VASCULAR BLEEDING DISORDERS

Interpreting screening results

Normal PT and APTT and Normal Platelet count
Vascular purpura
Screening tests are usually normal (i.e. bleeding disorders of
vascular tissue abnormalities)
Include:
o
Hereditary Hemorrhagic Telangiectasia
o
Ehlers-Danlos Syndrome
o
Osteogenesis Imperfecta
o
Scurvy
o
Steroid Induced-Purpura
o
Small Vessel Vasculitis
o
Purpura Associated with Presence of Paraprotein
INHERITED VASCULAR DISORDERS
Hereditary Hemorrhagic Telangiectasia
Rendu Osler Weber Syndrome
Dilated Superficial blood vessels that create small, focal red
lesions
Are fragile and prone to rupture
Confused with petechiae permanent bright red or purple spot on
the face, nose, lips and tongue
CFT: Abnormal (3+ or 4+)
B.T: Normal or Prolonged
P.C: Normal
Ehlers Danlos Syndrome
Hyper extensible skin, hypermobile joints and fragile tissue
Easy bruisability arterial rupture
Due to defects in skin collagen and structure
CFT: Abnormal (4+)
B.T: Depends on platelet count
OTHER INHERITED VASCULAR DISORDERS
Pseudoxanthoma Elasticum
Homocystinuria
Marfan Syndrome (Skeletal and ocular defects)
Osteogenesis Imperfecta
ACQUIRED VASCULAR DISORDERS
Allergic Purpura (Henoch Schonlein purpura)
o
Allergic Purpura
o
Anaphylactoid Purpura
Condition accompanied by transient arthralgia, nephritis,
abdominal pain, purpuric skin lesions
Affects boys (3-7 years old)
Skin rash (palpable purpura) and edema
Vasculitis mediated IgA ab (Autoimmunity to vessel walls)
May be accompanied by itching, tingling sensation erythema
found in buttocks and legs
Foods, drugs, cold, insect bites, vaccinations, after URTI (hemolytic streptococcus)
o
Renal lesions proteinuria, hematuria
Senile Purpura
Found in elderly, particularly on the areas exposed to sunlight
Caused by atrophy of dermal collagen
Loss of subcutaneous fat and weakening of the blood vessels of
the skin (ecchymoses)
Scurvy
Vitamin C deficiency
Petechiae: due to defect in the microvascular supporting tissue
caused by decreased synthesis of collagen
Presence of deeper hematoma
Drug Induced vascular Bleeding disorders
Due to toxic damage to the endothelium of immune complex-type
hypersensitivity
Allergic purpura erythematous and purpuric eruption as
hypersensitivity reaction to allopurinol
Purpura seen in SLE erythematous reaction
Mucosal hemorrhage from nasal and oral cavities
PURPURA ASSOCIATED TO INFECTION
Meningococcus septicemia
Typical purpuric skin lesions around the ankle with intravascular
coagulation
Herpes Zoster

Hemorrhagic herpetic skin eruption over the buttocks in patient

with acute leukemia
o
Herpes 1: affects upper portion (mouth, etc.)
o
Herpes 2: sexually transmitted
Infectious Mononucleosis
Extensive petechiae on the mucosa of the palate
Purpura fulminans
Large necrotic ecchymoses of leg and penis due to varicella
infection
PLASMA BLEEDING DISORDERS
Purpura Due to Abnormal Proteins
Paraproteinemia
Seen in multiple myeloma, benign, monoclonal gammopathy,
cryoglobulinemia or cryofibrinogenemia
Amyloidosis- deposition of amyloid in capillary that lead to
damage of normal cells
Interference platelet function and fibrin formations resulted from:
o
Hyperviscosity of blood
o
Damage to vessel due to precipitation of protein in cooler
parts of the skin
HEMORRHAGIC COAGULATION DISORDERS
Hemorrhage
Severe bleeding
Localized hemorrhage
Single location
May be due to trauma, infection, or isolated blood vessel defect
Generalized hemorrhage
Multiple sites
Spontaneous and recurring
Requires intervention and transfusion
Primary/ secondary hemostasis disorder
Mucocutaneous hemorrhage
Associated with thrombocytopenia, vWD, qualitative platelet
disorders, or vascular disorders
Symptoms:
Purpura- bruises, purple lesion of the skin caused by seeping of
RBC
a.
Petechiae- <3mm
b.
Ecchymoses- >3mm
c.
Menorrhagia- profuse menstrual flow
d.
Hematemesis- vomiting of blood
e.
Epistaxis- uncontrolled nose bleed
Anatomical hemorrhage
Seen in acquired/ congenital defects in secondary hemostasis

ACQUIRED DEFECTS
-

A.
-

Clinical manifestation first occurs in:

o
Adulthood
o
associated with another disease (liver disease, vit. k
deficiency, renal failure)
o
Not found in relatives
o
Due to drug exposure
Examinations: CBC, PT, PTT, fibrinogen, thrombin time
Acute Coagulopathy of Trauma and Shock
Fatal hemorrhage
Elements of systemic shock (ACOTS trigger):
o
Injury related acute inflammation
o
Platelet activation
o
Tissue factor release
o
Hypothermia
o
Acidosis
o
Hyperfusion
Plasma Expanders (5% dextrose)
o
Used to counteract hyperfusion
o
Intensifies coagulopathy
Massive Transfusion
o
4-5 RBC units within 1 hour or 8-10 units within 24 hours

Systolic - <110 mmHg

Pulse Rate - >105 beats per minute

pH - <7.25

Hematocrit - <32%

Hemoglobin - <10 g/dL

PT Prolonged (INR of 1.5)

Management of ACOTS

RBC transfusion
1.
Moderate Thrombocytopenia
Ordered when hemoglobin < 6.0 g/dL and are contradicted when
o
Plt count of 50,000-99,000 mg/dL
the hemoglobin concentration is between 6-10 g/dL
o
Causes:
If the Hemoglobin concentration is between 6-10 g/dL, the

Shortened plt survival

decision to transfusion is based on:

Sequestration associated with portal hypertension and

o
Physical evidence of blood los
hepatosplenomegaly
o
Current blood loss rate

Alcohol toxicity suppresses platelet production

o
Blood Pressure
2.
DIC
o
Arterial Blood Gas Values (pH and oxygen saturation)
o
Causes:
o
Urine Output

production of regulatory antithrombin, protein C and

o
Laboratory Evidence for Coagulopathy
protein S

During surgery coagulopathy is assessed through

Released of activated procoagulants from degenerating

microvascular bleeding
liver cells
Platelet concentrate

Liver does not clear activated procoagulants that are

Ordered when platelet count is <50.000/uL
normally produced from abdominal organs and present
Ineffective for those with ITP, TTP or HIT (Platelets are rapidly
in circulation
consumed)
o
Laboratory Examination
Fresh Frozen Plasma

Acute DIC
Thawed at 370C

PT- prolong
Transfused when there is:

PTT- prolong
o
Microvascular bleeding (till 30% coagulation factor activity

Thrombin time- prolong

is reached)

Fibrinogen (<100 mg/dL)

o
Prolonged PT (> 1.5 times mean of PT reference interval)

FDP, D-dimer
o
Prolonged PTT (>2 times)

Chronic DIC (Compensatory DIC)

o
Pre-existing single factor coagulation deficiency (when no

D-dimer
factor concentrate is available)

Therapy
o
Hemorrhage from warfarin overdose

FFP
10-15 mL/kg
Store at 1-60C up to 5 days (vWF, FV, FVIII declines upon storage)

Platelet concentrate
Adverse effects:

Activated protein C
o
TRALI

Antithrombin concentrates
o
TACO

Can often lead to Adult Respiratory distress syndrome

Hemostasis Test in Liver Disease

FP can also cause Anaphylaxis, Thrombosis, and

Multiple Organ Failure
1.
Factor V
Cryoprecipitate
2.
Factor VII
Indicated when there is:
3.
Reptilase Time
o
Microvascular bleeding
o
Reagent: Bothrops atrox venom
o
Fibrinogen concentration is <100mg/dL
o
Cleaves fibrinopeptide A
Lower risk of TACO
o
Insensitive to heparin
15-20mL cryoprecipitate= 150-280 mg/dL fibrinogen
Activated Prothrombin Compkex (FEIBA) (Autoplex)
Treatment to Resolve Liver Disease-Hemorrhage
Administered when RBC, Platelet, Cryoprecipitate, and FP fails to achieve
hemostasis
o
Vit. K
FEIBA contains activated coagulation factor = risk for DIC

Corrects bleeding associated with des-y-carboxyl FII, VII,

Dosage: 50 units/kg every 12 hours (not >200 units/kg in
IX, X
24hours)
o
FFP
Recombinant Activated FVII

Provides all coagulation factors

Used in treating hemophilia A & B when FVII/FIX inhibitors are

vWF, FV, FVIII may be reduced

present

Unlikely to return PT to normal

Dosage: 30mcg/kg

1 unit FFP = 200-280mL (typical dose: 2 units)

Effective in stopping microvascular bleeding in non-hemophilic

>30mL/kg may cause TACO

patients
Does not cause DIC
o
Cryoprecipitate
Possible link with arterial & venous thrombosis

For fibrinogen <50mg/dL

Anti-Fibrinolytic Drug (Tranexamic Acid-Cyklopan)

FP and Cryoprecipitate increases risk for viral

Dosage: 1 g over 10 mins followed by 1g over 8 hours
transmission
Prevents bleeding in hemophilic patients in invasive surgery

Allergic transfusion reaction is common with plasma

Effective for ACOTS
containing products
o
Platelet concentrate
Laboratory Tests
o
Prothrombin complex
Platelet count
o
Activated protein C
PT
o
Anti-thrombin concentrate
PTT
o
Novoseven
Platelet aggregometry (platelet function efficacy)
o
Cyklokapron
Coagulation assay
Hemostasis Laboratory Test in Liver Disease
Liver Disease
Liver produces nearly all of the plasma coagulation factors and
Fibrinogen Assay
>400mg/dL in early, mild liver disease
regulatory proteins
<200mg/dL in moderate to severe liver disease/
Esophageal Varices - enlarged and collateral esophageal vessels due to Chronic
dysfibrinogenemia
Alcoholic Cirrhosis
Affects vit. K dependent factors
Thrombin Time
Prolonged owing to dysfibrinogenemia,
Becomes des-y-carboxyl of
fibrinogen deficiency, or elevated fibrin
Markers of liver disease:
degradation products
o
FVII (First to decrease)
Reptilase Time
Prolonged in hypofibrinogenemia
o
FV (more specific for liver function test)
Significantly prolonged in dysfibrinogenemia
Dysfibrinogenemia: presence of fibrinogen with excessive sialic
PT
Prolonged in mild liver disease
acid residues caused by liver disease
PTT
Mildly prolonged in severe liver disease
Symptoms: mild tissue bleeding
Platelet Count
Mild thrombocytopenia in severe liver disease
Examination: thrombin time, RT
(<100,000 x 109/L)
Fibrinogen
Platelet
Mild suppression of platelet aggregation &
APR
Aggregometry
secretion but test is not clinically predictive of
Elevated in early/mild liver disease
bleeding
<100 mg/dL in end stage renal disease
FDP
>0.25mg/mL by semiquantitative immunoassay
D-Dimer
>240ng/mL by quantitative assay
Platelet Abnormalities in Liver Disease
B.
-

Euglobulin Lysis
Time

Lysis in<2hrs in primary or secondary systemic

fibrinolysis

C.

Renal Failure and Hemorrhage

Platelet adhesion and aggregation are suppressed because of

platelet coating by gunidisuccinic acid or dialyzable phenolic
compounds
Acute Renal Failure Failure - Associated with Gastrointestinal
Bleeding
Chronic Renal Failure - Associated with Platelet Dysfunction and
Mild to Moderate Mucocutaneous Bleeding
How
o
o
o
o

to correct bleeding?
Dialysis
EPO
RBC transfusion
IL-11 therapy

Hemostatic Activation Syndrome

o
DIC
o
HUS
o
TTP
o
Transplant rejection
o
Glomerulonephritis of the SLE
o
Deposition of fibrin in the renal microvasculature affecting
the glomeruli
Treatment
o
DDAVP (Desmopressin)- IV, increases vWF
o
Stimate- Intranasal
D.

Nephrotic Syndrome and Hemorrhage

Low molecular weight proteins are lost

Increased glomerular permeability associated with:
o
Chronic glomerulonephritis
o
Diabetic glomerulosclerosis
o
SLE
o
Amyloidosis (production of glycoproteins)
o
Renal vein thrombosis

What is lost? (@ urine)

o
FVII, FIX, FX, FXII
o
Antithrombin, Protein C

E.

Vitamin K Deficiency

Vitamin K is fat soluble and regulates bile salts for absorption

Causes of deficiency:
o
Biliary duct obstruction
o
Atresia (congenital absence/ undeveloped bile duct)
o
Fat malabsorption
o
Chronic diarrhea
o
Use of broad spectrum antibiotics
o
Breast feeding in newborns (antibodies present in the
mother are ingested by the baby causing loss of the normal
flora of the baby)
Vitamin K Agonists
o
Warfarin/ Coumarin (dysfunctional FII, VII, IX, and X, and
Proteins C, S, and Z PIVKA)
o
Brodifacoum (Superwarfarin)
Detection of Vitamin K Deficiency or Proteins in Vitamin K
Antagonism
o
PT- prolonged
o
PPP + Patient Plasma Normal PT and PTT results = factor
deficiencies

F.

Autoanti-VIII Inhibitor (Aquired Hemophilia)

Most common inhibitor

Autoantibody to Factor VIII - Diagnostic of Acquired Hemophilia
Associated with:
o
Rheumatoid Arthritis
o
Inflammatory Bowel Disease
o
Systemic Lupus Erythematosus
o
Lymphoproliferative Disease
Laboratory Tests:
o
PT, PTT, and Thrombin Time - Recommended tests
o
Factor VIII Inhibitor Presence - Prolonged PTT, Normal PT and
Thrombin Time; Factor VIII Assay <30%
o
PTT Prolongation corrected by NP:Specimen Ratio of 1:1
o
Quantitation of Autoanti-VIII: Bethesda Titer

CONGENITAL DEFECTS
-

Uncommon, occurs in 1 in 100 individuals

Diagnosed in
o
Infants, young children
o
With relatives who has similar symptoms
Leads to repeated hemorrhages
o
. Spontaneous
o
After minor injury (vWD, FXIII, FIX deficiency)

A.

HEMOPHILIA

Hemo meaning blood, philia meaning tendency toward

Known since the beginning of the first millennium, hemophilias
are among the most frequent inherited disorders of blood
coagulation and definitely the most severe
Blood of individual with hemophilia will not clot normally due to
deficiency of a particular coagulation factor.
Bleeding may occur spontaneously or follow an injury
Suspected in patients with:
o
Recurrent bleeding
o
Unexplained hemarthrosis
o
Prolong PTT
Most common bleeding disorders: vWD, hemophilia

Signs and Symptoms:

1.
Many large or deep bruises
2.
Joint pain and swelling caused by internal bleeding
3.
Muscle bleeding
4.
Blood in urine/ stool
5.
Prolonged bleeding from cuts or injuries or after surgery or
tooth extraction
Bleeding
1.
2.

3.

Mild (5%-30%)

Bleeding occurs only under severe stress (major

injuries)
Moderate (1%-5%)

Rare

Prolonged bleeding after surgery or trauma

May lead to spontaneous bleeding

Severe (less than 1%)

Rare

Spontaneous bleeding (bleeding without any

recognizable trauma)

Usually in joints

Hemophilia A
Classic Hemophilia
Hereditary bleeding disorder caused by lack of blood clotting
factor VIII
Gene: Chromosome X
Occurs in 1 in 10,000 people
7 times more common than hemophilia B
S/S
o
History
o
Newborns: continuous bleeding of umbilical cord
o
Child: Massive hematoma upon crawling
Note: Adults may develop bleeding disorder similar to hemophilia A
o
Post-partum in women with autoimmune disease
o
Individuals with cancer (most commonly leukemia and
lymphoma)
o
Idiopathic
Laboratory Diagnosis:
o
Unhemolyzed specimen (2mL)
o
PTT Prolonged
o
Thrombin Time and PT - Normal
Treatment for Hemophilia A
1.
2.

3.
4.

FVIII concentrate

Replaces missing clotting factor, amount needed

depends on the severity, size and site of bleeding
DDVAP

For mild hemophilia

Helps body release FVIII that is stored within the lining

of blood vessel
Immunization with Hepatitis B vaccine

Necessary for increased risk of exposure to hepatitis

due to frequent blood infusions
Other clotting factors (FVIIa)

For patients who developed an inhibitor to FVIII

Helps clotting even without FVIII

Hemophilia A and Factor VIII Inhibitors

-

Inhibitors - Immunoglobulin G4 and Non-complement-fixing,

warm-reacting antibodies
First step in detection of inhibitors - Factor VIII Assay
FVIII Activity >30% - No inhibitors
FVIII Activity <30% - Proceed to mixing studies
Prolonged PTT - Mix with 1:1 NP, incubate for 1-2 hours at 37C. If
no inhibitors - incubated mixture should produce a normal PTT
Result; if inhibitors are present - Prolonged or uncorrected PTT
Use Bethesda Titer for quantitation
o
Low Responder - Titers of 5 Bethesda Units or less
o
High Responder - Titers of 5 Bethesda units or more

Treatment of Hemophilia A in patients with Inhibitors

Low responders experience cessation of bleeding upon
administration of large doses of FVIII Concentrate
o
High Responders may gain no benefit from FVIII
Concentrates and instead are treated with Activated
Prothrombin Complex Concentrate (DOSAGE SHOULD NOT
EXCEED 200 UNITS/KG PER DAY)
Hemophilia B
Christmas disease
Factor IX Deficiency
Named after Stephen Christmas (1st patient diagnosed with
hemophilia B)
Occurs in 1 in 40,000 people
o

Treatment for Hemophilia B

1.
2.

FIX concentrate

Replaces missing clotting factor, amount needed

depends on the severity, size and site of bleeding
Immunization with Hepatitis B vaccine

Necessary for increased risk of exposure to hepatitis

due to frequent blood infusions

Pattern of Inheritance for Hemophilia A and B

-

X linked recessive trait

Females with 1 affected X gene does not exhibit the disease but
serves as a carrier
Males with 1 affected X gene exhibits hemophilia A or B
All female children of men with hemophilia carry the defective
gene

Affected fathers, normal mothers

o
all daughters of affected males are carriers of a mutant allele
o
all sons of affected males are unaffected

Carrier female have a:

o
25% chance of transmitting normal allele to a son
(unaffected)
o
25% chance of having an affected son
o
25% chance of having a daughter who carries a mutant gene
o
25% chance of having a daughter who received normal allele

Genetic mutations transmitted from an affected father to his

daughters:
o
daughter's son have 50% chance of inheriting mutation
o
daughters daughter have a 50% chance of being carriers
o
Note: genetic mutation is almost never transmitted from an
affected father to a son

Affected mother, Affected Father

o
100% chance of having an affected daughter
o
50% chance of having an affected son

Hemophilia C
Rosenthal Syndrome
Factor XI Deficiency
Rare
Predominantly occurs in Jews of Ashkenazi descent
Can be distinguished by Hemophilia A & B by absence of bleeding
into joints and muscles and by occurrence in individual of either
sex
Bleeding risk is not always influenced the severity of the factor
deficiency
More difficult to manage than Hemophilia A and B
1 in 100,000 population
Autosomal dominant
Physical findings are usually normal except when bleeding occurs
Signs and symptoms:
o
Pallor

o
Fatigue
o
Tachycardia with excessive bleeding
Acquired FXI deficiency occurs in patients who develop inhibitors
to the protein as sometime observed in patients with SLE and
other immunologic disease

B.

VON WILLEBRAND DISEASE

Named from Dr. Erik Adolf von Willebrand, finnish pediatrician

who 1st described the condition in 1926
vWD was first termed as pseudohemophilia
affects both sexes (autosomal dominant)

vWF
-

Glycoprotein (800,000-20,000,000 D)
Largest molecule
0.5-1.0mg/mL (plasma concentrate)
Synthesized in endoplasmic reticulum
Endothelial cells
Storage:
o
Weible Palade bodies
o
Alpha granules
Found in Chromosme 12
Protects FVIII from proteolysis
Type 1 VWD
60-80%
Quantitative defect
Exhibits asymptomatic to mild bleeding following surgery.
Menorrhagia is common in women
Type 2 VWD
Qualitative defect
a.
Subtype 2a VWD
o
10-20% of cases
o
Mutations in A2 structural domain that renders vWF more
susceptible to proteolysis
o
There is a predominance of small molecular weight
multimers
b. Subtype 2b VWD
o
Rare
o
Mutations in A1 domain
o
Increased affinity of vWF for GP Ib/V/IX
o
gain of function
o
Large vWF spontaneously binds to resting platelets and
unavailable for normal platelet adhesion
o
Moderate thrombocytopenia due to chronic platelet
activation
c.
Pseudo-VWD
o
Platelet mutation
o
GP Ib/IX/V affinity to normal VWF multimers
d. Subtype 2M VWD
o
Decreased platelet receptor binding but normal multimeric
pattern
e.
Subtyoe 2N VWD
o
Mutation impairs factor VIII binding site resulting in FVIII
deficiency
o
Autosomal hemophilia (clinical symptoms are
indistinguishable from hemophilia)
Type 3
Autosomal recessive vWF gene translation or absence
Severe mucocutaneous and anatomic hemorrhage
vWF is nearly absent, FVIII is dimished, Hemostasis is impaired
Acquired Von Willebrand Disease
Associated with
o
Hypothyroidism
o
Autoimmune disorder
o
Lymphoproliferative disorder
o
Myeloproliferative disorder
o
Benign monoclonal gammopathies
o
Wilms tumor
o
Intestinal angiodysplasia
o
Congenital heart disease
o
Pesticide exposure
o
HUS
Manifests moderate to severe mucocutaneous bleeding with no
significant medical history
Involves presence of autoantibodies
PTT Prolonged, PT normal
Diminished vWF activity, presence of vWF antigen, no bleeding
history
Treatment: DDAVP or FVIII/vWF concentrate
C.

CONGENITAL SINGLE FACTOR DEFIECIENCY

Factor V Deficiency
Bleeding time- prolonged

Platelet aggregation- normal

PT/PTT- prolonged
Treatment: Plasma concentrate
FV and FVIII Combined Deficiency
Defect in Chromosome 18
Factor VII Deficiency
PT- prolonged
Treatment: Novoseven, prothrombin complex concentrate
Factor X Deficiency
Described in Amyloidosis, Paraproteinemia, Antifungal Drug
Therapy
PT and PTT Prolonged
Russell Viper Venom Time Test
o
Activates the coagulation mechanism at Factor X
o
Prolonged in FX, FV, Prothrombin, and Fibrinogen
deficiencies
o
Useful in distinguishing FVII Deficiency
Treatment: FFP, prothrombin complex concentrate

Inhibits recurrence of DVT or PE

After AMI if the event is complicated by congestive
heart failure or coronary insufficiency
Dosage

5-mg daily oral dose

>70 y/o, debilitated, malnourished, or have

CHF,inherited warfarin sensitivity = 2 mg/day
First 2 or 3 days therapy = risk for thrombotic event

Administer UHF/LMWH for the first 5 days

Warfarin skin necrosis (severe reaction requiring

dbridement of dead tissue)
Monitoring (PT)

Reagent: TF +PL + Ionic calcium

First assay: 24 hours after therapy (Prolong within

24hours)

Therapeutic only when activity of FII & X decrease to

less than 50% in 4-7 days

Monitoring: every month (for 6 months, Atrial

fibrillation: lifelong)

Overdose: hemorrhage, underdose: thrombosis

Threapeutic Range (INR): 2-3

>4= hemorrhage

5= immediate communication

Factor XIII Deficiency

PT, PTT, Thrombin time- prolonged
5M Urea Solution- weak clots dissolve within 2 hours
Confirm through Chromogenic Substrate Assay
2-Antiplasmin and PAI-1 - Moderate to Severe Bleeding

Type

Incidence

Type I
Type II
Type III

Rare
Frequent
Rare

FXIII
Activity
Absent
Absent
Low

-Protein

-Protein

Absent
Normal
Absent

Absent
Low
Low

INR=

ANTICOAGULANT MONITORING

Coumadin

Unfractionate
d Heparin
(UFH)

Administration
Action

Oral
Vitamin K
antagonist

Effect
Duration

Slow acting
Long

IV
Catalyzes
inhibition of
thrombin, Xa,
& IXa by AT
Immediate
Short

Test for
monitoring

PT

APTT

Other

production
of F II,VII, IX, X

Requires AT to
be effective

Low Molecular
Weight
Heparin
(LMWH)
Subcutaneous
Catalyzes
inhibition of
Xa by AT

Immediate
Longer than
UFH
Monitoring
usually not
required
If needed Antifactor Xa
APTT is
insensitive to
LMWH

o
A.

WARFARIN THERAPY AND PT

o
Vitamin K

Found in fish, liver and meaty vegetables

Produced by intestinal Bacteroides fragilis & Escherichia

coli

Fat soluble

Necessary for -carboxylation of glutamic acid residues

in the GLA domains of proteins

Carboxylation is for binding of CF to negatively charge

phospholipids via Ca++
o
PIVKA

Protein induced by Vitamin K deficiency

Vit K dependent factors that lack COOH modification

Des-carboxy form (Lacks 2nd carboxyl group added

to carbon

Cannot bind Ca2+, cannot participate in coagulation

Basis for warfarin therapy

WARFARIN
o
Vitamin K Antagonist (affects F II, VII, IX, X)
o
member of the coumarin drug family
o
suppresses -carboxylation of glutamic acid by slowing the
activity of the enzyme vitamin K epoxide reductase
o
Uses:

Prevent strokes in patients with atrial fibrillation

Prevent VTE after trauma, orthopedic surgery, or

general surgery

Chronic medical conditions

reaction time of sample

( geometric
meanof 20 normal plasma )

INR unreliable during first five days, monitor PT result

only

Normal PT: 11.9-14.2

Normal INR: 0.9-1.19

Chromogenic Factor X Assay

Alternative to the PT/INR system, eliminates

normalization

Therapeutic range: 20% to 40% of normal factor X

activity
Effects of Diet in Warfarin Therapy

Dietary vitamin K- warfarin effect INR

Drugs metabolized through P450- warfarin effect

INR

Amiodarone, metronidazole, cimetidine- warfarin

effect INR

Pregnancy- contraindicates warfarin, use LMWK instead

Effect of Polymorphism

CYP2C9*2 and CYP2C9*3

Reduce cytochrome P450 pathway activity

Slow metabolic breakdown of warfarin

VKORC1

Slows vitamin K reduction ( sensitivity to

warfarin)

Warfarin receptor insufficiency

Can develop resistance to warfarin

Effect of Direct Thrombin Inhibitors on PT

Argatroban, lepirudin,, bivalirudin- prolong PT

Monitor with Chromogenic Factor X Assay

Reversal of Warfarin Overdose

Bleeding

INR
3-5

No significant
bleeding

5-9

>9

Serious
bleeding

Any

Life-threatening
bleeding

Any

Point of Care Testing

Permit near-patient PT testing at anticoagulation clinics,

bedside testing, self-testing, and testing of infants
UNFRACTIONATED HEPARIN THERAPY AND PTT
o

B.

Intervention
Reduce dosage/ omit 1 dose
Monitor INR frequently
Omit warfarin
Monitor INR frequently
Consider oral vitamin K (5 mg) if
high risk for bleeding (surgery)
Stop warfarin
5-10 mg oral vitamin K
Monitor INR frequently
Stop warfarin
Give 10 mg vitamin K by IV push
May repeat every 12 hr
Give thawed frozen plasma,
prothrombin complex concentrate/
recombinant FVIIa
Same as for serious bleeding
but stronger recombinant FVIIa

ISI

Mixture of sulfated glycosaminoglycans extracted from

porcine mucosa
o
3000 to 30,000 D
o
Binds antithrombin thrombin inactivation
o
Uses

IV to treat DVT & PE

Initial treatment of AMI

Prevent reocclusion after stent placement

Maintain vascular patency during cardiopulmonary

bypass surgery with extracorporeal circulation
o
Dosage

5000 to 10,000 units, followed by continuous infusion at

approximately 1300 units/hour, adjusted to patient
weight

Stopped after 5 days (risk for HIT & thrombosis)

o
Monitoring
PTT

Prolong:

Lupus anticoagulant

Factor deficiency

Hypofibrinogenemia

FDP

Paraproteins

First specimen: before therapy

Second specimen: 4-6 hours not longer than 24 hours

after bolus dosage

Repeated every day

Platelet count

>40% reduction= HIT

Replace with DTI therapy

ACT

Lee and white modification

Utilizes 2 mL tubes with 12 mg diatomaceous earth

Median of normal ACT: 98 secs

DVT: 180-240 secs

Bypass: 400 secs

o
Limitation of PTT in UFH

Heparin resistance (shortened PTT)

Inflammation accompanied by
hyperfibrinogenemia + FVIII activity

PF4
o
Neutralizes heparin
o
Begins to shorten 1 hour after collection
o
Remedy: Centrifuge & remove PPP
o
Reversal of UFH Overdose

Protamine Sulfate

Cationic protein extracted from salmon sperm

Neutralizes UFH at a ratio of 100 units of heparin

per mg

IV push

Neutralizes LMWH

Shortens APTT/ACT
LOW MOLECULAR WEIGHT HEPARIN THERAPY AND
CHROMOGENIC ANTI-FACTOR XA HEPARIN ASSAY
o
Prepared from UFH using

Chemical (Enoxaparin)

Enzymatic (Tinzaparin)
o
MW: 4500-5000 D
o
Provides little bridging surface and reduce antithrombin
response
o
Subcutaneous injection
o
Treats DVT, PE & unstable angina
o
Useful among pregnant women (contraindicates warfarin)
o
Advantage over UFH

No need for laboratory monitoring

Higher bioavailability

Longer plasma half life

LMWH: 4-6 hours, UFH: 0.5-1 hour

Less inhibition of platelet function

Less bleeding risk

Lower incidence of thrombocytopenia & thrombosis (HIT

syndrome)

Less interaction with PF4

Fewer heparin-dependent IgG antibodies

o
Monitoring

Usually unnecessary

Useful in:

Renal insufficiency (creatinine: >2.0mg/dL)

Major bleeding risk factor

PTT: not useful (low anti-IIa activity)

Anti-factor Xa assay

More appropriate
o

C.

D.

E.

F.

PENTASACCHARIDE THERAPY AND CHROMOGENIC ANTIFACTOR XA HEPARIN ASSAY

Fondaparinux sodium
o
Synthetic formulation of the active pentasaccharide
sequence in UFH and LMWH
o
Raises antithrombin activity 400-fold
o
Does not inhibit thrombin, IXa, XIa, or XIIa
o
Half-life: 12-17 hours
o
Dasage: once-a-day subcutaneous injections of 2.5 mg
o
Usage

Prophylaxis after surgery

Treatment of DVT and PE

o
Contraindicated in patients with creatinine clearance of < 30
mL/min
Chromogenic antifactor Xa heparin assay
o
Fondaparinux therapy in infants, children, obese or
underweight patients, patients receiving treatment for more
than 7 to 8 days, and pregnant women
o
Blood: collected 4 hours after injection
o
Therapeutic range: 0.14-0.19 mg/L
RIVAROXABAN, ORAL DIRECT FACTOR Xa INHIBITOR
o
Oxazolidinone derivative that directly inhibits factor Xa
independently of antithrombin
o
Inhibits Xa
o
More effective & convenient than UFH or LMWH
o
Uses:

VTE prophylaxis

Total knee replacement or total hip replacement surgery

o
Doasage: 10mg/day
o
Monitoring: seldom required
o
Chromogenic antifactor Xa heparin assay

Standardize with rivaroxaban in place of UFH, LMWH, or

fondaparinux
DIRECT THROMBIN INHIBITOR AND PTT/ ECARIN CLOTTING
TIME
ARGATROBAN
o
Reversely bind & inactivate free thrombin & clot bound
thrombin without involving antithrombin
o
Substituted for UFH or LMWH when HIT is suspected or
confirmed using the 4Ts assessment system
o
Non-protein L-arginine derivative with a (MW= 527 D)
o
Uses

Prophylaxis, treatment, and anticoagulation during

cardiac catheterization for patients with HIT
o
Dosage

IV 2mcg/kg/min

Hepatic disease: 0.5 mcg/kg/min

Percutaneous cardiac intervention: 350 mcg/kg/min

followed by 25 mcg/kg/min
o
Half life: 51 mins
o
Requires monitoring
LEPIRUDIN
o
Recombinant 7000-D protein DTI
o
Analogue of natural hirudin (produced by the leech Hirudo
medicinalis)
o
Binds free but not fibrin-bound thrombin
o
Dosage: 0.4 mg/kg/hr up to 110 kg over 15 to 20 seconds
followed by 0.15 mg/kg/hr as a continuous IV infusion for 2
to 10 days.
o
Requires monitoring
o
Discontinue: creatinine clearance <15 mL/min, serum crea
>6mg/dl
o
Half life: 20 mins
o
Antihirudin antibodies form in about 40% of HIT patients

anticoagulant effect
BIVALIRUDIN
o
Synthetic 20-amino acid peptide derivative of hirudin
(MW=2180D)
o
inactivates both free and clot-bound thrombin
o
Uses

Anticoagulant in patients with unstable angina at risk

for HIT who are undergoing percutaneous coronary
intervention
o
Dosage (use with aspirin)

325 mg/day

Decreased in patients with reduced creatinine clearance

o
Half life: 25 mins
DABIGATRAN
o
Oral prodrug that converts upon digestion to active
dabigatran
o
Binds both free and clot-bound thrombin
o
Half life: 12-17 hours
o
Not metabolized by liver cytochrome

Use: VTE prophylaxis during total knee replacement or total

hip replacement surgery

Monitoring DTIs
o
Prolong thrombin time, PT, PTT, ACT
o
PTT therapeutic range: 1.5-3.0
o
Ecarin Clotting Time

Alternative for monitoring DTI

Ecarin- extracted from Echis carinatus

7.

8.

DTIs bind meizothrombin and generate a linear, dosedependent prolongation of the ECT

Prolong:

DTI

fibrinogen & prothrombin activity

ANTIPLATELET THERAPY AND PLATELET ACTIVITY ASSAYS

G.

Aspirin
o
Inhibits cyclo-oxygenase and blocks the production of
thromboxane
Ticlopidine ,Clopidogrel, Prasugrel
o
Inhibit the binding of ADP to its platelet receptor and inhibit
platelet aggregation by blocking activation of the
glycoprotein IIb/IIIa pathway

Historical Perspective
18th Century- visual clot based testing
1822-1921- temperature control during clot formation using glass
capillary tubes to detect coagulation factors by measuring resistance
1900Length of time whole blood clot in glass tubes is measured
1910Coaguloviscometer (whole blood clot detection device)
Change in viscosity is measured thru voltage change
1920Gram added CaCl2to anticaoagulated plasma at 370C
Principle used by thromboelastography (TEG) & sonar clot
detection
Nephelometer (measure 900 light dispersion in colloidal
suspension)
20th century- Developments of manual loops
Electromechanical clot detection using movable lead/rolling
steel ball
Photo-optical clot detection
1950BBL Fibrometer (electromechanical clot detection
methodology)
Factors that Driven Automation

4.
5.
6.

Approaches to Automation
1.

Purpose: Reduce or eliminate manual tasks required to perform

analytical procedures

1.
2.
3.

9.

GP IIb/IIIa Inhibitors
o
Present on membrane of resting platelets
o
Block the binding of fibrinogen to glycoprotein IIb/IIIa
receptors on the platelet
o
Abciximab

Fab fragment of a mouse monoclonal antibody specific

for glycoprotein IIb/III
o
Eptifibatide

Heptapeptide that binds and blocks access to platelet

GP IIb/IIIa

Coadministered with aspirin and UFH

o
Tirofiban hydrochloride

Nonpeptide GPI that is coadministered with aspirin and

UFH

AUTOMATION & INSTRUMENTATION IN

HEMATOLOGY/COAGULATION LABORATORY

Turnaround times (TAT)

Specimen Integrity
Staff shortages
o
Economic factors
o
Less maintenance and calibration
o
Faster start-up times
o
24/7 uptime
To improve accuracy and precision
Random Access
Improved reagent handling
o
Reduce reagent/specimen volume
o
Open reagent system

Reagent Tracking maintaining a record of reagents lot no.

and expiration dates
Improved Specimen Handling
o
Closed tube sampling
o
Primary tube sampling

Primary Collection tube is used by the analyzer

Analyzers allow the use of multiple sizes of tubes

Significant amount of time save

Errors such as mislabeling is reduced

Flagging for Specimen Interferences
o
Lipemia Falsely prolonged clotting time, interferes w/ light
transmittance
o
Icterus falsely prolonged clotting time because of
inadequate clotting factor production
o
Hemolysis falsely shortened because of premature
activation of clotting factors
o
Abnormal clot formation- falsely elevated clotting time
because of instruments inability to detect end point
Automatic Dilutions
o

Continuous Flow (single channel analyzers)

-

2.
3.
-

One sample, one test (analyte)

Reagents, diluents and samples are pumped through a
system of continuous tubing
Disadvantage:
o
Significant carry over effect and wasteful use of
reagents

Batch Testing
All samples are loaded at the same time
Run multiple samples one test at time in a batch
Random Access testing
Variety of test can be run in any order on single or multiple
specimens
Measures only the test requested on a sample
* STAT testing available
Phases of Analytic Process

Preanalytic (Sample Processing)

Analytic (Chemical Analysis)
Post analytic (Data management)
A.

PREANALYTIC PHASE

Front End Automation (Robotics)

Pre-sorting
Centrifugation
Volume checks (QNS)
Clot detection
Decapping
Aliquoting
Destination Sorting
1.

2.

B.

Specimen Preparation
o
Use of whole blood/Plasma/PPP
o
Primary tube sampling/ closed tube sampling
o
Secondary tube labelling
o
Transferring of sample to the analyzer cup
Specimen Identification
o
Manual Labeling
o
Bar coding
ANALYTICAL PHASE

Specimen Measurement and Delivery

Standards, control and samples are aspirated from specimen
containers and dispensed into reaction chambers (by a probe) of
the analyzer
1.

Flagging for Specimen Interferences

o
Hemolysis, lipemia, bilirubinemia, clot
a)
b)
c)

Automatic dilutions
Reflex Testing a test that is done by the machine when it
gets an abnormal result right after the original test
*Prolonged ptt -> mixing -> single factor assay (e.g)
Improved flagging capabilities

Temperature error

Photo-optics error

Mechanical movement error

Probe not aspirating

C.

No end point detected

Specimen track error

POST ANALYTIC PHASE (Data Management)

Principles of Current Coagulation Instruments

1.
2.

Clot
o
o
Clot
o
o

Formation
Optical
Nephelometric
by Feel
Mechanical
Viscosity-based

Archiving
Uses bar coded specimens that are scanned and placed in
numbered positions in numbered racks
Retrieval
Features of Computer
1.

2.
3.

Systems of measurement
1.

2.

3.

4.

5.

Electromechanical end point detection

o
Fibrometer

Fibrin strands attach attach to a moving probe,

completing an electrical circuit and stopping the
interval timer

There is one stationary and one moving probe

During clotting, the moving probe enters and leaves the

plasma at regular intervals

Clot: fibrin strand conducts current between the probes

even when the moving probe exits the solution

Current completes a circuit and stops the time

o
Magnetic Sensor

Monitors the movement of a steel ball within the test

plasma

AMAX and Destiniy instruments

Photo-optical ability to observe graph of clot formation w/ some
instrumentation
o
500 nm and 600 nm
o
Could be used only for clot detection
o
Detect a change in plasma optical density (OD,
transmittance) during clotting
o
OD depends on the color and clarity of the sample
o
Formation of fibrin strands causes light to scatter, allowing
less to fall on the photodetector and thus generating an
increase in OD
o
Effects of lipemia and icterus are minimized.
Chromogenic ability to measure proteins that do not form a
fibrin clot as the end point.
o
405 nm
o
Sample protease cleaves chromogenic substrate (pNA)
free pNA (yellow) measure OD
o
Para-nitroaniline (pNA)

Colorless synthetic oligopeptide substrate conjugated

to a chromophore
o
Fluorogenic: fluorescent conjugate is used
o
Direct chromogenic measurement

OD is proportional to the activity of the analyte

Protein C activity
o
Indirect chromogenic measurement

Analyte being measured inhibits reagent enzyme that

has activity directed toward the synthetic substrate

Change in OD is inversely proportional to the analyte

Heparin in antifactor Xa assay

Immunologic ability to automate test previously available only
with manual such as ELISA
o
Based on antigen-antibody reactions similar to nephelometry
o
Coated latex microparticles + samples antigen Antigen +
antibody bridges agglutination wavelength light beam
light absorbance
o
Light absorbance is directly proportional to the samples
antigen level
Nephelometric ability to measure antigen-antibody reactions for
proteins present in small conc.
o
Modification of photo-optical end-point detection in which
90-degree or forward-angle scatter
o
600 nm
o
90 degrees (side scatter) and near 180 degrees (forwardangle scatter)
o
As fibrin polymers form, side scatter and forward-angle
scatter rise
o
First applied to immunoassay
o
Quantitative but not functional assay of coagulation factors
o
Used to produce high-volume multiple-assay coagulation
profiles
Data acquisition, calculations and displaying data
Display patient results and reference values
o
Clerical error would be avoided

Provides a means of communication between the analyzer and

operator
o
A computer has the ability to be linked to other computer LIS
o
TCP/IP (transmission control protocol/Internet protocol)
On board troubleshooting and training program
On board automatic inventory
o
XE2100
Selecting Coagulation Instrumentation

Instrumentation cost
Consumable cost
Service response time
Maintenance requirements/ time
Operator ease of use
Throughput (high volume testing) *
Ability to add new testing protocols
LIS interface
Special Specifications (water, waste drain)

AMAX DESTINY series

BCS XP system (siemens)
LH 780 CBC (flow cytometry)
CA 1500 PT & PTT
SP1000i Sysmex (w/in 30secs fully automated slide-maker stainer)
DM 96
Cell Avision DM1200- NEW
CS2100
STA compact
ACT plus automated coagulation timer system
TEG detect clot form
Multiplate for aggregometry
ESR 300
PFA-100 Platelet Function Analyzer
-

Employs blood flow and shear rate to assess platelet function

successful at detecting von Willebrand disease and the efficacy of
aspirin therapy

Accumetrics VerifyNow
-

Optical detection system that measures platelet-induced

aggregation by microbead agglutination
Employs a disposable cartridge that contains lyophilized
fibrinogencoated beads and a platelet agonist specific for the test
o
Aspirin assay using arachidonic acid as the agonist
o
GP IIb/IIIa inhibitor assay using TRAP as the agonist
o
P2Y12 inhibitor assay using ADP as agonist

TEG (Thromboelastograph Hemostasis Analyzer System)

-

Operator-dependent system that provides global hemostasis

assessment
Assess both bleeding and thrombosis risk in:
o
Patients with Hepatic disease
o
Cardiac surgery
o
Obstetric patients
o
Trauma patients
Predict and monitor clotting factor administration, platelet
transfusion, fibrinolytic therapy, and antiplatelet therapy
Pipet citrated whole blood cup oscillates by 4 degrees 45
minutes at a frequency of 0.1 Hz (cup contains pin) add kaolin
fibrin links pin to the cup viscoelasticity changes TEG
tracing

Multiplate Analyzer
-

Whole-Blood Multiple Electrode Platelet Aggregometer

o
Aspirin efficacy using arachidonic acid
o
Clopidogrel efficacy using ADP
o
GPIIb/IIIa efficacy using TRAP
Provides multiple channels with multiple electrodes per channel
for simultaneous analyses

Specialized Coagulometer Features

Random
access

A variety of tests can be performed on a single

specimen/ multiple specimens in any order as
determined by the operator.

Primary
tube
sampling
Cap
piercing
Bar coding
Graph of
clot
formation
Bidirection
al LIS
interface
Specimen
&
instrument
flagging
Liquid
level
sensing

On-board
quality
control
On-board
refrigerati
on of
specimens
&
reagents
On-board
specimen
storage
capacity
Patient
data
storage
Reflex
testing
Stat
capabilitie
s
Throughpu
t
Total
testing
(dwell)
time

Plasma is directly aspirated from an open or capped

centrifuged primary collection tube on the analyzer.
Analyzer aspirates plasma from the closed
centrifuged primary collection tube.
Reagents and specimens are identified with a bar
code; eliminates manual information entry.
Operator can visualize clot formation.

Analyzer queries the host computer (LIS) to

determine which tests have been ordered. Results are
returned to the LIS after verification.
Automated alerts indicate problems with specimen
integrity or instrument malfunction.

Operator is alerted when there is inadequate

specimen or reagent volume. An alert is also given
when the instrument fails to aspirate the required
sample volume. Volume is verified each time a
specimen or reagent is aspirated.
Instrument stores and organizes quality control data;
may include application of Westgard rules for flagging
out of range results; instrument may transmit quality
control data to the LIS.
Refrigeration maintains the integrity of specimens
and reagents throughout testing and allows reagents
to be kept in the analyzer for extended periods, which
reduces setup time for less frequently performed
tests
Indicates the number of specimens that can be
loaded at a time.

Test results can be stored for future retrieval; clot

formation graphs may be included
Instrument can be programmed to perform repeat or
additional
testing under operator-defined circumstances
Operator can interrupt a testing sequence to place a
stat specimen next in line for testing.
Number of tests that can be processed within a
specified interval, usually the number of tests per
hour; depends on test mix and methodologies.
Length of time from specimen placement in the
analyzer until testing is completed; depends on the
type and complexity of the procedure

Basic Components of Most Hematologic Analyzers

1.

2.
3.

Hydraulics
o
Aspirating unit
o
Dispensers
o
Dilutors
o
Mixing chamber
o
Aperture baths
o
Hemoglobinometer
Pneumatics
o
Vacuum and pressures for operating valves and moving the
sample through the system
Electrical System
o
Electronic analyzer and computing circuitry for processing
data

Primary Tube Sampling

-

The tube used in extraction is the same tube used in analysis

Makes automation more accurate
Prevents error
Automated Hematology Analyzer

Oscilloscope Screen
Display the electrical pulses in real time as the cells are counted
Visual guide to the size and number of particles being counted
Number of pulses is proportional to the number of cells counted

Basic Cell Counting Principles

The counting of the cellular elements of the blood can be based on the
two classical methods:
a.
b.

Electrical Impedance
o
Impedance effective resistance of an electrical circuit
o
Radio Frequency
Optical Scatter/ Detection
o
Uses laser and nonlaser light

Electrical Impedance
a.k.a. Electronic Resistance or Low Voltage Direct Current
Developed by Coulter in the 1950s
o
Coulter Principle
Most common method used
Cell counting and sizing is based on the detection and
measurement of changes in electrical impedance (resistant)
produced by a particle as it passes through a small aperture
Particles such as blood cells are non-conductive but are
suspended in an electrically conductive diluent
As a dilute suspension of cells is drawn through the aperture, the
passage of each individual cell momentarily increases the
impedance (resistance) of the electrical path between two
submerged electrodes that are located on each side of the
aperture.
The amplitude (magnitude) of the electrical pulse produced
indicates the cells volume.
Radiofrequency (RF)
a.k.a. Alternating Current (AC) Resistance
A modification sometimes used in conjunction with DC electronic
impedance otherwise known as RF resistance or
o
High voltage electromagnetic current; measures conductivity
Total volume of cell is proportional to the change in DC
Cell interior density (e.g. nuclear volume) is proportional to pulse
size change in the RF signal
Optical Scatter/Detection
Optical Scatter Systems = Flow Cytometers
Laser on nonlaser light may be used
A diluted blood specimen passes in a steady stream through a
quartz flow cell past a focused light source
Light sources:
o
Tungsten halogen lamp
o
Helium neon laser (laser = monochromatic light)
Take note: Laser light is the most common light source used in
flow cytometers because of the properties of:
o
Intensity
o
Stability
o
Monochromatism
Hydrodynamic Focusing prevents:
o
Recirculation
o
Re-measurement
o
Multiple cell entry
As each cell passes through the sensing zone of the flow cell, it
scatters the focused light
Scattered light is detected by a photodetector and converted to
an electrical pulse
The number of pulses generated is directly proportional to the
number of cells passing through the sensing zone in a specific
period
Three Independent Processes in Optical Scatter
1.
Diffraction bending of light around corners using small angles
2.
Refraction bending of light because of change in speed using
intermediate angles
3.
Reflection light rays turned back by obstruction using large
angles
Angles of Light Scatter
1.
2.
3.
4.

Forward-Angle Light Scatter (00) cell volume or cell size

Forward Low-Angle Light Scatter (20 to 30) can relate to size or
volume
Forward High-Angle Light Scatter (50 to 150) allows for
description of the refractive index of cellular components
Orthogonal Light Scatter (900C) otherwise known as Side Scatter
o
Correlates with degree of internal complexity
o
Structures such as nuclei and cytoplasmic granules
determine the intensity of light scattering at 900C (RightAngle Scatter)
Leukocytes
SIZE
RNA CONTENT

Immature
Big
High

Mature
Small
Low

GRANULARITY
FLUORESCENCE
Red
-

High
High

Low
Low

Cell Distribution Width

Provides an estimate of RBC anisocytosis
The normal reference range for the RDW is 11.5% to 14.5%
Abnormalities can be observed on the high side but no
abnormalities have been noted on the low side
The RDW is increased above the normal limits in Iron Deficiency,
Vitamin B12 and folic acid deficiency
It is calculated directly from the Histogram
A portion of the curve at the extreme ends is excluded from the
computation to exclude clumps of platelets, large platelets, or
electrical interference on the left side of the curve
The portion of the right side of the curve that is excluded
represents grouped or clumped erythrocytes

BLOOD CELL HISTOGRAMS

Provided by many high-volume instruments to provide size
distribution of the different cell populations
The volume given in cu. mm. or fL, is plotted against the relative
frequency for platelets, WBCs and RBCs
These types of histograms will provide an approximate number of
cells on the y-axis and the cell size on the x-axis
The instrument being used counts those cells with volume sizes
between 36fL and 360fL as erythrocytes
If the RBCs are larger than normal, the curve will shift to the right
If the RBCs are smaller than normal, the curve will shift to the left
If the histogram curve is bimodal, then there is two population of
red blood cells as might be seen when a patient received a blood
transfusion
Other conditions that will cause a bimodal distribution curve are:
o
Cold agglutinin disease
o
Hemolytic anemia with schistocytes present
o
Anemias with different size cell populations
The RBC histogram can measure cells as small as 24fL
Those cells that are counted in the 24 to 36fL range are rejected
as RBCs and not included in the RBC count
Leukocytes are present in the diluted fluid containing RBCs, but
their numbers are statistically insignificant in the count.
WBC Histograms
Provide a count and plot of cells in the WBC aperture bath larger
than 45 fL
Normal WBC histograms have three (3) distribution peaks
o
First peak (45 90 fL)

Small mononuclear population of cells (i.e.

lymphocytes)
o
Second Peak (90 160 fL)

Minor population of large mononuclear cells (i.e.

monocytes)

An increase in the number of cells in this size range can

also represent abnormal cell types (such as the
immature precursor of cell types found in patients with
leukemia)
Third Peak (160 450 fL)

Normal mature types of granulocytes

Abnormal WBC histogram patterns

Error Flags
Region code ( R ) Flags
Signal irregularities in the WBC distribution and will appear next
to the differential parameters that are in error
The R stands for the region.
o
R1 warns of increased interference in the area left of the
lymphocyte peak (approx. 35fL)

Typically caused by:

Sickled RBCs

Nucleated RBCs

Clumped and giant platelets being counted in the

WBC aperture bath
o
R2- warns of excessive overlap of cells populations at the
lymphocyte/mononuclear cell boundary (approx.. 90fL)
caused by the presence of abnormal cell types, such as:

atypical lymphocytes

blast

plasma cells
o
R3- warning is caused by excessive overlap of cell
populations at the mononuclear/granulocyte boundary
(approx.. 160fL) which is due to the increased presence of
immature granulocytes (i.e., bands, Metamyelocyte)
o
R4- warning is caused by the extension of the cell
distribution past the upper end of the WBC threshold
(approx. 450fL). This most commonly occurs when the
granulocyte population is very high
Platelet Histograms
-

Platelet derived histograms (via the electrical impedance method)

are obtained from volume sizes of 2 to 20fL.
The actual counting takes place in the RBC aperture