crystals when exposed to low oxygen tension.

• Deoxygenated Hb polymerizes into long fibers which may block different areas of the microcirculation or large vessels causing infarcts of various organs.

Clinical features:- Chronic haemolysis, leads to jaundice and anemia.- Vaso-occlusion of blood vessels leads to pain.- Infarction and infections.

1. Low Hb.2. Peripheral blood film shows, sickle cells,

target cells and howell-Jolly body appears.3. Positive Sickling test (screening test).4. Hb electrophoresis (confirmatory test) :

-Hb SS : 80 – 100% - no Hb A - Hb F : 5 – 15%

Howell-Jolly bodies

• These are basophilic nuclear remnants (clusters of DNA) in circulating erythrocytes.

• They are usually observed in hemolytic anemia, following splenectomy, and in cases of splenic atrophy.

The Sickling Test

• This is a wet preparation.• 5 drops of reagent (Sodium dithionite), are added

to 1 drop of anticoagulated blood on a slide. Cover glass is put on and sealed with petrollium jelly/parraffin wax mixture.

• The reagent is a reducing agent.• In Hb SS, sickling occur immediately, while it may

take 1 hour in Hb S trait.

Hb S solubility Test

• This is done after the Hb electrophoresis to differentiate between some hemoglobins that have the same electrophoretic mobility. (Differentiate Hb D & Hb G from Hb S)

• Only Hb S precipitate in the reduced state when placed in a high molarity phosphate buffer (as it removes oxyegen from test environment).

• 0.05 ml of blood is added to 1 ml of the buffer and mixed in a test tube. Positive results : presence of Hb S : cloudy solution . Negative results : other Hbs : clear solution .

This is a benign condition, where there is no anaemia and normal appearance of RBC on the BF. Haematuria is the most common symptom. Care must be taken with anesthesia, pregnancy and at high altitude.

Sicle cell trait

THALASSAEMIA

*There are two alpha genes on each of two chromosome 16 (four genes in the diploid state)

*Only 2 beta globin genes, one on each chromosome 11

**Excess alpha chains are unstable -precipitates in the cell which bind to cell membrane, causing membrane damage

**Excess b chains *High oxygen affinity – poor oxygen transporter

*unstable

Thalassaemias are a heterogeneous group of genetic disorders, which results from a reduced rate of œ (alpha) and ß (beta) chain synthesis.

• In alpha thalassaemia, there is no or little alpha-chain syntheses.

• In beta thalassaemia, there is no or little beta-chain syntheses.

ALPHA THALASSEMIA

1- Major alpha- thalassaemia or Hydrops fetalis or Hemoglobin Barts : four genes deletion, leads to complete suppression in the synthesis of alpha-chain. Alpha chain is important in formation of hemoglobin F in neonate, so in this case the formation of this haemoglobin which is important for fetal life will fail, leading to death in uterus.

2- Three genes deletion: leads to moderate to sever microcytic hypochromic anaemia, with splenomegaly. This is known as Hb H disease.

3- Two genes deletion: alpha-thalassaemia trait or minor, associated with mild anaemia.

4-One gene deletion: silent thalassemia usually asymptomatic.

Alpha-Thalassemia minor

*Two alpha genes either on same or opposite chromosomes are missing

*Unaffected globin genes are able to compensate for the affected genes

*Mild anemia – significant microcytosis*Normal lifespan

*Hb. Electrophoresis is normal.

Normal Hemoglobin Electrophoresis

*Hgb F (N = < 1% after age 1 year) *Hgb A2 (N = 2-3.5%)

*Hgb A1 (N=95.5-100%)

Causes: deletion

Type genotype Clinical

0 deletion Normal / --------

One deletion Thal : + heterozygous

-/ Silent carrier: mild hypochromic microcytic

anemia

Two deletions

Thal trait: + homozygous or 0 heterozygous

-/-

/--

Minor: mild hypochromic microcytic anemia

Three deletions

Hb H disease:0/+ double heterozygous

-/-- variable severity, but less severe than Beta Thal Major

deletion of all four a globin genes

Hb Bart’s; - homozygous

--/-- Hydrops Fetalis:In Utero or early neonatal death

complete absence of a globin synthesis

BETA-THALASSEMIA

Classification:1. ß- Thalassaemia major, very

sever.

2. Intermediate ß- thalassaemia

3. ß- Thalassaemia minor or trait.

1. Sever anaemia (2-3 g/dl)2. Enlargement of liver and spleen.3. Expansion of bones, leads to Bone

deformities.

Beta-thalassemia Major

Causes: one point mutation

Type genotype Clinical

0 mutation Normal /

Minor point mutation

Minor: Trait0 heterozygousOr+ heterozygous

/0

/+Minimal anemia; no treatment indicated

Two mutations

IntermediaDouble distinct mutation

0/+ severe heterozygoteCan be a spectrum; most often do not require chronic transfusions

Severe gene mutations

Major+ homozygous(double) Or0 homozygous (double)

+/+

0/0

Cooley’s AnemiaHomozygous minor point mutation Need careful observation and intensive treatment

The classification of Beta Thalassemias

• Laboratory findings:*Hemoglobin as low as 2-3 g/dl

*Markedly microcytic/hypochromic*Marked anisocytosis and poikilocytosis*Basophilic stippling and polychromasia

*Hemoglobin electrophoresis –90% Hb F and increased Hb A2

*HPLC: confirmatory test

Of moderate severity (Hb 7-10 g/dl). The patient may show bone deformity, enlarged liver and spleen.

Beta-thalassemia intermedia

-This is usually symptomless, but mild anaemia may occur.

- Normal iron, ferritin, TIBC- Prenatal counseling.

(25% risk rate if both partners carry beta thalassemia minor).- Hb. Electrophoresis:

4-7 % Hgb A2 92-95% Hgb A1

2-6 % Hgb F