MTAP Basic Hematology Concepts Laboratory Procedures

Hematology Rationale #1

Insufficient centrifugation will result Which of the following is the preferable site for bone marrow
A. False increase in hematocrit aspiration and biopsy as an adult?
(Hct) value A. Iliac crest
B. False decrease in hct value B. Sternum
C. No effect on hct value C. Tibia
D. All of the above depending on the patient. D. Spinous processes of a vertebra
Rationale: Insufficient centrifugation does not pack down the red Rationale : this site is the safest and most easily accessible, with
blood cells, therefore, the hct will increase. the bone just beneath the skin and neither blood vessels nor
nerves are in the vicinity

HEMATOCRIT BONE MARROW ASPIRATE

• Packed cell volume • Embryonic and Fetal Hematopoiesis
• Ratio of the volume of erythrocytes to that of whole Mesenchyme of yolk sac → liver → + spleen and
blood. lymph nodes → BM
• Anticoagulants: Dried heparin or EDTA • Shortly after birth the marrow becomes the only site of
• Methods for hct determination: production of erythrocytes, granulocytes and platelets.
1. direct centrifugation via macromethod or • Later in childhood , only the flat bones and the proximal
micromethod parts of long bones are sites of blood formation.
2. indirectly via automated instruments : MCV X Note: During the first month of prental life, the first blood cells
RBC ct arise outside the embryo in the mesenchyme of the yolk sac as
Note: Before taking a sample from a tube of venous blood for a blood islands, cells are predominantly primitive erythroblasts. At
hematologic dterination, mix blood thoroughly. If the tube has the 6th week, hematopoiesis begins in the liver and becomes the
been standing, his requires atleast 60 inversions. (JBH) major hematopoietic organ of early and mid fetal life. In the latter
half of fetal life, the BM becomes progressively more important
Macromethod = wintrobe method which is not anymore used.
as a site of blood cell production and liver’s role diminishes.
• Micromethod • Considerations : difficulty of access, risks involved, , the
• Equipment: 7cm capillary hematocrit tube with a 1 mm ease of obtaining a good biopsy specimen and the
bore. discomfort of the patient
• Centrifugation : 5 mins at 10,000 to 12,000 g • Posterior iliac crest as the preferred site
• Reference value: • Different preparations: Direct films, Imprints, Crush
adult males – 0.41 – 0.51 preparations.
adult females – 0.36 -0.45 • The appearance of fat as irregular holes in the films
gives assurance that marrow and not just blood has
• Sources of error: centrifugation, excess EDTA, technical been obtained.
errors ( failure to mix blood prior to sampling, improper • M:E is the ratio of total granulocytes to total normoblasts
reading of erythrocyte volume) • Normal value in adults may range from 1.2:1 to 5:1
NOTE: Excess EDTA would result in falsely low hct as a result • Increased M:E ratio may be found in patients with
of cell shrinkage but hgb and cell ct will not be affected. infections, chronic myelogenous leukemia

A bite cell seen on a Wright’s stained peripheral blood smear

Erythrocytes that vary in size from the normal 6-8 um are
exhibits
described as exhibiting:
A. Poikilocystosis
A. Anisocytosis
B. Anisocytosis
B. Hypochromia
C. Hypochromia
C. Poikilocytosis
D. Polychromasia
D. Pleocytosis
Rationale: variation in shape of the erythrocytes on a peripheral
Rationale: A mature RBC is ~7 um in diameter. Hypochromia is
blood smear is poikilocytosis
a term that indicates increased central pallor. Poikilocytes
denotes variation in red cell shape.
POIKILOCYTOSIS
• Elliptocytes : elliptical in shape; most abundant in
ERYTHROCYTES
hereditary elliptocytosis, also common in IDA,
• Appear as circular, homogenous discs of nearly uniform
myelofibrosis with myeloid metaplasia, megaloblastic
size, ranging from 6-8 um
anemia and sickle cell anemia,
• Anisocytosis is a feature of most anemia
• Spherocytes: does not exist as a biconcave disc, lacks
• The depth of staining furnishes a rough guide to the
central pallor, seen in hereditary spherocytosis and AHA.
amount of hgb in red cells: normochromic,
• Target cells/leptocytes : when stained show a peripheral
hyperchromic, hypochromic
rim of hemoglobin with a dark, central hgb containing
• Polychromatophilia or polychromasia : blue gray tint to
area; found in obstructive jaundice
the red cells
• Schistocytes (cell fragments): indicate the presence of
- combination of the affinity of hgb for acid stains
hemolysis
and the affinity of RNA for basic stains, indicating
• Acanthocytes: irregularly speculated red cells in which
that it is a young red cell.
the ends of the spicules are bulbous and rounded; seen
in abetalipoproteinemia, and certain cases of liver
disease.
RTRM
CERO, F.M.
F
 MTAP Basic Hematology Concepts Laboratory Procedures
Hematology Rationale #1

• Crenated or echinocytes: regularly contracted cells that  Monocyte

commonly occur as an artifact during preparation of films. - largest cell of normal blood.
- has 1 nucleus, partially lobulated, deeply
indented or horseshoe shaped, may
sometimes appear round or oval.
- fine chromatin, cytoplasm is blue-gray and
has a ground glass appearance and
often contains fine red to purple granules
• Lymphocyte
- mononuclear cell without specific cytoplasmic
granules
- single nucleus containing heavy blocks of chromatin.
- the chromatin stains dark blue with Wright stain.
• Plasma Cells
- Have abundant blue cytoplasm, often with
light streaks or vacuoles, an eccentric
round nucleus.
- nucleus has heavily clumped chromatin
which is sharply defined, often arranged
in a radial or wheel-like pattern.
- not present normally in blood
When an erythrocyte containing iron granules is stained with
Prussian blue, the cell is called a
A. Spherocyte
B. Leptocyte
C. Schistocyte
D. Siderocyte
Rationale : siderocytes are red cells containing inorganic iron
granules and is made visible with Prussian blue
Note: if stained with Wright stain they are called Pappenheimer
bodies.

What phagocytic cells produce lysozymes that are bactericidal

A. Eosinophils
B. Lymphocytes
C. Platelets
D. Polymorphonuclears
Rationale: Neurtrophils are highly phagocytic and release A decreased osmotic fragility test would be associated with
lysozymes, peroxidase and pyrogenic proteins. which of the following conditions
A. Hereditary spherocytosis
LEUKOCYTES B. Sickle cell anemia
• Neutrophils C. Hemolytic disease of the newborn
- average diameter of 12 um D. Acquired hemolytic anemia
- smaller than monocytes and eosinophils but Rationale: Osmotic fragility test is decreased when numerous
slightly larger than basophils. sickle cells and target cells are present and is increased in the
- the nucleus stains deeply and is irregular. Nucleus presence of spherocytes.
is segmented which are connected by delicate
filaments. The MT on duty used a buffer with a p of 6.0, what would be its
- cytoplasm is colorless, is packed full of tiny effect on the cells of a wright’s stained smear
granules that stain tan to pink with Wright. A. White cell cytoplasm would be stained too blue
 Eosinophils B. Red cells would be stained too blue
- 13 um in diameter C. Red cells would be stained too pink
- Bilobed D. Red cells would lyse
- their cytoplasm contains larger round or oval Rationale: The pH of buffer is critical in Romanowsky stains.
granules with a strong affinity for acid When the pH is too low < 6.4, the red cells take up more acid
stains, which stains bright red with eosin. dye and become too pink. Leukocyte also show poor nuclear
details when the pH is decreased.
 Basophils
- resembles neutrophils except that the
STAINING
nucleus is less segmented
• Most Romanowsky stains are dissolved in methyl
- granules are larger and have a strong affinity
alcohol and combine fixation with staining. Among the
for basic stains which stains deep purple
best known methods are Giemsa and Wright stain.
often obscuring the nucleus
- granules are water soluble!

RTRM
CERO, F.M.
F
 MTAP Basic Hematology Concepts Laboratory Procedures
Hematology Rationale #1

• Wright stain is a methyl alcoholic solution of eosin and HEMOGLOBIN

a complex mixture of thiazines including methylene • Main component of the red blood cell
blue and azure B. • 1 gram Hgb = 1.34 mL of Oxygen
• The buffer solution is at a pH of 6.4 • 2 pairs of globin chains, 4 heme groups, 4 atoms of
• Film stained well must have: pink color via naked eye; ferrous iron
under LP the cell should be evenly distributed; red Note: The red cell mass of adult contains ~ 600 g of hgb
cells are pink, minimum precipitate; blood cells should
be free from artifacts; nuclei of leukocytes are purple
Note: Buffer is amixture of potassium phosphate, sodium • Reduced Hemoglobin – iron unassociated with oxygen
phosphate and distilled water. • Oxyhemoglobin – each heme group is associated with
one molecule of Oxygen
• Excessively blue: thick films, prolonged staining time, - carry the reduced state of iron (Ferrous)
inadequate washing, too alkaline stain, too alkaline • Methemoglobin – hgb in which the ferrous iron is
buffer. oxidized to the ferric state, loses its capacity to carry
• Excessively pink: oxygen or carbon dioxide
• Precipitate on the film: unclean slides, drying during - decreased NADH-cytochrome b5 reductase activity
the period of staining, inadequate washing of slide, • Sulfhemoglobin – mixture of oxidized, partially
inadequate filtration of stain or dust settled on the slide denatured forms of hgb that form during oxidative
or smear. hemolysis, sulfur is incorporated into heme rings
• Carboxyhemoglobin – hgb bound to carbo monoxide
Interpret given the following RBC indices obtained on a patient - 210 x greater affinity
are as follows: MCV 88 fL; MCH 30 pg; MCHC 34%. - increasing concentration shifts the Hgb-oxygen
A. Microcytic, hypochromic dissociation curve increasingly to the left.
B. Microcytic, normochromic - at 20%-30% blood saturation, symptoms may
C. Normocytic, normochromic begin to appear.
D. Normocytic, hypochromic
What is the normal WBC differential lymphocyte percentage
RBC INDICES range in the adult population
A. 20-50%
B. 10-20%
C. 5-10%
D. 50-70%

25/M trauma patient was seen in the EMD, pale, large artery
was transected in his lower leg, active bleeding noted. CBC
results revealed the following: Hgb 12 g/dL, Hct 36 %, RBC 4.0 .
Solve for the blood indices

In which stage of erythrocytic maturation does Hgb formation

begin
A. Reticulocyte
B. Pronormoblast
C. Basophilic normoblast
Which of the following is considered a normal hemoglobin (hgb) D. Polychromatophilic normoblast
A. Carboxyhemoglobin
B. Methemoglobin
C. Sulfhemoglobin
D. Deoxyhemoglobin
Rationale:
Deoxyhemoglobin is the physiological Hgb that results from the
unloading of oxygen by hgb.

RTRM
CERO, F.M.
F
 MTAP Basic Hematology Concepts Laboratory Procedures
Hematology Rationale #1

C. Romanowsky staining
D. Cytochemical staining

Which of the following hgb configurations is characteristic of

HGB H
A. 4 gamma
B. 2 alpha and 2 gamma
C. 4 beta
D. 2 alpha and 2 beta

Autoagglutination of red cells at room temperature can result in

which of the following
A. Low RBC count
B. High MCV
C. Low hematocrit
D. All of the above

What staining method is used most frequently to stain and count

reticulocytes
A. Immunofluoresence
B. Supravital staining

RTRM
CERO, F.M.
F