Topic: staining of blood smears (20-21, lab man)

Romanowsky group of stains:

Wrights stain

Leishmans stain

Giemsa stain

Jenner-Giemsa stain

May-Grunwald-Giemsa stain
Romanowsky contains:
Methylene blue (or oxidation prods like Azure B)
Eosin B or Y
Wrights stain:
Contains eosin and methylene blue
Powder form dissolved in glycerine then mixed with acetone
free meth-OH
Meth-OH is also a fixative for bld. film
Phosphate buffersets pH after fixation and staining
o pH 6.8-for bld and bone marrow staining
o pH 7.2-for malarial para, Schuffners granules
Well stained Bld smear:
appear evenly pink or purple macro
@OIF,
o RBCs - orange to salmon pink
o WBCs (nuclei) - purple to blue
o Neutrophils violet or lilac in a pink to tan cytoplasm
o Eosinophil bright orange or reddish-orange
Thin (feathery) edgemorph should be done; RBCs are not
overlapping but touching each other
Meth-OH fixative: container should be capped or covered meth-OH
readily takes up water
pH acidic: RBCs bright red to red; WBCs pale blue or colorless
pH basic/alkaline: RBCs deep blue; WBCs structures hardly discerned
Procedure:
1. Bld smear to methanol fixative for 5 quick consecutive times
2. Eosin jar 5 quick consecutive times; then 25 quick consecutive
times in methylene blue jar
3. Phosphate buffer solution. Then drain slide
4. Air dry, dont blow

Topic: Microhematocrit (179-181, Rodak)

Hctvol. of packed RBCs that occupies a given vol. of whole bld;
Packed Cell Vol. (PCV).
reported as % or L/L
Procedure:
1. Fill 2 plain cap. Tubes three quarters w/ anti-coag bld (EDTA
or heparin); Mylar-wrapped recommended; Bld. for
heparinized tube collected by skin puncture
2. Seal colored end of tube w/ nonabsorbent clay; hold
horizontally and seal by placing dry end into tray w/ sealing
cmpd @ 90 deg angle; rotate and remove; plug should be
@least 4mm long
3. Balance tubes in centri, clay outside, touching rubber gasket
And so on . . .
Sources of error:
Increase Hct
Not mixed
properly

Decrease Hct
Improper sealing
Inc. anti-coag
conc.

Insufficient centri
or delay in
reading

Sickle cell
anemia,
macrocytic
anemias,
hypochromic
anemias,
spherocytosis and
thalassemia.

RBC shrinkage

Not mixed
properly

Buffy coat is
included in the
reading
MHct reader is
not used properly

Loss of bld
during centri

MHct reader is
not used
properly
Plasma is
trapped in
RBC layer, 13% higher

(temporary)
blood loss

Inc. in plasma
vol.

Plasma is
replaced faster
that RBCs

Intro. Of
interstitial fluid
from cap
puncture or
improper flushing
of catheter

Fluid loss assoc.

w/ dehydration

1|Hema Topic Summary

Rule of three:
Applies only to specimen w/ normocytic normochromic
erythrocytes
Val. Of Hct should be 3 times the val of Hb +/- 3 [Hb x 3= Hct
+/- 3]
Discrepant may indicate abnormal RBCs or first indication of
error
Topic: Hemoglobin metabolism (115-124, Rodak)
Hb1st CHON whose structure was described using x-ray
crystallography.
Conj. Globular CHON w/ 4 heme grps and 2 heterogeneous
pairs of polypeptide chains
Main cytoplasmic component of RBCs
Free Hb from RBC hemolysis, short half-life, rapidly salvaged,
catabolized or excreted renally
Conc of Hb in RBC: approx. 34 g/dL, MW-64,000 daltons
Transpo of O2 lungs to tissues
Modulates vascular dilation by transpo of Nitric oxide (NO)
Transpo of CO2 tissues to lungs
Heme structureconsists of:
o Ring of carbon
o H2
o Protoporphyrin IX, (N2 atoms)
4 heme grps is positioned in a pocket of PP chain
near the surface of Hb molecule
Combines reversibly with 1 O2 molecule
Renders bld cell, double bonds
Globin structure4 each Hb molecule
Consist of 2 identical pairs of unlike PP chains
141-146 AA each
Designated by Greek letter
Globin chain is divided into

8 helices (A-H w/ num subgroups) &

7 nonhelical segments (connect

helical segments, flexible)
o NA-bet N-terminus & A helix

o
o
o

AB-bet A & B helices

Others-BC,CD,DE, EF, FG, GH
HC-H-helix and C-terminus

Complete Hb Molecule:
Primary structure: AA sequence of polypeptide
Secondary structure: Chain arrangements of helices and
nonhelices
Tertiary structure: helices into pretzel-like configuration
o Globin chainsloop to form cleft pocket for heme;
suspended bet E&F helices
o Iron of protoporpyrin IX ringpositioned bet 2 His
radicals forming proximal (bond in F8) and distal (in
E7) histidine through linked O2.
o Distal histidineswing in and out to permit the
passage of O2 in Hb molecule.
o Globin chain AA in cleft are hydrophobic while AA
outside is hydrophilic that makes Hb H2O soluble;
helps iron be in its ferrous (divalent) form
Quaternary structure (tertrameric molecule): complete Hb
molecule
o Spherical
o Has 4 heme molecules attached to 4 PP chains
o May carry 4 molecules of O2
o
o Has 1 heme grp attached
o Heme mol is capable of carrying 1 mol of O2
HEMOGLOBIN BIOSYNTHESIS
Heme Biosynthesis:
Mitochondria and cytoplasm of bone marrow RBC
precursors
Pronormoblast to polychromato[hilic RBC
1.
2.

Condensation: glycine + succinyl CoA cat by

aminolevulinate synthase aminolevulinic acid (ALA)
ALA cat by ALA dehydratase (aka ALA dehydrase,
porphobilinogen synthase) porphobilinogen (PBG)

2|Hema Topic Summary

3.
4.

PBG cat by porphobilinogen deaminase (aka

hydroxymethylbilane synthase) hydroxyl methylbilane
Fe2+ + protoporphyrin w/ ferrochelatase/heme synthase
HEME

Transferrincarries iron in ferric form (Fe3+) to developing RBCs

Globin Biosynthesis:
Six structural genes: Chrom 16-- and genes; Chrom 11:
, , , and .
All are 2 genes/person except and (4 genes/person)
Takes place in pronormoblast to polychromatophilic RBC
but not in mature RBC
Hb assembly:
Hb A
chains + 4 heme molecules; predominant
Hb in postnatal life.
Hb A2
in RBCs
Hb F
present in F cells
HB ONTOGENY:

Hb F is predominant at birth

Hb A in adults w/ small amts of Hb A2 and Hb F

Glycationposttranslational modification formed by

nonenzymatic binding of various sugars w/ globin chain
amino grps
o Hb A1cMost glycated

Glu attach to N-terminal Valine of

chain.

4-6% of Hb A in this form

Increased in uncontrolled DM
Stage
Intrauterine
Early embryogenesis (product of yolk sac RBC)

Begins in early embryogenesis; peaks during

midgestation and declines rapidly just before

Globin
Chain

Hb

2 + 2
2 + 2
2 + 2

Gower1
Gower2
Portland

2 + 2

birth
Birth

2 + 2
2 + 2

Adulthood

2+

2 + 2

2 + 2

F
F, 6090%
A, 1040%
F, 1-2%
A2,<3.5%
A, >95%

HB PRODUCTION AND REGULATION:

Heme regulation
Key limiting is rxn of glycine to succinyl CoA to form ALA by
ALAS
ALAS gene is inhibited by heme w/c decreases heme
production (negative feedback)
Others inhibited by heme: ALA dehydrase & PBG deaminase/
hydroxymethylbilane synthase
Ferrochelatase (heme synthase)inhibited by heme ( neg.
feedback) and PBG IX (substrate inhibition)
Globin regulation:
Regulated by the rate at w/c the DNA is transcribed to
mRNA.
Amt of specific globulins is proportional to globin mRNAs
Heminthe Fe3+ oxidation prod of heme
o Impt. In ctrl of rate of globin synthesis in intact
polychrome RBC and various cell-free systems
Hb regulation:
Stimulated by tissues hypoxia (inc. EPO in kidney)
Gen reference:
o Men: 14-18 g/dL (140-180 g/L)
o Women: 12-15 g/dL (120-150 g/L)
o Newborn: 16.5-21.5 g/dL (165-215 g/L)
@ high altitudesslightly elevated Hb for compensatory
mechanism to provide more O2 to tissues
HB FUNCTION:
Bind O2 in lung and transpo
Approx.. 1.34 mL of O2 is bound by each gram of Hb

3|Hema Topic Summary

Partial pressure of oxygen

P50 value
Sigmoidal curve
Bohr effect
27mmHg normal
Reference: arterial=80-100mmHg; venous=30-50mmHg
Shift to the rightlow oxygen affinity, Hb absorbs less oxygen
but deliver more oxygen (Hb S)
2,3-bisphosphoglycerate
Sigmoidal oxygen curve : myoglobin hyperbolic curve
Shift to the left
Shift to the right

low body temp.

inc body temp.

multiple transfusions of

inc 2,3-BPG
stored bld w/ depleted

inc H+ (dec pH)

2,3-BPG

abnormal Hb w/ low

alkalosis
affinity for oxygen

presence of Hb variants

high fever

acidosis

hypoxia: high altitude,

pulmonary insufficiency,
congestive heart failure,
severe anemia, and
cardiac right-to-left shunt

VARIANT HB:
Methemoglobincontains iron in ferric state
<1% of total Hb
brownish to bluish
elevated when there are: nitrites (oxidants), dec activity of
MHb reductase (genetic), Hb M dse (abnormality in globin
portion),
CO-oximeter, 620-640nm pH 7.1
Treatment: removal of offending substance, admin of
Ascorbic Acid or methylene blue
Sulfhemoglobinoxidation og Hb by sulfonamides, phenacetin,
acetanilide, or phenazopyridine
In vitro, add hydrogen sulfidegreenish pigment
Present with cyanosis

Treatment: avoidance of the offending agent

620nm, no shift when cyanide is added
Carboxyhemoglobincombi of carbon monoxide w/ heme iron
Affinity is 240 times that of oxygen and 10,000 slower release
CO: silent killer, odor and color less gas, quickly become
hypoxic
Reference interval: 0.2-0.8% endogenous
Exogenous: from pollutants
Smokers: 4-20%
10-15%--headaches and dizziness are experienced
>50%--coma and convulsions
541nm, blood cherry red color
Treatment: hyperbaric oxygen therapy.
HB MEASUREMENT:

Reference method: cyanmethemoglobin method

o Potassium ferricyanideferrous to ferric form
o Potassium cyanidew/ methemoglobinto form
stable pigment cyanmethemoglobin
o Color is proportional to Hb conc
o 540nm, compared w/ standard
o Others use sodium lauryl sulfate (SLS)SLS metHb
(doesnt produce toxic wastes

Hb electrophoresis: to separate types of Hb ( Hb A, A2, and F)

4|Hema Topic Summary