laboratory approach to anemias

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Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: [email protected] website: pathologybasics.wix.com/notes

APPROACH TO ANEMIAS

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OVERVIEW

1. Definition with normal range of Hb and PCV 2. Establishing the presence and severity of anemia

a. determination of hemoglobin (STEP 1) b. Methods of estimation

1. colorimetric methods i. Visual

- Tallquist blotting paper - Sahli’s acid hematin - WHO Hb color scale

ii. Using colorimeter - Cyanmeth Hb method - Oxy Hb method - Alkaline method - Haldane method

2. Gasometric methods Van slyke method 3. Chemical methods 4. Specific gravity method

c. Grading of anemia (STEP 2) d. determination of hematocrit (PCV)

i. Wintrobe method ii. Microhematocrit method

3. Determining the cause of anemia (STEP 3) a. Peripheral blood smear b. Reticulocyte count c. Red cell indices (MCV,MCH,MCHC,RDW) d. Serum iron studies

4. Morphological types flow charts (STEP 4) a. Macrocytic anemia b. Microcytic hypochromic anemia c. Normocytic anemias d. Hemolytic anemias

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*Definition Reduction in concentration of Hemoglobin below that expected for age and sex matched healthy controls Or Reduction in oxygen carrying capacity of blood Normals#:

Hb PCV Adult males 13 – 17 gm/dL 40-50 % Adult females 12 – 15 gm/dL 38-45 %

#Values vary with age, sex, geographical area and from textbook to textbook. Advisable to determine and set reference values for own lab according to local conditions

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*STEP 1: Determination of hemoglobin

(i) Tallquist blotting paper method

Allow blood to absorb into one of the test papers and compare with the color scale to determine the percent and weight of hemoglobin in blood under normal and anemic conditions

(ii) WHO Hb color scale

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The color of a finger prick blood sample, soaked into chromatography paper, is compared with the color of known hemoglobin content depicted on the scale in 2 g/dl increments from 4 g/dl to 14 g/dl.

(iii) Sahli’s acid hematin method

Method:

1. The diluent is N/10 Hydrochloric acid (HCL). Add it from the dropping bottle provided to the graduated tube, up to mark 2.

2. Measure 0.2 ml of well-mixed blood, with the provided micropipette (Sahli’s pipette) and transfer it to the HCL in the tube.

3. Thoroughly mix blood and acid using a fine glass rod (HCL will react with the haemoglobin and convert it into acid-haematin, which has a brown color).

4. Wait up to 3 minutes to allow the color to develop sufficiently to achieve an accurate comparison.

5. Add distilled water gradually to the mixture and mix the solution with glass rode. 6. Place the tube in the haemoglobinometer and compare it with the standard. 7. Continue to add distilled water until the sample firstly appears to be detectably pallor

than the standard. 8. Note the level of the liquid in the tube.

Disadvantages 1. It is tedious and time consuming to perform, especially with large number of samples. It is not accurate (its accuracy is of the order 15 %).

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(iv) Cyan meth hemoglobin method (Drabkin’s method) Recommended by ICSH

Method: 5 ml Drabkin’s reagent + 0.5 ml anticoagulated blood 5 min Check absorbance (potassium cyanide+potassium Ferricyanide) Principle: Hemoglobin meth hemoglobin cyanmethhemoglobin K ferricyanide K cyanide Cyanmethhemoglobin is a colored compound. After reaction, absorbance is measured at 540 nm. Absorbance is converted to Hb levels using calculating tables. All forms of hemoglobin (oxy Hb, Carboxy Hb, Meth Hb) except sulphmeth hemoglobin are measured by this method. Drabkin’s reagent is a colored compound, so zero is set using distilled water.

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(v) Oxy Hb method Ammonium hydroxide (0.04ml / dl) is used to hemolyse the red cells and convert the hemoglobin to oxyhemoglobin for measurement in the spectrophotometer. This conversion is complete and immediate and the resulting colour is stable.

(vi) Alkaline hematin method Ammonium hydroxide (0.04ml / dl) is used to hemolyse the red cells and convert the hemoglobin to oxyhemoglobin for measurement in the spectrophotometer. This conversion is complete and immediate and the resulting colour is stable.

(vii) Haldane method In this method, hemolysis of red cells is produced by mixing blood with a hypotonic solution like distilled water. Carbon monoxide is added to the mixture. The colour of the solution is compared with the standard one.

(viii) Gasometric method / Van Slyke method / Manometric method Gasometric method of estimation of hemoglobin by using van Slyke apparatus is the most accurate method. But it is not used routinely in clinical laboratories because it is time-consuming and the process of estimation is complex. It is used as a reference method to obtain the hemoglobin concentration in blood samples used for standardization of hemoglobin estimation procedures. This is the preferred method for research.

If interested in details, please refer to the following article available free online: http://www.jbc.org/content/91/1/307.full.pdf

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(ix) Chemical method It is an indirect method based on assumption that 1 gm Hb contains approximately 3.47 mg of iron. Value of Hb is calculated indirectly from value of iron.

(x) Specific gravity method This is the most commonly used method in blood banks for screening blood donors. Principle: The method is based on the fact that plasma or whole blood dropped into a solution of copper sulphate of known gravity is encased in a sack of copper proteinate and the gravity of this discrete drop is not changed for about 15 sec. The rise or fall of the drop during this interval (within 15 sec) shows whether it is lighter or heavier than the solution.

Method: 1. The copper sulfate solution is placed into a clear, several inch high test tube that is kept at room temperature and covered to prevent evaporation. A new tube is made daily or after 20-30 tests. 2. A small amount blood is produced from the side of an alcohol swabbed finger using a lancet followed by pressure at the stick site. The blood drop is then drawn into a small capillary tube by capillary action. The finger prick site has gauze applied to it to stop any bleeding. 3. A latex dropper bulb is then attached to the capillary tube containing the blood. The dropper bulb is squeezed slightly to expel a blood drop half an inch above the now opened copper sulfate test tube. The blood drop automatically forms a pellet upon contact with the copper sulfate. The used capillary tube is disposed of as biohazardous waste. Result: The blood drop is observed for a short time (15 sec) to determine whether it sinks (donor hemoglobin above 12.5 g/dL cut-off) or swims (donor hemoglobin MAY be below 12.5 g/dL cut-off). Since the test is just an estimate, many false-negatives tests (hemoglobin is not <12.5 g/dL) are produced and hemoglobin may be checked in another more accurate manner if available.

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*STEP 2 : Grading of anemia according to Hb levels

Normal Mild Moderate Severe Hb levels (gm/dL)

13-17 Males 12-15 Females

>10 7-10 <7

*Determination of Hematocrit (PCV) Definition:

1. It is the percentage of blood volume that is occupied by red cells. 2. It is expressed as a percentage.

Uses:

1. It is used to detect anemia and polycythemia 2. To calculate red cell indices such as MCV or MCHC 3. To check accuracy of Hb value

Methods:

1. Wintrobe’s method 2. Microhematocrit method Wintrobe’s method: 1. Anticoagulated blood is centrifuged for 30 min at 2300 g in a wintrobe’s tube 2. Blood gets separated as shown below 3. Wintrobe tube is 110 mm long and has an internal bore of 3mm diameter and is closed

at one end Wintrobe tube rack after centrifugation

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Microhematocrit method: 1. Capillary tubes with coated heparin (75 mm long, 1 mm internal bore) are filled about

3/4th with blood, sealed at one end with bees wax. Bee’s wax plate

2. They are centrifuged in a capillary centrifuge for 5 min

3. Readings are obtained either via a microhematocrit rube reading device or Arithmetic graph paper

Microhematocrit tube Arithmetic graph paper

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Normals:

Normal RBC column (PCV)(%) 40-53 males

36-48 females WBC column (buffy coat) (%) 0.5-1 Plasma column (%) 50-55, straw colored

Abnormals:

Value RBC column (PCV)(%) Anemia <40 males <36 females

Polycythemia >53 males >48 females WBC column (buffy coat) (%) >1 – leukocytosis, thrombocytosis, leukemia

This layer can be pipetted and used to demonstrate malarial parasites and blast cells

Plasma column (%) Pink – hemolysis Yellow – jaundice Colorless - anemia

RULE OF THREE

RBC COUNT (NORMAL 5) X 3 = HEMOGLOBIN (NORMAL 15)

HEMOGLOBIN (15) X 3 = PCV (NORMAL 45)

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*STEP 3 Determining the cause of anemia Peripheral Blood smear Reticulocyte count Red cell indices Serum iron studies

(i) PERIPHERAL BLOOD SMEAR Only salient points related to RBCs will be discussed here. For details please refer to separate notes on Peripheral blood smear examination.

Microcytic, hypochromic Iron deficiency anemia, thalessemia Sickle cells Sickle cell anemia Oval macrocytes Megaloblastic anemia, alcoholism Spherocytes Heriditary spherocytes, autoimmune hemolysis Target cells Thalessemia, jaundice, HbC disease Schistocytes Microangiopathic hemolytic anemia Burr cells Uremia Bite cells G6 PD deficiency Tear drop cells Myelofibrosis, myelopthisic anemia Polychromatic RBC Hemolysis, blood loss Basophilic stippling Lead poisoning (coarse) megaloblastic anemia (fine) Howel jolly bodies Megaloblastic anemia, thalessemia, post splenectomy Rouleaux formation Multiple myeloma, hypergammaglobulinemia nRBC Hemolytic anemia Autoagglutination AIHA (cold antibody type)

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(ii) RED CELL INDICES (MCV, MCH, MCHC AND RDW) MEAN CORPUSCULAR VOLUME (MCV) 1. Volume of a single RBC 2. Only MCV is determined by a cell counter, other parameters are calculated from PCV

and MCV MCV = PCV X 10 RBC COUNT Normals:

MCV 80-100 fL Classification of anemias based on MCV

Microcytic anemia <80 fl

Normocytic anemia 80-100 fl

Macrocytic anemia >100 fl

1. Iron deficiency anemia

2. Thalessemia 3. Sideroblastic anemia 4. Anemia of chronic

diseases

Decreased retic count 1. Aplastic anemia 2. anemia of chronic

diseases 3. Chronic renal failure 4. hypothyroidism 5. myelopthisic anemia

Increased retic count

1. Acute blood loss 2. Hemolytic anemia

Megaloblastic anemia 1. Vit B12 deficiency 2. Folate deficiency

Non megaloblastic

1. Liver disease 2. Alcoholism 3. MDS 4. hypothyroidism

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MEAN CORPUSCULAR HEMOGLOBIN (MCH) 1. It is the average hemoglobin in each RBC MCH = Hb (gm/dL) x 10 RBC count (millions/µl) Normals:

MCH 27-32 pg Abnormals:

Low MCH High MCH Microcytic hypochromic anemia Macrocytic anemia

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MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (MCHC) Definition: Average concentration of Hemoglobin in a given volume of packed red cells MCHC = Hb (gm/dL) x 100 PCV Normals:

MCHC 32-36 gm/dL Abnormals:

Low MCHC High MCHC Microcytic hypochromic anemia 1. hereditary spherocytosis

2. >40 gm% - cold agglutinin disease, MPD, viral infection

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RED CELL DISTRIBUTION WIDTH (RDW) 1. Indicates degree of variation in red cell size 2. Apart from anemias causing variation in cell size, also useful to differentiate iron

deficiency anemia from thalessemia minor (RDW raised, MCV low – iron def) (RDW normal, MCV low – thalessemia minor)

Normals:

RDW 11.6-14.6 %

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(iii) Reticulocyte count 1. Reticulocytes are young RBCs that contain RNA remnants 2. They are stained by supravital stains like brilliant cresyl blue or new methylene blue 3. used to find out the erythropoietic activity of bone marrow and to differentiate aplastic

anemias from other types of anemias $$ RETIC COUNT Retic count = Reticulocytes counted x 100 No. of RBCs counted Normals:

Adults 0.5 - 2.5% New born 2 - 5%

Abnormals:

Reticulocytosis Reticulocytopenia 1. acute blood loss 2. hemolytic anemia 3. response to therapy in nutritional

anemias

Decreased production: 1. Iron deficiency anemia 2. Anemia of chronic disease 3. Aplastic anemia 4. Anemia due to marrow infiltration

(leukemia, lymphoma, mets) Ineffective erythropoeisis:

$$ CORRECTED RETICULOCYTE COUNT 1. Reticulocyte count depends on PCV of the patient, low PCV can give falsely low retic

count and vice versa. 2. Hence retic count is corrected for normal average PCV for age of the patient Corrected Retic count = Retic count x PCV Avg PCV for age $$ ABSOLUTE RETICULOCYTE COUNT

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ARC = Reticulocyte percentage x RBC count in millions/µl $$ RETICULOCYTE PRODUCTION INDEX (RPI) 1. After formation normally reticulocytes spend 2 days in bone marrow and one day in

peripheral blood before fully maturing 2. When there is over production , they are released prematurely and they require more

time for maturation in peripheral blood. This results in doubling of reticulocytes in blood 3. So RPI is calculated to get an idea about the actual erythropoeitic activity of bone

marrow. Reticulocyte production index = corrected reticulocyte count Maturation time in days MATURATION TIME DEPENDS ON PCV

PCV TIME (DAYS) >35 1 25-35 1.5 15-25 2 5-15 2.5

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(iv) Serum iron studies See notes on iron deficiency anemia

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*STEP 4 MORPHOLOGICAL TYPES flow charts

(i) Macrocytic anemias

MCV >100 fl

Reticulocyte count

Normal (0.5-2.5%) Increased Bone marrow Reticulocytosis in hemolytic anemia Megaloblastic Normoblastic dysplastic Hypocellular Folate/B12 def Liver disease MDS Aplastic Hypothyroidism anemia

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(ii) Microcytic hypochromic anemia

MCV < 80

Serum ferritin

Low Normal High (<12 µg/L) (15-300 µg/L) (>300 µg/L) Iron deficiency anemia Electrophoresis Sideroblastic Anemia Increased HbA2/HbF Normal

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(iii) Normocytic normochromic anemia

MCV 80-100 fl

Retic count

High Normal/Low Post hemorrhagic Bone marrow Post hemolytic Normal Abnormal Anemia of chronic aplastic anemia Diseases Myelopthisic Chronic renal anemia Failure

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(iv) Hemolytic anemias

Features suggestive of hemolytic anemias (increased retic, increased indirect bilirubin, low hemoglobin)

Examine peripheral smear

Malaria Normal Sickle Bite Schistocytes Spherocytes Microcytic Cells cells cells Hypochromic G6pd G6PD MAHA DAT Thalessemia PNH Unstable Hb Positive Negative AIHA Spherocytosis DIC HUS TTP RENAL NEUROLOGIC HEMOGLOBIN ELECTROPHORESIS

laboratory approach to anemias

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