Laboratory approach to anemias

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APPROACH TO
ANEMIAS
Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

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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#:

Adult males
Adult females

Hb
13 – 17 gm/dL
12 – 15 gm/dL

PCV
40-50 %
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
(potassium cyanide+potassium
Ferricyanide)

Check absorbance

Principle:
Hemoglobin

meth hemoglobin
K ferricyanide

cyanmethhemoglobin
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 timeconsuming 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

Hb levels
(gm/dL)

Normal
13-17 Males
12-15 Females

*Determination of Hematocrit

Mild
>10

Moderate
7-10

Severe
<7

(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:

RBC column (PCV)(%)
WBC column (buffy coat) (%)
Plasma column (%)

Normal
40-53 males
36-48 females
0.5-1
50-55, straw colored

Abnormals:

RBC column (PCV)(%)
WBC column (buffy coat) (%)

Plasma column (%)

Value
Anemia
<40 males
<36 females
Polycythemia >53 males
>48 females
>1 – leukocytosis, thrombocytosis, leukemia
This layer can be pipetted and used to demonstrate malarial
parasites and blast cells
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
Sickle cells
Oval macrocytes
Spherocytes
Target cells
Schistocytes
Burr cells
Bite cells
Tear drop cells
Polychromatic RBC
Basophilic stippling
Howel jolly bodies
Rouleaux formation
nRBC
Autoagglutination

Iron deficiency anemia, thalessemia
Sickle cell anemia
Megaloblastic anemia, alcoholism
Heriditary spherocytes, autoimmune hemolysis
Thalessemia, jaundice, HbC disease
Microangiopathic hemolytic anemia
Uremia
G6 PD deficiency
Myelofibrosis, myelopthisic anemia
Hemolysis, blood loss
Lead poisoning (coarse) megaloblastic anemia (fine)
Megaloblastic anemia, thalessemia, post splenectomy
Multiple myeloma, hypergammaglobulinemia
Hemolytic anemia
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
RBC COUNT

10

Normals:
MCV

80-100 fL

Classification of anemias based on MCV
Microcytic anemia
<80 fl
1. Iron deficiency
anemia
2. Thalessemia
3. Sideroblastic anemia
4. Anemia of chronic
diseases

Normocytic anemia
80-100 fl
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

Macrocytic anemia
>100 fl
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
Microcytic hypochromic anemia

High MCH
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
Microcytic hypochromic anemia

High MCHC
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
New born

0.5 - 2.5%
2 - 5%

Abnormals:
Reticulocytosis
1. acute blood loss
2. hemolytic anemia
3. response to therapy in nutritional
anemias

Reticulocytopenia
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
>35
25-35
15-25
5-15

TIME (DAYS)
1
1.5
2
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
Hypothyroidism

MDS

Aplastic
anemia


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(ii) Microcytic hypochromic anemia
MCV < 80

Serum ferritin

Low
(<12 µg/L)

Normal
(15-300 µg/L)

Iron deficiency anemia

High
(>300 µg/L)

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
Post hemolytic

Bone marrow

Normal

Abnormal

Anemia of chronic
Diseases
Chronic renal
Failure

aplastic anemia
Myelopthisic
anemia


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

Examine peripheral smear

Malaria

Normal
Cells

G6pd
PNH
Unstable
Hb

Sickle
cells

Bite
cells

G6PD

Schistocytes

Spherocytes

MAHA

Microcytic
Hypochromic

DAT

Thalessemia

Positive

AIHA

DIC

Negative

Spherocytosis

HUS

TTP

RENAL

NEUROLOGIC

HEMOGLOBIN ELECTROPHORESIS


Laboratory approach to anemias

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