RBC Indices

RBC indices

Using RBC count, Hb, Hct calculate:

• MCV

• MCH

• MCHC

• RDW

RBC count

TOtal erythrocyte count per mm³ or ul or per L

10^6/ul or 10^12/L (SI)

Determination of:

• RBC production

• OXygen transport

• Identification of anemia

• Response to treatment

Reference Values:

• Men - 4.6 - 6.2 × 10^6 / uL

• Women - 4.2 - 5.4 × 10^6 / uL

• Children - 4.0 - 5.5 × 10^6 / uL

Procedure:

Electronic Impedance

Flow Cytometry

ASCP combined Reference Range - 4.0 - 6.0 × 10^6 / uL

Electronic Impedance

Cells flow through a small aperture between two
electrodes in an ionic solution
 As cells pass between the electrodes resistance between
the electrodes increases
 Resistance R increases, Voltage, V, increases
 Producing a voltage pulse of short duration
 The number of voltage pulses is proportional to the
frequency of cells passing through the aperture
 The magnitude of voltage pulse is proportional to the cell
volume and size (MCV)

Flow cytometry

technique for counting, examining and sorting microscopic cells suspended in a stream of fluid

• sheath fluid

simultaneous analysis of the physical and/or chemical characteristics of single cells flowing through an optical/electronic detection apparatus

 Each suspended cell, passing through the beam,
scatters the light in some way both forward and side
 This combination of scattered light is picked up by the
detectors in flow cytometry
 By analyzing fluctuations in brightness at each detector
it is possible to deduce the size and complexity of each
individual particle.
 FSC correlates with the cell volume (size)
 SSC measures the inner complexity of the cell
 i.e. shape of the nucleus, the amount and type of
cytoplasmic granules or the membrane roughness

Flow cytometry analysis principles

in flow cytometry analysis a beam of light (usually laser light) of a single frequency (color) is directed onto a hydrodynamically focused stream of sheath fluid

A number of detectors are aimed at the point where the stream passes through the light beam:

• One in line with the light beam (forward scatter, FSC)

• one in perpendicular to it (side scatter, SSC)

RBC Histograms

Macrocytosis

Shift to Right

Microcytosis

Shift to left

Decreased RBC values

 Anemia
 Aplastic anemia
 Iron deficiency anemia
 Hemolytic anemia
 B12 deficiency
 Folic acid deficiency anemia
 Anemia of blood loss
 Anemia of Chronic Diseases
 RA & SLE
 Leukemia
 Hypothyroidism
 Liver disease
 Kidney disease

Increased RBC values

 Polycythemia
 Primary
 Secondary
 Heart failure, causing low blood O2
 Lung disease: emphysema, COPD, pulmonary fibrosis
 Hypoxia
 Carbon monoxide (CO) exposure
 Erythrocytosis
 (increased RBC production)
 Congenital heart disease
 Severe dehydration
 Smoking
 Living in high altitude
 Performance enhancing drugs
 Anabolic steroids, testosterone
 Erythropoietin
 Hemoconcentration

Physiological variations on RBC counts

 Posture:
 Blood samples is obtained from a
healthy persons in a recumbent
position
 RBC count is lower than normal

 Exercise:
 Extreme exercise and excitement

 Dehydration:
 Hemoconcentration
 Severe burns
 Severe, persistent vomiting or diarrhea

Physiological Variations

 Age:
 Normal RBC of a newborn is higher than
that of an adult
 Drop to the lowest point in life at 2 to 4
months
 Will increase to adulthood
 Ultimately lead to decreases RBC count
 Altitude:
 The higher the altitude the greater the
increase in RBC
 Decreased oxygen content of the air
stimulates the RBC to rise (erythrocytosis)

 Pregnancy:
 There is a normal decrease in RBC
count
 Associated with body fluid increases in
pregnancy
 Normal number of erythrocytes
becoming more diluted
 Drugs:
 Gentamicin and methyldopa
 Increase RBC count

Hematocrit (HCT)

Red blood cell mass

Packed red blood cell volume

Percentage of red cells in whole blood

hematocrit means to separate blood

plasma and blood cells are separated by centrifugation

Hematocrit Reference values

Men - 40-54% or 0.40-0.54

Women - 37-47% or 0.37-0.47

Newborn - 50-62% or 0.50-0.62

ASCP combined adult range - 35-50%

Microhematocrit

Done on small amount of blood

usually drawn from finger lance

Men:

• 40-54% or 0.40-0.54

Women:

• 37-47% or 0.37-0.47

Newborns:

• 44-65% or 0.44-0.65

Microhematocrit Procedure

A capillary micro-hematocrit tube

Anticoagulant added

Filled two thirds with venous blood

the tube is then centrifuged

separate the cellular elements from the plasma

the height of the packed cells in the tube is measured

indicates the hematocrit

automated hematocrit

calculated by

Hct = [RBC x MCV] / 10

Decreased Hct Values

 Anemia
 Aplastic anemia
 Iron deficiency anemia
 Thalassemia
 Hemolytic anemia
 B12 deficiency
 Folic acid anemia

 Anemia of blood loss
 Anemia of Chronic Diseases
 RA & SLE
 Leukemia
 Hypothyroidism
 Liver disease
 Kidney disease

 Reaction to chemicals or drugs
 Reaction to infectious agents such as Clostridium sp.
 Reaction to physical agents
 Prosthetic heart valves
 Severe burns

Increased Hct Values

Polycythemia

• Primary and Secondary

Erythrocytosis (increased RBC production)

severe dehydration

shock

hemoconcentration

Rule of Three

The Hct should be approx. 3 x Hb +- 3% if the patients RBC are normocytic and normochromic

if it does not agree it should be repeated

may be indicative of automated error

Hemoglobin (Hb or Hgb)

 Hemoglobin
 The main component of erythrocytes
 Transportation of oxygen and carbon dioxide
 An important buffer for hydrogen ion

 Procedure
 Automated methods
 Cyan-met-Hb principle
 Hgb is converted to cyanmethemoglobin
 Absorbance of the cyanmethemoglobin at
540 nm is directly proportional to the
hemoglobin concentration

Hemoglobin Reference Values

Women: 12-15 g/dl

Men: 14-18 g/dl

Newborn (both genders): 14-20 g/dl

ASCP combined adult Reference Range: 12-18 g/dl

Purpose of Hemoglobin tests

Diagnose Anemia

Oxygen Transport capacity

Follow up to treatment for anemia

Evaluate polycythemia

Decreased Hb Values

 Anemia
 Aplastic anemia
 Iron deficiency
anemia
 Thalassemia
 Hemolytic anemia
 B12 deficiency and
folate acid anemia
 Anemia of blood loss
 Anemia of Chronic
Diseases
 RA & SLE
 Leukemia
 Hypothyroidism
 Liver Disease
 Renal Disease

 Hemolytic reactions
 Transfusion reactions of
incompatible blood
 Reaction to chemicals or
drugs
 Reaction to infectious agents
 Clostridium sp.
 Reaction to physical
agents,
 severe burn
 prosthetic heart valves

Increased hemoglobin levels

Polycythemia

• primary and secondary

hemoconcentration of the blood

chronic obstructive pulmonary disease

congestive heart failure

variance in hemoglobin levels

post transfusion

• 1 unit of packed RBC

• raise the Hgb by 1 gram

physiological factors:

• high altitudes

• excessive fluid intake

• age/newborns

• pregnancy

• smoking

red blood cell indices

 Mean corpuscular volume (MCV)
 Mean corpuscular hemoglobin (MCH)
 Mean corpuscular hemoglobin concentration (MCHC)
 Red cell indices characterize RBC morphology

 RBC indices characterize erythrocytes size as:
 Normocytic
 Microcytic
 Macrocytic

 Hemoglobin content is classified as:
 Normochromic
 Hypochromic

Mean corpuscular Volume (MCV)

Reference value:

• 80-100 fl

measures the average volume of a RBC

is the best index for classifying anemia

categorizes red blood cells by size

MCV

if the MCV is normal

• the red blood cells are normocytic

• look for normal size RBCs

if the MCV < 78 fl

• the red cells are microcytic

• look for small RBCs

if the MCV > 100 fl

• the red cells are macrocytic

• look for large RBCs

MCV procedure

Electronic impedance

• measures pulse height of the electrical signal

• as the red cells pass between two electrodes

Flow cytometry

• forward scatter - cell size

manual calculation

• MCV = Hxt x 10 / RBC

Clinical implications of MCV (decreased MCV < 78 fl)

decreased MCV (<78 fl)

Microcytic anemia

• iron-deficiency anemia

• Thalassemia

• Anemia of chronic disease/inflammation

• Sideroblastic anemias

• lead poisoning

• Porphyria

Normal MCV (80-90 fl)

The patient has no sign of BM failure

Normocytic Normochromic anemias

 Acute blood loss anemia
 Hemolytic anemia
 Aplastic anemia
 Pure red cell aplasia
 Kidney disease
 Decreased erythropoietin production
 Malnutrition
 Stem cell related anemia

Elevated MCV (>100 fl)

 Megaloblastic Anemia
 (MCV 100-160 fl)
 Vitamin (B12 ) Deficiency
 Folic Acid Deficiency

 Pernicious anemia
 Impaired B12 absorption:
 Intrinsic factor deficiency
 Gastrectomy (total and partial)

 Anemia of liver disease
 Increased reticulocyte production

Mean corpuscular hemoglobin (MCH)

Measures the average concentration of
hemoglobin in a red blood cell
 Manual Calculation Formula
 MCH = Hb (g/dl) x 10 / RBC
 Reference Values
 Combined Adult Range: 26-34 picograms
(pg)/cell
 (Normally higher in newborns and infants)

Clinical Implications of MCH

 MCH normal:
 Normochromic anemia
 Hemolytic anemia
 Blood loss anemia
 Anemia of chronic disease/inflammation

 Increased MCH
 Megaloblastic anemia
 Anemia of liver disease

 Decreased MCH (Hypochromic anemia)
 Fe deficiency anemia
 Thalassemia
 Lead poisoning, Porphyria, Sideroblastic anemia

Mean Corpuscular Hemoglobin Concentration (MCHC)

 The average concentration of hemoglobin per volume of
red blood cells (100 ml)
 Categorizes red blood cells according to their
concentration of hemoglobin
 Categorized RBCs as hypochromic or normochromic
 MCHC relates to the color of the cells
 Used to give a rough guide to what shade of red RBC will
be
 Lower MCHC = Paler than normal
 Cells with too little hemoglobin are lighter in color with a
larger pale central area

Procedure for MCHC

manual calculation formula

MCHC = Hgb (g/dl) x 100 / Hct (%)

Reference values: 32-36%

MCHC normal

 Normal MCHC
 Acute blood loss
 Hemolytic anemias
 Aplastic anemia
 Megaloblasic anemia
 Anemia of liver disease
 Anemia of chronic disease/inflamation

Clinical Implications of MCHC

 Decreased values
 Hypochromic anemia
 Iron deficiency
 Thalassemia
 Lead poisoning
 Porphyria
 Sideroblastic anemia
 Increased values
 An MCHC > 38%
 Indicates Hereditary Spherocytosis
 MCHC is not increased in pernicious anemia

RDW-Red cell Distribution width

 Reference Values: 11.5-14.5%
 Numerical expression which correlates with
the degree of anisocytosis
 Derived from RBC Histogram
 Useful in differentiating Thalassemia from
iron deficiency anemia
 Low MCV, High RDW = Iron Deficiency anemia
 Low MCV, Norm RDW = Thalassemia, ACD

MCV and RDW with clinical importance Table