CHAPTER IV: ERYTHROCYTE (b) Confirmatory Test: [“culling” = removal of the whole

METABOLISM Quantitative PK assay RBC] -> HEMOLYTIC ANEMIA.)

Embden Meyer Pathway Hexose Monophosphate Shunt Methemoglobin Reductase Pathway
 Non-oxidative anaerobic  Aka Phosphogluconate  Maintains the iron in the Hb in
pathway Pathway or Pentose Phosphate its reduced/ferrous state (Fe+
 Use 2 ATP Pathway +)
 Product: 4 ATP and 2 NADH  Aerobic or Oxidative glycolysis  Heme iron is constantly
molecules  Functionally dependent on G-6- exposed to oxygen and
 Requires glucose to produce PD/Glucose-6-Phosphate peroxide that it oxidizes heme
ATP Dehydrogenase iron from the ferrous (Fe++) to
 ATP is used by RBC in 3 ways:  G-6-PD Deficiency the ferric (Fe+++) state, the
1. Maintenance of RBC shape - Most common red cell affected Hgb is called
and deformability enzymopathy associated “Methemoglobin”.
2. Gives Energy for the active with hemolysis  Methemoglobin
transport cations - Abnormal cells in PBS: Heinz - Oxidized Hgb
3. Helps in modulating the Bodies and Bite Cell - Heme is in ferric state
amount of 2,3 DPG/BPG - Tests: - Cannot bind O2
(diphosphoglycerate/ (a) Recommended Screen  NADPH slowly converts Ferric
biphosphoglycerate) Test: G6PD Fluorescent Spot State iron to Ferrous form
 Pyruvate Kinase (PK) Deficiency Test  Methemoglobin reductase
- most common deficiency in (b) Confirmatory Test: (cytochrome b5 reductase)
EMP Quantitative G6PD assay renders the NADPH conversion
- Abnormal cells in PBS: Burr more efficient.
(G6PD Deficiency -> Hemoglobin
Cell (Oxidized, Denatured, Precipitated) ->
- Tests: Heinz Bodies -> Spleen (“pitting” =
(a) Screening Test: PK removal of a portion of RBC) -> Bite
fluorescent spot test Cells -> Spleen (Splenic Macrophages)
 Miller Disk = Miller disk placed
in the ocular of microscope
Rapaport-Luebering Shunt/Pathway Reticulocyte
* Large square/Square A = use
 For the production of 2,3 DPG  Young RBC’s with residual RNA
for counting reticulocyte
(binds to Hb and decreases the  Non-nucleated cell which
O2 affinity of Hb). contains ≥2 blue-stained, *Small Square/Square B = use
 2,3-DPG competes with oxygen granulofilamentous materials for counting RBC (1/9 of square
in the oxygen-binding site of after staining (supravital stains) A)
Hgb. When 2,3-DPG binds
Reticulocyte Count  Supravital Stains
heme, oxygen is released,
- Brilliant Cresyl Blue (BCB)
which enhances delivery of O2  Permits effective assessment of
Major components:
to the tissues. RBC production by the bone
(1) Sodium Citrate =
 Two variables affecting the marrow.
prevents coagulation
degree of association and  Increase Reticulocyte Count:
(2) Sodium Fluoride =
dissociation between oxygen Considered as the first sign of
provides isotonicity
and Hb: accelerated erythropoiesis and
- New Methylene Blue (NMB)
1. Partial Pressure of Oxygen observed in hemolytic anemias,
(more preferred than BCB)
2. Affinity of Hb for Oxygen individuals with iron deficiency
Major components:
(ph, partian pressure of C02, anemia receiving iron therapy,
(1) Sodium Oxalate =
concentration of 2,3-DPG, thalassemia, sideroblastic
prevents coagulation
temperature, presence of anemia, and in acute and
(2) Sodium Chloride =
other Hgb species that ae non chronic blood loss.
provides isotonicity
functional)  Decreased Reticulocyte Count:
Observed in aplastic anemia Important Reminders:
Bohr Effect= relationship and in conditions which the  NMB is chemically different
between blood pH and O2 bone marrow is not producing from methylene blue.
affinity of Hgb RBCs.
 Materials:  Brilliant Cresyl Blue also stains
reticulocytes but shows too
 much unpredictability in therefor settle on top of red d) Reticulocytes
staining for routine use. blood cells in the mixture. should be also
counted as RBC’s.
 If a patient is very anemic or
e) Continue counting
polycythemic, the proportion
 Methods of Counting until 1000 RBC’s
of dye to blood should be
Reticulocytes have been
adjusted accordingly. For best
1. Routine Light Microscope observed.
results, a larger proportion of
Method
blood should be added to the Calculations:
a) Use the oil
stain when the patient’s
immersion Retics (%) = No. of retics
hematocrit is low. Add a
objective observed over 1,000 RBC’s
smaller amount of blood to the
b) Select an area observed, multiple to 100
stain when the patient has a
where RBC’s are
very high hematocrit.
close but not
 The time allowed for staining overlapping and 2.Calibrated Miller Disk
of the reticulocytes is “not reticulocytes Method
critical”. It should not however, appear to be well- - Count 500 RBCs in square B.
be less than 10 minutes. stained. - A reticulocyte in square B is
c) Count the counted as both an
 Increased blood glucose or the
reticulocytes and erythrocyte and
use of heparin as anticoagulant
RBC’s in each field reticulocyte.
may cause the reticulocytes to
using the same - At this point, theoretically,
show pale staining.
patter normally the number of retics in 4500
 The blood and stain should be used for RBCs have been counted.
mixed well prior to making performing a
smears. The reticulocytes have leukocyte Calculations:
a lower specific gravity than differential count. Retics (%) = Total Retics in Square A
mature erythrocytes and over Total RBCs in square B x 9, x 100
 3. Sysmex R-1000 Hct of 0.45L/L to allow  Counting Chambers
correction for the degree of - According to type:
- Automated reticulocyte
patient’s anemia.  Open Type
analyser
(Examples: Spencer,
- Uses flow cytometry to Burker, Levy, Levy-
determine the reticulocyte Hausser)
count (% and absolute number)  Closed Type (Ex.:
Absolute Reticulocyte Count Thoma-Zeiss)
 Addis
 Actual number of reticulocytes  Exton
Reticulocyte Production Index (RPI)
in 1 litre of whole blood.  Petroff
 Aka Shift Correction - According to ruling:
 provides a further refinement  Thoma
of the CRC.  Tuerk
 Reference Range: 25 to 75 x
 A general indicator of the rate  Fuchs-Rosenthal =
109/L
of erythrocyte production used for absolute
Corrected Reticulocyte Count (CRC) increase above normal in eosinophil count.
anemias.  Neubauer
 Also referred to as Reticulocyte
Index or Hematocrit  Improved Neubauer
Correction.  Bass-Jones
 Percentage of reticulocytes Improved Neubauer Counting
 RPI >3 = adequate BM
may appear increased because Chamber
response
of early reticulocytes release
RPI <2 = inadequate BM  Most commonly used counting
into the circulation because of
response chamber/hemocytometer.
a decrease in the number of
mature cells in circulation.  Chamber is 3mm by 3mm
(divided into 9 square
 The CRC corrects the observed Microscopic Hemocytometry (RBC millimeters)
reticulocyte count to a normal Count)
  The 4 corner(large)  RBC Diluting Fluid = ideal is (1) Glacial Acetic Acid,
squares – subdivided isotonic 2% (v/v)
into 16 squares [ for  Formol-citrate/Dacie’s (2) Hydrochloric Acid, 1%
manual WBC count] fluid (v/v)
 The middle(central)  Others: (3) Turk’s Solution =
square – subdivided into (1) Hayem’s enhances leukocyte
25 squares [for manual (2) Gower’s nuclear definition.
RBC count] (3) Toisson’s  Platelet Diluting Fluid = must
 This hemocytometer has (4) Bethel’s preserve platelet integrity
two identical sides and (5) Formol Citrate/ while inhibiting their
both sides are counted. Dacie’s fluid aggregation
 Depth = 0.1 mm (6) NSS – ideal to use in
case of excessive
Pipets
Rouleaux formation and
 RBC Thoma Pipet autoagglutintion Hemoglobin
 Markings: 0.5,1,101 (7) 3.8% Sodium Citrate
 Bead: Red  WBC Diluting Fluid = ideal is  Single most common complex
 Bulb Volume: 100 hypotonic organic molecule in
 WBC Thoma Pipet  Will hemolyzed RBC vertebrates.
 Markings: 0.5,1,11 except metarubricyte  1 gram of Hgb can carry 1.34
 Bead: White/Colorless (nucleated red cells) mL of O2 and a constant 3.47
 Bulb Volume: 10  Diluents for counting mg of Iron.
only leukocytes must be  Complete adult Hgb molecule
capable of lysing is composed of four different
Diluting Fluids erythrocytes without constituents:
destroying leukocytes A protein component (globin)
 Used mainly to disperse blood
 One of the following composed of two sets of two
cells to facilitate counting of
diluents may be used: different polypeptide chains.
cells.
  Four molecules of nitrogenous - Normal daily diet 1. Ferritin – Soluble form
protopotrphyrin IX. contains about 15mg of storage iron
 Four iron atoms in the ferrous iron (only 1-2 mg are (measured in blood).
form (Fe++) that combine with absorbed) 2. Hemosiderin –
protoporphyrin IX to create the - Duodenum and upper insoluble form of stored
four heme molecules. jejunum (sites of iron.
 One 2,3-DPG molecule as a maximal absorption of  EXCRETION
sometime resident in the iron) - Average daily loss of
center of the Hgb unit.  TRANSPORTATION: iron: 1-2mg (through
 Primary functions of Hgb: - Transferrin – transport sweat, urine and feces)
(1) To deliver O2 to the tissues protein for iron; Can be  Hepcidin:liver
and organ. measured in plasma. But - Master regulatory
(2) To carry the waste product not as clinically hormone of systemic
CO2 away to the lungs. significant as TIBC (Total iron metabolism
Iron Binding Capacity –
an indirect measure of
transferrin Increase iron accumulation (blood
Heme Synthesis
concentration). and tissues):
 Also known as - Iron uses transferrin for  Hemosiderosis - characterized
Ferroprotoporphyrin IX transportation bcs it by LITTLE parenchymal cell
 Belongs to class of pigments can’t travel by itself injury.
known as porphyrins  UTILIZATION  Hemochromatosis - iron
 Site of heme synthesis: - Mitochondrial iron: accumulation and consequent
Mitochondrion incorporated rapidly into injury to the tissues and
protoporphyrin to form organs.
Iron Metabolism
HEME. o Bronze Diabetes a.k.a.
 Source of iron: food  STORAGE “Hereditary
 ABSORPTION: - Two forms:
 Hemochromatosis” circulating polychromatic  Used to measure haemoglobin
[absorbed excess iron]. erythrocyte, but not in the concentration
mature erythrocyte.  Cyanmethemoglobin (HiCN)
Globin Synthesis
 Transcription of the globin Method
 Site: ribosomes in the genes to messenger ribonucleic  Reference method for
normoblast cytoplasm acid (mRNA) occurs in the hemoglobinometry (Best
 Globin chains are assembled nucleus. method)
from two pairs of polypeptide  Translation of mRNA to the  Uses Drabkin’s Reagent
chain. globin polypeptide chain occurs o Major
 Chromosome 16 = dictates the on ribosomes in the cytoplasm. components:
production of ALPHA and ZETA o Although transcription of (1) Potassium
globulins/globin chains. the a-globin genes ferricyanide –
o Alpha and Zeta Globin produces more mRNA converts the Hgb
genes are on the short than the b-globin gene, in the sample into
arm of chromosome 16. there is less efficient methemoglobin.
translation of the a- (2) Potassium
globin mRNA.2 cyanide – provides
 Chromosome 11 = dictates the Therefore, the a and b the cyanide.
production of BETA, EPSILON, chains are produced in  Cyanmethemoglobin is
DELTA, & GAMMA globin approximately equal measured at 540 nm.
chains amounts. After  All types of Hgb may be
o Epsilon, Gamma, Delta, translation is complete, measured except
and Beta Globin genes the chains are released Sulfhemoglobin (Shb)
are on the short arm of from the ribosomes in  Turbidity in the mixture will
chromosome 11. the cytoplasm cause falsely elevated values.
 The production of globin chains  Some of the causes of
takes place in erythroid specimen turbidity:
precursors from the Hemoglobinometry
pronormoblast through the
 - Lipemia -  Slowest = HbC, HbE,  Not bound to oxygen
accumulation of HbA2, and Hb O-Arab  Found in venous blood
lipoprotein particles
(dark red)
- Extremely ↑WBC Note: HbS, HbD and HbG
 Conformation of Hb in
count (>20 x 109/L) migrate to the same area
deoxygenated blood (T
- Extremely ↑ at cellulose acetate
State – Tense State)
Platelet count electrophoresis.
 Carboxyhemoglobin (HBCO)
(>900x109/L)  Citrate Agar (pH 6.0-6.2)  Hb with Fe++
- Presence of HbS  Use to differentiate HbS  Bound to Carbon
and HbC from HbD and HbG Monoxide (CO)
- Easily precipitated  Complementary test.  CO affinity for Hgb is 240
globulins (such as
times stronger than O2
seen in
 Color of blood and skin
Waldenstroms
in HbCO poisoning:
macroglobulinemi
Cherry Red
a and Multiple
 Considered as a silent
Myeloma)
killer (odourless and
Hemoglobin Electrophoresis Types of Hgb (Hgb derivatives) colorless gas)
(test that measures the different types of  Sources: exhaust of
 Oxyhemoglobin (HBO2)
hemoglobin in the blood) automobiles, tobacco
 Hb with Fe++
smoke, from industrial
 Hgb has a net negative charge  Bound to oxygen
pollutants such as coal,
(alkaline pH).  Found in Arterial Blood
gas, and charcoal
 Migrate towards the (+) pole (bright red)
burning.
[anode]; if (-) pole termed  Conformation of hgb in
- Smokers have
cathode. oxygenated blood (R
higher Hct and
 Cellulose acetate (pH 8.4-8.6) state – relaxed state)
have polycythemia
 Fastest = HbH, in normal  Deoxygenated Hemoglobin
to compensate for
individual fastest is HbA1  Hb with
hypoxia.
 - Exposure to CO  Not bound to oxygen phenacetin,
may be (chocolate brown). nirites, and
coincidental  HI cannot carry O2 phenylhydrazine
(smoke from because the oxidized - Exposure to sulfur
house fires), ferric iron cannot bind it. chemicals in
accidental or  <25% of Hgb: industrial or
intentional asymptomatic environmental
(suicidal).  >30% of Hgb: Cyanosis settings
 Levels of up to 40% of (bluish discoloration of - Bacteremia (C.
total Hgb may cause skin and mucous perfringens)
coma, seizure, membranes); Hypoxia - Enterogenous
hypotension, cardiac (dyspnea, headache, cyanosis
arrhythmias, pulmonary vertigo, change in  Color of Blood: Mauve-
edema and death. mental status). Lavender
 Increase in Hi is called  Irreversible form of Hgb
 Manifestations: methemoglobinemia/tox derivative.
- Sleepiness ic methemoglobinemia.  In-vitro, sulfhemoglobin
- Hypoxia - a state in  It can be acquired or forms when hydrogen
which oxygen is not hereditary. sulfide is added to Hb,
available in sufficient
amounts at the tissue
 Sulfhemoglobin (SHB) thus the name
level to maintain  Mixture of oxidize, Sulfhemoglobin.
adequate homeostasis partially denatured  In-vivo, sulfhemoglobin
 Methemoglobin (HI) forms of Hb. forms in the occasional
 HiCN =  Some of the causes of patient as a result of Hb
Cyanmethemoglobin formation of SHb: oxidation by certain
 A.k.a. - Prolonged drugs and chemicals.
“Ferrihemoglobin” / constipation
Common Test for RBCs
“Hemiglobin”. - Exposure to
 Hb with Fe+++. sulfonilamides, Hematocrit
“Packed cell Volume” (2) w/ Red Band: with 8. Spin for 5 minutes at 10,00
anticoagulant (Heparin); use RPM.
“hematocrit” – actually pertains to
for collection on non-AC blood 9. Note: RPM must be checked
the instrument used to determine
(direct blood from finger). periodically with a tachometer.
packed cell volume (PCV).
10.After centrifugation, read the
1. Summarized procedure (using
After centrifugation of an Hct (the buffy coat layer should
non anticoagulated whole
anticoagulated whole blood not be included).
blood)
specimen, the red blood cells along
2. Perform skin puncture. Reminders:
with other formed elements (WBCs
3. Wipe of the first drop of blood.
and Platelets) will settle at the Trapped plasma may cause the Hct to
4. Fill two heparinized capillary
bottom of the tube. be falsely increase by as much as 0.02
tubes two-thirds with blood.
L/L.
The volume of the RBCs that have 5. Note: Air bubbles denote poor
settled is called packed cell volume skills but do not actually affect When determined by fully automated
otherwise known as hematocrit. the test results. methods, the Hct may be 0.01 to 0.03
6. In a vertical position, carefully L/L lower than microhematocrit
Micro Hematocrit tube:
seal the dry end of the method because it is electronically
o Approx. = 75 mm long heparinized capillary tubes calculated and therefor is unaffected
o Inner bore = 1.2 mm with the sealing clay and the by trapped plasma. The difference in
o Can hold 0.05 mL of blood. plug should be 4-6 mm long. the Hct results is usually significant
o Plug = 4 to 6 mm long (seal the 7. Place the two heparinized unless there is more severe case of
capillary tubes at the end of capillary tubes in the radial poikilocytosis and anisocytosis.
the tube with colored ring) grooves of the micro centrifuge Automated hematocrit – a calculated
o 2 Types: with their heads exactly value from RBC and MCV.
(1) w/ Blue Band: plain tube opposite of each other. The
sealed end should be away Trapped plasma – small amount of
[no anticoagulant]; use for plasma that remains in the
collection anticoagulated blood from the center of the
centrifuge. erythrocyte portion of the spun Hct
(EDTA blood) even when proper centrifugation is
used.
More trapped plasma in the ff.:
Hemolysis
o Sickle Cell Anemia
Introduction of Interstitial Fluid
o Hypochromic Anemia
Increase Anticoagulant
o Spherocytosis
Concentration
o Macrocytosis
Improper sealing of tube
o Thalassemia
Certain abnormal RBC shapes (e.g.,
spherocyte and sickle cell) inhibit
complete packing.
Immediately after a blood loss, Hct is
not a reliable estimate of the degree
of anemia because plasma volume is
replaced faster than RBC volume,
therefore causing a temporarily lower
Hct.
Potential causes of errors:
o May cause falsely increase
HCT:
Dehydration
Hemoconcentration
Insufficient Centrifugation
Buffy Coat Inclusion
o May cause Falsely Decrease
Hct: