CLINICAL LABORATORY
DIAGNOSTICS OF HEMOBLASTOSIS
Etiology is Unknown: Possible Causes:
B. Marrow damage due to irradiation increases
the frequency
of some leukemias, but not others.
D. Possible genetic factors have been
implicated, especially in Chronic Lymphocytic Leukemia.
2. If the prominent cell line is of the lymphoid
series it is a lymphocytic leukemia
Therefore, there are four basic types of leukemia.
II. Acute lymphocytic leukemia – ALL - (includes T
cell, B cell, and Null cell)
III. Chronic myelocytic leukemia – CML - (includes
myelocytic and myelomonocytic)
The acute leukemias are divided into 2 categories, depending
upon their cell of origin. Leukemia evolving from the myeloid/granulocyte cell
line is called acute
myelogenous leukemia (AML). Lymphocytic precursors
give rise to acute lymphocytic leukemia (ALL). Each year in the
AML is the most common type of acute leukemia in
adults, accounting for 80% of new cases. AML is un-common in children. The
incidence increases steadily with age, with a sharp increase after 45 years.
ALL is the most common malignant disease affecting chil-dren, accounting for
approximately 30% of all child-hood cancers.
ALL has a bimodal age distribution, peaking in
children between 3 and 5 years of age and again in persons older than 65 years.
Acute leukemias can occur in all age groups
n
ALL is more
common in children
n
AML is more common in adults
Chronic leukemias are usually a disease of adults
n
CLL is extremely rare in children and unusual before
the age of 40
Age Distribution: optimum ages
for development:
5. Monocytic: middle age (rarely before 30)
Comparison of acute and chronic leukemias:
Clinical onset sudden insidious
Course (untreated) 6 mo. or less 2-6 years
Leukemic cells immature >30% blasts more mature cells
Thrombocytopenia prominent mild
Lymphadenopathy mild present; often
prominent
Splenomegaly mild present; often
prominent
Malignant transformation of a stem cell leading to
unregulated proliferation and
arrest in maturation at the primitive blast stage.
Remember that a blast is the most immature cell that can be recognized as
committed to a particular cell line.
Pathophysiology of the clinical manifestations of
acute leukemias
1. Marrow failure due to infiltration
-thrombocytopenia
-bleeding, spontaneous bruising
-neutropenia-infections, sepsis
2. Infiltration
of other organs
-liver, spleen, lymph nodes (particularly in ALL)
-gums –gum hypertrophy (monocytic subtype of AML)
-bone pain, esp. in children with ALL
3. Leukostasis (only seen with WBC >>50x109
/L)
-lungs -pulmonary infiltrates, hypoxemia
-exposure of substances that can initiate coagulation
can cause DIC
At the time of presentation, the leukocyte count
in a patient with acute leukemia is generally elevated, but it may be normal or
reduced. Fewer than 20% of patients have a leukocyte count greater than 100
×103/mm3.
Peripheral blood smears show blasts in most
cases.
Other notable findings include anemia and
thrombocy-topenia; these result from the increased percentage of blasts in the
bone marrow, which leaves little room for erythroid and megakaryocytic
precursors. Severe throm-bocytopenia (fewer than 50 ×103platelets/mm3) is
pres-ent in more than half of patients presenting with acute leukemia. Abnormal
results of coagulation tests (ie, hypofibrinogenemia, elevated fibrin split
products, defi-ciency of coagulation factors) are seen in patients pre-senting
with signs and symptoms of DIC.
Serum electrolyte levels are typically
normal in patients with acute leukemia. Lactate dehydrogenase and uric acid
levels may be elevated due to rapid cell
turnover; this can lead to urate
nephropathy and acute renal failure. Rapid lysis of tumor cells, especially
when chemotherapy is instituted, can result in tumor lysis syndrome, manifested
by hypocalcemia, hyperkalemia, hyperphosphatemia, and hyperuricemia.
DIAGNOSIS OF LEUKEMIA
The diagnosis of acute leukemia requires
that blasts comprise 30 % or more of bone marrow cells or circu-lating white
cells.
(For AML, the new WHO classifica-tion
proposes to change this to 20 % blasts.) For differ-entiating ALL and AML, a
bone marrow aspirate and
biopsy are necessary.
A peripheral blood smear may provide clues
to the type of acute leukemia. Myeloblasts exhibit great vari-ability in size,
abundant pale blue cytoplasm with
azurophilic (bluish) granules, and distinct
nucleoli. The presence of Auer rods, appearing as pink strands within the cytoplasm
of the myeloblast, is characteristic of AML. Lymphoblasts tend to be small,
with scant cytoplasm and indistinct nucleoli.
Because it is extremely difficult to
characterize leu-kemia as lymphoblastic or myeloid based on morpho-logic
appearance of blasts, additional analyses of the
blasts are necessary, including
cytochemical staining, phenotypic analyses via flow cytometry, and molecular
evaluation for chromosomal abnormalities
(ie, cytogenetic studies).
The most important cytochemical stains for
deter-mining lineage are myeloperoxidase, Sudan black B, and the esterases.
Positive myeloperoxidase reaction or staining with Sudan black B in 3% or more
of blast cells indicates myeloid origin. Acid phosphatase is present in early T
cells, and demonstration of its activity can dif-ferentiate T-cell ALL from
non–T-cell ALL. Generally, lymphoblasts stain with terminal
deoxynucleotidyltrans-ferase (Tdt), although a small percentage of myelo-blasts
may be positive as well.
Immunophenotyping by flow cytometry will
con-firm the diagnosis of leukemia or establish the diagno-sis in cases in
which morphology and cytochemical
stains are equivocal. Cytogenetic studies
are important because two thirds of patients diagnosed with AML or ALL and 90 %
of patients with secondary leukemia will have leukemic blasts showing clonal
chromosomal abnormalities.
The chromosomal abnormalities differ
between AML and ALL and among the various sub-types. Immunophenotyping and
cytogenetic analyses
assist in risk stratification and provide information
that has important clinical, prognostic, and treatment implications.
The lab diagnosis is based on two things:
2. Identification of the cell lineage of the leukemic
cells
Anemia (normochromic, normocytic)
The degree of peripheral blood involvement determines
classification:
n
Leukemic – increased WBCs due to blasts
n
Subleukemic – blasts without increased WBCs
n
Aleukemic – decreased WBCs with no blasts
Classification of the immature cells involved may be
done by:
Cytochemistry
Cytochemistry –
help to classify the lineage of a leukemic cell (myeloid versus lymphoid). Myeloperoxidase – is found in the
primary granules of granulocytic cells starting at the late blast stage. Monocytes may be weakly positive.
Rare positives occur in lymphoid cells. Nonspecific esterase – is used to
identify monocytic cells which are diffusely positive. T lymphocytes may have focal staining.
Acid phosphatase may be found in
myeloblasts and lymphoblasts. T lymphocytes have a high level of acid
phosphatase and this can be used to help make a diagnosis of acute
T-lymphocytic leukemia.
Leukocyte alkaline phosphatase – is located in
the tertiary granules of segmented neutrophils, bands and metamyelocytes. The
LAP score is determined by counting 100 mature neutrophils and bands. Each cell
is graded from 0 to 5. The total LAP score is calculated by adding up the
scores for each cell.
The differential diagnosis of acute leukemia
The differential diagnosis of acute leukemia
in-cludes other conditions in which patients present with an elevated leukocyte
count, anemia, and thrombocy-topenia. These include leukemoid reactions and
deep-seated infections, which may be associated with an ele-vated leukocyte
count and a left shift. Infection with Epstein-Barr virus may cause severe
lymphocytosis with atypical lymphocytes present on peripheral smear. The
diagnosis can be made easily by the absence of blasts in these conditions.
Patients with acute leukemia may also present with
low leukocyte counts together with anemia and throm-bocytopenia. The
differential diagnosis of this presen-tation includes the primary bone marrow
diseases of myelodysplastic syndrome and aplastic anemia. Infil-tration of the
bone marrow by other neoplasms, in-cluding solid tumors and hematologic
malignancies, may also present with anemia and/or thrombocytope-nia. Immature
forms of leukocytes resembling blasts may be seen in severe megaloblastic
anemia due to folate and vitamin B 12 deficiency.
Chronic Lymphocytic
Leukemia
Neoplastic
proliferation of small mature-appearing lymphocytes
>99 % are B-cell
derived
Older patients
(> 40)
Disease may be
discovered incidentally
Fatigue, weakness,
weight loss, anorexia, and/or recurrent infections may occur
Variable
splenomegaly and non tender lymphadenopathy
Leukemic counterpart
of small lymphocytic
Lymphoma
Lab diagnostics
Absolute
lymphocytosis (> 4000/mm3)
Smudge cells
Variable anemia,
neutropenia, and thrombocytopenia
Hypercellular bone
marrow with lymphocytic infiltrates
Hypogammaglobulinemia
Elevated serum LDH
Chronic Myelocytic Leukemia
(CML)
15-20 % of all
leukemias
Young or
middle-aged patients
Asymptomatic,
fatigue, abdominal fullness, early satiety, weight loss, anorexia
Splenomegaly, bone
pain, bone tenderness
Proliferation of
myeloid cells in blood and bone marrow (mostly myelocytes polys)
Slow progression (3
yr survival without Rx)
Ph 1 chromosome in
> 95 %
Bone Marrow in CML
Hypercellular bone
marrow
High Myeloid :
Erythroid Ratio
Myeloid hyperplasia
Relatively few
blast cells
Mostly mature
neutrophils
Increased basophils
and eosinophils
Increased
megakaryocytes
Multiple Myeloma
Multiple
myeloma, also known as plasma
cell myeloma or Kahler's disease,
is a cancer of plasma cells, a type of white blood cell normally responsible for
producing antibodies. In multiple myeloma,
collections of abnormal plasma cells accumulate in the bone marrow, where they interfere with the
production of normal blood cells. Most cases of myeloma also feature the
production of a paraprotein
— an abnormal antibody which can cause kidney problems. Bone lesions and hypercalcemia (high calcium levels) are
also often encountered.
Myeloma is diagnosed with blood tests (serum protein
electrophoresis, serum free kappa/lambda light chain assay), bone
marrow examination, urine protein electrophoresis, and X-rays of
commonly involved bones.
The presence of unexplained anemia, kidney
dysfunction, a high erythrocyte
sedimentation rate (ESR), lytic bone lesions, elevated beta-2
microglobulin, and/or a high serum protein (especially raised globulins or immunoglobulin) may prompt further
testing. The globulin level may be normal in established disease. A doctor will
request protein
electrophoresis of the blood and urine, which might show the
presence of a paraprotein
(monoclonal protein, or M protein) band, with or without reduction of the other
(normal) immunoglobulins. One type of paraprotein is the Bence Jones
protein which is a urinary paraprotein composed of free light
chains. Quantitative measurements of the paraprotein are necessary to establish
a diagnosis and to monitor the disease. The paraprotein is an abnormal immunoglobulin produced by the tumor
clone. Very rarely, the myeloma is nonsecretory (not producing
immunoglobulins).
In theory, multiple myeloma can produce
all classes of immunoglobulin, but IgG paraproteins are most
common, followed by IgA and IgM. IgD and IgE myeloma are very rare. In addition, light and or heavy
chains (the building blocks of antibodies)
may be secreted in isolation: κ- or λ-light chains or any of the five
types of heavy chains (α-, γ-, δ-, ε- or μ-heavy
chains).
Additional findings include: a raised calcium (when osteoclasts are breaking down bone,
releasing calcium into the bloodstream), raised
serum creatinine due to reduced renal function, which is mainly due to
casts of paraprotein deposition in the kidney, although the cast may also contain complete
immunoglobulins and albumin.
Cancer of plasma cells
Disease of older men and women (> 60 years)
Produce abundant useless monoclonal Ig
(paraprotein, M-protein)
Decreased normal Ig, infections occur
Lytic bone lesions, bone pain, pathologic
fractures, hypercalcemia
Tumorous masses of plasma cells (spine, skull,
ribs, pelvis)
Renal failure may develop
Investigation of a patient with suspected myeloma
should include the screening tests, followed by further tests to confirm the
diagnosis. Electrophoresis of serum and concentrated urine should be performed,
followed by immunofixation to confirm and type any M-protein present.
Immunofixation and SFLC assessment are indicated in patients where there is a
strong suspicion of myeloma but in whom routine serum protein electrophoresis
is negative.
Quantification of serum M-protein should be
performed by densitometry of the monoclonal peak on electrophoresis;
immunochemical measurement of total
immunoglobulin (Ig) isotype level can also be used
and is particularly useful for IgA and IgD M-proteins. Quantification of
urinary total protein and light chain excretion can be performed directly on a
24-hour urine collection or calculated on a random urine sample in relation to
the urine creatinine.
Quantification of SFLC levels is an additional tool
for the assessment of light chain production. The serum tests are particularly
useful for diagnosis and
monitoring of light chain only myeloma and patients with oligosecretory /
non-secretory disease and in requests
for which urine has not been sent to the laboratory. In renal impairment the
half life and thus serum concentration of SFLC can increase ten fold and there
is often an increased kappa: lambda ratio. A diagnosis of myeloma should be
confirmed by bone marrow (BM) assessment. It is recommended that an adequate
trephine biopsy of at least
Lymphogranulomatosis
Lymphogranulomatosis – is a malignant tumor of lymphoid tissue with
lesion of lymph nodes and organs, characterized by growth of the giant cells
called Reed-Sternberg- Beresovsky cells, large and small atypical cells
(Hodgkin,s cells) and inflammatory infiltration.
Hodgkin's
lymphoma, also known as Hodgkin
lymphoma and previously known as Hodgkin's disease, is a
type of lymphoma, which is a cancer originating from white blood
cells called lymphocytes.
Patients with Hodgkin's lymphoma may present with the following symptoms:
§
Lymph nodes: the most common symptom of Hodgkin's is the painless
enlargement of one or more lymph nodes, or lymphadenopathy. The nodes may also feel rubbery and swollen when examined. The nodes of
the neck and shoulders (cervical and supraclavicular) are most frequently involved (80–90 % of the time, on average). The lymph
nodes of the chest are often affected, and these may be noticed on a chest radiograph.
§
Unexplained
weight loss
§
Splenomegaly: enlargement of the spleen occurs in about 30 % of people with Hodgkin's
lymphoma. The enlargement, however, is seldom massive and the size of the
spleen may fluctuate during the course of treatment.
§
Hepatomegaly: enlargement of the liver, due to liver involvement, is present in about 5 % of
cases.
§
Hepatosplenomegaly: the enlargement of both the liver and spleen caused by the same disease.
§
Red-coloured
patches on the skin, easy bleeding and petechiae
due to low platelet count (as a result of
bone marrow infiltration, increased trapping in the spleen etc. – i.e.
decreased production, increased removal)
Although the bone marrow cytology findings
from the aspirate are sufficient or even preferable for most hematological
questions, it is regarded as good practice to obtain a sample for bone
marrow histology at the same time, since with improved instruments the
procedure has become less stressful, and complementary cytological and
histological data are then available from the start. After deep local
anesthesia of the dorsal spine and a small skin incision, a histology cylinder
at least 1.5cm long is obtained using a sharp hollowneedle (Yamshidi). A Klima
and Rossegger cytology needle is then placed through the same subcutaneous
channel but at a slightly different site from the earlier insertion point on
the spine and gently pushed through the compacta. The mandrel is pulled out and
a 5- to 10-ml syringe body with 0.5ml citrate or EDTA (heparin is used only for
cytogenetics) is attached to the needle. The patient should be warned that there
will be a painful drawing sensation during aspiration, which cannot be avoided.
The barrel is then slowly pulled, and if the procedure is successful, blood
from the bone marrow fills the syringe. The syringe body is separated from the
needle and the mandrel reintroduced. The bone marrow aspirate is transferred
from the syringe to a Petri dish. When the dish is gently shaken, small,
pinhead-sized bone marrow spicules will be seen lying on the bottom. A smear,
similar to a blood smear, can be prepared on a slide directly from the
remaining contents of the syringe. If the first aspirate has obtained material,
the needle is removed and a light compression bandage is applied. If the
aspirate for cytology contains no bone marrow fragments (“punctiosicca,” dry
tap), an attempt may be made to obtain a cytology smear from the (as yet
unfixed) histology cylinder by rolling it carefully on the slide, but this
seldom produces optimal results.
Cell composition in the bone marrow:
normal values (%)
Cell densities vary widely, 0.5–2 per view field during
screening at low magnification. |
Bone Marrow:
Medullary Stroma Cells
These procedures, less invasive than bone marrow
biopsy, are a simple and often diagnostically sufficient method for lymph node
enlargement or other intumescences. The unanesthetized, disinfected skin is
sterilized and pulled taut over the node. A no. 1 needle on a syringe with good
suction is pushed through the skin into the lymph node tissue. Tissue is
aspirated from several locations, changing the angle of the needle slightly
after each collection, and suction maintained while the needle is withdrawn
into the subcutis. Aspiration ceases and the syringe is removed without
suction. The biopsy harvest, which is in the needle, is extruded onto a
microscopy slide and smeared out without force or pressure using a cover glass
(spreader slide). Staining is done as described previously for blood smears.