HEMATOLOGY NOTES

LYMPHOCYTES

NK-CELL
TH1
CD4+ T-HELPER CELL
TH2
LYMPHOCYTES T-CELL

CD8+ T-CYTOTOXIC CELL

B-CELL -- Differentiate into antibody-producing PLASMA CELLS

T and B cells cannot be differentiated down a microscope. Further tests are needed such as
immunophenotyping or markers.

DIFFERENCES BETWEEN T AND B CELLS

T CELLS B CELLS

% FOUND IN BLOOD : 65 – 80 % 15 – 28 %

ANTIGEN RECOGNITION : T-cell Receptor Surface Immunoglobulin

ACTIVATED FORM : Secretes Cytokines Plasma cells produce

antibody

ANTIGEN EXPRESSION OF CD2: Sheep rosettes CD19 / CD20: B-cell

THE VARIOUS CELL-TYPES CD3: T-cell Receptor (TCR) Surface
(MARKERS) : CD7: Pan T-cells Immunoglobulin
CD4: Helper T-cells
CD8: Cytotoxic T-cells

CD = Cluster Designation. (Term for classification of unique protein markers on cell surfaces)
 IMMUNODEFICIENCY / LYMPHOPAENIA

B-cells: Congenital

Congenital Hypogammagloblinaemia:

X-linked inheritance; therefore mostly in males.

Presents in early infancy with recurrent infections.

Laboratory:
Low immunoglobulin levels: IgA, IgM, IgG.
T-cells are normal.
B-cells are reduced.

Treatment:
Immunoglobulin replacement therapy.

Isotype and Subclass Deficiencies:

Clinical:
Mainly infections.

Laboratory:
Patient is deficient in certain immunoglobulin isotypes.
IgA is the most common.
IgG may also be deficient.
The fault lies in the isotype switching process which takes place after activation.

Treatment:
Immunoglobulin replacement therapy.

T-cells: Congenital

Di George Syndrome:

The patient lacks both thymus and parathyroid glands. This results in absence of T-cells.

Clinical:
Infections, cardiac abnormalities and mental retardation.

Laboratory:
Normal B-cells and Immunoglobulin.
Natural Killer cells are normal. (Develop in bone marrow.)
Reduced T-cells.

Therapy:
? transplant of infant thymus.

Other

Severe Combined Immune Deficiency (SCID):

Defect is in stem cell development which leads to an absence of T-cells, B-cells and Natural killer
cell function.
The cause is not clear.
Treatment: ? Bone marrow transplant.

Ataxia telangiectasia:

Autosomal recessive. Very rare.

Caused by a failure in DNA repair mechanisms leading to numerous chromosomal breaks.

Clinical:
Cerebella Ataxia.
Telangiectasia of the mouth.
Numerous chest infections.
Patients often develop lymphoid malignancies.

Laboratory:
Lymphopaenia (Lymphopenia).
Absence of B-cells and decreased IgA.
X-ray: Thymic hypoplasia.

Wiskott Aldrich Syndrome:

Autosomal recessive.
Patients have eczema, bruising and bleeding.
Lymphopaenia.
Hypogammaglobinaemia.
Low platelets.
Bone marrow transplant only hope of cure.

Acquired Immunodeficiency

HIV / AIDS:

Reduced CD4+ helper T-cells due to virus.

Acquired Lymphopaenia:

Secondary to:
Bacterial infection – usually relative decrease.
Response to certain drugs – e.g Steroids.
 LYMPHOCYTOSIS

Lymphocytosis occurs when the lymphocyte count is > 3.5 X 109 / l. Note that absolute lymphocyte
count is more important than the % of total WBC – e.g. in neutrophilia, lymphocytes will be lower
% of overall WBC, yet may be normal absolute count.

Causes of lymphocytosis:

Infections:
– Pertussis: Lymphocytes are small round and often have tiny cleft.
– Infectious lymphocytosis: Rare, mainly seen in children. May be due to an enterovirus.
– Granuloma disease : TB, Syphilis. Lymphocytes are large, irregular and vacuolated.
– Infectious mononucleosis and various viral infections.

Malignant conditions:
Chronic Lymphocytic Leukemia (CLL).
Lymphoma.

Infectious Mononucleosis:

Pathophysiology:
Caused by Epstein Barr virus.
Usually found in saliva. (“Kissing disease”)
Occurs in patients under age 40.
Virus usually affects the B-cells causing activation and proliferation.
T-cells mount an immune response to suppress the uncontrolled B-cell proliferation.
CD8+ cytotoxic T-cells kill infected B-cells.
The atypical reactive lymphocytes seen in the blood are T-cells.
Once the T-cells succeed in controlling B-cell proliferation, the illness subsides.

Clinical:
Sore throat, fever, malaise.
Lymphadenopathy. (hence, “glandular fever”.)
Pharingitis.
Splenomegaly / palpable liver (rare).

Laboratory:
FBC: White cells – variable, counts rise as disease progresses.
Anemia and thrombocytopaenia are rare features.
Smear: Increased lymphocytes.
Lymphocytes are usually activated and thus are atypical. They have :
Eccentric or irregular nuclei,
Increased cytoplasm,
Increased basophilia / blue cytoplasm,
Prominent nucleoli,
A range or spectrum of atypical lymphocytes are present – vs. malignancy where all are
clones.
Lymphocytes seen which “hug” red blood cells.

Heterophile antibody test (monospot):

The EBV stimulates the B-cells to produce a variety of antibodies.
The monospot test is based on the principle that one of these antibodies, the “heterophile antibody”,
agglutinates horse red cells.
However, horse red cells contain both Forssman and the infectious mononucleosis (diagnostic)
antigen. In order to differentiate, the patient's serum is incubated in both guinea-pig kidney
(contains only Forssman antigen) and bovine cells which contain only infectious mononucleosis
antigen.
Detection is diagnostic of infectious mononucleosis and will be positive in +_ 95 % of patients.

Therapy:
No specific therapy as of 2002.
Disease usually subsides after 2 – 3 weeks.
Malaise and atypical lymphocytes can persist.

If the T-cells do not manage to control the B-cells a lymphoma can develop. This is usually
associated with other immunodeficiencies such as HIV.

PLASMA CELLS

Plasma cells are terminally differentiated B-cells and usually develop after B-cell activation.
After activation B-cells will first secrete IgM and then switch to IgG, etc. after second exposure.
Plasma cells are not normally found in peripheral blood.
Plasma cells lack surface immunoglobulin.
The function of plasma cells is to produce antibodies during an infection.
When increased amounts of plasma cells are found in the peripheral blood and bone marrow there
are often abnormal serum proteins which are usually immunoglobulins.
This usually suggests a diagnosis of Myeloma or Waldenstroms macroglobinaemia.

Tests reflecting abnormal serum proteins:

● Erythrocyte Sedimentation Rate (ESR)

The ESR measures the rate of settling red cells in diluted plasma.
Trisodium citrate is the anticoagulant of choice and an exact dilution of 4 volumes blood to 1
volume anticoagulant is used. Red cells settle as blood stands. The reported value is obtained by the
measured drop in the red cell meniscus after 1 hour. The rate at which this occurs depends on the
concentration of plasma substances.
– Fibrinogen
– Immunoglobulin
Fast sedimentation rate seen in infection, myeloma.

● Globulin and Albumin levels

Increased serum globulin indicates increased immunoglobulins.

● Serum Protein Electrophoresis

Albumin moves very fast. Immunoglobulin slower.
Normal:
ALB α1 α2 β γ
Reactive State: Polyclonal peaks :

ALB α1 α2 β γ

Monoclonal peak: NB feature of malignancy: Myeloma, Lymphoma or Waldenstroms

Macroglobinaemia :

ALB GLOB

● Immunoelectrophoresis
Separation of immunoglobulins.
Use an antibody to the different immunoglobulins – e.g. anti-IgG or anti-IgA to differentiate
between the different subtypes.

Myeloma:

Myeloma is a fatal malignancy in which plasma cells proliferate in the bone marrow.
They can also take over the liver, spleen and lymphnodes.
When the plasma cells infiltrate the peripheral blood and appear in increased numbers, the patient
has plasma cell leukemia. This is usually fatal.

Pathogenesis:
The clinical manifestations of the disease are caused by the following:
The release of immunoglobulin from the plasma cells is deposited in the tissues causing severe
damage.
Light chains (Bence Jones proteins) are deposited in the kidney causing renal failure.
Osteoclast activating factor and other cytokines (IL-1β, TNF, IL-6) are released from the plasma
cell. These activate osteoclasts, which in turn release IL-6, leading to reactivation of plasma cells
and bone destruction.
Impaired haemopoeisis due to plasma cells taking over the bone marrow.
Impaired immunity leading to increase in infections.
Clinical findings:
Seen in patients between 30 and 70 years.
Skeletal pain with pathological fractures and lytic lesions is often a feature.
Anemia, Uremia (renal failure) and hypercalcaemia.

Laboratory features:
FBC: Low Hb
Often low White cell count.
ESR: Increased due to increase in plasma proteins.
Smear: Normocytic normochromic anemia.
Infrequent plasma cell is seen.
Rouleaux formation.
May have a leucoerythroblastic picture – i.e. immature white cells and erythroid cells (nucleated
RBC + erythroblasts).
Bone marrow: Soft bones.
Increased plasma cells which are bizarre, large and multinucleated.
Plasmablasts, proplasmacytes and flame cells (red flame-like cytoplasm) are often present.
The plasma cells in myeloma differ from normal in that they have a more open nuclear chromatin,
large nucleoli, lack of a perinuclear halo and less intensely staining cytoplasm.
Russel bodies – cytoplasmic mucoprotein inclusions that stain pink with romanowski stains are also
present.
The Mott cell is also seen. Mott cells contain numerous blue inclusions which are thought to be
accumulations of immunoglobulin.
X-ray: Lytic lesions.
Serum proteins: Plasma cells may synthesize whole immunoglobulin or just light chains.
Hypergammaglobulin on electrophoresis.
Hypogammaglobulin if only light chains are produced.
Urine proteins: Excretion of Bence Jones protein.
Light chains may cause renal damage.
In renal failure: Albumin and whole immunoglobulins are excreted.
Blood chemistry: Hypercalcaemia due to bone decalcification.
Raised urea and creatinine due to renal failure.
Hyperviscosity: Increased viscosity of the serum and plasma due to increase in plasma protein.

Diagnosis:
If the bone marrow contains increased plasma cells the diagnosis of myeloma can usually be made.
The following tests are usually done to confirm:
Protein electrophoresis showing monoclonal peak.
Bence Jones proteinuria.
Lytic lesions on the X-ray.
Immunoperoxidase stain to demonstrate monoclonality.

Treatment:
Chemotherapy: patients can improve initially. However they usually die of complications e.g. renal
failure.
? Bone marrow transplant.
Waldenstroms Macroglobulinaemia:

This disease is characterised by plasmacytoid cells (have features of plasma cells; likely not fully
differentiated plasma cells) which secrete IgM.
IgM antibodies are large and can cause problems such as cold agglutinin disease and loss of vision.
Patients with cold agglutinin disease often develop Waldenstroms.

Clinical:
Usually seen in older men.
Weakness and fatigue.
Abnormal bleeding.
Hyperviscosity of the blood causing retinal problems which include haemorrhage.
Lymphadenopathy and Splenomegaly.
Confusion.

Disease can transform to an aggressive lymphoma.

Laboratory:
FBC and Smear:
Normocytic normochromic anemia.
Normal to low retic count.
White cells: normal; occasional plasmacytoid lymphocyte.
Platelets: mildly decreased.
Bone marrow:
Increased numbers of lymphocytes and plasmacytoid lymphocytes.
Protein electrophoresis:
Narrow band of homogenous protein.
Coagulation:
Bleeding tendency due to antibody coating platelets.
ESR:
Very raised. Increased plasma viscosity.
X-ray:
No lytic lesions.

Treatment:
Hyperviscosity causes many problems: bleeding, heart failure, visual disturband confusion.
Plasmapheresis controls viscosity.
Chlorambulicol can control disease for many years.

Notes from Cape Peninsula University of Technology (Fmr. Cape Technikon): Biomedical
Technology, 2002.
Related notes at:

http://www.scribd.com/my_document_collections/2374607