CLINICAL PATHOLOGY LECTURE 5 Generalities of Blood & Its Changes in RBC shape

Elements 1. Poikilocytosis
Notes from Lecture of Dr. Ng/Dr. Lim - indicates a variation in RBC shape (abnormal
USTMED 07 Sec C Anne Mayoralgo erythropoeisis)
- high poikilocytosis in
Changes in the process of RBC formation: a. bone marrow deficit (vit B12 deficit)
1. Progressive diminution of cell size b. abnormal RBC destruction
2. Ripening of cytoplasm 2. Spherocytes
3. Ripening of nucleus - almost spherical in shape
- not biconcave like a normal RBC & dont have the
For normal erythrocyte production, the following are central area of pallor which a normal RBC shows
required: - has less surface are for its size
1. protein - associated with
2. iron a. hemolytic anemia (HA)
3. vitamin B12 b. ABO hemolytic disease of the newborn
4. Folic acid c. Hereditary spherocytosis
5. Vitamin B6 3. Crenation (echiocytes)
6. Metal traces - have blunt spicules evenly distributed over the surface
of the RBC & are usually artifactual due to faulty drying
RBC of the blood smear, or may be a result of
- 120 days hyperosmolarity
- biconcave - seen in very anemic patients
- no nucleus or organelles 4. Acanthocytes
- contains membrane - abnormally crenated RBCs caused by deficiency of
- 30% hemoglobin ________?
- stroma - Defect in cell membrane
- Seen in:
Hemoglobin a. congenital/acquired abetalipoproteinemia
- most important component of RBC b. certain liver & lipid metabolism disorders
- consists of heme + globin 5. Burr cells
- functions: - regular curved or scalloped shape
a. transport of O2 from lungs to body (transport of - RBC with irregular projections
O2) - RBC with uniformly spaced, pointed projections on
b. assists acid-base balance (elimination of CO2) their outer edges
- Occur in:
Morphoology of abnormal erythrocytes can be determined by: a. uremia
1. changes in size b. acute blood loss
2. changes in shape c. cancer of the stomach
3. degree of hemoglobinization d. pyruvate kinase deficiency
6. Schistocytes
Changes in size & Hgb content can be determined by use of: - red cell fragments
1. blood indices - cause: trapping b/w violently opposed mechanical
2. peripheral smear (also used to determine changes in surfaces
RBC shape) - may occur in:
a. microangropathic HA
Changes in erythrocyte size can be determined by MCV b. uremia
c. severe burns
MCV = Hct x 10____ d. HA caused by physical agents, as in DIC
RBC in millions 7. Ovalocytes (elliptocytes)
Normocytic : = 76-96 fL - oval shaped RBCs
Microcytic: < 76 fL (seen in IDA & secondary 8. Target cells (leptocytes)
anemia) - abnormally thin resembling arranged target
Macrocytic: >96 fL (seen in Vit B12 deficiency - show a centrally stained area within a thin outer rim of
& folic acid deficiency) Hgb
- Anisocytosis pathologic variation in cell size - associated with:
a. hepatic disorders
Changes in Hgb content can be determined by: b. certain hemoglobinopathies (abnormal Hgb)
Hgb x 10 c. sickle cell anemia
1. MCH = -------------------------- d. Hgb CC, E and SC disease
RBC in millions 9. Stomatocytes
- normal range: 28-32 pg - RBC with central stoma/mouth which appear unstained
- Show a slitlike (rectangular) area of central pallor)
2. MCHC = (Hgb/Hct) x 100 - Have lost the indentation
- normal range: 32-36% - May be found in
a. liver disease
Conditions associated with changes in Hgb content b. alcoholism
1. Hypochromasia c. eletrocyte imbalance
- low HgB concentration thus giving central pallor to RBC d. hereditary stomatocytosis
- seen in 10. Sickle cell (drepanocytes)
a. iron deficiency anemia (IDA) - crescent shape due to formation of rodlike polymers of
b. Thalassemia Hgbs within the cell
2. Anisochromasia - seen in sickle cell anemia
- morphology of red cells which stain unequally with
ERYTHROCYTE INCLUSIONS
only portion of cell hypochromasia
1. Basophilic stippling
- seen in patients after transfusion due to IDA
- fine or coarse gray-black granules in the red cells
3. Hyperchromasia
- seen in lead poisoning
- unusually deep staining, not related to Hgb over
2. Howell-jolly bodies
saturation in cell (indicated by upper limit of MCHC)
- Remnant of nuclear material (chromatin within
- important aspect to be able to assess mean cell
the red cell)
thickness
- Disorder of spleen
- ex. Spherocyte
3. Cabot rings
4. Polycromasia
- Red violet structures appearing as rings,
- indicates young red blood cells which contain residual
incomplete rings or figure of eight
RNA
- Seen in severe anemia
- cells are generally larger than normal & stain pinkish
4. Sederocytes
gray to pinkish blue color
- Red cells containing bright blue non-hemoglobin
- basophilic in Romanowsky stain
iron granules
- when stained supravitally with brilliant cresyl blue they
- Seen in hemolytic anemia and after splenectomy
show up as reticulocytes
ERYTHROCYTES DISORDERS - NV = 0.5 1.5% (5-15 x 10-3)
1. polycythemia vera - in the number of red cells
2. anemia in the number of red cells Osmotic Fragility test
can be determined by doing RBC count - reagent 0.5% NaCl (hypotonic solution)
- cells are suspended in a series of tubes containing
hypotonic NaCl varying from 0.9% to 0.0%, incubated at
ANEMIA room temperature for 30 mins and centrifuged. The %
- Reduction in the concentration of Hgb in the peripheral of hemolysis in the supernatant solution is observed
blood below normal for age and sex of the patient and measured for each NaCl concentration
- Causes - cells that are more spherical, with a decreased surface
1. impaired red cell production volume ratio, have a limited capacity to expand in
2. blood loss hypotonic solution and lyzed at higher concentration
3. accelerated red cell destruction (hemolysis) in than normal cells and are said to have increased O.F. it
excess of the ability of bone marrow to replace is increased in hemolytic spherocytosis.
these losses - Conversely cells that are hypochromic and flatter have
- Anemia Panel a grater capacity to expand in hypotonic solution, lyzed
1. CBC with indices at a lower concentration than normal cells. They have
2. Reticulocyte count decreased O.F.
3. Osmotic fragility test - NV = hemolysis begins in tube 33 containing 0.44$ or
4. Sucrose hemolysis test 0.42% NaCl and is complete in tube 17 containing 0.34%
5. Hams test NaCl.
6. Iron
7. Total iron binding capacity Sucrose Hemolysis Test
8. Ferritin - used to diagnose PNH, hypoplastic anemia,
9. B12 megaloblastic anemia
10. Folate - AIHA
11. Electrophoresis - Principle: isotonic sucrose solution provides a medium
- anemia considered to be present if the Hgb of low ionic strength which promotes binding of
concentration or hematocrit is below the lower limit of complement to red cells. In PNH, a portion of red cells
95% of reference interval for individual age, sex and is abnormally sensitive to complement mediated lysis
geographical location (altitude) - Patients washed RBC are mixed with sucrose, incubate
for 30 mins, centrifuged and observed for hemolysis.
Complete Blood Count Make also a control using NSS with patients blood the
- hemoglobin, hematocrit and RBC values are low control tube should be negative.
- smear shows RBC morphology
- Anisocytosis, poikilocytosis, target cells, sickle Hams Test (Acidified Serum Test)
cell, spherocytes, hypochromic, microcytic, - for definitive diagnosis of paroxysmal nocturnal
macrocytic, etc. hemoglobinuria (PNH)
- in acidified serum, cooplement is activated by
Blood indices alternate pathway, binds to RBC and lyses the PNH cells
- Hgb, hct and RBC count are those needed for which are unusually susceptible to complement. The
computation of indices washed RBC are mixed with ABO compatible normal
- These will give the characteristics of red cells as to serum (fresh) and acid, after an hour incubation at
size, Hgb content and Hgb concentration. 37oC, the PNH cells are lyzed
- the patients own serum may or may not result in lysis,
Mean corpuscular volume (MCV) depending on the residual complement and the other
- is the average volume of individual RBC tubes provide control.
- allow classification of cells into normacytic, macrocytic
and microcytic
IRON
MCV = HCT x 10 - it is essential to living organism for cellular oxidation
RBCs (x 1012/L) mechanism and transport of oxygen to tissues
- diurnal variation demonstrates normal values in the
- NV = 87 5 cu microns (fl) morning, dectreasing in the afternoon
- patient preparation; moning specimen (red top)
Mean corpuscular hemoglobin (MCH) - NV = 0.5-17 ug/dl
- is the amount of hemoglobin by weight in an average Method of Determining Values:
WBC - Based on the principle that when pH is decreased in
- in new born and macrocytic anemia, MCH is high and in serum, iron is released from transferring, Fe 3 to Fe2 and
deficiency anemia, it is low complexes
Total Iron Binding Capacity (TIBC)
MCH = Hgb gm% x 10 - transferring is usually measured indirectly by the
RBCs (x 1012/L) amount of iron that it can bind, this is the TIBC
- % saturation of TIBC is the ratio of serum iron to TIBC
- NV = 27-31 ug (pg)
COBALAMIN (Vit B12)
Mean corpuscular hemoglobin concentration (MCHC)
-Vit B12 is the only vitamin exclusively synthesized by
- is the concentration of hemoglobin in an average RBC
microorganism
- if below normal, hypochromic
- Stored in the liver, released by digestion of proteins by
- highter than normal concentration are not possible as
animal origin and then is bound by gastric intrinsic
normal RBC contains the maximum amount of Hgb
factor
- Normochromic, within normal MCHC
- Absorbed cobalamine is deliveredb to the liver,
hemopoietic cells and other dividing cells
MCHC = Hgb gm% x 100
- NV = 200-900 ng/L (pg/mL)
Hct
Cobalamin deficiency due to :
1. inadequate intake (vegetable)
- NV = 33-38%
2. defective production of intrinsic factor (gastrectomy)
RETICULOCYE
3. pernicious anemia (failure of gastric mucosa to secrete
- young red cells that have matured enough to have lost intrinsic factor)
their nuclei leaving their cytoplasmic RNA content, 4. defective absorpition (tapeworm infestation)
which are detected by supravital stain (new methylene - deficiency results in megaloblastic anemia
blue) - serum cobalamine assay, therapeutic trial, urinary
- increased when there is increase etythropoiesis as in methyl maloric assay and deoxyuridine suppression test
blood loss, dectreased in aplasia of bone marrow are also used to diagnose cobalamine deficiency
- decreased iron values are observed in menstrual cycle,
Reticulocyte in % = # of reticulocyte counted x 100 pregnancy, inflammatin, MI, malignancy, and iron
1000 RBC deficiency anemia
 - increased iron values are seen in hepatitis, oral - The Prafon Hemoglobin Electrophoresis kit is intended
contraceptive usage and increased ingestion for electrophoretic separation of human hemoglobins to
screen for clinically important Hgb variants
Laboratory Findings: - Alkaline hemoglobin electrophoresis on agarose gel is
1. macrocytosis used as a screening procedure for Hgb A, F, S and C
2. presence of hyperlobulated neutrophils (5-6 lobes in
more than 5% neutrophils) audsmartinez@gmail.com
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3. High MCV
4. reticulocyte and NRBC elevated
5. Vit B 12 decreased
- in pernicious anemia there is decreased cobalamin
intrinsic factor and megaloblastic anemia

FERRITIN
- absorbed iron becomes attached to plasma protein.
Excess protein combines with the protein apoferritin to
form ferritin, these are stored form of iron in the liver,
bone marrow and spleen
- if the amound of apoferritin is insufficient to bind the
remaining iron, it is deposited in the tissues as iron
oxide granules called hemosiderin
- BTR iron overload (excess blood 100 units) used as in
thalassemia. Daily excretion of a patient who is not
bleeding is 1mg/day. Since there is no way to eliminate
iron except through blood loss, excess iron accumulates
in the mitochondria of cells of organs like liver, heart
and endocrine glands leading to organ failure
- Check diagnosis by requestin ferritin
- NV = 12-300 ug/L (adult); 1-142 ug/L (infant)

FOLIC ACID (Pteroyl Monoglutamic Acid)

- Source: food like eggs, milk, leafy vegetables, yeast,
liver, fruits and formed in the intestine absorbed in
the jejunum plasma cells tissue for ulitization
- NV = 5-21 ng/mL
Laboratory findings:
- Cobalamine deficiency are also seen in folate
deficiency except in leukopenia and thromocytopenia
- Seen also in pernicious anemia
Folic Acid Deficiency Due to:
1. inadequate intake of folic acid
2. Increased demands as in pregnancy, infancy, infection
or hemolytic anemia, liver disease associated with
alcoholism
3. Defective absorption as in malabsorption syndrome
To diagnose deficiency thru laboratory methods needed are:
1. Serum folic acid
2. Serum folate
3. Red cell folate

Evaluation of Hemolytic Anemia

Hematology Testing
- CBC: check for spherocytes, sickle cells, target cells,
evidence of infection or leukemias, malarial parasites
- Reticulocyte count: increased production of RBCs
Urine Testing
- urobilinogen
- Urobilin
- Urine hemosiderin
Special Testing
- Plama hemoglobin
- Bilirubin Fractionation (direct, indirect)
- Direct antiglobulin test (DAT)
If hereditary or congenital anemia is suspected:
- osmotic fragility
- autohemolysis test
- G6PD
- Glutathione stability
- Hemoglobin electrophoresis and hemoglobin F
- Heinz body stain
Suggested testing if autoimmune process is suspected
- direct and indirect coombs test using polyvalent IgG
and C sera
- Cold agglutinins and hemolysis
- Antibody elutions from patients cells
- Donath-Landsteiner test for paroxysmal cold
hemoglobinuria
- Hams test, sucrose hemolysis test for PNH
- Serum protein electrophoresis or immunofixation
- Rapid plasma regain (RPR) or venereal disease reaserch
laboratory (VDRL) test to rule out syphilis

Electrophoresis
Methodology