RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX

1. HEMOBLOGBIN (Hgb) (: common question

 Evaluation of erythrocytes is an important part in Board!)
of complete blood count (CBC)  Provides an estimate of the oxygen- carrying
 Complete blood count (CBC): capacity of the blood
o Screening procedure that is helpful in  RECOMMENDED METHOD:
diagnosing disease (: first step that is Cyanmethemoglobin (HiCN) Method (using
helpful identifying what’s wrong with this all forms of hemoglobin will become
your body) cyanmethemoglobin)
o Indicator of the body’s ability to fight  PRINCIPLE: Whole blood is mixed with
disease (: example WBC- means Drabkin’s reagent (for Hbg only!) to
there is an infection) produce a colored mixture
o Monitor the effects of drug and (cyanmethemoglobin). The color intensity of
raddiation therapy the mixture is measured in a
o Indicator of patient’s progress in spectrophotometer at a wavelength of 540
certain disease such anemia or nm. All forms of hemoglobin are measured,
infection except sulfhemoglobin.The absorbance of
COMPLETE BLOOD COUNT syanmethemoglobin at 540 nm is directly
proportional to the concentration of
A. Red blood cell parameters hemoglobin.
 NOTE: The sulfhemoglobin can’t be measured
because it is irreversible – we can’t change
1. Hemoglobin it into something hbg—it will stay until the life
2. Hematocrit span of the RBC
3. Red blood cell count  DRABKIN’S REAGENT:
4. Red blood cell indices I. Potassium ferricyanide
5. Blood smears (always pag nay CBC req.) o Converts hemoglobin (Fe2+)
to methemoglobin or
hemiglobin (Fe3+)
II. Potassium cyanide

B. White blood cell parameters o Converts methemoglobin to

cyanmethemoglobin

1. White blood cell count III. None-ionic detergent

2. Differential count (surfactant)

o Improve lysis of red blood cells
and decreases amount of
turbidity
SPECIAL TESTS
o NOTE: Lysis is important
because hemoglobin will not
1. Erythrocyte sedimentation rate (ESR)
be converted if not freed
2. Osmotic Fragility Test
IV. Sodium Bicarbonate
o pH buffer: waiting time of 15
NOTE: these two are not part of complete blood minutes
count!
 MODIFIED DRABKIN’S REAGENT:
A. Potassium ferricynide
o Converts hemoglobin (Fe2+)
to methemoglobin or
hemiglobin (Fe3+)
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX
B. Potassium cyanide IV. Cells with Hgb S and Hgb C
o Converts methemoglobin to (resistance to lysis)
syanide methemoglobin  Make 1:2 dilution of distilled
C. Non-ionic detergent water and multiply results with 2
(surfactant)
2. HEMATOCRIT (HCT)
o Improve lysis of red blood cells
and decreases amount of
 Used to determine packed cell volume (PCV)
turbidity
 Denotes the percentage of erythrocytes in a
D. Potassium dihydrogen
known volume of whole blood
phosphate
 Reflects concentration of red blood cells, not
o pH buffer: waiting time of 3
the specific count
minutes
 METHOD: Microhematocrit Method
o Prevents precipitation of
(RECOMMENDED IF MANUAL)
globulins
 PRINCIPLE: A small amount of whole blood
is centrifuged to determine maximum
 0.02 mL of (EDTA) whole blood sample is
packing of erythrocytes, which is expressed
added with 5.o mL of Drabkin’s reagent
in percentage or liters/liter (L/L).
(weather modified or not)
 10,000 to 15.000 x g (RCF) or 10,000 to
 Ratio of sample to reagent - 1:251
12,000 rpm – 5 minutes
 After centrifugation, results should be read
within 10 minutes.
 K2Fe(CN)- Potassium  Results should agree within 1-2% (+/- 0.01
ferricyanide to 0.02 L/L) (In performing hematocrit it
 KCN- Potassium cyanide MUST be collected in 2 tubes and you should
read it both and results should agree within
 SOURCES OF ERROR:
1 to 2%  example: 1st tube is 34% the 2nd
tube should only fall between 34-48% ---if
I. Drabkin’s reagent is sensitive to
layo na repeat!
light
 Microhematocrit tube measures
 Store reagent in a Brown bottle or
approximately 75 mm long (or 7.5 cm), with
in a dark place
inner bore size of approx. 1.2 mm; sealing
II. High WBC count (>20 x 10 9/L)
clay should be at least 4-6 mm long.
– Turbidity
 Two types of microhematocrit tubes:
 ( pag turbid mag increase ang
Heparinized (ring color red)
absorbance that lead to false
and plain tubes (ring color
increase of hemoglobin--- that’s
blue)
why need tanggalon ang tubidity
 Microhematocrit tubes hold
to prevent error)
approximately 0.05 mL of
 Centrifuge reagent- sample
whole blood
solution and measure Hgb using
the supernatant
 SOURCES OF TECHNICAL ERRORS:
III. Lipemia (turbidity)
 Add 0.01 mL of patient’s plasma I. Excess anticoagulant
to 5mL of reagent and use it as a. Falsely decrease HCT value
the reagent blank because of RBC shrinkage (or
mog gamay/kuyos)
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX
o Not used today as it requires larger
amounts of blood and consumes
II. Insufficient mixing of blood
more time
a. Falsely increased or decreased
HCT  RULE OF THREE:
III. Improper sealing of tubes
 RBC count x 3 = Hgb
a. Falsely decreased HCT because of
o Ex. 4 x 3 = 12
leakage
 Hgb x 3 = HCT
IV. Inadequate centrifugation
o Ex: 12 x 3 = 36
a. Falsely increased HCT
 HCT/3 = Hgb
V. Allowing tubes to stand for too
o 36/3 = 12 Hgb
long/delay in reading
 Only applicable in normocytic,
a. Falsely increased HCT (:if mag
normochromic red blood cells
delay sa reading ang pagtapok is
mabungkag so instead nag settle
3. RED BLOOD CELL COUNT
na sila it will mixed again with the
plasma if mo mix sya with plasma  Cell counts are usually automated
it will increase the HCT reading )  Manual cell counts exceed linearity of
VI. Including buffy coat in reading instrument
a. Falsely increased HCT o Automated counts exceed linearity of
instrument
 SOURCES OF PHYSIOLOGIC ERRORS
o Instrument is nonfunctional with no
backup
I. Trapped plasma may cause
o Remote laboratories in poor countries
a. Falsely increased HCT (Dili maka
o When testing is done in the field
pili ang RBC since nay plasma sa
o When doing manual counting, a
tunga usually happen when there
laboratory technologist needs
are abnormalities in the shape)
a. Hemacytometer or counting
II. Abnormal erythrocyte shapes
chamber
inhibit complete packing
b. Dilution pipettes
a. Falsely increased HCT
c. Diluting fluids
III. Dehydration
d. Microscope
a. Falsely increased HCT (pag
dehydrated gamay nalang ang
A. HEMACYTOMETER
plasma and daghan tan awon ang
RBC)  Most commonly used: Levy chamber with
IV. Hemoconcentration Neubauer rulings
a. Falsely increased HCT  Composed of two raised surfaces, each with
V. Contamination of interstitial fluid a 3 x 3 mm grid
a. Falsely decreased HCT  One grid is made up or nine 1 by 1 mm
VI. Hemolysis squares
a. Falsely decreased HCT  Four corner squares is subdivided to 16
squares each (WBC counting) ( :in four
 METHOD: MACROHEMATOCRIT
corners you have 64 overall squares that is
METHOD
used to count WBC)
o Uses Wintrobe tube
 Central square is composed of 25 smaller
o Centrifuged in 2000 to 2300g for 30
squares (RBC COUNTING)
minutes
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX
Each of the 25 smaller squares measured allow lysis of red blood cells) (allow lysis kay
0.04 mm para dilli magsamok samok si RBC and sure
na WBC nalang ma count)
Example:
D. RED BLOOD CELL COUNT

*RBC- in 25 smaller square only 5 smaller square

will be use in counting RBCs and that is the 4 corner  NOTE: The best RBCs diluting fluid is the
square in the center and the center square of the
central square that measured 0.04 mm Formol citrate/Dacie’s fluid
 NOTE: Best WBC diluting fluid is Turk’s
B. DILUTION PIPETTES
solution
 Important in making dilutions in
o Turks solution is compose of glacial
preparation for counting
acetic acid and crystal violet  kaya

 Depth factor is constant (0.1mm) sya best diluting fluid kay may STAIN
sya—ma stain ang wbc that make
Pipette: Important to create dilution counting them easy to count

HOW DO WE COUNT?

Naa kay sample, magsip ka ug sample gamiton

nimo ang diluting pipette either ang sample up
to the 0.5 (concentrated) or 1.0 (wala kaayoy
red cell—tend na nadaghanon nimo imong
 Has 0.5 mark and 1.5 mark and 101 mark sample) mark of the RBC sample

After you sip the sample up to 0.5 mark; mag

sip na pd ka na naa japon imong sample ug best
diluting fluid like Formol citrate/Dacie’s fluid
mamix sya together with the sample upon >>
the diluting fluid should up to the 101 mark and
didto nimo sila imix.

The mixed solution or the sample with the

diluting fluid mao tuy ibutang sa Neubauer
chamber – idrop (1 drop) >> then ipasettle para
dili molihok anf RBCs pag imong basahon—the
entire process called CHARGING from the drop
up to the settlement >> after charging the
Neubauer chamber ibutang sa microscope.
C. DILUTING FLUIDS

 Mixed with blood sample in preparation for

cell counting
 For RBC counting, the diluting fluid must be
ISOTONIC (Because Isotonic solution
preserves RBCs)
 For WBC counting, the diluting fluid must be
HYPOTONIC (Because Hypotonic solution
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX
1ST EXAMPLE PICTURE OF THE UPPER FIRST 4TH LOWER LEFT CORNER OF THE SQUARE
SQUARE IN THE LEFT

RBC COUNT: 23
5TH LOWER RIGHT CORNER OF THE SQUARE

RBC COUNT: 23

NOTE: LAHAT ICOUNT; EXCEPT SA NI GAWAS SA

LINE AND TANAN NI TOUCH AND NI GAWAS SA RED
LINE OR TANAN NO TOUCH OR NIGAWS SA
INVERTED L DILI APILON SA COUNTING 
APPLICABLE TO ALL SQUARES (RBC OR WBC)

2ND SQUARE OF THE UPPER RIGHT CORNER

RBC COUNT: 21
TOTAL RBC OF 5 SQUARES: 106

RBC COUNT: 21
3RD SQUARE IN CENTER SQUARE NOTE: either of the two gamiton

NOTE: 2 lang nag pweding dilution factor ang 1:100

and 1:200 (identify lang ito base sa problem
RBC COUNT: 18 NOTE: In identifying dilution factor base sa problem
remember the “the blood asa kutob gipa sip
NOTE: Kung ang blood gisip up to 0.5 mark the
diluting is 1:200 and DF is 200.
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX
NOTE: If ang blood gipa sip sya ug upto 1.0 mark 4. RED BLOOD CELL INDICES (plural of RBC
the dilution would be 1:100 and DF is 100 index)
NOTE: One RBC square is equivalent to 0.04 mm –  Consist of:
base sa problem nag count sya ug up to 5 therefore A. Mean Cell volume (MCV)
you multiply 0.04 by 5 = area of 0.2 B. Mean Cell Hemoglobin (MCH)
GIVEN C. Mean Cell hemoglobin Concentration
o Cells counted = 106 RBCs (MCHC)
o Dilution factor = 200 (1:200)  Calculated to determine the average
o Area counted = 0.2 𝑚𝑚2 volume, hemoglobin content, and
o Depth = 0.1 mm (for.1) or 10 (for.2) hemoglobin concentration of the red blood
FIRST FORMULA cells in the sample
 May be used for the initial classification
A. MEAN CELL VOLUME (MCV)
o Average volume of red blood cells,
expressed in femtoliter (fL) or 10−15 𝐿
o Values:
NOTE: per uL is not the SI unit so u need to convert  <80 fL – microcytic
from 106 /𝑢𝐿 into 1012 /𝐿  80-100 fL – normocytic
SECOND FORMULA  >100 fL – macrocytic
FORMULA AND EXAMPLE:

GIVEN
o Cells counted = 400 RBCs
o Dilution factor = 100 (1:100)
o Area counted = 0.12 𝑚𝑚2 from 0.04 x 3 RBC
square
o Depth = 0.1 mm
FIRST FORMULA
B. MEAN CELL HEMOGLOBIN (MCH)
(used for the correctness of MCHC result)
o Average weight of hemoglobin in a
red blood cell, expressed in
picograms (pg) or 1012 𝑔

NOTE: Atleast 2 decimal places and didto na mag o Picogram is also called

round off and it will happen in the FINAL ANSWER Micromicrogram (uug)

SECOND FORMULA o Generally, not considered in

classification of anemias
o NOTE: Ang ginagamit sa initial
classification of anemias is the
MCV & MCHC >> Ginagamit si
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX
MCH computation to correlate to
the MCHC result (e.g., if taas si
MCH dapat taas pd si MCHC)
o Values:
 26- 32 pg – normal value
FORMULA AND EXAMPLE:

NOTE: Same formula with MCV but MCV uses

HEMATOCRIT while MCH uses HEMOGLOBIN

C. MEAN CELL HEMOGLOBIN

CONCENTRATION (MCHC)
o Average concentration of
hemoglobin in individual red blood
cell, expressed as g/dL or %
o Values:
 <32 g/dL – Hypochromic
 32-36 g/dL – normochromic
 >36 g/dL – Hyperchromic
(only the spherocyte ang
mo abot ani not RBC)
NOTE: Normal RBC dili mo lampas sa 36 MCHC but
in the event na lumampas ng 36 up to 38 >> check
for the presence of SPHEROCYTE because sya lang
dapat ang mo abot ana na values --- if walay
spherocyte tas ni lagpas kag 36 there might be
something wrong with the computation of values.
FORMULA AND EXAMPLE:
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX

RED BLOOD CELL INDICES

 Blood picture is based on the MCV and MCHC result example: (or the interpretation of MCV and
MCHC; so pag tinanong ang blood picture – tan awa ang MCV & MCHC values)

NORMOCYTIC (80-100), MICROCYTIC (<80), MACROCYTIC (100),

NORMOCHROMIC (32-36) HYPOCHROMIC (>36) NORMOCHROMIC (32-36)
“Gamay na in size luspad pa gyd!”

 Aplastic Anemia  Sideroblastic anemia  Megaloblastic Anemia

 Hemolytic Anemia  Iron Deficiency Anemia i. Pernicious anemia
 Acute blood loss  Thalassemia ii. Sprue
 Sickle cell anemia  Chronic Blood loss iii. D. Latum Infection

Mnemonics: AHAS (imong hate na Mnemonics: SIT-C  Non-megaloblastic Anemia

tao normo-normo) i. Liver disease

5. BLOOD SMEAR (PAG UUSAPAN SA FINALS

TOGETHER WITH STAINING WITH WBC
COUNT)
SPECIAL TESTS
1. Erythrocyte sedimentation Rate (ESR)
2. Osmotic Fragility Test
 ESR is affected by three factors:
NOTE: BOTH ARE NOT PART OF THE COMPLETE
a. Erythrocytes
BLOOD COUNT
b. Plasma Composition
1. ERYTHROCYTE SEDIMENTATION RATE
c. Mechanical/Technical factors
 A nonspecific measurement used to detect
1. ERYTHROCYTES
and monitor an inflammatory response to
 Red cell size/mass in an important
tissue injury
factor in determining the distance of
 Not specific test for inflammatory disease
the red cells fall (mas bug at mas dali
(not used to diagnose but it helps para
mo baba)
makadiagnose)
 Larger RBCs tend to settle more
 A simple procedure that allows a specific
rapidly than smaller RBCs
amount of blood sit in a vertical for a period
 Red cells are negatively charged-
of time (hayaan mo lang sya magsettle
RBCs repel each other
compared sa centrifuge na imoha syang
 In inflammation, negative charge of
pugson; allow the cells to sediments on their
RBCs reduces because of the changes
own)
in the plasma protein concentration-
 The distance in which the red cells fall during
leads to increased sedimentation
the time period is the erythrocyte
(normally rbc are negatively charge
sedimentation rate, expressed in mm/hr 
and they repel each other >> since
(for example: 5 mm lang iyang gi baba for 1
dili sila mag dikit normally so dugay
hr then the ESR  5mm/hr)
ang sedimentation>> but if mawala
ang uban negative charge sa RBC
mapulihan ug positive charge >>
magkakaroon ng attraction –SANA
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX
ALL>> SO THEY TEND TO DIKIT
LEADS TO HEAVINESS and increase
sedimentation
 Alterations in RBC shape- decrease ESR
 Decrease RBC count- increase ESR
2. PLASMA COMPOSITION  ESR tubes with narrower bore yield
 Single most important factor in lower sedimentation rate (narrow
determining the ESR bore payat and tube so mag guot sila
 Rouleaux and aggregation of red pa baba unlike pag dako ang tube dali
blood cells are influenced by plasma makababa ang RBCs)
proteins  PRINCIPLE OF ESR: When anticoagulated
o Promotes rouleaux and blood is allowed to stand at room
aggregation temperature undisturbed for a period of
 Increased concentration of albumin time, the blood cells settle towards the
o Decrease ESR bottom of the tube. ESR is the distance (mm)
 Increased plasma viscosity that the red blood cells fall in 1hr. ESR is
o Decrease ESR (pag lapot ang directly proportional to RBC mass and
plasma dugay mag settle ang inversely proportional to plasma viscosity
RBC)  STAGES OF ESR:
3. MECHANICAL/TECHNICAL FACTORS i. Lag phase (10 minutes)
 ESR tubes must be placed  Rouleaux formation, slight
perpendicularly sedimentation
 A tilt of 3 degrees can cause error up ii. Decantation phase (40 minutes)
to 30%  More rapid and constant rate
 Vibrations and movements must be iii. Final settling phase (10 minutes)
avoided  Slow sedimentation rate
 Increased temperature = Increased
ESR

 METHODS:
i. Westergren
ii. Wintrobe
iii. Modified Westergren (same with
Westergren but not the same
ANTICOAGULANT
NOTE: Westergren uses Na Citrate while MW uses EDTA
Some conditions that ESR is not a specific tool to diagnose
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX
2. OSMOTIC FRAGILLITY TEST
 A measure of the ability of the red
cells to take up fluid without lysing
 Primary factor affecting the test is the
shape of the red blood cell, which
depends on the volume, surface, and
functional state of the red cell
 Larger surface area = more resistant
(decreased fragility)
(: Those larger surface area is
capable of holding more
entering the cell makes them dugay
mo burst)
 PRINCIPLE: If red cells are placed
in an isotonic solution (0.85 % NaCl),
fluid neither enter nor leave the cell;
If red cells are placed in a hypotonic
solution, fluid enters the cell until the
cell ruptures. Red cells decreased
surface are-to-volume ration ruptures
quickly than normal ones.
 SPHEROCYTES
o Have decreased surface are-
to-volume ratio
o A spherocytes swells up in
hypotonic solutions
ruptures much more quickly
than cells having a normal or
large surface area
volume- increased osmotic
fragility. (Spherocyte
problem/defects in
membrane makes
fragile >> pag masudlan lang
silage gamayng water mag
burst dayun sila kay dili
flexible ilang membrane)
o NOTE: Increase fragility =
Dali mabungkag; Decrease
fragility= dili dali mabungkag
 Fragility test of red cells increase
when rate of hemolysis is increased
 Lysis in hypotonic solutions depend
on stability of membrane
fluid
 General procedure: Whole blood is
added to varying concentrations of
buffered sodium chloride (NaCl)
solution and allowed to incubate at
room temperature. The amount of
hemolysis in each saline
concentration is then determined by
reading the supernatants on a
spectrophotometer
 NaCl concentration used: 0.85% to
0.1%
and  Normally, Hemolysis starts at 0.45%
NaCl and is complete between 0.35%
and 0.30% NaCl (normaly RBC stats
per to lyse at 0.04; spherocytes starts at
0.65)
have  In HEREDITARY SPHEROCYTOSIS,
their initial hemolysis starts at 0.65% NaCl
them and is complete at 0.45% NaCl
(the more mababa ang concentration
ng NaCl the more hypotonic ang
solution; the more hypotonic anf
solution padulong na sya maWater
that tend to RBCs to lyse)
 RED BLOOD CELL PARAMETERS – VIDEO LECTURE- SIR GLENFLIX

Actual Example of OFT:

Perfect example of those cells that have large surface area; they also have decrease fragility that
means dugay sila mabungkag