Laboratory investigation of

Haemophila, von-willebrand &

Christmas diseases
 Laboratory findings Haemophila

Assay Results.
Bleeding time (BT) ?

Clotting time (CT) ?

aPTT ?

Prothrombin time (PT) ?

Thrombin time (TT) ?

Platelet count ?

Factor VIII Assay ?

 Laboratory findings Haemophila

Assay Results.
Bleeding time (BT) Normal

Clotting time (CT) Prolonged

aPTT Prolonged

Prothrombin time (PT) Normal

Thrombin time (TT) Normal

Platelet count Normal

Factor VIII Assay Reduced (confirmatory test)

 Laboratory finding in von-willebrand
disease
Assay Results
Bleeding time (BT) ?
Clotting time (CT) ?
Prothrombin time (PT) ?
aPTT ?
Thrombin time (TT) ?
Platelet count ?
vWF: Ag ?
vWF: Ristocetin ?
Factor VIII ?
 Laboratory finding in von-willebrand
disease
Assay Results
Bleeding time (BT) Prolonged
Clotting time (CT) Prolonged
Prothrombin time (PT) Normal
aPTT Prolonged
Thrombin time (TT) Normal
Platelet count Normal
vWF: Ag Reduced (confirmatory test)
vWF: Ristocetin Reduced (confirmatory test)
Factor VIII Reduced
 Laboratory finding in Christmas disease

Assay Results
Bleeding time (BT) ?
Clotting time (CT) ?
Prothrombin time (PT) ?
aPTT ?
Thrombin time (TT) ?
Platelet count ?
Factor IX assay ?
 Laboratory finding in Christmas disease

Assay Results
Bleeding time (BT) Normal
Clotting time (CT) Prolonged
Prothrombin time (PT) Normal
aPTT Prolonged
Thrombin time (TT) Normal
Platelet count Normal
Factor IX assay Reduced (confirmatory test)
Cytopenias.

Definition?
 Cytopenia.

• The simultaneous presence of

– Anemia
– Leukopenia
– Thrombocytopenia

• Hb< 11.5 g/dL (adult females),< 13.5 g/dL (adult males)

• WBC < 4x10^9/L (4000/mm3)
• Plt. < 150 x10^9/L (150.000/mm3 )
 Causes of Pancytopenia
1. Aplastic anemia 3. Paroxysmal nocturnal
hemoglobinuria (PNH)
2. Bone marrow 4. Myelodysplastic syndrome
infiltration by 5. Hypersplenism
– Hematologic 6. Vit B12 or folate deficiencies
malignancies. 7. S. Lupus erythematosus
– Storage cell 8. Cytotoxic agents and
disorders antimetabolites
9. Radiotherapy
– Osteoporesis
10. Overwhelming infections
– Myelofibrosis 11. other
 Symptoms/Findings
• Related to pancytopenia
– The presenting symptoms are related to
anemia or thrombocytopenia
– Leukopenia may sometimes be life
threatining (eg: late in the course of AA,
severe neutropenia)
– Anemia develops slowly ( long life span of
RBC’s)
 Symptoms/Findings
• Related to pancytopenia
– Thrombocytopenic type bleeding may occur
and
– severity depends on the plt. number.
eg: spontaneous bleeding indicates
plt<20.000/mm3
– Infections are related to neutropenia , with
severity depending on the decrease in
neutrophil counts.
 Symptoms/Findings
• Related to the cause of pancytopenia
eg:
– Splenomegaly: Hypersplenism, lymphoma,
leukemia, myelofibrosis etc
– Lymphadenomegaly:Lymphoma , leukemia,SLE etc
– Atrophic glossitis: Megaloblastic anemia
– Others
Investigation of Pancytopenia
(Outline)
• Physical exam:
– Splenomegaly
– Hepatomegaly
– Lymph node enlargement
– Signs of liver failure
– Evidence of malignancy
Investigation of Pancytopenia
(Outline)

• Essential tests
–CBC:Pancytopenia
– Reticulocyte count
–MCV
–Peripheral blood smear
»Anisocytosis, poikilocytosis,
»leuko-erythroblastosis
»atypical cells
–Bone marrow exam. (aspiration +
biopsy)
 Aplastic Anemia (AA)
• The term AA is first used by Ehrlich in 1888
• Describes a disorder of unknown etiology
characterized by
– pancytopenia with
– hypo or acellular bone marrow.
• It is one of the stem cell disorders.
 Classification of AA

I-Inherited AA – Dyskeratosis
–Fanconi’s anemia: congenita
•Autosomal. recessive inheritance •Skin, nail and hair
•Skeletal and renal defects abnormalities
•Hyperpigmentation •Telangiectasia
•Small stature
•Mental retardation
•Hypogonadism
•Chromosomal changes •Growth failure and
–Familial AA (non-Fanconi) hypogonadism
–Shwachman–Diamond
•Familial but without features of
syndrome
Fanconi’s anemia
 Classification of AA
II-Acquired AA
1. Idiopathic
2. Radiation
3. Drugs/chemicals
• Chloramphenicol
• NSAID:
(phenylbutasone,indomethacin,gol • Antineoplastic and
d etc) cytotoxic drugs
• Oral hypoglycemic drugs • Pesticides
(chlorpropamide,tolbutamide) • Solvents and glues:
• Antithyroid drugs, phenothiazines, benzene,toluene,xylene,na
antimalarials, phtalene
diuretics,antiepileptics • Dyes and industrial toxins
• Others
Classification of AA (continued)

4. Infections
– Hepatitis 5. Paroxysmal nocturnal
– E.Barr virus hemoglobinuria(PNH)
– Rubella
6. Immunological disorders
– CMV
– SLE
– HIV
– Graft- versus- Host Disease
– Parvovirus
– Hypoimmunoglobulinemia
– Brucellosis
– Chronic TB
– Toxoplasmosis 7. Pregnancy
 Epidemiology of AA
• A disease of the young
– Median age: about 25 yrs
• 1.5 – 2 /1.000.000-year
• Equal sex ratio
Clinical Features of AA

• History:
– Bleeding
– Symptoms of anemia
– Infections
– Drugs, chemicals or other etiologically important
exposures have to be questioned.
Clinical Features of AA

• Physical exam:
– Petechiae, ecchymosis
– Retinal bleeding
– Pallor
– Fever and other signs of infection
– Presence of lymphadenomegaly and /or
splenomegaly are unusual (indicate other
diagnoses).
• LAB:
– Pancytopenia
– Reticulocytes: Low or absent
– RBC: normochromic-normocytic,or slight
macrocytosis
– Neutropenia and relative lymphocytosis
– Red and white cell precursors are almost
never seen in the peripheral smear
LAB:
– PNH tests may be positive (Ham’s or sucrose lysis
tests,others)
– Serum iron is increased
– Bone marrow :
• Aspiration; Dry tap
• Biopsy; all three cell lines are reduced or absent,
raplaced by fatty tissue, residual lymphocytes, rarely
hot spots of hematopoesis
 Course and prognosis of AA

• Definition of severe aplastic anaemia:

1-hypocellular bone marrow
2-neutrophils <500/mm3
3-platelets<20.000/mm3
4-reticulocytes<20.000/mm3 (<1%)
• Survival in severe disease is about 1 year if it is
treated with transfusions only.

• Very severe AA:

Severe AA criteria + Neutrophils:< 200/mm3
 Treatment of AA (2)
• Stem cell transplantation:
– Young patients with severe AA and a HLA matched
donor should undergo a stem cell transplantation

– Risks: GVHD, engraftment failure, other

 Treatment of AA (4)
Supportive treatment
• Hemopoetic Growth Factors:
GM-CSF, G-CSF:
• Transfusions:
– Only when indicated
– Exclude family members as transfusion donors
– Cellular blood products must be irradiated before transfusions
– May cause:Iron overload (Repeated RBC’s), alloimmunization,
infection transmission
(eg : HIV,HCV,HBV,CMVetc),
• Infections: Preventive care, early diagnosis and treatment
 Pure Red Cell Aplasia
• Anemia + Erythropoetic hypoplasia/aplasia occuring in a
normocellular bone marrow
Classification
• Congenital (Diamond-Blackfan Syndrome)
• Acquired
– During chronic hemolytic anemia (aplastic crisis)
– Infections(eg: Parvovirus B19)
– Malnutrition
– Drugs (Alpha Methyl Dopa, Azathioprine, Carbamazepine,Gold, NSAID,RMP
,Chloramphenicol etc)
– Malignancy
– Idiopathic
Myelodysplastic syndrome (MDS)
• Definition:
• Myelo = marrow in Greek
• Dys = irregular in Greek
• Plasia = proliferation in Greek
• MDS comprises a group of clonal
stem cell disorders characterized by
ineffective blood cell production
resulting in peripheral Cytopenias of
all the three lineages.
 Prevalence
• MDS is mainly a disease of the elderly
with Median age at diagnosis of 60-70yrs
• with male predominance and its rare in
children.
• 10,000 cases annually in US
• 4.1 MDS per 100,000 population
• Risk of Development increases with Age
• Incidence increases with age:
• 0.5/100,000 in individuals < 50 years,
• 5.3/100,000 “ “ 50 - 59 years
• 15.0/100,000 “ “ 60 - 69 “
• 49.0/100,000 “ “ 70 -79 “
• 89.0/100,000 “ “ > 89 “
 Myelodysplasia (MDS)
Patho-physiology

• Since the stem cell is diseased any or

all of the erythroid, myeloid or
megakaryocytic cell lines may be
affected.
• In addition to peripheral cytopenia,
there is also shortened survival of
the affected cells as well as
qualitative deficiencies of these cells:
• e.g. deficiencies in Chemotaxis of
granulocytes, bleeding, anaemia etc
 Myelodysplasia (MDS)
Nucleo – Cytoplasmic asynchrony
Hemoglobinized orthochromic Giant metamyelocyte
erythroblast with still immature Immature nucleus,loose
Chromatin.
Alk. PO4-ase granules

Nucleus with visible Peroxidase granules

Chromosomes ready to
Divide.
 symptoms
• Many patients may be asymptomatic
and may be diagnose accidentally
during routine blood test.
• This disease grows slowly and may
take many years undetected
• Others may have symptoms of bone
marrow failure, this includes
symptoms of anaemia, shortness of
breath, chest pain, fatigue, fever,
weakness
• Thrombocytopenia (easy bleeding)
• Neutropenia ( frequent infections)
• Physical findings are often minimal,
but may include pallor, purpura and
rarely Splenomegaly.
• There is a recognized association
between exposure to occupational
chemicals
Acquired causes
• Benzene
• Chemotherapy
• Radiotherapy,
• stem cell transplantation
• Exposure to alkylating agents e.g.
Melphalan, cyclophosphamide etc
• Vit B12/folate deficiency
• Chronic liver disease
• Initial steps in the pathogenesis
involves damage to a Pluripotent
stem cell
• This leads to the development of a
Myelodysplastic clone, which has
advantage over other haemopoietic
cells.
• Certain congenital disorders such
• Fanconi Pancytopenia
• Down’s syndrome
have unstable genes and are at high
risk of developing mutations that can
cause MDS
 Classification of MDS
• French American British (FAB)
• World Health Organization (WHO)
• The classification of both depends on the
presence of % blast cells and ringed
sideroblast in the peripheral blood film and
the Bone marrow film.
• However WHO further classifies it to subtypes
 FAB classification of MDS

• MDS
• Refractory Anemia ( RA )
• Refractory Anemia with
Sideroblasts ( RAS )
• Refractory Anemia with
Excess of Blasts ( RAEB )
• Refractory Anemia with
Excess of Blasts in transformation
( RAEB – T )and
• chronic myelomonocytic leukaemia
 American French British (FAB)
classification of MDS
Blood film Bone marrow Ringed sideroblast
Refractory anaemia <1 % Blast <5% Blast <15%
Refractory anaemia ≤1 % < 5% >15
With ringed
sideroblast (RARS)
Refractory anaemia < 5% 5-20 >15
With excess blast
(RAEB)
Refractory anaemia ≥ 5% 20-30 <15
With excess blast
(RAEB-T) in
transformation

Chronic < 5% 5-20 <15

myelomonocytic
Leukaemia (CMML)
 Classification of MDS
• The current WHO classification
system recognizes several major
subtypes of MDS
• This subtypes are distinguished from
each other by the degree to which
normal blood cells production is
affected
• The number of blast cells present
and the likelihood of transformation
to acute myeloid leukaemia (AML)
 MDS – WHO classification
Subtype of MDS % Blasts BM % Blasts
PB
Refractory Anemia (RA) <5 None/rare
Refr.Anemia w. R. S. <5 None/rare
Refr. Cytop. Multil. Dysplasia <5 None /rare
(RCMD)
Refr. Cytop MD-RS <5 None /rare
Refr.Anemia w. Excess Blasts 5–9 <5
(RAEB – 1 )
RAEB - 2 10 -19 5 -19
MDS – unclassified MDS-U <5 None /rare
MDS – Del 5 q (5 q - ) <5 <5
 Lab investigations.
FBC
• Normo – or – macrocytic anemia, aniso-
poikilocytosis.
• Normal B12, folate.
• Leuco-erythroblastosis may be present, when
MDS advanced.
• Basophilic stippling, Howell-Jolly bodies, giant
bands and hypogranular granulocyte., Pelger-
Huett anomaly, hypersegmentation
• Bone marrow biopsy
• (hypercell., megaloblastoid = nucleo-
cytoplasmic asynchrony, micro- and-
very polyploid megakaryocytes)
 Cytogenetic tests

• Deletion of the long arm of

chromosome 5 Known as 5q or del
(5q) confirms MDS
 MDS – Necessary diagnostic criteria

• Persistent, unexplained cytopenia

with the known morphologic
anomalies.
• Older adults with normo- or –
macrocytic anemia without B12,
folate deficiency.
• Hyper ( rarely hypo ) cellular BM with
megaloblastoid and asynchronous
maturation features. Ineffective
hematopoiesis.
• Cytogenetic anomalies in 40-60% of
pts.: 5q
• Blast count: Myeloblasts or
monocytes over 1,000 / ul., CD34 is
proof of blast, but not all blasts are
CD 34 +
 Note
• It is very important to diagnose and
correct Vit B12 or folate deficiencies
before considering the diagnosis of
Myelodysplastic syndrome.
 Myelodysplasia - Therapy

• A. Supportive
• Early stage and low IPSS do not
require treatment for some time if
slowly evolving.
• RBC Transfusions according to
symptoms:
• Hb.< 90 g/l in elderly will cause
dyspnea, angina, CHF.
• EPO and G-CSF have been used
successfully in a series of pts., but on
occasion have accelerated leukemic
transformation
• Regular observation
• Good Nutrition
• Supportive care
• Blood transfusion
• Chemotherapy
• Stem cell transplantation
END