What's In a Red Blood Cell?

The Laboratory Investigation of
Hemolytic Anemia : A Logical
Approach
Mr. Andrew McFarlane
Supervisor Genetic Services
Hamilton Regional Laboratory Medicine Program
McMaster University Medical Centre

Conflicts of Interest
• Financial Conflicts of Interest: NONE
• Non-Financial Conflicts of Interest:
NONE
• Unlabelled/ unapproved Use Disclosure:
NONE

Objectives
• Review normal red cell physiology
• Discuss the pathophysiology and
clinical presentation of hemolytic
anemia
• Construct a practical diagnostic
approach to hemolytic anemia

A Red Blood Cell
A very flexible and deformable cell able to negotiate the 3 u
sinusoids of the spleen.

What’s In A Red Cell?
Intrinsic Components
‣ Phospholipid Membrane
connected to
‣ Protein cytoskeleton
contains
‣ Hemoglobin

Life Cycle of an RBC
~120 days
GLUCOSE
ANTIOXIDANT
PROTECTION
METHEMOGLOBIN
REDUCTION
4-6 days 3 days Glycolysis
HEMOGLOBIN
FUNCTION
MEMBRANE
iNTEGRITY

Normal erythrocyte destruction
120 days
1% of RBCs destroyed
every day
Reticulocytes produced to
replace RBCs

Hemolysis
• Heme is Greek for “blood”.
• Lysis means “Break down” in Greek.
• Hemolysis is the end stage of the
erythrocyte 120 life span which occurs
normal in the Reticulo Endothelial System
(RES) of the spleen.

Why do we see overt hemolysis?
Extracorpuscular defects
-acquired hemolysis
• ANTIBODIES against RBC
membrane (e.g., AIHA, DHTR,
some drug-induced anemias)
• SPLENIC TRAPPING
• TRAUMA (e.g. MAHA,
thermal burns)
• OXIDANT exposure (e.g.,
aniline dyes, dapsone,
pyridium)
• PATHOGEN destruction by
(e.g., malaria, babesiosis,
clostridium perfringens)

Why do we see overt hemolysis?
Intracorpuscular defectsinherited
hemolysis
• HEMOGLOBIN DEFECTS -
unstable variants (e.g. Hb Köln),
defective globin chains (HbS,
thalassemia)
• MEMBRANE DEFECTS –
altered or missing proteins (e.g.
hereditary
spherocytosis,elliptocytes,
abnormal hydration (e.g.
xerocytosis)
• ENZYME DEFECTS – defective
glycolysis (e.g. PK deficiency),
defective reducing power (e.g.
G6PD deficiency)

Increased hemolysis happens in 2 sites
1. Extravascular - localized in the spleen
• Intrinsic red blood cell defects
– Hemoglobin, membrane or enzyme problems
• Extrinsic red blood cell defects
– Liver disease
– Hypersplenism
– Infections (eg, bartonella, babesia, malaria)
– Oxidant agents (eg, dapsone, nitrites, aniline dyes)
– Autoimmune hemolytic anemias
2. Intravascular - in the circulation
• Direct trauma, shear stress, thermal burns
• Complement-induced lysis (i.e., PCH)
• Lysis from osmotic change, bacterial toxins

EXTRAVASCULAR
• RED CELLS REMOVED VIA RE
SYSTEM
BONE MARROW
Fe++
RBC
RE SYSTEM
globin
BILIVERDIN
L
I
V
E
R
Glucoronal
transferase
UNCONJGATED
BILIRUBIN
GUT
UROBILINOGEN
acted on by
bacteria
CONJUGATED
BILIRUBIN
STERCOBILINOGEN
excreted in feces

INTRAVASCULAR
• CELLS LYSED IN VESSELS
FREE HB
HAPTOGLOBIN
b globulin
HB
HAPTOGLOBIN
Binds at 1:1 ratio (NR 0.3-
0.75 g/l )
Excreted by phagocytic cells-
30min half life
FREE HEME
HEMOPEXIN
FREE HEME
HEME
ox
albumin
HEMOPEXIN
a globulin Binds at 1:1 ratio(NR
0.6-1.0 g/l)
Excreted by hepatic cells - 3.5hr half
life
Excreted by
phagocytic cells -half
METHEMALBUMIN life 20hrs

Excess Extravascular hemolysis

Case: A post-operative patient with a
sudden hemoglobin drop
• 47 yo Italian-Canadian woman, sphenoid tumour
resection and post-op right MCA stroke
• Past medical history:
– Diabetic
– Depression/anxiety
• Sudden hemoglobin drop on post op day 2 and 32
– Admission: Hb 158
– Post op day 1 : Hb 114 dropped to 83 transfused
– Post op day 29: Hb 110
– POD 32: Hb 68 –Hematology consulted

Identifying hemolysis
• Clinical clues
– Rapid fall in hemoglobin with no evidence of blood
loss
– Jaundice
– Dark urine, pale stools, pigmented gallstones
– Splenomegaly
• Laboratory clues
– Reticulocytosis
– Increase in serum lactate dehydrogenase (LDH) and
indirect bilirubin concentrations

Does the patient have increased
hemolysis?
1. Increased erythrocyte destruction
2. Increased erythrocyte regeneration

HEMOLYTIC SCREEN
• CBC - include Retic count
• RBC MORPHOLOGY- fragments,
membranes
• SUPRAVITAL STAINS
– Incubated BCB
– Methyl violet
• BILIRUBIN TOTAL AND DIRECT
• HAPTOGLOBIN ELECTROPHORESIS
• COOMB’S TEST (DAT)

Haptoglobin Electrophoresis
• Protein electrophoresis utilizing agar matrix
• Barbituate buffer – alkaline pH
• Electrophoresed @ 4 o C for 1hr at
10 mamps / plate
• Stained with a heme specific dye
• Assesses presence of free hemoglobin, hemopexinheme
complex, methemalbumin
• Gives a semi-quantitative measurement of
haptoglobin

1volume
Plasma/Serum
1 volume
0.6g/l free
Hb solution
Completely binds all
free Hb added
Partially binds free
Hb added
Incubated @
room temperature
for 30 mins
Does not bind any free
Hb added

Haptoglobin Agar Gel
Plasma
+
Controls
Normal Reduced Absent
1:1 mix with
0.6g/l Hb
Free Hb
Hemopexin/Heme
complex
Hb/Hapt
complex
Methemealbumin
_
Lane 1 2 3 4 5 6 7 8

Haptoglobin Electrophoresis
MHA
Hb/HAPT
Heme/Hx
Free Hb

Case: A post-operative patient
with a sudden hemoglobin drop
• Clinical assessment
– No evidence of blood loss
– Jaundice, pallor, dark urine
– Abdominal ultrasound
• Gallbladder stones and sludge, thickening of gall bladder wall
• Splenomegaly not noted
• Laboratory assessment
– Ferritin elevated at 2029 ug/L
– Reticulocyte count elevated at 246 x 10 9 /L, RPI 2.6
– LDH elevated at 1977 U/L (ref 110-220 U/L)
– Bilirubin elevated at 28 umol/L (ref

Further hemolytic testing
Haptoglobin
Free Hb
Hemopexin-Heme
Methemalbumin
Osmotic fragility
Hb electropheresis
G6PD Assay
Pyruvate kinase assay
• Absent
• Absent
• Absent
• Normal
• Abnormal = Hb Setif [HBA2c.283G>T]
• Normal
• 1.59 U/mL RBC (ref 1.87-3.90)

GLUCOSE
Met Hb
reduction
Pyruvate kinase
Glucose-6 P
Fructose-6 P
Fructose-1,6 BP
Glyceraldehyde-3 P
1,3 BP-Glycerate
3 P-Glycerate
2 P-Glycerate
Phosphoenol –
Pyruvate (PEP)
Pyruvate
Lactate
ADP
ATP
Hexose
monophosphate
shunt
Rapoport-
Luebering
shunt
Adapted from Israels et al.
Mechanisms in Hematology.

Impact of pyruvate kinase deficiency
on the red cell
• Red cell can’t produce enough energy to maintain
integrity, causing hemolysis
• Metabolites upstream to PK accumulate
– PEP
– 2,3 BPG (from Rapoport-Luebering shunt)

Genetics of PK Deficiency
• Gene frequency is 1-2%,
distributed worldwide
• Autosomal recessive
inheritance
• Phenotype varies!
• 158 mutations reported
Zanella A et al. Blood Reviews 2007:21,217-231.
Anastasiou D et al. Nature Chemical Biology 2012: 8, 839–847.

Genetic testing in post-operative
• PKLR gene analyzed by
PCR and direct
nucleotide sequencing
of coding exons
• Patient heterozygous
for pARG486Trp
(c.1456C>T)
• Missense mutation of
pyruvate kinase gene
• Mild clinical phenotype
patient
Beutler E, Gelbart T. Blood 2000, 95(11):3585-3588.

Clinical consequences of pyruvate
kinase deficiency
• Lifelong chronic hemolysis may have anemia
• Splenomegaly
• Gallstones
• Iron overload
– from chronic transfusion therapy, ineffective
erythropoiesis, synergistic effect of
hemochromatosis mutations
• Rare outcomes
– transient aplastic crises, folate deficiency,
neonatal jaundice and kernicterus
Zanella A et al. Br J Hem 2005.

Treatment of PK deficiency
• No specific therapy available
• Red cell transfusions for severe, symptomatic
anemia
• Splenectomy for severe, symptomatic anemia
and transfusion dependence
– Does not stop hemolysis, but can increase Hb
– No way to predict therapeutic efficacy
• Iron chelation may be needed
• Gene therapies on the horizon
Zanella A et al. Br J Hem 2005.
Tanphaichitr VS et al. Bone Marrow Transplant. 2000;26(6):689.
Tani K et al. Blood. 1994;83(8):2305.

Hb Setif: Unstable Hb Testing
• Isopropanol Instability Test
• Heat Denaturation Test

HEINZ BODIES
• Denatured hemoglobin - intracellular body
stained by supravital stains
• Seen in oxidative hemolysis
• Usually shows typical “bite cells”
‣G6PD deficiency
‣Unstable Hemoglobin
‣Oxidative drugs

Methemoglobinemia
• Several chemical and drugs can increase
MetHb levels.
• An acquired MetHb is suggested when
sudden cyanosis appears with the absence
of cardiopulmonary pathology.

Methemoglobin
• Normal levels of less than 1%.
• Increase of MetHb causes cyanosis because
of the decreased O2 delivery.
• Can be genetic causes- Hb M or deficiency
of NADH-dependent cytochrome b5
reductase (b5R)
• Exogenous oxidizing agents.

Glucose-6-phosphate
dehydrogenase deficiency
(G6PD)
• The most common enzymatic disorder of
the human erythrocyte.
• estimates =>200-400 million people world
wide.
• Most are asymptomatic some will have
hemolytic episodes or chronic hemolysis.

G6PD Deficiency
• Lack of G6PD means decreased NADPH
and therefore an inability to regenerate GSH
• Globin denaturation and precipitation -
Results in Heinz bodies and bite cells
• Damaged membrane, non-deformability
• Extravascular hemolysis
• X-linked disease

Hexose Monophosphate Shunt
• Production of NADPH, which is used to produce
GSH (Glutathione) the main protection vs oxidative
injury

WHO Classification of G6PD
• Class I- Severe deficiency with chronic anemia.
• Class II- Severe deficiency with intermittent
hemolysis.
• Class III- moderate deficiency with intermittent
hemolysis- associated infections and drugs.
• Class IV- No enzyme deficiency or hemolysis
• Class V- increased enzyme activity.

Fluorescent spot test for G6PD
• Small amount of blood incubated with G6P
and NADP .
• G6PD enzyme source is patients whole
blood
• Spotted every 5 minutes on filter paper.
• Normal will show fluoresence in 5-10
minutes.
Glucose-6-Phospate +NADP
No Fluorescence
G6PD
6-Phosphogluconate +NADPH
Fluorescence

0
TIME
5 10 15 20
Mediterranean
G6PD
Normal
Intermediate
G6PD
Reagent

Glucose 6 Phosphate Dehydrogenase
Deficiency Assay
Buffer containing:
Glucose 6 Phophate, NADP
Patients Whole
Blood
Inc @ 30oC
Using average
change in OD
/min,millimolar
absorptivity of
NADP and patients
Hb level
Calculate units of
activity in U/g Hb
OD read each minute at 340nm
for 10 minutes

G6PD Assay
• Same reaction and principle as screen.
• Performed at specific temperature and
measured on a spectrophotometer at 340
nm.
• Measure rate of change in Absorbance.
• Reference = 146-376 U/10 12 RBC
4.6-13.5 U/g Hb

Other Enzyme Deficiencies
• Pyrimidine 5’ Nucleotidase Deficiency
– Marked basophilic stippling due to deficiency.
– Prevents RNA degradation products from leaving the maturing reticulocyte.
– Results in a build up of insoluable pyrimidines.
• Hexokinase deficiency
– Inability to phosphorylize glucose and therefore keep it
intracellular
• Glucose Phosphate Isomerase Deficiency
• Phosphofructokinase deficiency
• Triosephosphate isomerase deficiency
• Phosphoglycerate kinase deficiency
• Adenylated Kinase deficiency
• Aldolase Deficiency
• Lactate Dehydrogenase deficiency
• Biphosphoglycerate Mutase deficiency

Basophilic Stippling
• Pyrimidine 5’ nucleotidase deficiency

Conclusions: Take-home
message
• A systematic approach to for investigation of
unexplained anemia is be useful – CBC including the
retic count and red cell morphology.
• Think about hemolysis in all patients with anemia
– Careful review of basic labs are useful for identification of
hemolytic anemia
– Consider causes of hemolysis congenital -inside the RBC
and acquired - outside the RBC.
• Patients with chronic, sometimes well compensated
and stable hemolytic conditions can develop acute
exacerbations with other medical conditions