CASE 1

 25 yr old Patient with history of fatigue,

 RBC: 3.7 x 106/µl
 Hb: 8.0 g/dl
 MCV: 62 fl
 MCH: 19.0 pg
 MCHC: 27 g/dl
 WBC: 5.3 x 109/l
 Platelets: 400 x 109/l

 Impression?
 MICROCYTIC HYPOCHOMIC ANEMIA
 CASE 2

 30 yr old, Male patient with fatigue

 RBC: 4.5 x 106/µl
 Hb: 11.0 g/dl
 MCV: 115 fl
 MCH: 25.0 pg
 MCHC: 30 g/dl
 WBC: 7 x 109/L
 Platelets: 350 x 109/L

 Impression?
 MACROCYTIC ANEMIA
 CASE 3

 23 yr old, Male patient

 RBC: 4.5 x 106/µl
 Hb: 11.0 g/dl
 MCV: 87 fl
 MCH: 30.0 pg
 MCHC: 33 g/dl
 WBC: 6.5 x 109/L
 Platelets: 350 x 109/L

 Impression?
 NORMOCYTIC NORMOCHROMIC ANEMIA
DIAGNOSTIC UTILITY OF
RBC INDICES

DR. MANAN SHAH

POST GRADUATE
MD PATHOLOGY
 OBJECTIVES

 To define & calculate the RBC Indices

 To describe the application of RBC indices in

diagnosing various disorders.

 To interpret RBC Histograms.

 RBC INDEX

 It is a quantitative measurement of red blood cell,

the amount and concentration of hemoglobin in
them.

 They were first introduced by Wintrobe in 1929 to

define the size (MCV) and hemoglobin content
(MCH & MCHC) of red blood cells
 DEFINITIONS:

 Mean cell volume (MCV)

 It is the measure of average volume of RBCs

 Mean cell Hemoglobin (MCH)

 It is a measurement of the average weight of hemoglobin in

individual erythrocytes.

 Mean Cell Hemoglobin Concentration (MCHC)

 It is the average concentration of hemoglobin in erythrocytes

 Red cell distribution width (RDW)

 It is a measure of variability of erythrocyte size

 WHY RBC INDICES REQUIRED?

 To classify the erythrocytes by their volume and

Hemoglobin content

 This indices suggest how the RBC’s appear

microscopically and provide significant information
(most commonly for Anemia diagnosis)

 Laboratory professionals correlate the indices with

Hct, Hb and RBC count to ensure that technical
problems are identified when they occur
 MEAN CELL VOLUME

 MCV = Hct(L/L) x 1000

RBC count ( x 1012/L)
 Normocytic: 80-100 fL
 Microcytic: Red cells with reduced volume(<80fL)
 Macrocytic: Red cells with an increased
volume(>100 fL)
 Remember MCV is a measurement of volume
whereas estimation of size of flattened cells is a
measurement of cell diameter. Cell diameter and
cell volume are not same
Fig. 1

Causes?

MCV Decreased

Fig. 2

MCV Increased

Causes?
MEAN CELL HEMOGLOBIN CONCENTRATION

 It is the average concentration of hemoglobin in a

deciliter of erythrocytes and expressed in g/dl
 It is the ratio of hemoglobin mass to volume in which
it is contained
 MCHC = Hb (g/dl) x 100
Hct (L/L)

 Normochromic: 32-36g/dl
 Hypochromic: <32g/dl
 Hyperchromic: >36g/dl
MEAN CELL HEMOGLOBIN CONCENTRATION

 Hypochromic: If the area of central pallor is >1/3rd

of the cell size

 Hyperchromic: The only erythrocyte that is

hyperchromic with an MCHC of > 36g/dl is the
spherocyte

Apparent hyperchromasia ( high MCHC) is usually

due to an artifactual increase in the haemoglobin
result, due to haemolysis, lipaemia, or large numbers
of Heinz bodies
Fig. 3

Causes?

MCHC Increase..??

Fig. 4
MCHC Decrease

Causes?
 MEAN CELL HEMOGLOBIN

 It is a measurement of the average weight (in

picograms 10 -12 g)of hemoglobin in individual
erythrocytes. It is calculated by:
 MCH = Hb (g/dl) x 10
 RBC( x 10 12/L)
 MCH varies in direct linear relationship with the
MCV. Cells with less volume contain less Hb and
vice versa
 Normal value for the MCH : 28 to 34 pg
 MEAN CELL HEMOGLOBIN

MCH Increase MCH Decrease

 B12 deficiency  Iron deficiency anemia

 Folic acid deficiency  Thalassemia
 Reticulocyte  Anemia of chronic
 Hemolytic anemia disorder
 alcoholism
Fig. 5

• Comment on size of RBC..?

Anisocytosis
 CASE 4

 25 yr old Patient with history of fatigue

 RBC: 3.0 x 106 /µl
 Hb: 8.0 g/dl
 MCV: 62 fl
 MCH: 19.0 pg
 MCHC: 30 g/dl
 WBC: 5.3 x 109/l
 Platelets: 400 x 109/l

 Impression:
 MICROCYTIC HYPOCHOMIC ANEMIA

 Differential diagnosis?
 CASE 4

 25 yr old Patient with history of fatigue

 RBC: 3.0 x 106 /µl
 Hb: 8.0 g/dl
 MCV: 62 fl
 MCH: 19.0 pg
 MCHC: 30 g/dl
 WBC: 5.3 x 109/l
 Platelets: 400 x 109/l
 RDW: 18%

 Impression:
 MICROCYTIC HYPOCHOMIC ANEMIA

 Importance of RDW here?

 RED CELL DISTRIBUTION WIDTH

 RDW is used because MCV is less reliable in describing

the erythrocyte population when considerable variation
in erythrocyte size occurs.
 RDW is a coefficient of variation in size distribution of
RBCs
 Measured as : RDW = Standard deviation of MCV × 100
MCV
 Normal value:11.5-14.5%
 Increased value indicates ANISOCYTOSIS.
RDW and MCV
 RDW

 RDW is increased in Iron deficiency anemia.

 While RDW is normal in Thalassaemia minor.

 Combination of low MCV and high RDW is one of the

best screening test for the Iron deficiency anemia.

 Mentzer index..?
 Sideroblastic anemia..?
 Further evaluation..?
 FURTHER EVALUATION

 For IDA
 S. ferritin level (low)
 TIBC (Increased)
 Transferrin saturation (Decreased)
 PBS: pencil cells, anisocytosis
 RBC count: Decrease
 For Thalassaemia
 PBS: target cells, polychromatic cells, basophilic stipling
 Rbc count: Normal / Raised.
 HbA2: Raised(>3.5)
 For Sideroblastic anemia
 S. iron increased
 S. ferritin level increased
 Dimorphic anemia and pappenheimer bodies seen
Fig. 6

Causes..?

Codocytes/Target cells

Fig. 7

Elliptocytes/ pencil
cells

Causes..?
 Fig. 8

Causes..?

Acanthocytes/ spur cells

Fig. 9

Echinocytes/ Burr
cells

Causes..?
Fig. 10

Causes..?

Drapanocytes/
sickle cell

Fig. 11

Stomatocytes/
mouth cells or cup
cells

Causes..?
 Fig. 12

Causes..??

Spherocytes

Fig. 13

Dacryocytes/
Teardrop cells

Causes..?
 CASE 5

 26 yr, Patient of low socio economic status comes with history

of weakness since 6 months, history of chronic abuse of PPI.
 RBC: 3.0 x 106 /µl
 Hb: 11.0 g/dl
 MCV: 75 fl
 MCH: 24.0 pg
 MCHC: 29 g/dl
 WBC: 5.3 x 109/l
 Platelets: 400 x 109/l
 RDW: 17%

 Interpretation?
 Microcytic hypochromic anemia
 Is Vit B12 def possible in this patient? If yes - explain MCV?
 A case of dimorphic anemia or combine anemia.
 CAUSES?

 Folate/ Vit B12 and Iron deficiency

 Malnutrition

 Malabsorption

 Pregnancy

 Alcoholism

 Atrophic gastritis (Vit B12)

 Thalassaemia and Vit B12 / Folate deficiency

 Iron deficiency with hemolysis. (prosthetic valve)
 Peripheral blood smear examination is very critical.
 CASE 6

 32 yr old patient with GI Bleed/hemorrhoid, no history

of drugs or hematological disorder
 RBC: 3.0 x 106 /µl
 Hb: 11.0 g/dl
 MCV: 102 fl
 MCH: 26.0 pg
 MCHC: 32 g/dl
 WBC: 5.3 x 109/l
 Platelets: 400 x 109/l
 RDW: 12%

 Impression?
 Macrocytic Normochromic anemia
 RETCULOCYTES

 Premature RBC

 They contain remnants of Ribosomal RNA

 Number of reticulocytes in PBS is a fairly accurate

reflaction of erythropoietic activity

 It is most useful and cost effective test in monitoring and

response to iron therapy
 RETCULOCYTES COUNT

 It can be done by two methods,

 Manual method
 Automated method

 Manual method.
 Romanowsky stain can not stain reticulocytes effectively.
 Suspect reticulocyte when Polychromatophilic cells on
PBS with Romanowsky stain seen (erythocyte with
bluish tinge)
 A supravital stain such as NEW METHYLENE BLUE,
BRILLIANT CRESYL BLUE must be used to identify
reticulocytes.
 RETCULOCYTES COUNT

 Automated methods
 By Fluorescent flow cytometry
 Or by scattered light with methylene blue staining
 In automated method >30,000 RBCs are assessed
 So the method is more precise and has high degree of accuracy than
the manual method. (where only 1000 RBCs are assessed)
 It is also more accurate when reticulocyte count is low
 RETCULOCYTES COUNT

 Corrected reticulocyte count

 Used to adjust the reticulocyte count in proportion to the
severity of anemia

 Corrected retic count= patient hematocrit

Normal hematocrit x % reticulocyte

 Its practical importance is to assess the degree of

erythropoiesis in anemic patient.

 In anemic patient <2% of corrected retic count associated with

hypo cellular bone marrow
 Reticulocyte production index
 This index is used to correct the time of prolongation of
maturation of reticulocyte due to sever anemia

 Calculated by = patient hematocrit x retic count (%)

normal hematocrit x retic maturation time(days)

 Also known as SHIFT CORRECTION INDEX

PCV% MATURATION DAY(S)
45% 1
35% 1.5
25% 2
15% 2.5
REICULOCYTE SUPRAVITAL STAIN
Fig. 14

Howell-jolly bodies

Fig. 15

Heinz bodies
 RETICULOCYTES

 A higher reticulocytes count may indicate:

 Anemia due to RBC being destroyed earlier than normal
(hemolytic anemia)
 Bleeding (GI Bleeding)
 Blood disorder in a fetus or newborn (erythroblastosis fetalis)
 Kidney disease, with increased production of erythropoietin
 Reticulocyte count may be higher during pregnancy.

 A lower reticulocytes count may indicate:

 Bone marrow failure (eg. drug, tumor, radiation Rx or infection)
 Cirrhosis of the liver
 Untreated patient of pernicious / megaloblastic anemia
 Chronic kidney disease
STRESS RETICULOCYTE OR STRESS ERYTHROPOIESIS

 In sever anemia when human body is in need of

reticulocyte or when there is a overproduction of
RBC, these is increase in reticulocyte in blood.

 This is called as Shift reticulocyte or stress

reticulocyte.

 This is similar to shift to left in WBC.

 CASE 7

 65 yr, Male, chronic cigarette smoker. H/o chronic cough

and confusion since few weeks.

 Complete blood count of the patient done…

 RBC: 7.0 x 106/µl
 Hb: 20 g/dl
 MCV: 70.o fl
 Hct: 60%
 WBC: 16 x 109/l
 Platelets: 625 x 109/l
 Reticulocyte: 3.5%

 Differential diagnosis..?
 CASE 7

 Detail history…
 Presented with no change in voice
 No h/o haemoptysis, weight loss.
 No change in pattern of cough, and not affecting his sleep.

 Enlarged spleen, slightly purple-red discoloration of

facial skin
 Chest X-ray: hyperinflation, no evidence of lung cancer

 Differential Diagnosis..?
 Diagnosis: Polycythemia vera
 RBC COUNT

 It is total number of RBC per µL or a liter of blood.

 The test can help to diagnose anemia with low

number of RBCs and other conditions affecting red
blood cells.

 Conditions require RBC count are,

 Kidney diseases

 Anemia

 Bonemarrow disorder like mylofibrosis

 RBC COUNT

 Mature RBC is a biconcave disc about 7-8 µ in

diameter.

 Normal volume is 80-100 fl, normal hemoglobin

content is 28-34 pg/dl.

 Normal RBC count

 Male: 4.3 – 5.9 (x 1012/l or 106/µl)

 Female: 3.5 – 5.0 (x1012/l or 106/µl)

 RBC COUNT

 Higher numbers of RBCs may be due to:

 Cigarette smoking

 Failure of the right side of the heart (cor pulmonale)

 Dehydration (for example, from severe diarrhea)

 Kidney tumor (renal cell carcinoma)

 Low blood oxygen level (hypoxia)

 Bone marrow disease that causes abnormal increase in RBCs

(polycythemia vera)
 Drugs like gentamycin / methyldopa
 RBC COUNT

 Low number of RBC may be due to:

 Anemia
 Bleeding
 Bone marrow failure (for example, from radiation, toxins, or tumor)
 Deficiency of a hormone called erythropoietin (due to kidney disease)
 RBC destruction (hemolysis) due to transfusion, blood vessel injury,
or other cause
 Malnutrition
 Multiple myeloma
 Deficiency of iron, copper, folic acid, vit B6, or vit B12 in the diet
 Over hydration
 Pregnancy
 POLYCYTHEMIA

 Polycythemia is a general term used to describe

erythrocytosis resulting in an increase in both hemoglobin
concentration and hematocrit ie increase in number of RBC
 It can be due to,
 Relative polycythemia
 Due to decrease plasma volume
 There is normal or even decrease red cell mass
 It is generally mild polycythemia
 Due to dehydration, hemoconcentration

 Secondary polycythemia
 Physiologic stimulus is present hense the name secondary
 Elevated plasma EPO levels (unlike polycythemia vera)

 Polycythemia vera
 POLYCYTHEMIA VERA

 Increased in Red Cell Mass

 Acquired myloproliferative disorder
 Over production of all 3 cell line, mostly RBC
 Increased HB
 > 18.5 g/dl in Male
 > 16.5 g/dl in Female
 Increased Hematocrit
 > 52% in Male
 >48% in Female

 Also known as 1° polycythemia, Polycythemia rubra vera

and Osler Syndrome.
 Result from the mutation in JAk2, a signaling
molecule in 95% of cases.
 FURTHER EVALUATION OF PV

 EPO level are normal or low in PV

 On PBS
 RBC overcrowding and with feathered edge seen

 Leukocyte aggregate in PBS of PV patient.

 Increased WBC count and platelet number

 BONE MARROW BIOPSY

 Shows red cell hyperplasia
 CASE 8

 A 20 yr old male, with history of weakness since few

month
 RBC: 1.0 x 106/µl
 Hb: 6 g/dl
 MCV: 110.o fl
 Reticulocyte count: <1%
 WBC: 0.4x 109/l
 Platelets: 10 x 109/l

 Impression..?
 PANCYTOPENIA
 Causes of Pancytopenia?
 APLASTIC ANEMIA

 It is condition of pancytopenia with hypocellular

bonemarrow
 Diagnostic criteria:
 Bone marrow cellularity: <25% (hypocellularity)
 Plus two of following
 Granulocyte count: <0.5x109/L
 Platelet count: <20x 109/L
 Anemia with corrected reic count: <1%
 HEMATOCRIT

 Hematocrit measures the volume that the RBC occupy

within whole blood.

 It is expressed in percentage or L/L.

 In automated analyzer, the hematocrit is usually

calculated by the measured MCV and RBC count by
using formula

 Hematocrit = MCV (fl) x RBC count (x1012/l)

1000
 HEMATOCRIT

 Elevated hematocrit  Low hematocrit

 Dengue fever (sign of an  Iron deficiency anemia
increased risk of DSS)  Leukemia
 Polycythemia vera
 COPD
 False low hematocrit
 Hemolytic anemias
 Hemodilution
 False high hematocrit  Volume overload
 Dehydration  pregnancy
 Burns patient
 Sever vomiting
 If Sample is not mixed
properly
 HEMATOCRIT

 One should interprets accuracy of RBC count,

hematocrit and hemoglobin value using quick formula
called the “RULE OF THREE”.
 RBC Count x 3 = hemoglobin x 3 = hematocrit(%)
 If error > 3% of the measured value, a measurement error
or instrument malfunction suspected.

 DIURNAL VARIATION
 RBC Count, hematocrit and hemoglobin concentration shows
diurnal variation
 These values are higher in the morning
 MICRO HEMATOCRIT

 Definition:
 A procedure for determining the ratio of the volume of packed
red blood cells to the volume of whole blood by centrifuging a
minute quantity of blood in a capillary tube coated with
heparin. (RED color marked capillary tube).

 Uses:
 Sample is very less

 Pediatric age group

 LIMITATIONS OF RBC INDICES

 Though the test for RBC INDICES is cost effective

and simple, it has its own limitations. Such as,
 It is a screening test not a diagnostic test
 MCV is not reliable when Anisocytosis is present
 In combine or dimorphic anemia again MCV is not reliable
 MCV does not reflect the diameter of the RBC, It is a volume of
RBC
 False high or false low value, which should be evaluated
further
 Automated Reticulocyte count is not standardized yet. And
manual method has inter-observer bias.
 RBC HISTOGRAM

PRICE – JONES CURVES

X axis: RBC volume Y axis: RBC Number
 Salient Features

 RBC Histogram is a bell shaped curve

 The instrument counts cell as erythrocyte with volume

sizes between 25 fl to 250 fl

 Area of the peak is used to calculate the MCV and the

RDW. This area represents 60-125 fl.

 Shift to right- RBC are larger (Macrocytes)

 Shift to left- RBC are smaller (Microcytes)

 SUMMERY

 RBC index (MCV, MCH, MCHC) and RBC count

helps in diagnosis of anemia

 Combination of various RBC indices (MCV, MCH,

MCHC) especially with RDW is useful in differential
diagnosis of anemia

 Reticulocyte count is a important indicator of bone

marrow activity and it can also be useful in
monitoring and response to iron therapy
 REFERENCES

 McKenzie SB, Williams JL. Clinical laboratory

hematology. 3rd edition. New jersey: Pearson
education inc; 2015
 Bain BJ, Bates I, Laffan MA, Lewis SM. Practical
hematology. 11th edition. Netherlands: Elsevier; 2012
 Lokwani DP. Interpretation of complete blood count
and histograms
 McCann S, Foa R, Smith O, Conneally E. Clinical
cases uncovered: Hematology. New jersey: Wiley-
Blackwell; 2009