HEMOGLOBIN MEASUREMENT  Ferric thiocyanate is the end product

and it will be measured

General Basis of spectrophotometry.
Methods measurement  Beer’s law: The intensity of the color of
Gasometric Oxygen Indirect a solution. The end product is directly
Method Method proportional to the solution of the
Chemical Iron (Fe2+) substance.
Method  Iron is being measured
Gravimetric Specific gravity  Equation: 1 gm hb = 3.47 mg iron
Method
 Fe →Hgb
Colorimetric Hemoglobin Direct
Method
B. ASSENDELFT METHOD
1. GASOMETRIC METHOD
(VAN SLYKE OXYGEN CAPACITY) 2. GRAVIMETRIC
‫־‬ VAN SLYKE APPARATUS ‫־‬ Based on specific gravity
‫־‬ This method is based on the oxygen carrying capacity of
‫־‬ Uses a standard CuSO4 solution (in 40 tubes)
hemoglobin.
‫־‬ Measures the oxygen only with specific gravity of 1.035 to 1.075 at interval
 PRINCIPLE: of .001
○ A given sample of blood can be equilibrated ‫־‬ A drop of patient’s blood is placed into each
with O2 under standard conditions of tube. The drop of blood becomes coated with
temperature and pressure. copper proteinate and for about 12 seconds, the
○ It measures directly the amount of oxygen blood drop may either:
present in a blood sample. After measuring the o Sink (if heavier/denser): the blood sp.
oxygen, the O2 concentration can be converted Gravity is greater than the solution present
into Hgb. in the tube.
 EQUATION: 1g Hgb = 1.34 mL O2 o Float (if lighter/less dense), the blood has
○ HUFNER FACTOR: every gram of hgb can the sp. Gravity lighter than the solution.
actually bind to 1.39 ml of oxygen. Once the o Remain (if it has same density and the
oxygen is already known, we can convert it in CuSO4 solution) in the suspension.
hemoglobin. NOTE: NORMAL VALUES
 LIMITATION: It cannot measure hemoglobin that FEMALE 1.053 = 12.5 g/dL hb
cannot bind with oxygen. Measures active MALE 1.055 = 13.5 g/dL hb
hemoglobin only.
NOTE:
1. CHEMICAL METHOD  Gravimetric is no longer performed in routine
A. WONG METHOD clinical laboratory, but it remains to be the
 Iron is liberated by H2SO4 and potassium acceptable method in the mass blood donation.
persulfate  Only one reagent having a specific gravity of
 Hemoglobin: contains heme & 1.053 is needed in mass blood donation.
globin.
 Heme: contains FeH 3. GRAVIMETRIC
 Globin: protein A. DIRECT VISUAL COLORIMETRIC (TDAA)
 Separation of heme by globin by 1. TALLQUIST METHOD
treating the sample with sulfuric acid  Patient’s undiluted blood is absorbed unto
& potassium persulfate, the globin an absorbent pad and the color is compare
will precipitate by tungstic acid. with a litographed color scale representing
 PROTEINS: precipitated by tungstic acid values from 10-100%
 Iron → Ferric thiocyanate  Inaccurate: gives as much as 50% error
 2. DARE’S HEMOGLOBINOMETER
 Blood is drawn by capillary action between
2 glass plates and the color of blood is
matched with a rotating disc of red tinted
glass with varying thickness and color.
 Consists of different colors and it is
arranged in rotating disc.
 Each color has corresponding
concentration.
 Inaccurate: gives as much as 30% error.
 The automated version is acceptable

3. ACID HEMATIN
 PRINCIPLE: hemoglobin is
converted into alkali
hematin upon addition of an
alkali. Abnormal hb are
converted to alkali hematin
and are thus measured.
 Hb+acid →Acid hematin
 The reagent (acid) is 0.1N
HCl
 Hb+alkaline → alkali hematin
 The intensity of the brown color depends on
the amount of hemoglobin.
o More Hgb: darker the color of
brown.
o Less Hgb: lighter the color of
brown.
 The end product which is the brown color
which will be diluted with distilled H2O until
the brown color matches the color of the
comparator black. (g/L,g/uL)
 DISADVANTAGE: does not measure inactive
forms
 B. PHOTOELECTRIC COLORIMETRIC
1. OXYHEMOGLOBIN METHOD
 Oxyhb- oxygen containing Hgb
 What is measured in this method is the
entire hemoglobin wth oxygen.
 There is instances that instead of oxygen,
crbon dioxide is attached in hemoglobin.
The best thing to do in that case is to allow
the hmoglobin to oxygente first, so that all
the hgb will be converted into an
oxygenated form.
 PROCEDURE:
R 0.02 mL whole blood is reacted w/ 5
mL of 0.07 N NH4OH/0.1%Na2CO3
then mixed for proper oxygenation.
NOTE: R Oxyhemoglobin→ Absorbance is read @

Allow the solution to stand

for 3-5 mins.
Methods: Sahli-Hellige; Sahli- 540 nm against reagent blank.
adams; Sahli-hayden hayden-hauusser; Newcomer;  LIMITATION: it does not measure
Osgood-haskin. other derivatives such as methgb,
caboxyhgb, & sulfhgb.
 This is relevant in th diagnosis of acute
hemolysis.
 Methods:
 Harboe: 415 nm (Soret bad: 400-
430 nm)
 Naumann: 540 nm --- hgb
catalyzes the rapid oxydation of
benzidne by H2O2 (more sensitve
but less accurate)

2. CYANMETHEMOGLOBIN

4. ALKALI HEMATIN
 PRINCIPLE: hemoglobin is converted into alkali
hematin upon addition of an alkali. Abnormal hb
are converted to alkali hematin and are thus
measured.
 Produces a more stable pigment but is not ideal
for infants and children
 Wherein the major hgb of infants & children is
HgbF, one major characteristic of ftal hgb is, it is
resitant to acid and alkali.
 Methods: Wu; Klegg & King
 Phosphate - Incubation time is 10 mins
KH2PO4 - In modified Drabkin’s reagent,
 Gold standard/ Reference method for Hgb (NaCO3- the sodium carbonate has
measurement ORIG been replaced by KH2. The
 Most accurate Drabkin’s) effect of KH2 will accelerate
the reaction, shortening now
 PRINCIPLE: When blood is mixed w/ a
the incubation from 10 mins to
solution of potassium cyanide and
3 mins.
potassium fericcyanide, the erythrocytes
- Advantage: All type of
are lysed thereby producing hgb. hemoglobin is transformed to
Potassium ferricyanide oxidizes hgb into cyanmethmoglobin (except
methgb that combines w/ potassium sulfhemoglobin)
cyaide to produce hemiglobincynide
(cyanmethemoglobin) whose absorbance is
measured @ 540nm against reagent blank.
 ADVANTAGE: all type of Hgb is
transformed to cyanmethemoglobin
(except sulfhemoglobin)

DRABKIN’S REAGENT
Non-ionized - Detergent reagent: enhances lysis
detergent - When blood is mixed with a solution
(sterox of potassium cyanide and potassium
[Harleco] or ferricyanide, the erythrocytes are
Triton) lysed thereby producing hemoglobin.
- The hgb is needed to be hemolyzed
for it to react to another reagent.
Potassium - HbFe+2 → HbFe+3
Ferricyanide Methemoglobin
(K3Fe[CN]6) - Potassium ferricyanide oxidizes hgb
into methemoglobin that combines
with potassium Cyanide PRINCIPLE OF ABSORBANCE: The darker the color of the
- Methemoglobinemia: end product, the more light will be absorbed. Any light
Potassium - In combination of potassium that is not absorbed by the solution will be transmuted.
Cyanide (KCN) ferricyanide and potassium
cyanide it will produce CHEMICAL REACTION:
hemiglobincyanide
‫־‬ The more hgb that reacts with the reagent, the
(cyanmethemoglobin) whose
more amount of the product is produced. The
absorbance is measured at
540 nm against the reagent more end product, the darker the color.
blank. ‫־‬ The darker the color, more hemoglobin.
Sodium - Gives off cyanide to combine Color = conc. Of the unknown
Bicarbonate with:
 Methemoglobin to PROCEDURE:
make the end product a. 1:251 blood-reagent
Cyanmethemoglobin b. 20 ul of blood + Drabkin's reagent
 Hemiglobin (Hi) to c. 3 minutes at room temperature
make the end product d. Absorbance at 540 pm against reagent blank
Hemiglobincyanide e. Determine concentration
(HiCN)
f. Standard curve
Dihydrogen - Uses sodium carbonate as the
Potassium buffered solution g. Calculate (Beer's Law)
PREPARATION OF STANDARD CURVE:
NOTE: REFERENCE VALUES
a) Will use standard reagent of hgb 15 g / dl
b) Prepare dilutions to achieve certain conc.
Male 140-175 g/dL
Female 123-153 g/dL
c) Read abs/ at room temperature of each dilutions
At birth 150-200 g/dL
@ 540 nm
d) Plot absorbance at room temperature against its - Physiologic error is in the blood.
concentration.  Turbidity
- Will cause more light to be absorbed.
o Lipemic blood: high lipids, milky,
chylous. Fats cannot be separated
through centrifugation
 Remedy: Use patient blank
o High leuokocyte count: High WBC
count 50,000/uL will cause
turbidity of the sample. This can be
separated through centrifugation.
 Remedy: Centrifuge and use
supernatant.
o Abnormal hemoglobin: Hgb S and
NOTE: why is there a higher amount of hgb at birth? Hgb C will become resistant to
At birth, both flat and long bones are active in lysis, it will not release the hgb and
hematopoiesis. Whenever there is an RBC production, it cannot react with potassium
hemoglobin production also occurs. ferricyanide and the other reagent.
 Remedy: dilute sample 1:1
SOURCES OF ERROR: CYANMETHEMOGLOBIN distilled water (Hypotonic)
- Considered as reference procedure for both o Easily precipitated proteins:
manual & automated.  0.1 gm of K2CO3/L orig.
- Most accurate method because it is stable. Drabkin’s
- It measures all hemoglobin except  Multiple myeloma
sulfhemoglobin.  Produces bence jones protein
- Sulfhemoglobin is an abnormal hemoglobin and large amount of IgM and it
formed when a sulfide radical reacts with hgb, will affect the plasma cells
the addition is irreversible. when mix in drabkins reagent,
the solution will become
TECHNICAL/HUMAN ERROR turbid.
 Inaccuracy of the pipet  Waldenstro
- Dilution is inaccurate on pipets with cracks on macroglobinemia
its tip.
 Pipetting error QUALITY CONTROL OF DRABKIN REAGENT
 Use of unmatched cuvette  Use fresh reagent
- Should be clean & free from any finger prints.  Reagent: pale yellow with a pH of 7.0 – 7.4
 Deteriorated reagent - Once the color of the reagent changed into
- Cyanmethemoglobin uses 2 reagents (Drabkins dark or pale yellow, it is an indication that
and standard reagent) the reagent is deteriorated.
- The standard reagent is not added to the  Abs= 0 @ 540 nm
sample.  Storage: amber bottle at room temperature
 Oxidation of the reagent - Exposure to light can oxidize the reagent,
*CLERICAL ERROR: most common error in the lab and some analytes in the blood.
 Standard: 15 g/dL
PHYSIOLOGIC ERROR
 Stable in a polyethylene bottle at 2 – 8°C.
 now the hgb level.
CLINICAL SIGNIFICANCE ‫־‬ Decreased in oxygen is compensated in increase in
INCREASED HEMOGLOBIN: RBC by the increase of hemoglobin.

 Polycythemia QUANTITATION OF FETAL HEMOGLOBIN (HbF)

- Hgb increases in blood cells due to abnormal - Adults have 2% HgbF. Higher than 2% is considered abnormal.
increase of blood production.
 Dehydration (burns, diarrhea) A. ALKALI DENATURATION
- Burns & diarrhea has massive loose bowel,  Quantitation of the percentage of HbF in the
which contains more water and also plasma blood
water.  A hemolysate is alkalinized and then
- Loose of plasma water will result to cell neutralized, and the deatured adult Hb is
concentration and will lead to precipitated by ammonium sulfate.
hemoconcentration.  A filtrate will then contain only alkali-resistant
DECREASED HEMOGLOBIN: Hb, which is measured spectrophotometrically
and expressed as a percentage of the total.
 All types of Anemia  PRINCIPLE: When added in alkali, all
- Anemia is a group of condition, and all are hemoglobin except HbF will suffer from
characterized in decreased hemoglobin. denaturation of alkali reagent.
 Leukemia  The methods of measuring or quantitating
- Disease of the bone marrow, wherein there is hgbF are all based on characteristics of hgbF
an excessive production of leukemic cells and being resistant to both acid and alkali
the normal cell production will decrease. treatment.
- Decrease RBC count leads to a decreased  Using the alkali the A1 and A2 will denature
hemoglobin. then the F will still remain.
- Because of the excess production of leukemic a. BETKE METHOD
cells, the patient develops anemia, this is one of b. SINGER METHOD
the reasons why the hgb will decrease.  The purpose of this method is to
determine the percentage of F in the
NOTE: HEMOGLOBIN blood.
 Slightly decreases after 50 years of age  (+) control: drop of blood from fetal
 Hemoglobin is lower if lying down
umbilical blood.
- The interstitial fluid will shift inside the circulation
 PROCEDURE:
resulting now in hemodilution, the blood cells
 RBC hemolysate + Drabkin’s NaOH +
will be diluted in more plasma fluid.
 Higher in the morning and lower in the (NH4)2SO4→ ppt denatured HbA will
evening be removed by filtration.
- Circadian effect, there are some tests that affect  Filtrate(HbF)-measured
the time of collection. spectrophotometrically
 Increased in smokers  % HbF
- It is increased especially in chronic smokers;
they have a higher value of abnormal hgb REFERENCE VALUE:
(carboxyhemoglobin).  Mod by Betke: 0.2% - 1.0%
- 10% of the hemoglobin of the chronic smokers - (Less than 1 %)
is the carboxy hgb.  National Committee for Clinical Laboratory
 Increased among males Standards (NCCLS): HbF quantitation (2-40%)
 Increased in high altitude  RID: <2% (most preferred method)
‫־‬ The higher the altitude, the atmospheric oxygen is  Column Chromatography: >40%
lower resulting now to hypoxia. Hypoxia triggers  High Performance Liquid Chromatography
the erythropoietin. As a result, there is an - most acceptable method but not a routine test
increase in the RBC production which will increase in clinical laboratory
  Elevated HbF: some hemoglobinopathies
 B-thalassemias: Hereditary Persistence of
Fetal Hemoglobin. In adults: almost all red cells appear ghost cells
Test: patient sample + KOH  CLINICAL SIGNIFICANCE:
 Hereditary Persistence of Fetal
Hemoglobin (HPFS)
B. ACID ELUTION METHOD (Modification of Kleihauer-Betke -even distribution of hbF among red
by Shepard) cells
 To assess whether the distribution of  Thalassemia and
HbF in all red cell is the same. (homo Hemoglobinopathy
or hetero distribution) -heterogenous distribution of the HbF
 For detection of fetal red cells in among cells.
maternal circulation
 EXAMPLE: Bloody Amniotic fluid- fetal
red ceels appears red. And if those red
cells are A then its maternal red cell.
 Useful in diagnosis of Hemolytic HEMOGLOBIN ELECTROPHORESIS
Disease of Fetus & the New born. A. CELLULOSE ACETATE ELECTROPHORESIS (pH 8.4-8.6)
 PROCEDURE: Method of separating substances or molecules on the basis
 Prepare blood smear of their net charge.
 Air-dried blood film Movement of molecules on the electromagnetic field
 Elution: citric acid-phosphateLIMITATION: cannot be easily identified.
buffer (pH 3.3)
 Stain-Ehrlich acid Hematoxylin
 Counterstain-Erytrosin:
Standard test for quantitating
fetomaternal hemorrhage
 Prepare blood smear
 Air-dried blood film
 Elution: citric acid-phosphate
buffer (pH 3.3)
 Stain-Ehrlich acid Hematoxylin  Anion: negatively charge (-)
 Counterstain-Erytrosin:  Cation: positively charge (+)
Standard test for quantitating  Electrode:
fetomaternal hemorrhage  Cathode: negatively charged
 INTERPRETATION: (-)
 Maternal red cells: appears  Anode: positively charged (+)
as ghost cells
 Fetal red cells: appears as rose
pink in color
  Separates hgb that migrate together on cellulose
acetate.
 SEPARATION OF HgBs:
a) interactions among Hb variants,
b) Interaction of agar and citrate buffer ions;
c) Altered electrical charge of the various
HgbS at acid pH
- They are not all positive or negative charge,
 Proteins are bipolar, it means they have their charge is altered.
both cathode and anode electron  PROCEDURE
“AMPOTHERIC”  Place the specimen exactly at the center
 Therefore, Hgb is neutral  Once the current is applied, some goes to
 But if the protein is placed in a medium cathode and some are going to the anode
(cellulose acetate), it wil have an altered  We cannot compare the mobility of one
electrical charge. It will have net negative another because they have a different pattern
of migration
chrage.
 Relevance: S and C migrates alone
NOTE:
The importance of cellulose acetate is in separation
of four major and significant hemoglobin. Which are
hgbA, hgbF, hgbS (ABNORMAL,most common hgb
variant. Related in sickling RBC), HgbC (third most
common Hgb variant.
Both of the abnormal hgb is associated in
crystallization of the hemoglobin.
Since the movement is towards the anode, the
mixture of hemoglobin need to place in an area
nearer the cathode to observe how far do they go
and how
fast do they go. NOTE:
Cellulose acetate electrophoresis
Slowest to fastest arrangement C
SFA
Mnemonics:
Crawl Slow Fast Accelerate

Citrate Agar electrophoresis Cathode: F A Anode: S

C Anode: CS Cathode: AF

NOTE: CHARACTERISTIC OF HbF

 Resistant to alkali denaturation and
acid elution
 Slower on electrophoresis than HbA
 Has high affinity with O2
 The more negatively charged ions, the faster
 Has a decreased affinity with 2,3 DPG or
it is to migrate to the anode.
Diphosphoglycerate
 The movement of the hemoglobin is
-- 2,3 DPG is a molecule that
dependent on net negative charge. affects affinity of hgb to oxygen.
 The Amino acid component of globin gives the
negative charge of the hemoglobin.
HEMATOCRIT (PACKED CELL VOLUME)
B. CITRATE AGAR ELECTROPHORESIS (pH 6.0-6.2) HEMATOCRIT (HCT)
 - Otherwise known as the Packed Cell Volume
(PCV). In some, they define it separately that  Used in the calculation of absolute indices/
Hct is the methodology of doing it and Packed constants or Red Cell Indices (MCV, MCHC)
Cell Volume is the test that we are to report.  In Rough Quality Control calculations
But this time, we can interchange. We may be o 1 Hct point= 0.34 gm Hb/ 100mL
referring to the method or to the test itself. o 1 Hct point= 107,000 RBC/cumm of
Either Hct or PCV is used. whole blood
- Ratio of the volume of erythrocytes to that of o Buffy coat= WBC count
the whole volume.  The buffy coat portion can be used in the
- Refers to the proportion of whole blood that preparation of buffy coat smears.
consists of red blood cells.
- Expressed as a percentage (%) of the total REFERENCE VALUES:
blood volume. Male 0.42-0.50 / 41.5-50%
- Hct provides clinicians with an estimate of the Female 0.36-0.45 / 35.9-44.8%
body’s red cell volume and thus, the blood’s
oxygen-carrying capacity. NOTE:
- Hct determination is useful in screening for Buffy coat contains WBCs and platelets. We can also
diagnosing, or monitoring a number of make use of the buffy coat layer to estimate for the WBC
conditions and diseases that affects the of a patient. For every buffy coat layer, there are
approximately 5,000 -10,000 WBC.
proportion of blood (e.g. Anemia).
NOTE:
- Unit/ Reported:
If we are to prepare purely buffy coat to see mainly the
o Percentage (%)
WBCs and platelets, then do not use whole blood in
o Decimal fraction (L/L) preparing the blood smear. Rather, use the buffy coat
only and we can make use of the capillary tube to
collect the buffy coat to be smeared.

METHODS OF HEMATOCRIT (HCT) MEASUREMENT

INIDRECT HCT
- One of the automated principles.
- Automated Hct can be measured through
CONDUCTIVITY or in INDIRECT WAY.
- The Hematology Analyzer/ Hemoanalyzer will
not centrifuge the blood sample. It will only
CALCULATE.
- Derived from the values of RBC count and
Mean Cell Volume (MCV). Depending on the
Hct value, you can divide it by 10.

2. DIRECT (MANUAL/ SPUN HCT)

- Requires centrifugation
- Hematocrit Centrifuge/ Hemofuge: a special
centrifuge used in this method.
- Can be performed using:
OTHER USES OF HCT
 o Micro Method: small volume of blood = no. of cell x 𝟏𝟎𝟗 /L (WBC)
o Macro Method: e.g. method by = no. of cells x𝟏𝟎𝟏𝟐 /L (RBC)
Wintrobe.
 WINTROBE METHOD- DOUBLE OXALATE Hgb= gm % ; gm/L ; gm/dL
- Macro method Hct= % or L/L
- Makes use of the Wintrobe’s tube which
has two graduation (1 in left, 1 in right) SUMMARY:
wherein we read the Hct using the right MICROHEMATOCRIT MACROHEMATOCRIT
side graduation. METHOD METHOD
Microhematocrit Tube Wintrobe Tube
- Double Oxalate: anticoagulant of choice  Lenth: 70  Length: 11cm or
when using Wintrobe Method. or 110 mm
 HADEN’S METHOD: 1.1% Na Oxalate 75mm  Diameter: 3mm
 VAN ALLEN: 1.6% Na Oxalate  Bore: 1mm  Blood column:
 SANFORD MAGATH METHOD: 1.3% Na Oxalate  Blood column: at 100mm or
 BRAY’S METHODS: Heparin least 5cm 10cm
 ADAMS MICROMETHOD: makes use of  Plus/Seal:  Graduations:
the hematocrit centrifuge. Left: 0-10;
clay and paraffin ESR
 Centrifugation Right:10-0; Hct
(RCF):  Centrifugation
(RCF): 3,000 rpm
10,000- for 30 minutes
15,000 g for 5
minutes

NOTE: LAYERS FORMED IN SPUN HCT

RBCs: the densest or has the highest EQUIPMENT NEEDED IN PERFORMING MICROHEMATOCRIT
Sp. Gravity of all the cells that is why METHOD
they settle at the most bottom upon 1. HEMATOCRIT READER
centrifugation. 2. SEALANT/ CLAY SEALANT
3. PARAFFIN SEAL
Buffy Coat: second layer; consists of 4. BLOOD COLLECTION MATERIALS
WBCs and platelets. WBCs lies at the 5. MICROHEMATOCRIT CENTRIFUGE/ HEMOFUGE
bottom of the buffy coat due to 6. CAPILLARY TUBES
higher sp. Gravity (denser) while - Internal diameter: 1mm
platelets lie right above the WBCs. - Length: 70 or 75 mm / 70cm or 7.5 cm
- Types:
Plasma: liquid portion that is
o Heparinized: red band; used when
separated
directly collecting blood from skin
Note: If we are to arrange these cells, mature/non- puncture.
nucleated RBCs lie at the most bottom part. If there are o Non-heparinized: blue band; used
immature RBCs, they lie just above the mature RBCs. when the blood is already in an EDTA
The more nucleated the cell is, the less dense it is. tube or mixed in an EDTA coagulant
in order to prevent shrinkage of the
NOTE: WAYS OF REPORTING RBCs and decreased volume of Hct
Cell count= cells/ cumm and MCV
= no. of cells/ µL
NOTE: PROCEDURE (MICROHEMATOCRIT)
 a) Tubes are filled with blood on the opposite side
of the band. Fill the capillary tube with blood in
¾ full. When it is in ¾ full already, the tube
approximately contains 0.5mL or 50µL.

Note: Appearance after centrifugation.

b) When using heparinize, invert it upside down in

order for the blood and the heparin to be
mixed.
c) Let the blood move downward until the edge
of the tube to start sealing. Do not seal if blood
is placed on the other side of the band to
prevent spillage or displacement of the blood. f) Measure total volume, height of packed cell
Start sealing with clay first which is the one in volume excluding the buffy coat. Inclusion of
direct contact with the blood because it is the buffy coat will result to an erroneously
denser and will totally seal or prevent the increased Hct reading.
blood from leaking.
d) To hold the clay in place, follow it up with
paraffin so that the clay will not be washed out
because during centrifugation, the force will be
going outwards. The clay will be washed off if it
is too soft. The length of the seal (clay+paraffin)
should be at least 4-6mm.
NOTE:
 Foot rule: can be used to measure the PCV
 Microhematocrit Reader: made up of two
rotating plates

e) After sealing, balance the centrifuge and

centrifuge the capillary tube at 10,000-15,000 g
for 5 minutes.

How to use:
a. Set capillary tube into the groove of the sliding
Note: To prevent the blood from spilling yet allows the curve
red cells to settle, the sealed area should be pointing b. Set bottom of blood cell in tubes into 0% line.
outwards. Once the machine rotates, the centrifugal c. Move cursor and set upper end of blood
force is to push the blood outwards. plasma in tubes onto 100% line
 d. Move scale and set red line onto upper end of DEHYDRATION
blood layer and read the measure value.
NOTE
MACROHEMATOCRIT Hematocrit (PCV) is one of the values or tests that is
MACRO METHOD (WINTROBE) greatly affected when blood specimen collection is
wrong particularly the prolonged application or tight
 Length: 11cm or 110 mm application of tourniquet. Because of this, the fluid
 Blood column: 100 mm or10 cm inside the plasma will be pushed outwards but the cell
 Graduations: will remain in the circulation. However, some of the
○ Left: 0-10; ESR plasma fluid will move outside resulting now to
○ Right: 10-0; hct hemoconcentration.
 Diameter: 3mm
 Centrifugation (RCF): 3,000 rpm for 30 minutes Change in patient’s position from supine to upright or
sitting position can cause hemoconcentration. The
plasma volume of the patient will move outside the
interstitial space resulting to hemoconcentration.

In this situation, the effect on PCV is not great. Around

REFERENCE RANGES: 8% will be the erroneous effect in the PCV.
Male 0.42-0.50 / 41.5- 50.4 %
Female 0.36-0.45 / 35.9- 44.6 %
At birth 0.45-0.60

NOTE: MACROHEMATOCRIT
 Unlike for the micro method wherein the
centrifugation is only for 5 minutes, in macro
method, the more blood you use, the longer is
the centrifugation time yet lower centrifugal
force. NOTE: HCT are expected to DECREASE in the following
conditions:
 Manual calculation is done in Macro method
unlike in micromethod where hematocrit ANEMIA
reader is used for measuring. - Group of conditions characterized by
 It is more accurate to read in millimeters (mm) decreased in RBC count, Hgb, and Hct.
because of the small graduations. Decrease in RBC is the one that leads to the
decrease in the Hgb concentration and Hct.
NOTE: HCT are expected to be ELEVATED in the following - Total volume is not changed. However, the PCV
conditions: has decreased because of the decrease in
POLYCYTHEMIA VERA production.
- One condition wherein Hgb is elevated because HYDRATION
in this condition, the RBC count is high due to - Hydration will lead to diluted PCV resulting to a
an increase production or too much production deceased Hct.
of RBC. In fact, all blood cells (WBCs, platelets, - The effect of hydration is hemodilution. By
RBCS) are increased in this condition. increasing the fluid inside the circulation (Shift
- The measure of this RBC volume is the Hct. of extravascular fluid) will cause dilution of the
Therefore, Hct is also elevated. The higher RBC, suspended RBCs resulting now to a decrease in
the higher PCV. the measured PCV.
- When a patient is given an IV fluid, this too can
HEMOCONCENTRTION result to hemodilution.
- Increase in Hct is not due to an increase in cell - Other cause is the change in patient position
prodution but rather the decrease of plasma that if a patient is in a lying position, the fluid
concentration resulting to outside the blood vessel will move inside the
hemoconcentration which can also be due to blood vessel resulting to hemodilution.
dehydration.
INTERPRETATION:  Prolonged-standing of blood sample prior to
test
- Will cause RBCs to crenate and swell. When a
cell is swollen especially at the 6th hour of
letting the sample to stand, it will result to an
elevated Hct.
- EDTA: most common anticoagulant used but it
From left to right:
is most preferred to use immediately within
1st tube: decreased plasma volume due of
dehydration. the first 2-3hrs from collection.
- Remedy: gently invert the sample 60x to
nd
2 tube: increased in RBC and decreased in plasma ensure adequate distribution of the blood
volume because of the abnormal production of RBC; cells.
“Polycythemia Vera”
 Insufficient mixing of blood
rd
3 tube: normal - Will cause either a decrease or increase in Hct
depending on which blood component
4th tube: Normal; despite the total reduction in blood collected.
volume, the reduction is proportional. This type of - In prolonged standing, the red cells will lie
condition is somewhat an unreliable condition or is at the bottom of the tube. The upper
unreliable to evaluate and interpret the Hct of this portion of a prolonged standing blood will
condition. This happened during acute blood loss. In
result to decreased Hct. But if you will
acute blood loss, there is an increased loss of blood but
aspirate blood at the bottom, it will then
in proportion resulting now to normal Hct.
result to an elevated Hct.
Compensatory: blood cell undergo vasoconstriction so
that at least the amount of blood being lost is limited.
But the effect of this is the decrease in circulation  Improper sealing of tube
because the blood is somewhat obstructed resulting to - Some of the components will be lost.
a decrease in oxygenation. To expand the blood vessel, - Improper sealing will cause the RBCs to
the blood volume should also expand and the blood escape from the tube during
volume will come from the bone marrow but it is too centrifugation.
long for the bone marrow to compensate blood loss. To
immediately compensate blood loss is the shift of fluid  Inadequate centrifugation/ allowing the
from the interstitial tissues but the effects would be tubes to stand too long after
hemodilation. centrifugation
- 5 minutes: required time for
Note: taking the Hct of a patient immediately after an
microhematocrit centrifuge
acute blood loss is one of the sources of errors.
- 30 minutes: required time for macrohematocrit
5th tube: decreased RBC, increased plasma volume - Inadequate centrifugation will cause the
because of the hemoconcentration. cells to disperse resulting to an increase of
height formed. Allowing the cells to stand
6th tube: normal vol of RBC, but increased in plasma after centrifugation for too long will lead to
volume, because of the hydration. erroneously increased.
- Protocol of the laboratory:
HEMATOCRIT SOURCES OF ERROR o >55% Hct value:
TECHNICAL ERRORS/ HUMAN ERRORS -will cause polycythemia vera
condition. To prevent
 Excess anticoagulant
misdiagnosis, before reporting
- Will cause the RBC to shrink and shrinkage will
very high Hct, we have to make
lead to decrease in Hct.
sure that we followed the
procedure correctly.
 -Protocol: centrifuge it again. - Slowing down/stoppage
o If in 2nd centrifugation is still be greater - Example: Prolonged application of tourniquet,
than 55%, the result is ready to be the circulation was slow down in the arm
reported. because of the prolonged application of the
o Re-centrifugation is required if the tube tourniquet.
is not read 10 mins after initial
centrifugation because letting it stand RULE OF THREE
for 10 mins will cause the RBC to - To check the accuracy of the RBC, Hgb, and
disperse again. Thus, erroneously Hct values generated by an automated
elevated analyzer or by manual methods.
 Reading errors (Parallax) - The rule of three applies only to RBCs that
- Parallax Error: error in reading volumes are NORMAL in size and in hemoglobin
due to the different position of the eyes or content.
the volume being read.
- It is more accurate to read the volume in
eye level.

PHYSIOLOGIC ERRORS
 Trapped plasma (poikiolocytes & anisocytes)
- It refers to the volume of plasma, trapped in
between red blood cells.
- Spun hematocrit: higher compared to
automated hematocrit. It is higher for about 1-
3%, but it will be greater than 3% in the
presence of poikilocytes and anisocytes. Thus,
it will lead to higher hematocrit reading.
- In the presence of abnormal size or shape of
cells, the more spaces would be left in
between that will be filled with plasma, that
will trap plasma.
 Values taken immediately after acute blood INTERPRETATION
loss
- It will show a proportional decrease in PCV and
the plasma. It will result to normal hematocrit
count, but after few minutes shift of fluid from
the extravascular into intravascular occurs
then that is the true hematocrit of the patient.
The values taken immediately are not reliable,
because there is no change in the result.
- True reflection of acute blood loss will be
observed after 48 hours.
- If the values coincide, and the results obtained from
 Dehydration the blood smear evaluation appears to exhibit normal
RBCs or appears to be normocytic and normochromic,
- The hematocrit will appear elevated not
the results obtained are ready to be reported. The
because of true abnormal increase in
results should not be reported if the acquired values
production, but only because of an alteration match with each other yet the blood smear shows
of the body fluid of the patient. abnormal RBCs. Possible causes of a falsely low
hemoglobin concentration or a falsely high hematocrit
 Stasis should also be looked into.
- If the values do not coincide and the RBC appears to - To make the classification more
be abnormal, the results obtained are ready to be complete together with the hgb
reported. On the other hand, the values acquired are concentration
not reliable when the readings do not agree with each  RED CELL DISTRIBUTION WIDTH (RDW)
other yet tend to have normal cells visible in the blood - The use of MCV & MCHC in the
film. morphologic classification of anemia
If the values do not correlate, more research should be applies only when the red cells are normal
done to find the source of the problem. When a
in size or when they belong only in one
discrepancy is found, a control specimen should be
population.
examined. In this case, random error may have
happened if appropriate results for control are - If the cells are diverse or varied in size, the
achieved. When inexplicable inconsistencies are MCV is not reliable. What is more reliable
discovered, the specimens tested before and after the is the RDW or index of anisocytosis.
specimen in question should be analyzed to see if their
results are consistent with the rule. OTHER INDICES
 Color index (CI)
RED CELL INDICES/ ABSOLUTE INDICES  Volume index (VI)
- Used to define the size and hemoglobin  Saturation index (SI)
content of the red blood cell.  Mean corpuscular Diameter
o The normal appearance of RBC is salmon  Mean
pink, the central pale area does not contain
MEAN CORPUSCULAR VOLUME (MCV)
hgb.
‫־‬ Refers to the average VOLUME of the individual
o Normal RBC with normal Hgb: the size of
red cells in a given blood sample
the central palor should be 1/3 the diameter
‫־‬ Hematocrit packed cell volume test
of the RBC
o Hypochromic: If the central palor is greater - For the estimation of the average SIZE of
than 1/3, it means that the rbc lacks hgb. the RBC
o We can describe the shape and the hgb - Size and volume are directly proportional.
content of the rbc through the examination Since it is directly proportional, the value of
of smear, but not always accurate. the MCV is also used to estimate the average
size of RBC.
 MEAN CORPUSCULAR VOLUME (MCV) - Not dependable when RBC vary markedly in
- Used in classifying types of Anemia. size. The relationship of value and size
- With the computation of MCV alone, we can becomes unreliable and undepandable.
already morphologically classify anemia as to  Dimorphism: dual morphology
whether the anemia is Normocytic, Microcytic, - Ex. Mixture of normal and macrocytic cells
or Macrocytic.  Reticulocytosis
- The single best index aid in morphologic - Is a condition where reticulocyte volume is
classification of anemia. higher, are immature cells and they are larger
than the normal cells and we can see again
the dimorphism.
REFERENCE RANGE:
80-100 fL (1fL=10-15L)-normocytic
<80 fL – Microcytic
>100 fL- Macrocytic
 MEAN CORPUSCULAR HEMOGLOBIN (MCH)
- It is not very well important.
 MEAN CORPUSCULAR HGB
CONCENTRATION (MCHC)
 REFERENCE RANGE:
32-36 g/dL or 320-360 g/L
 32-36 g/dL- Normochromic
 <32 g/dL – Hypochromic
 >36 g/dL- Spherocytic

MEAN CORPUSCULAR HEMOGLOBIN (MCH)

- The average weight of Hgb per RBC
- Always correlates with the MCV and MCHC
- Directly proportional to the size of the RBC & the
concentration of hgb in the cell.
- Does not always give us an accurate information, MCV & MCHC
because it always follows the value of the MCV. There are 3 general classifications of anemia
- Whenever the value of MCV and MCHC is low then according to morphology:
the value of MCH is also low.
- We do not interpret the MCH. 1. Normocytic & Normochromic RBC Anemia
- Normal size and hemoglobin
- Main Cause: lack of production
- Example: Aplastic Anemia
2. Microcytic & Hypochromic anemia
- Example: Iron Deficiency Anemia
- A patient with IDA does not always
show microcytic hypochromic red

27-33 pg (1pg= 𝟏𝟎−𝟏𝟐)

REFERENCE RANGE: blood cells. There is a transition
wherein when we develop anemia, we
don’t immediately have the IDA.
<27 pg (microcytic) Rather, we start to develop it. As the
>33 pg (macrocytic)
IDA develops further, the cells change
NOTE: from normocytic, normochromic into
microcytic hypochromic
“Normocytic but Hypochromic”
It is possible that we can see an abnormal cell with a 3. Macrocytic & Normochromic Anemias
normal size but it lacks hgb. - Example: B12 deficiency anemia &
folate deficiency anemia, it
MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION describes as megaloblastic.
(MCHC) NOTE:
- Refers to average Hgb concentration of red Some books, there are 4 general classification wherein
cells in a given volume of blood. the 4th one is, the cells are normocytic but
hypochromic.
MCV & MCHC