Haematology

HAEMOGLOBIN ESTIMATION

Haemoglobin is an iron containing oxygen transporting metalloprotein of the red blood cells. 

Its main function is to transport oxygen from lungs to the tissues and to carry the carbon dioxide from the tissues to the lung.

Haemoglobinometry is the measurement of the concentration of haemoglobin in the blood. 

Normal Values Of Haemoglobin

Adult female – 13 to 15 gm/dl

Adult male – 14 to 16 gm/dl

Methods of estimation:

I. Colorimetric methods: The principle is to convert haemoglobin into acid haematin, alkaline haemation, oxyhaemoglobin, carboxyheamoglobin or cyanmethaemoglobin and compare the colour of the compound produced with that of a standard. The method of comparison may be visual (comparing with a glass comparator) or photoelectric (i.e., photo colorimeter), based on which there are different methods:

a.   Visual comparison

        1. Acid haematin method (Sahli’s)

b.   Photo colorimetric  

        1. Alkali haematin method

        2. Oxyhemoglobin

        3. Cyanmethaemoglobin

II. Gasometric method: The oxygen carrying capacity of blood is measured and Hb is estimated indirectly.

III. Chemical method: Heamoglobin is determined from iron content.

IV. Specific gravity method: Haemoglobin concentration is >12.5 gm/dl if a drop of blood sinks when allowed to fall in copper sulphate solution of specific gravity 1.053 from a height of 1 cm

V. Coulter Counter Method: Hb is measured in a designated channel using sodium lauryl sulphate (SLS) which combines with methemoglobin to form SLS hemichrome molecule. The absorbance of this molecule is measured at 540nm to determine Hb.

Estimation of Erythrocyte Sedimentation Rate

Principle:

     When anticoagulant mixed blood is allowed to stand undisturbed for a period of time, the erythrocytes tend to settle (sediment) to the bottom. Two principal layers are formed:(a) an upper plasma layer, (b) a lower RBC layer .The rate of red cell sedimentation (estimated under standard conditions) is called as erythrocyte sedimentation rate (ESR). It is expressed as mm in one hour. 

Sedimentation takes place in following 3 stages:

1. Stage of rouleaux formation (10 min), 2. Stage of sedimentation (40 min), 3. Stage of packing (10 min)

Methods: It is done by 2 methods 

1. Westergren’s Method

2. Wintrobe’s Method

Normal Values

In males = 3 to 5 mm in first hour

In females = 4 to 7mm in first hour 

Increase in ESR

 Physiological conditions 

1. Old age 

2. After exercise 

3. Pregnancy, particularly in 2nd and 3rd trimester.

Pathological conditions 

1. Tuberculosis

2. Myocardial infarction

3. Anaemia except sickle cell anaemia and hereditary spherocytosis

4. Rheumatoid fever 

5. Rheumatoid arthritis

6. Macroglobulinaemia

7. Multiple myeloma 

8. Temporal arteritis

9. Hodgkin’s disease, where ESR value is above 60mm per hour indicate bad prognosis.

Decrease in ESR

1. Due to abnormal shaped RBCs as in sickle cell anaemia or spherocytosis.

2. Polycythaemia

3. Congestive heart failure

4. Hypofibrinogenaemia

PACKED CELL VOLUME (PCV)

Background

Packed cell volume (Haematocrit) is the ratio of volume of packed red blood cells to that of whole blood. It is expressed as percentage.

Besides packed blood cells and plasma, a buffy coat composed predominantly of leucocytes, is also seen at the junction of red blood cells and plasma.

Normal value

42 to 52% in adult males.

36 to 47% in adult females.

Calculation 

PCV=(Height of RBC Column x 100) / Height of whole blood specimen

Increase in haematocrit values is observed in:

1. Polycythemia

2. Dehydration

3. Emphysema

4. Congenital heart disease

5. Burns and shock

Decrease in haematocrit values is observed in:

1.  Anemia 

2. Excessive fluid volume of blood as in pregnancy

BLOOD GROUPS AND Rh TYPING

Immunohaematology is an application of the principles of immunology to the study of red cells antigen and their corresponding antibody in the serum.There are certain lipoproteins and /or glycolipids on the surface of red blood cells which act as antigen .On the basis of these inherited antigens a number of blood group systems are known. The most important are “ABO” system and “Rh”system .Examples of other systems are P system, MNS system, Lutheran system, Kell system, Duffy system, Lewis system etc.

Uses of blood grouping:

1. Blood grouping is of extreme importance for blood transfusion 

2. In haemolytic disease of newborn (if the mother and child’s blood group or Rh type are at variance )-for diagnosis and treatment 

3. Disputes over paternity of a person 

4. Susceptibility to various diseases. Eg: Peptic ulcer 

5. Medicolegal uses 

ABO system :

Lansdsteiner discovered the ABO blood group system. This system depends on the presence or absence of A and B antigen on the cell membrane of RBCs.According to ABO system all human beings can be divided into 4 major blood groups i.e. “A”,”B” ,“AB” and “O” group. The Landsteiner’s law states that when a particular antigen is present on the RBC, the corresponding antibody will be absent in serum.

Determination of ABO blood group

 The blood group can be determined in two ways: 1. Forward grouping or 2. Reverse grouping 

Both these methods can be done by:

      1) Slide method

      2) Tube method 

      3) Microplate method

      4) Microtyping method 

       5) Gel card method

CROSS MATCHING

The aim of cross matching  is to prevent transfusion of incompatible blood, which will have reduced survival in recipient and can provoke a hemolytic transfusion reaction. Most serious is ABO incompatibility in particular when A or B cells are transfused to group O person. So cross matching is necessary in all patients who need transfusion.

It is done in 2 parts-

Major cross matching: Donor cells are mixed with recipient serum.

Minor cross matching: Done by mixing donor serum/plasma and recipients cells. It is obsolete now.

Methods:

1. Saline tube method

2. Immediate spin method

3. Coombs (AGT) cross matching

Adverse transfusion reaction

       A transfusion reaction can be defined as many untoward reactions that occur as a consequence of infusion of blood or one of its components.

List of Transfusion Transmitted Diseases

1. Viral infections:

a) HIV

b) Hepatitis B

c) Hepatitis C

d) Human T-Lymphotropic Virus (HTLV I/II)

f) Cytomegalovirus

g) West Nile Virus

h) SARS

2) Parasitic and Bacterial infection:

 a) Malaria

 b) Babesiosis

 c) Chagas disease

 d) Leishmaniasis

 e) Syphilis

 f) Lyme disease

  3) Prions, which cause Creutzfeldt-Jakob disease, are also transmissible through transfusion

STUDY OF ANAEMIAS

Definition: the term ‘anaemia’ refers to a reduction in the normal concentration of haemoglobin or red blood cells in blood.

Etiological Mechanisms Responsible for Anaemia

1. Blood loss (hemorrhagic anaemia)

2. Diminished erythropoiesis(dyshaemopoietic anaemia)

3. Increased haemolysis (haemolytic anaemia)

Morphological Classification of Anaemia

1. Normocytic normochromic 

• Anaemia due to acute blood loss

• Anaemia associated with leukemia

• Aplastic anaemia

2. Microcytic hypochromic

• Iron deficiency

• Thalassaemia

• Sideroblastic anaemia

• Anaemia due to chronic disorder

3. Macrocytic normochromic

a. Megaloblastic anaemia

    Vitamin B12 and folate deficiency

b. Non megaloblastic anaemia

    Liver disorder

Normocytic Normochromic Anaemia

Clinical features

General features

1. Easy  fatigability, generalized muscular weakness, dyspnoea & palpitation on exertion, headache, vertigo, tinnitus

Laboratory findings

Peripheral blood

1. Haemoglobin: haemoglobin levels are below the normal values for the age and sex of the person

2. Hematocrit: below the normal value

3. Red cell indices – are generally within normal limits with occasional cases showing mild increase in RDW.

• Mean corpuscular volume (MCV)- 82-100 fl

• Mean corpuscular haemoglobin MCH)- 27-32 pg

• Mean corpuscular haemoglobin concentration (MCHC)- 31- 35 gm/dl

• Red cell distribution width (RDW) 11.5 - 14.0 %

• RBC histogram – generally normal, occasionally show mild broad base curve histogram correlating well with the increased RDW.

Peripheral smear:

• RBCs : Red blood cells are reduced in number, they are  normocytic and normochromic 

• WBCs: Total leukocyte count and differential count are normal.

• Platelets: Adequate

Reticulocyte count: Normal

Bone Marrow

• Cellularity: Bone marrow is moderately hypercellular/ hypocellular.

• M:E ratio: Variable

• Erythropoiesis: Increased/ decreased with Normoblastic maturation

• Myelopoiesis: Normal/ reduced/ replaced by abnormal cells

• Megakaryopoiesis: Normal/ reduced

• Bone marrow iron: Normal

Microcytic Hypochromic Anaemia

Clinical features

1. Insidious onset

2. Easy fatigability, generalized muscular weakness, dyspnoea & palpitation on exertion, headache, vertigo, tinnitus

3. Pharyngeal / oesophageal webs-folds of mucosa are formed at the junction of the hypopharynx with oesophagus.

4. Congestive heart failure

5. Appearance of pica, which is unusual craving for certain substances.

Laboratory findings

Peripheral blood

1. Haemoglobin: hemoglobin levels are decreased and usually in the range of 6 to 10 gm/dl

2. Hematocrit: is reduced (13-30%)

3. Red cell indices – there is a decrease in MCV, MCH and MCHC 

• Mean corpuscular volume (MCV) reduced (<80fl)

• Mean corpuscular haemoglobin (MCH) reduced (<25 pg)

• Mean corpuscular haemoglobin concentration (MCHC) reduced (<27gm/dl)

• Red cell distribution width (RDW) increased. It is the earliest sign of iron deficient erythropoiesis.

• RBC Histogram - low MCV will show a shift towards left and increased RDW shows a mild broad base histogram

Peripheral smear:

Diagnostic features of megaloblastic anaemia:

1. Macro ovalocytes in peripheral smear

2. Hypersegmented neutrophils

3. Megaloblastic erythropoiesis in bone marrow

4. Giant metamyelocytes and band forms

5. Response to B12 and folate therapy.  

Dimorphic Anaemia

Laboratory findings

Peripheral blood

1. Hemoglobin: Hemoglobin levels are decreased

2. Hematocrit: Decreased

3. Red cell indices: There is a dual population of microcytic and normocytic or normocytic and macrocytic red cells, or an admixture of small, normal, and large cells of different sizes and forms. The MCV, MCH and MCHC are either normal or variable with  increased RDW due to high degree of anisopoikilocytosis.

4. Histogram: shows two peaks representing dual population.

Peripheral smear

RBC: Dual population of RBCs in the peripheral blood smear with both microcytic hypochromic and normocytic or macrocytic RBCs.

WBC: White cells are decreased (leukopenia)

Platelets: Decreased (thrombocytopenia)

Reticulocyte count: It is normal or low. 

Bone Marrow

• Cellularity: Marrow is moderately to markedly hypercellular.

• M:E ratio: Because of marked erythroid hyperplasia, M:E ratio is reversed ranging from 1:1 to 1:6 (normal 2:1 to 4:1) 

• Erythropoiesis: show megaloblastic maturation .

• Megaloblasts: These are large, abnormal counterparts of normal normoblasts.

• Ineffective erythropoiesis: with preponderance of more primitive cells.

• Iron stores- increased

Study of Leukemias

Leukemia is a progressive neoplastic disease of hematopoietic system characterized by unregulated proliferation of uncommitted or partially committed stem cells. 

Based on cell of origin, they are classified into Myeloid and Lymphoid leukemias which are further classified into acute and chronic leukemias based on clinical course.

CHRONIC MYELOID LEUKEMIA

Neoplastic growth of primarily myeloid cells in the bone marrow with an extreme elevation of these cells in the peripheral blood. 

It is characterized by presence of distinctive molecular abnormality- a translocation involving the BCR gene on chromosome 9 and ABL gene on chromosome 22 (Philadelphia chromosome).

It is common in middle aged adults 

CLINICAL FEATURES: The peak incidence is seen in middle aged individuals with mean age occurrence of 45years. Generally patients present with massive splenomegaly.

It is important to understand granulocytic leucopoiesis to interpret the blood smear. It comprises of following stages.

• Haemocytoblast – it is an undifferentiated cell present in the bone marrow.  It is the primitive cell which differentiates into granulocytic precursor cells which undergo maturation to form other cells.

• Myeloblast : 

• Size- 11-18 microns in diameter

• Cytoplasm is deep blue and doesn’t contain any granules (except sometimes fine pink or red Auer rods or Auer bodies, which are abnormal lysosomal structures.)

• Nucleus is round /oval and occupies 7/8th portion of cell.

• The nuclear chromatin is finely reticular. Nucleoli are 2-5 in number with indistinct margins.

• The nucleoli appear as clear, round, hollow spaces in the nucleus

• Promyelocyte: 

• Slightly larger than myeloblasts (12-20 microns in diameter).

• A few pale blue or fine purple staining non specific azurophilic granules appear in the cytoplasm.

• Nucleus contains remnants of nucleoli.

• Myelocyte: 

• Smaller than myeloblast (11-16 microns)

• Cytoplasm contains specific granules on the basis of which they can be divided into neutrophils, basophils and eosinophils.

• Nucleus is oval/plano convex.

• Metamyelocyte: 

• Cytoplasm contains more specific pinkish granules

• Nucleus shows a notch or indentation.

• Band forms/ Stab cells: Nuclear indentation deepens so that 2 lobes of nuclei appear connected through band, therefore often called as band form.

• These cells further mature to form mature granulocytes, viz., neutrophils, eosinophils and basophils. With maturation size of the individual matured cell is reduced

Peripheral blood smear in chronic myeloid leukemia (Chronic phase)