ROUTINE

LABORATORY
EVALUATION OF RBC
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Evaluation of RBC is essential in CBC

Manual RBC Count - no longer performed - inaccurate
and unreliable
Used to be performed in a thoma pipet using isotonic
soln, Hayems or Gowers solution and read in
hemocytometer

HEMOGLOBINOMETRY measurement of the

concentration of haemoglobin in the blood
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COLORIMETRIC METHODS
A. DIRECT
VISUAL 1. TALIQUERT
METHOD
Uses undiluted blood from a finger prick that
(OBSOLETE) is blot onto the absorbent paper ad directly
compared with the color scale (book) ranging
from 10-100 %

2. Dares Hemoglobinometer
Employs direct visual comparison of undiluted
blood drawn between two giant plates by
capillary action of the hemoglobinometer, and
the color is matched with a rotating glass disc
with varying depths of red color
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COLORIMETRIC METHODS
A. DIRECT
VISUAL 3. Acid Hematin Method
METHOD
Depends upon conversion of hemoglobin in
(OBSOLETE) whole blood, after the addition of 0.1 N HCl
into an acid hematin, which is brownish
yellow in color, and is diluted until it matches
with the color of the standard comparator.

4. Alkaline Hematin Methods

Depends upon the conversion of Hb in blood
into an alkaline hematin, after the addition of
excess alkaline (0.1 N NaOH) solution.
Methods: a. Wub. Clogg and King
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Hemoglobin Determination
International Committee for Standardization in
Hematology & NCCLS recommend
1. CYANMETHEMOGLOBIN (Hemiglobincyanide, HiCN)
Principle: Whole blood is diluted in a solution of potassium
ferricyanide (K3FE(CN)4 and potasssium cyanide (KCN).
The (K3FE(CN)4 oxidizes hemoglobin to hemiglobin (Hi,
methemoglobin).
The KCN provides cyanide ions to form hemiglobincyanide
(HiCN). The absorbance of the solution in a
spectrophotometer at 540 nm and compared with that of a
standard HiCN solution.
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Specimen is venous blood in EDTA less than 24 hrs old

Reagent is commercially known as Modified Drabkins
solution
Measured via spectrophotometer
NONIONIC DETERGENT enhances red cell lysis and
decrease turbidity from protein precipitates

-The method measures all forms of hemoglobin but not

SULFHEMOGLOBIN.
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Microhematocrit
Principle: small amount of WB is centrifuged to
determine max. packing of RBC, expressed as PCV or
HCT
Spx Req.- whole blood anticoagulated w/ EDTA or
heparin or capillary blood collected in heparinized
capillary tubes.
Reagents & equipment: sealing clay, capillary tubes,
reader device, microhematocrit centrifuge(10K-15K g of
RcF)
Note: read it w/in 10 mins after centrifugation = decreased
EXCESS ANTICOAGULANT (EDTA) WILL GIVE
FALSELY LOW VALUE DUE TO CELL SHRINKAGE.
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Rule of three- checking only

Basic equations
3 x RBC = Hb
3 X Hb = Hct 3% (common)

Note- applies only to normochromic normocytic cells.

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Erythrocyte Indices
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RETICULOCYTE COUNTS
Used to assess BM erythropoietic activity
Principle: ribosomal RNA of Retic must be stained
supravitally; that is, w/ the RBC in the living state. A retic
is defined as any nonnucleated RBC that contains two or
more particles of blue-stained, granulofilamentous
material after staining (NMB or BCB)
Spx: WB anticoagulated w/ EDTA
Reagents & Equipment: no special equipment needed but
enumeration may be facilitated by a calibrated disk
(miller disk).
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RETICULOCYTE COUNTS
Routine Light microscopy method
OIO
Select the best area-well stained no overlapping
Count retic per 1000 RBC
Retic % = number of retics counted
--------------------------------- x100
1000 RBC observed
Result is expressed in percentage
NV: ________________ Newborn is higher - _____ %
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Absolute Reticulocyte Count

P: Reflects the actual number of reticulocyte in 1 L of
blood.

Retic (%) x RBC count x (1012/L)

_______________________ x1000
100

NV: 25-75X 109/L

E.g. Retic count is 4 %, RBC count is 3. 30 x 1012, ARC
would be:
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Corrected Reticulocyte Count

Sometimes referred to as Retic Index or Hct Correction
Principle: % of retic may appear increased because of
early retic release into the circulation or decrease number
of mature cells in circulation (degree of anemia)
CRC= retic % X Hct (L/L)
-------
0.45L/L

NV- approx 1%
If patients retic count is 4.5 %, the hematocrit is 0. 30 L/L,
the CRC is
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RETICULOCYTE PRODUCTION INDEX

- ALSO KNOWN AS SHIFT CORRECTION; SIMPLEST
MEASURE OF EFFECTIVE EPO
HEMATOCRIT (L/L) RETICULOCYTE MATURATION
TIME (DAYS)
1.0
0.45 (+/- 0.05)
1.5
0.35 (+/- 0.05)
2.0
0.25 (+/- 0.05)
2.5
0.15 (+/- 0.05)
RPI = CRC/ MATURATION TIME IN THE PERIPHERAL
BLOOD
e.g. Retic = 5 %, Hct is 28 L/L, RPI is ________
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- REVIEW ESR
Reading Assignment: Review
Hematology Books (e.g Lotspeich-
Steininger, C., Steine-Martin, E. A.,
Koepke, J., Clinical Hematology,
Principles, Procedures and
Correlations, JB Lippincott Company,
East Washington Square,
Philadelphia, Pennsylvania, USA,
1992, pp 88 122 and other
reference books)