CLINPATHLEC| RED BLOOD CELLS

Tutor: DR. RACELIS | Lecture Date: MARCH 10 2021 | 2 ND SEM

 Smoking
Chapter Outline  Genetic causes (altered oxygen sensing,
I. Complete Blood Count abnormality in hemoglobin oxygen release)
II. RBC Parameters  Polycythemia vera
A. Hemoglobin(Hb)
B. Hematocrit(Hct)
C. Reticulocyte Count II.RBC PARAMETERS
D. ESR
E. Osmotic Fragility Test A. Hemoglobin (Hb)
F. RBC Indices B. Hematocrit (Hct)
G. RBC Morphology C. Reticulocyte Count
- Abnormalities in Colors and Size
- Abnormalities in shape D. ESR
- Abnormal Inclusions E. Osmotic Fragility Test
- Abnormalities in Cell Size F. RBC Indices
H. Megaloblastic Anemia
I. Pernicious Anemia G. RBC morphology

Note the bold especially the underlined.

A. HEMOGLOBIN
Determination of concentration of hemoglobin in
I. COMPLETE BLOOD COUNT
whole blood
 A complete blood count (CBC) is an important and Uses:
readily available investigation that focuses on Red  Anemia DIAGNOSIS and SCREENING — most
Blood Cells, White Blood Cells and Platelets and common use
their various parameter.  Anemia severity (if you will require transfusions
 It can help to serve as a screening test for many or not)
disorders and as prognostic or follow up tool.  Anemia treatment response monitoring
 Expressed in g/dL (will be used for our exams) or
Components g/L
WBC RDW Normal Values
RBC Platelers  Male: 14-16 g/dL
Hemoglobin (Hgb) Neutrophils  Female: 12-14 g/dL
Hematrocrit (Hct) Lmyphocytes  Newborn: 16-18 g/dL
MCV Monocytes
MCH Basophils A. HEMOGLOBIN
MCHC Immature Granulocytes Differs according to age, sex, location and race
Reticulocyte Count  Females have lower values due to regular
menstruation
RBC Normal Values:
 Newborns have high values due to lower oxygen
Males 4.7 to 6.1 million cells per microliter
supply in utero
Females 4.2 to 5.4 million cells per microliter
 In high altitudes, Hb tends to be of higher in
Low RBC Count value due to low oxygen concentration
 Known as Anemia  Race is also a factor, such as Caucasians having a
 Acute or Chronic bleeding higher concentration than Filipinos
 RBC destruction (e.g. hemolytic anemia, etc.)
 Nutritional deficiency (e.g. iron deficiency, Method of hemoglobin determination: Colorimetric
vitamin B12 or folate deficiency)  Add potassium ferricyanide to the blood
 Bone marrow disorders or damage producing a brown color, hence,
 Chronic inflammatory disease ↑ color (intensely brown) = ↑ Hemoglobin
 Kidney failure  (Hb)Fe + KCN -> K3Fe(CN) [K3Fe(CN) gives the
brown color)
High RBC count
 Known as polycythemia
 Dehydration
 Pulmonary disease
 Kidney or other tumor that produces excess
erythropoietin

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 4 — (S= Sickle cell anemia — Sickle cell anemia is
diagnosed when there is purely HbS, as compared to
sickle cell trait where there is A)
6 — sickle cell trait (still has another Hb other than S)
7 — Hb SC; milder form of sickling
8 — sickle cell trait

B. CHROMATOGRAPHY
Another way to distinguish hemiglobinopathies

The only time HbA2 is increased is that you have a

hemoglobinopathy such as thalassemia where you
don't have beta. If you don't have beta, you have an
increase in your non-B portion of hemoglobin, so you
can increase either HbA2 (which is alpha delta) or HbF
(which is alpha gamma)

Recall that when a child is born, 60% of the

hemoglobin is HbF. Therefore, any hemoglobinopathy
related to HbA is not reflected at the birth of the
baby. Symptoms usually occur 6 months after, when
HbF becomes decreased or absent in the blood.

HEMOGLOBIN TESTS
A. HEMOGLOBIN ELECTROPHORESIS
 Makes use of an electric current to separate
normal and abnormal types of hemoglobin in the
B. HEMATOCRIT
blood which will aid in diagnosing
hemoglobinopathies  Ratio of the volume of erythrocytes to that of
 The presence of significant levels of abnormal the whole blood (not plasma)
hemoglobin may indicate Hemoglobin C (HbC)  Measures the volume of packed RBC after the
disease, rare hemoglobinopathy, sickle cell centrifugation of blood:
anemia or thalassemia HCT= Height of red cell L1
 The best way to know what type of hemoglobin Height of whole blood L 2
you have is to do electrophoresis  Ratio of RBC volume to whole blood
 Corresponding mark on A, F, S or C indicates the  Gives a rough, indirect estimate of Hemoglobin
type of Hb  Hematocrit is 3x the Hemoglobin
 Not always true
 Normal Values
 Male: 40-54%
 Female: 36-47%
 Generally, normal adult hematocrit values range
from about 36% to 45%
 Normal values for females are generally slightly
lower than those for male.

Interpretation
1 and 5 — control (has all the red marks on A, F, S and
C); all the different types of Hgb is positive(not a
patient)
2 — purely A, therefore normal individual
3 — F is more intensely colored than A (seen in
newborn)

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CLINICAL CONSIDERATIONS  Leukemia and lymphoma due to bone marrow
 Since hematocrit is a reflection of hemoglobin, replacement of the tumor cell
the values will also depend on sex, location, and  Chronic blood loss — most common microcytic
race anemia in elderly
 Hematocrit may NOT be reliable in the following  Excessive fluid administration (overhydration)
conditions:  Cardiac failure (water retention)
a. Immediately after blood loss  Anemia
Evaluate hematocrit after blood loss, because you
both lose serum and RBC. If you do Hct, it will Factors affecting hematocrit:
appear normal, such as in obstetric  Volume of RBC (↑ volume = ↑ Hct) ↓
complications. Hct only goes down when the  Volume of plasma (↑ volume = ↓ Hct)
interstitial fluid goes into the intravascular space,  Shape of RBC (smaller = ↓ Hct)
and thus Hct will decrease. In the case of
immediate blood loss, however, Hgb would be Sources of error (TTOIL)
better. A few hours after, Hct may reflect true  Time and speed of centrifugation (overcentrifuge
values in the patient. = ↓ Hct)
b. Immediately after transfusion  ↑ Trapped plasma = ↑ Hct (Sickle Cell = trapped
After transfusion, check Hct AFTER 24 HOURS. plasma ↑)
Otherwise, you will get the Hct value of the blood  Over coagulation = ↓ Hct
being transfused. A value higher than usual may  Incomplete sealing of Hct tube = ↓ Hct (due to
result. leakage)
c. Abnormal size and shape of red cells  Long-standing = ↑ Hct (hence, READ THE TUBE
 It is necessary for red cells to pack properly in RIGHT AWAY, because the RBCs level off and the
order to get the correct Hct value reading becomes higher than it should be.
 If the red cells cannot pack properly, it is
called trapped plasma such as in sickle cell The standard method for taking a hematocrit reading
anemia. Due to this, despite the patient involves lancing a finger and then pulling a blood
having anemia, Hct may be falsely high. Hgb sample into a capillary tube. The tube is then spun in a
then, is a better parameter of measure. centrifuge. The amount of red blood cells per unit
volume is defined as the hematocrit level.
CAUSES OF ABNORMAL HEMATOCRIT LEVELS
Table 2. Abnormal hematocrit levels
DECREASED INCREASED
 Anemia  Hemoconcentration
 Nephrotic syndrome  Polycythemia
 Leukemia, lymphoma Figure 5. Hematocrit determination
 Physiologic: pregnancy
 Chronic blood loss C. RETICULOCYTE COUNT
 Recovery phase  Used as an index of production of RBC by the
following acute blood bone marrow in response to anemia
loss  Reticulocytes are immature non-nucleated red
 Excessive fluid cells that contain ribonucleic acid (RNA) and
administration continue to synthesize hemoglobin after loss of
the nucleus
Increased Hct  Because reticulocytes are immature red cells
 Hemoconcentration (decreased serum such as in that lose their RNA a day or so after reaching the
vomiting and diarrhea) blood from the marrow, a reticulocyte count
 Polycythemia vera (increased RBC by tumor) or provides an estimate of the rate of red cell
physiologic increase (high altitudes) production
 An absolute reticulocyte count or reticulocyte
Decreased Hct production index is more helpful than the
 Pregnancy (physiologic — fetus needs RBC) percentage
 Initial recovery phase after acute blood loss  ↓ reticulocyte count in the presence of anemia
 Nephrotic syndrome (loss of erythropoietin + – bone marrow is not functioning = POOR
management includes increase in fluid intake = prognosis
decrease in Hct)

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  ↑ reticulocyte count in patients with anemia to see the reticulum which corresponds to the
indicates that the bone marrow is functioning disintegration of your nucleus.
well

 Indications
o Basic diagnostic workup in all types of
anemia
o Baseline and therapeutic monitoring in Table 3. Reticulocyte count
nutritional anemia (IDA or B12 Deficiency, or INCREASED RETICULOCYTE COUNT
anything lacking in the diet) HaT PLEASE
 Reticulocyte count increased ONLY upon  Hemolytic anemia
initiation of treatment  Thalassemia
o Bone marrow function monitoring in patients  Physiologic (decreased amount of oxygen)
under treatment – reticulocyte count goes  Lead poisoning
down in chemotherapy  Erythroblastosis fetalis
 Normal value: 0.5% - 1.5%  Acute bleeding
 Sideroblastic anemia
¿ of reticulocytes Effect of treatment of nutritional anemia (B12
reticulocyte count= x 100
1000 mature RBC deficiency, folic acid deficiency and iron
deficiencies) – expect an increase in retic count in
pt .hematocrit nutritional anemia with therapy
Reticulocyte index ( RI )=reticulocyte count x DECREASED RETICULOCYTE COUNT
45
 Aplastic anemia
o Study how to compute.  Bone marrow failure
o Correction factor
o When you have anemia and you use the D. ERYTHROCYTE SEDIMENTATION RATE
reticulocyte count as your basis, there is a  Rate at which RBC FALLS in a specified time (no
tendency for it to give a falsely increased centrifugation)
result. Why? Because you’re counting per  Methods
thousand (recall retic count formula). But o Westergren method
since the patient has anemia, (↓ RBCs), o Wintrobe method
instead of counting the 1000 RBCs needed
in one field, you end up counting several  When well mixed venous blood is placed in a
fields (just to complete the 1000 RBCs) vertical tube, erythrocytes will tend to fall
hence it gives a falsely high result. In cases toward the bottom. The length of fall of the top
like anemia, it is necessary to correct the of the column of erythrocytes in a given interval
reticulocyte count, hence the reticulocyte of time is the ESR
index, which is more or less the normal
hematocrit of the anemic patient.
 Normal Value
o Will usually result in a lower value than
o Female: 0—20 mm/hr
what is actual
o Male: 0—15 mm/hr
o As clinicians, you should correct this once
o Children: 0—10 mm/hr
you receive your results
o The more RBCs (in newborn), the slower the
o Reticulocyte is increased in all forms of
fall (lower value). The less RBCs (in females
anemia EXCEPT for anaplastic and bone
and anemic patients), the faster the fall
marrow failure
(higher value)

(left, A) in Wright’s Stain, reticulocyte appears

polychromatophilic (arrow) which is difficult for the Figure 7. Type of tube used in ESR (either
medtech and will result in an error. (right, B) In Westergren or Wintrobe)
methylene blue stain (supravital stain) you are able

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  Vibration or jarring - speeds up the rate of RBCs
falling o Temperature - temp, ↑ ESR
 Too short/narrow tube - ↓ ESR

E. OSMOTIC FRAGILITY TEST

 Test to deretmine susceptibility of the RBC to

rupture
REMEMBER:  Shape of RBC is the main determining factor of
↑ESR = FASTER FALL, ↓ESR = SLOWER FALL the test
 Use is for the diagnosis of spherocytosis and not
Factors affecting ESR for sickle cell
 RBC  INCREASED OFT (0.7) in spherocytosis
o Size: big size , ESR  DECREASED OFT in SCD/hypochromic
o Agglutination: ESR
o Changes in shape, sickling; ↓ ESR (umbrella-
shaped RBCs would have a harder time falling
o Number of cell: ↑ cells, ↓ ESR
↓ cells, ↑ ESR

 Plasma charges
o Rouleau & agglutination will ↑ ESR Figure 9. Osmotic Fragility Test: Osmotic fragility test
 Rouleaux formation also has a includes a series of test tubes with decreasing values
decreased surface area/volume ratio (it of NSS (0.9-0.0%). Put 1 drop of blood on all test
becomes "heavier") and accelerate the tubes. Normally, blood will rupture at 0.5, 0.4.
fall of RBC, hence ESR increases. Hence, Spherocytes are small cells that are rounded;
cells that hinder with rouleaux (decreased surface/volume ratio) rather than discoid
formation (such as sickle cell or in shape therefore you will need only a small amount
spherocytosis) usually have lower ESR. of fluid (higher concentration of NSS) for that red cell
to rupture (limited capacity to expand in hypotonic
o Presence of fibrinogen, α-1/α-2 globulin will solutions) compared to a sickle cell or a hypochromic
↑ESR cell wherein lysis occurs later at a lower
concentration. Madaling mag rupture kasi hindi
Conditions with ELEVATED ESR (FASTER FALL) discoid - parang perfect circle at hindi flexible yung
 Inflammation, Infection wall niya
o Presence of immunoglobulins which will
remove the positive charge of red cell thus F. RED CELL INDICES
causing agglutination of red cells  Used to demonstrate RBC characteristics
o Rheumatic fever, TB, acute hepatitis, PID,  Used for parameters of quality control in
Appenditis hematology
 Autoimmune disorders
o RA, temporal arteritis, SLE A. MEAN CORPUSCULAR VOLUME (MCV)
 Tumors  Normal value: 80-98 fl (size)
o Plasma cell dyscracia, Hodgkins lymphoma o < 80: microcytic
 Anemia o In between: normocytic
o macrocytic
Sources of Error  Indicates average volume of Hb in each RBC
 ↑ conc. of anticoagulant - ↓ ESR  Hct / RBC count x 10
 Prolonged standing > 60 mins - ↑ ESR
 Tilting - ↑ ESR (RBC does not fall the whole B. MEAN CORPUSCULAR HB CONCENTRATION
distance [top to bottom], thus it falls to the (MCHC)
sides of the tube [shorter distance) and then  Normal value: 32-36% (color)
slides downward, making it faster o < 32: hypochromic
 Bubbles / fibrin - interferes with the packing of o In between: normochromic
RBCs o > 36: hyperchromic

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 You should not get values above 40 because  Elevated bilirubin
hemoglobin and hematocrit are a related value,  Elevated hemosiderin
therefore there should only be a little change.  Examples: thalassemia, spherocytosis
 Indicate average concentration of Hb
 Hb / Hct x 100
Note: Your MCV is micro/macrocytic, while your
MCHC is hypo/hyperchromic.

C. MEAN CORPUSCULAR HB (MCH)

 Normal value: 25-34 pg
 Indicates average wt./amt of Hb
 Hb / RBC count x 10 G. RBC Morphology
MORPHOLOGIC CLASSIFICATION OF ANEMIA
A. ABNORMALITIES IN COLOR & SIZE
 Helps in eliminating the diagnosis for anemia
 Normochromic normocytic
o N MCHC, N MCV, N MCH
o (N = normal)
 Hypochromic, microcytic
o ↓ MCHC, ↑ MCV, ↓ MCH
o IDA, thalassemia, sideroblastic anemia (not
common in the Philippines) Figure 13. Normal RBC. 1/3 central pallor and size
 Macrocytic anemia similar to smallest lymphocyte in the smear.
o ↑ MCV, ↑ MCH, N MCHC
o B12 & folic acid deficiency

LABORATORY EVALUATION OF HEMOLYTIC ANEMIA

(HA)
 Determine if intravascular or extravascular
 Increased RBC production Figure 14. Microcytic, hypochromic RBCs. Central
BM → increased erythroblasts: In HA, there is an pallor is more than 1/3 and RBC is much smaller than
increase in your Erythroid: Myeloid ratio (normal the lymphocyte. (70% or 80% pallor)
EM ratio is 1:4 or 1:5 with the myeloid being
more than the erythroid). In HA, you expect an
elevation of the EM ratio to 1:1 or 1:2

INTRAVASCULAR HEMOLYSIS
 Presence of free Hb seen as Hemoglobinemia,
Hemoglobinuria (tea-colored urine) WITHOUT
Figure 15. Macrocytic Hyperchromic RBCs. Larger than
jaundice
lymphocytes and <1/3 central pallor or none at all.
 Methemoglobinemia and Methalbuminemia
 ↓ serum haptoglobulin
B. ABNORMALITIES IN SHAPE
 Example: malaria, G6PD, Erytroblastosis fetalis
1. Spherocytosis

Figure 16. Spherocytes.

Figure 12. Hemoglobinuria Important Conditions in newborns:
Congenital Spherocytosis / Hereditary Spherocytosis
EXTRAVASCULAR HEMOLYSIS  No jaundice; Symptomatic when the patient
 Occurs in the spleen hence fed cells stay in the turns a little older
spleen and is destroyed there → cause; jaundice  Negative Coombs test - not antibody induced
WITHOUT tea-colored urine

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 Abnormality of membrane protein Spectrin → seen after exposure to strong reducing agents or ↓
sphere instead of discoid shape → inflexible in 02 pressure
capillaries especially in the spleen → hemolysis
 (sphere shape = not flexible) a. Sickle cell anemia (HbSS) — HOMOzygous
recessive; present with the disease
ABO Hemolytic Disease of the Newborn b. Sickle cell trait (HbAS) — HETEROzygous; usually
 Very symptomatic asymptomatic/carriers only but can be
 Seen as progressive jaundice such that you symptomatic in certain cases
should be very careful that the baby should not c. HbSC — these also produce sickle cells but
produce kernicterus present with a milder form of anemia so
 Positive Coombs test because there is patients seldom seek consult
Antibodies against the red cells coming from the
mother
Note: Your Congenital Spherocytosis results to
Negative Coombs test, while in ABO Hemolytic 3. Target cells/Codocytes/Mexican hat cells
Diseases results to Positive Cooms test.

Table 7. HS vs ABO HDN.

HEREDITARY ABO HDN
SPHEROCYTOSIS
Symptoms Asymptomatic at Symptomatic at
birth birth Figure 18. L. Target cells. R presence of rod-like crystals
Jaundice Mild to none Rapidly progressive (HbC crystals)
Coombs Negative Positive  Central condensation of Hgb
Retic ct N - sl. high Very high  First thing that comes to mind should be
MCHC High Normal thalassemia, second one should be Hemoglobin C
Ο Thalassemia — SEVERELY symptomatic,
RDW High Normal
anemic
Ο Hemoglobin C — very mild, Check for
 Other conditions:
Hemoglobin C crystals (rod-like) in the PBS
Ο Stored blood — Can be seen in transfused
Ο Other conditions: Post-splenectomy,
blood as they are stored in the refrigerator
Obstructive Jaundice
thus losing their discoid shape
Ο History of multiple transfusion
LABORATORY DIAGNOSIS FOR TARGET CELLS
Ο Acquired Hemolytic Anemia
a. Peripheral Blood Smear
Ο Hypochromic, microcytic anemia with marked
 Laboratory Diagnosis
anisocytosis and poikilocytosis
Ο PBS: RBC is spherical with loss of central
Ο Target cells, schistocytes and basophilic stipling
pallor
b. Hb electrophoresis (diagnostic procedure of
Ο ↑ MCHC; ↓ MCV
choice)
Ο ↑ osmotic fragility (at 0.7 conc.) - cell
Ο ß thalassemia major — marked é in HbF/HbA2,
hemolyses at this level of concentration of
no HbA
NSS
Ο ß thalassemia minor — slight é HbA2 (4-8%) +
Ο Evidence of extravascular hemolysis
HbA
Ο a thalassemia — adult: HbH; pedia: Barts Hb
2. Sickle Cells/ Drepanocytes/ Meniscocytes
c. Evidence of Hemolysis
Ο RBC destruction with severe Fe overload:
bilirubin, hemosiderosis, siderocytes

4. Schistocytes/Fragmented RBCs

Figure 17. Sickle Cells

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 Figure 19. Schistocytes
 RBCs that are irregularly shaped, jagged, and
asymmetrical
 Identify/check the patient’s probable
condition. – May denote emergency.
 Also known as “helmet cells”

CLINICAL CONDITIONS
1. Microangiopathic Anemia Figure 21. Burr cells
o DIC (Disseminated Intravascular Coagulation)  Red blood cells that look like acanthocytes, but
 All coagulation factors are elevated have more moderate spiculations that are
 Identify the cause uniform/regular in height and distribution
 Prepare blood products – pt is expected to  Highly associated with KIDNEY disease
bleed
o Burns CLINICAL CONDITIONS
o TTP (Thrombotic Thrombocytopenic Purpura)  Uremia
 Immediate plasmapheresis is required.  Kidney-related disease
 Decreased platelet count and bleeding  Pyruvate kinase deficiency
time  Acute blood loss
 Has neurologic features  Old Blood: Crenated cells
o HUS (Hemolytic Uremic Syndrome) 7. Dacrocytes
 Immediate plasmapheresis is required.
 Usually renal clinical picture
 Almost same as TPP although no
neurologic features
o Prosthetic ♥ valves
 Damage the RBCs as they pass through it,
thus the fragmented RBCs
2. Hemolytic Anemia
3. Thalassemia

5. Acanthocytes Figure 22. Dacrocytes

 Teardrop-shaped RBCs w/ single elongated

point or tail

CLINICAL CONDITIONS
 Myeloid metaplasia
 Thalassemia
 Megaloblastic anemia
Figure 20. Acanthocytes  Hypersplenism
 Red blood cells that are spiked, or possess o Pancytopenia (or Thrombocytopenia
various abnormal thorny projections +/- neutropenia +/- anemia, in any
 Sharp tips, spur or thorn cells that are irregular in variant), proliferative response of the
height and irregularly distributed Bone marrow, splenomegaly, and
 Presence of spicules or spurs in the RBC correction by splenectomy
 Tells you about a problem in the LIVER  The first three conditions have RBCs that will
 Present in abetalipoproteinemia – a congenital have difficulty during splenic passage,
disorder wherein there is an absence of the resulting to squeezing and partial
carrier protein of your cholesterol fragmentation

6. Burr Cells/Echinocytes 8. Elliptocytes and Ovalocytes

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  Use of Anti-Malarial drugs

LABORATORY DIAGNOSIS
 Initial phase: Intravascular hemolysis + Abnormal
PBS, N/N (Normocytic, Normochromic) Anemia
 Recovery phase (10-14d): Reticulocytosis,
Increased Hb/Hct

2. Basophilic Stippling
Figure 23. Elliptocytes
 Bipolar arrangement of Hb
 Abnormal cytoskeleton proteins

CLINICAL CONDITIONS
 Healthy individuals
 Myelofibrosis
 Sickle cell
 Iron Deficiency anemia
Figure 25. Basophilic Stippling

Manifest as small dots in the periphery of RBCs →

slightly abnormal unstable RNA in young cells

C. ABNORMAL INCLUSIONS TYPES

1. Heinz Bodies a. Fine – thin round dark blue granules →
polychromasia in reticulocytes
b. Coarse – medium-sized → represents impaired
erythropoiesis

CLINICAL CONDITIONS
 Thalassemia*
 Lead poisoning*
 Severe anemia
Figure 24. Heinz Bodies (L) Bite Cells (R)  G6PD Deficiency
*most common causes
 Precipitated oxidized hemoglobin that appear as
small round inclusions within the cells body. 3. Howell-Jolly Bodies
 Oxidized hemoglobins creates reactive oxygen
species (ROS), → Damaged RBC membranes are
removed by splenic macrophages, leading to the
formation of Bite Cells
 Stain used: Methyl violet or Wright stains
o Needs a supravital stain to be able to
appreciate
 Similar with Howell-Jolly bodies. But Heinz bodies
composed of methemoglobin, therefore they are
hemoglobin and are red in color. They are usually Figure 26. Howell-Jolly Bodies
darker than the usual cytoplasm of red cells and  Basophilic nuclear fragments (DNA/Nuclear
are seen at the periphery of the red cells and they remnants) that were not expelled during the RBC
tend to protrude. They are multiple in number maturation process
unlike the Howell-Jolly bodies that are solitary.  Nuclear fragments, solitary, blue in color
(Wright’s), clean outlines, like a ballpen dot, and
CLINICAL CONDITIONS they don’t approach the outline of the red cell
 G6PD Deficiency and they don’t protrude (vs Heinz bodies)
 Methemoglobinemia (intravascular hemolysis)  Stain used: H&E

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  They are non-specific changes in red cells and are
seen in severe forms of anemia

CLINICAL CONDITIONS
 Post splenectomy
 Severe anemia
 Thalassemia
 Accelerated erythropoiesis
Figure 29. Ringed Sideroblasts
4. Cabot Rings  Nucleated RBCs that contain non-heme Fe
particles (siderotic granules or ferritin) arranged
in ring form
 Excessive iron overload in the mitochondria of
normoblasts
 Due to incomplete heme formation in the
mitochondria
 Stain used: Prussian Blue Stain (for iron)

CLINICAL CONDITIONS
Figure 27. Cabot Rings
 Sideroblastic anemia – not common in the
 Thin, red-violet staining, threadlike strands in the
Philippines
shape of a loop or figure-8 in RBCs
 Myelodysplastic Syndrome (MDS) – precursor to
 Due to abnormalities in mitotic spindle
leukemia
 Remnants of microtubules from mitotic spindles

CLINICAL CONDITIONS Summary of Abnormal Inclusions:

 Lead poisoning  Heinz bodies – Oxidized hemoglobin
 Pernicious anemia  Howell-Jolly bodies – Nuclear remnant
 Basophilic stippling – Unstable RNA
5. Pappenheimer Bodies  Cabot rings – Mitotic spindle
 Pappenheimer bodies – Unused Fe
 Ringed sideroblasts – defective Fe deposits

 Only Heinz bodies are significant because it is

specific to G6PD. All others are non-specific
but once present, you have to look for an
abnormality existing in the patient related to
some amount of anemia or myelodysplastic
Figure 28. Pappenheimer bodies
syndrome.
 Small 2-3 µm irregular basophilic inclusions that
aggregate in small clusters near the periphery
 From unused iron deposits seen as several dark D. ABNORMALITIES IN CELL SIZE
blue staining material that coalesce 1. Microcytes
 Stain used: Wright’s stain  Abnormally sized RBCs that are 5-µm or
 Needs an iron stain, if iron stain was mentioned smaller in diameter
it is for sure Pappenheimer bodies  Usually due to impaired hemoglobin synthesis
 Clinical Conditions (ATIS)
CLINICAL CONDITIONS o Anemia of chronic disease (ACD) – iron
 Thalassemia transfer mechanisms are affected despite
 Post-splenectomy relatively normal iron levels
o Thalassemia
6. Ringed Sideroblasts o Iron deficiency
o Sideroblastic anemia
 Laboratory Diagnosis: Iron Deficiency Anemia
(IDA)
o PBS: Hypochromic, microcytic anemia

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 o Bone Marrow: Sideroblasts are decreased,  Increase membrane cholesterol and lecithin
platelets, WBC, reticulocytes and (Liver disease and alcoholism)
normoblasts are normal.  Clinical Conditions
o Iron Studies: o Megaloblastic anemia
 Serum Fe: Decreased o Pernicious anemia
 Serum Ferritin (Storage form): o Liver disease – can cause folate deficiency
Decreased o MDS (Myelodysplastic Syndrome) – due to
 Transferrin (Transport form): mutations in totipotent bone marrow stem
Decreased cells
 TIBC (Total Iron Binding Capacity): o Acquired causes:
Increased  Hypothyroidism
o Elevated RDW  Abnormalities in cholesterol and
lecithin
Table 8. Evaluation of Microcytic Hypochromic  Alcohol or drugs
Anemia
Serum Ferritin TIBC RDW
Fe
IDA Low Low High 90%
Thalassemia High Normal- Low 50%
High
ACD Low High Low N
Sideroblastic High High Low High Figure 30. Macrocytes

 Both IDA and thalassemia cause hypochromic,

microcytic anemia wherein if you give iron to one
(thalassemia) you can kill the patient and if you
give iron to the other, you treat it (IDA). MEGALOBLASTIC ANEMIA
Therefore Iron Studies can be helpful to
differentiate the different causes of anemia.  Due to B12 & folic acid deficiency
 IDA – expect a decrease of serum iron, decrease  Loss of substances needed for nuclear
in transport form resulting to a high capacity to maturation and division → nucleus remains
bind (TIBC) immature thus are abnormally large.
 Thalassemia – elevation of serum iron, elevation
of storage form and transport form is full, thus
the capacity to bind iron (TIBC) is decreased
 Anemia of Chronic Disease – there is low iron
blood because there are no enzymes because the
person is weak, there are no proteins because the
person is sick. There is a high storage of iron (high
ferritin), there is high transport (low TIBC), but it
cannot be transported into the serum.
 Sideroblastic Anemia – basically similar to
Figure 32. B12/FA Pathways. B12 is needed in 2 pathways:
thalassemia; congenital problem wherein iron
methylmalonic pathway (methylmalonic acid to succinyl-
cannot be used CoA) and methionine synthase pathway (homocysteine to
 RDW – represents red cell distribution width, the methionine). In B12 deficiency, there is an elevation of
more anisocytosis you have in a patient, the homocysteine (causing thrombosis and MI) and
higher the RDW. In a case of hypochromic methylmalonic acid (causing demyelination or neurologic
microcytic anemia, you have to look at RDW. If problems). FA enters the pathway through methionine
the RDW is high, you probably have IDA rather synthase pathway, thus FA deficiency only elevates
than Thalassemia. homocysteine not methylmalonic acid. Homocysteine is
elevated in both B12 and FA deficiencies, but
methylmalonic acid is only elevated in B12 deficiency. That
2. Macrocytes
is the reason why neurologic problems are only seen in B12
 Impaired DNA synthesis – vitamin B12 of Folic deficiencies.
acid deficiency  Folic Acid deficiency → Increase Homocysteine
 Can be seen in accelerated erythropoiesis

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  B12 deficiency → Increase homocysteine
(destructive to CVS) and Methylmalonic acid
(destructive to nerve)

A. LABORATORY DIAGNOSIS OF MEGALOBLASTIC

ANEMIA
Peripheral Blood Smear
 Pancytopenia: decrease RBC, WBC, platelet (all
are affected because they contain nucleus and
has DNA PERNICIOUS ANEMIA
 RBC: macrocytic normochromic RBC Pathogenesis:
o (+) Large nucleated RBC in PBS, decrease Antibody to intrinsic factor → chronic atrophic
reticulocyte count gastritis → loss of intrinsic factor (IF) for B12
 WBC: “macropolys” large hypersegmented absorption
Neutrophils
Bone marrow  Cause is immunologic, hence can identify
 Hypercellular, E:M 1:1 antibodies in serum of the patient. These
 “megaloblasts” all stages of RBC dev are large antibodies can eat up the parietal cells of the
 Nucleo-cytoplasmic asynchronism/dissociation stomach which can decrease the intrinsic factor.
RBC Indices In pernicious, there is an atrophy of the stomach.
 ↑ MCV, HI Normal MCHC; increase RDW
Abnormal Chemistry  Note:
 Elevation of homocysteine (folic acid & B12 def)  Impaired Hemoglobin/cytoplasm: Microcytic
 Elevated methylmalonic acid (B12 def)  Impaired DNA/Nucleus: Macrocytic
 Elevation of LDH – elevated if there is red cell  How would you know if it is Micro or Macro?
abnormality Compare it with WBC, same size or bigger =
Macro; slightly small = Normo; half the size of
WBC = Hypo
B. MEGALOBLASTIC ANEMIA
Response to treatment: Remember this in exact order Laboratory Diagnosis
2 days Fall of serum LDH (earliest  Achlorhydria
improvement)  Decreased serum B12
3-4 Reticulocytosis (it will take 3-5 days from  Positive serum antibodies – against intrinsic
days pro-normoblasts to mature re blood factor; can cause death of parietal cells →
cells) Atrophy of stomach and achlorhydria – Basic
10 days Rise in Hgb (then normalizes in 8 weeks difference from regular B12 deficiency
with correction of MCV)  Marked elevation of LDH
10-14 Hypersegmented PMNs disappear
days Macrocytes
Must watch closely for severe hypokalemia during  Impaired DNA synthesis – vitamin B12 or Folic
early response due to marked K use in hematopoiesis acid deficiency
 Can be seen in accelerated erythropoiesis
 Increase membrane cholesterol and lecithin
(Liver disease and alcoholism)
 Clinical Conditions:
o Megaloblastic anemia
o Pernicious anemia
o Liver disease – can cause folate deficiency
o MDS (Myelodysplastic Syndrome) – due to
mutations in totipotent bone marrow stem
cells
Figure 33. (R) Megaloblasts (L) Hypersegmented nucleus o Acquired causes:
 Hypothyroidism
 Abnormalities in cholesterol and
lecithin
 Alcohol or drugs

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 OTHER TESTS

COOMB’S TEST
 Test for presence of antibodies in the blood
 E.g. Positive for Coomb’s test: Autoimmune
Hemolytic Anemia (AHA) → antibodies against
RBC; ABO Incompatibility
 aka Anti-human globulin
o antibody against blood antibody resulting in
greater degree agglutination

 This test will help differentiate hemolytic disease

of the newborn versus spherocytosis (see Hb, Hct and RDW are all elevated in the NB.
appendix) o Notice the Hb is very high in the NB and starts to
 DIRECT Coombs Test – antibodies attached to the decrease in 6 months and normalizes in
red cell → antiglobulin reagent → coagulation adulthood.
 INDIRECT Coombs Test – antibodies in serum → o MCV is also high and then you see normoblasts
incubate red cells with antibodies → antibodies occasionally on PBS or an elevation of reticulocyte
attach to the red cell count because of the decrease in oxygen in the
 In cases of cross matching (Indirect Coomb’s) → uterus.
incubate red cells of the donor with the recipient
WBC & Differential Count
Hemolytic Disease of the Newborn (Rh or ABO
Incompatibility)
 “Warm” AHA (Autoimmune Hemolytic Anemia) –
IgG; extravascular hemolysis, cancer, drugs, SLE;
Intense jaundice, very progressive may lead to
kernicterus
 “Cold” AHA – IgM; intravascular hemolysis,
infectious mononucleosis, Mycoplasma
pneumonia

Paroxysmal Nocturnal Hemoglobinuria (PNH)

 Acid serum test = Ham’s test
 Sucrose hemolysis test  Elevation of segmenters with stab in NB which
shifts at about 6 months of age into
Hematologic Alterations in the Newborn lymphocytosis
 Hemoglobin – increased
 Mild reticulocytosis (2.5 – 6.5%);
normoblastemia
SAMPLEX
o Becomes normal in 2 weeks
Matching Type:
 WBC
1. Cabbot Rings
o 1-3 days – 10,000 to 35,000 (U appears
2. Heinz bodies
extremely high; but this is often normal in
3. Pappenheimer bodies
newborn; abnormal if it is very low)
o 1 wk – decrease, normalizes in 5-8 yrs
A. G6PD deficiency
 Neutrophilia – NB
B. Thalassemia
 Lymphocytosis – infancy & early childhood
C. Pernicious Anemia
 Myelocytes and stabs present
MCQ:
Hematology Values 1. Which of the following is considered in the
laboratory diagnosis of Pernicious anemia?
a. RBC destruction with severe iron overload
b. Increased osmotic fragility
c. Decreased serum ferritin (storage form)

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 d. Positive serum antibodies against IF
e. B and D

2. Which of the following is a sign of earliest

improvement in the treatment of megaloblastic
anemia?
a. Rise in hemoglobin
b. Reticulocytosis
c. Fall of Serum LDH
d. Presence of hypersegmented PMNs
e. None of the above

3. All are true regarding Hematocrit EXCEPT:

a. It is the ratio of the volume of erythrocytes
to that of the plasma
b. Gives a rough, indirect estimate of Key Answers
hemoglobin
c. Normal values for male is 40-54% , while for Matching Type:
female: 36-47% 1. C
d. NOTA. All are true of hematocrit 2. A
3. B
4. HCT may not be reliable in the following
conditions EXCEPT: MCQ:
a. Immediately after blood loss 1. D
b. Immediately before transfusion 2. C
c. Immediately after transfusion 3. A HCT is the ratio of the volume of erythrocytes to
d. Abnormal size and shape of red blood cells that of the whole blood (not plasma)
4. B
5. HCT levels are decreased in the following 5. D
conditions EXCEPT: 6. True
a. Anemia 7. True
b. Nephrotic Syndrome
c. Leukemia Short answers:
d. Polcythemia 1. Males 4.7 to 6.1 million cells per microliter
Females 4.2 to 5.4 million cells per microliter
True or false 2. due to regular menstruation
1. T/F Increased reticulocyte count in patients with 3. due to lower supply of oxygen in utero
anemia indicates that the bone marrow is 4. gamma-4 and beta-4 respectively
functioning well.
2. T/F During Hemoglobin Electrophoresis, an HbF
that is more intensely colored than HbA is
normally seen newborn

Short Answers
1. What is the normal value of RBC for both male
and females?
2. Why do female have lower hemoglobin?
3. Why do newborns have higher hemoglobin?
4. what is the composition of alpha-thalassemia in
fetus? how about in adults?

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