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generalized enema of the entire body. Enlargement of the liver is common in children but is
not commonly seen in adults. Primary infections can often pass unnoticed; however, severe
infections may be fatal. The disease is transmied by triatomine bugs which become infected
by ingesting the blood-infected humans or animals. The parasite multiplies in the intestine of
the triatomine bug. There is a serious risk that Chagas disease may be transmied via blood
transfusion if proper precautions are not taken.
Laboratory diagnosis
Motile trypanosomes are found in the blood during the acute phase of the disease, and rarely
thereafter. During the chronic stage, the diagnosis is based essentially on immunological
methods. The technique of laboratory diagnosis is similar to African trypanosomiasis although
trypanosomes that cause Chagas disease are dicult to nd in the blood. The laboratory
procedures have been described above (see section on sleeping sickness) which include:
• Examination of wet mount.
• Examination of blood smear—thick and thin lms.
• Examination of blood lm prepared from centrifuged blood sample.
• Examination of dried blood samples for IgM and specic antibodies (card test).
IdenticationcharactersofT. cruzi
Fifteen micrometre broad and 20 µm in length—slender fonn, broad forms are C-shaped,
slender forms are generally S-shaped—pale blue-coloured cytoplasm with a central large
nucleus red in colour. Kinetoplast, which is a network of circular DNA inside a large
mitochondria are only found in protozoa. These large and round granules, dark red or purple
are located near the posterior extremity of the protozoa. The undulating membrane is narrow
and reddish-pink. The agellum is pink and extends beyond the undulating membrane.
Medical Laboratory Technology: Volume 1
Kala Azar (Leishmaniasis)
Kala azar is the second largest parasitic killer in the world—only malaria is more deadly.
Along with Chagas disease and sleeping sickness, kala azar is one of the most dangerous
neglected tropical diseases (NTDs).
A agellate protozoan of the genus Leishmania causes kala-azar. It can aect the skin
(cutaneous leishmaniasis), mucous membranes (mucocutaneous leishmaniasis) and the
reticuloendothelial system (visceral leishmaniasis or kala azar). In India, L. donovani is the
most common cause of kala azar. Charles Donovan, who was a professor of Medicine at
Madras Medical College, India, discovered this infectious agent and reported it in 1903.
Clinical significance
A remient type of fever, massive enlargement of the spleen and generalized weakness
characterize the disease. Untreated patients will die within two years. The clinical symptoms
often overlap with other diseases.
Epidemiology of the disease has unique features in each region and varies from one
geographical area to another. In the Americas, infection is spread to humans by the bite of the
phlebotomine y Luomyia longipalpis. In India, the vector feeds on dogs, wild animals and,
less frequently, humans. It can be found both in the countryside as well inside dwellings. The
disease occurs mainly in rural areas.
The incubation period of the infectious agent is 2–6 months, but can vary from 10 days
to several years. In some patients a primary lesion forms several months before other
symptoms appear. Amastigotes are the intracytoplasmic, non-agellated leishmanial form of
the Leishmania sp. These multiply slowly in macrophages near the site of inoculation. Some
infected macrophages enter the bloodstream and reach the viscera, whereas amastigotes
multiply rapidly.
Special Haematological Tests
329
Laboratory diagnosis
The single most important test in the diagnosis of kala azar is the microscopicexamination.
This, however, is performed at reference laboratories or by the histology department.
The smear is stained with May-Grünwald-Giemsa stain or any other appropriate stain
described in cytology (Chapter 37). Non-agellar forms of Leishmania sp. are found within
the reticuloendothelial cells. These are called Donovan bodies. They are round, measuring
2–4 μm, with a small dot-like nucleus (Figure 11.10).
Figure 11.10 Laboratory diagnosis of leishmaniasis (kala azar): (a) Donovan bodies in monocyte —
non-agellated oval body, (b) Leishmania donovani in Kuper cells of liver
Cutaneous leishmaniasis
Skin ulcers, which can be single or multiple, characterize cutaneous leishmaniasis. In certain
forms of cutaneous leishmaniasis plaques, papules or nodules may appear in dierent parts
of the body.
Principle
Cutaneous leishmaniasis is diagnosed by demonstrating the typical amastigote stage of the
organism from slit skin smears of ulcers. Typical leishmaniasis ulcers are cratered with a
raised edge. Slit skin specimens are collected from the edge of the ulcer.
Equipment and supplies
• Microscope, timer
• Microscopic slides, scalpel, gauze, slide rack, diamond pencil or grease pencil (marker)
Reagents
• Ethanol 70%
• Methanol
• Giemsa stain (See Chapter 10 for details)
• Phosphate buered water at pH 6.8 (See Appendix С at the end of Volume III for the
preparation of phosphate buer)
For use, dilute the Giemsa stain in phosphate-buered water (1 volume of stain to
19 volumes of buered water).
Procedure
1. Collection of specimen
a. Clean the edge of the ulcer using a swab soaked in ethanol. Using the gauze pad,
compress the edge of the ulcer as rmly as possible to obtain a bloodless area.
b. Use the scalpel to make a supercial incision along the edge of the ulcer about
0.5 cm long and 2–3 mm deep. Still rmly holding the ulcer in place, turn the scalpel
330
on to the at side and gently scrape the base of the incision with the point of the
blade. Collect tissue cells, but avoid drawing blood.
с. Spread the material collected from the tip of the blade on to a slide in a circular
motion to cover an area of 5–7 mm in diameter. Allow the smear to air-dry and label
the slide with a diamond pencil or grease pencil.
2. Staining of smears (Figure 10.22)
a. Fix the air-dried smears by ooding the slide with methanol for 2 min.
b. Tip o the methanol and ood the slide with the diluted Giemsa stain for 20 min.
c. Rinse the slide with phosphate-buered water and place it upside down in a slide
rack to drain and dry.
3. Microscopicexamination:Examine the slide under l00× oil-immersion objective. The
amastigotes of Leishmania sp. may be found intracellularly in the macrophage cells
or lying separately between the cells. They measure 2–4 µm and have a prominent
nucleus and a rod-shaped kinetoplast. The nucleus and the kinetoplast stain red and
the cytoplasm stains pale blue.
4. Report: The microscopic observation is reported as ‘amastigotes of Leishmania sp.
present‘ in case of positive nding. Negative result is reported as amastigotes of
Leishmania sp. not found.
Medical Laboratory Technology: Volume 1
Visceral leishmaniasis
Clinically, the early phases of visceral leishmaniasis are characterized by chronic intermient
fevers, cough and diarrhoea. It can also cause bleeding of mucous membranes and secondary
infections. Later, progressive enlargement of the spleen, liver and, occasionally, lymph
nodes, weight loss and—in some patients—patchy hypopigmentation of the skin occurs.
An increase of a non-specic gamma globulin is seen in the serum of most patients with
visceral leishmaniasis. This forms the basis of this chemical diagnostic test. It is convenient,
inexpensive and a good screening tool.
Principle
Kala azar infection increases the gamma-globulin fraction of serum. The protein forms a
solid gel when reacted with formaldehyde (formalin). This test is a non-specic indicator,
commonly referred to as serum aldehyde test or formal-gel test. It is widely used for a quick
screening of kala azar. Positive Sia water test (described below) is also associated with kala
azar infection but may also be related to macroglobulinaemia.
Specimen
Serum
Equipment and supplies
• Centrifuge
• Centrifuge tube, test tubes and test tube rack
Reagent
1. Formalin 37–40% (commercial grade, no dilution necessary).
Note Formalin vapour is injurious to health. Work inside a hood or near the window.
Procedure
1. Collect 2–5 mL of blood into a centrifuge tube and allow it to clot.
2. Separate the serum by centrifuging the tube for 3 min at 5000 G or leaving the tube
overnight in a refrigerator or on the bench.
3. Take a small test tube (5-mL) and transfer a few drops of patient’s serum to the boom
of the tube with the help of a Pasteur pipee.
4. Add to the test tube containing serum an equal volume of formalin.
Special Haematological Tests
Figure 11.11 Formal-gel test for diagnosis of Kala azar (leishmaniasis): (a) Take I mL of patient’s
serum in a test tube, (b) Add 1 drop of formalin, (c) Wait for 24 h, (d and e) Then tilt the
tube. If the serum is still uid, it is considered to be normal and if the serum goes solid
and sometimes white in colour, the serum is reported as positive for possible Leishmania
infection. In case of a positive reaction, a white-coloured solid gel will develop within
20 min.
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review Questions
1. What causes sickle cell anaemia and how does a clinical laboratory help in its diagnosis?
Which laboratory test will be able to conrm sickle cell disease?
2. Explain the dierence between: sickle cell trait and sickle cell disease.
3. Explain the clinical conditions of haemoglobinopathies? How are they diagnosed in a
laboratory?
4. Why is it necessary to examine thick and thin smears of blood for the laboratory
diagnosis of malarial infections?
5. State the characteristic features of Plasmodium falciparum and Plasmodium vivax.
6. What is the principle behind RDT (Rapid Diagnostic Test)?
7. Explain the technique of immunochromatograpy. Give an example of its application in
laboratory diagnosis.
8. State the routine tests conducted for the laboratory diagnosis of sleeping sickness and
kala azar.
9. Review the presumptive and conrmatory tests for sleeping sickness and kala azar.
10. What causes Chagas disease? How is this diagnosed in the laboratory? How is this
dierent from kala azar?
Interpretation of Laboratory Findings in Haematology
Chapter Outline
• Overview
• Anaemias
▪ Microcytic hypochromic anaemia
▪ Macrocytic normochromic anaemia
▪ Normocytic normochromic anaemia
▪ Haemolytic anaemias
▪ Haemoglobinopathy
▪ Hereditary spherocytosis
▪ Drug-induced anaemia
• Leukaemias
▪ Acute Myelocytic Leukaemia (AML)
▪ Chronic Myelocytic Leukaemia (CML)
▪ Acute Lymphoblastic Leukaemia (ALL)
▪ Chronic Lymphocytic Leukaemia (CLL)
▪ Lymphomas
▪ Multiple myeloma
• Miscellaneous Disorders
▪ Polycythaemia vera
▪ Infectious mononucleosis
▪ Genetic anomalies in white cells
▪ Acquired abnormalities of white cells
• Review Questions
12
Anuradha Chakravarthy
Overview
Physicians routinely request haematological reports as part of the patient’s evaluation. This
is because the blood picture represents the general health of a patient, and it may also reveal
inherent blood diseases that have remained undiagnosed. Blood diseases are broadly classied
under anaemias, leukaemias and miscellaneous diseases. Some of the important laboratory
ndings regarding these diseases will be discussed in this chapter. Although the physician
is responsible for the diagnosis of these diseases, you should be aware of the signicance of
your ndings so that discordant results can be checked on the spot.
Interpretation of Laboratory Findings in Haematology
333
AnAemiAs
Anaemia means lack of red blood cells. All anaemias lead to a fall in the red cell count,
haemoglobin concentration and haematocrit value. Anaemia can arise from various defects of
the red cells—production defect (aplastic anaemia), maturation defect (megaloblastic), defect
in haemoglobin synthesis (iron deciency anaemia), genetic defect of haemoglobin maturation
(e.g., thalassemia) or synthesis of abnormal haemoglobins (haemoglobinopathies—sickle cell
anaemia, thalassaemias and others), physical loss of red cells (haemorrhagic anaemia) or
internal destruction of red cells (haemolytic anaemias).
Microcytic Hypochromic Anaemia
In this type of anaemia, the red cells are small and pale. The Mean Cell Volume (MCV),
Mean Cell Haemoglobin (MCH) and Мean Cell Haemoglobin Concentration (MCHC) are
decreased. This is commonly associated with iron deciency.
Macrocytic Normochromic Anaemia
The red cells are larger in size in this type of anaemia. The MCV and MCH are high, but MCHC is
normal. This is commonly associated with megaloblastic anaemia caused by vitamin B12 and/or
folic acid deciency. Megaloblasts are immature-nucleated RBCs, which are normally not seen in
circulation except in case of megaloblastic anaemia. Megaloblasts are bigger in size (macrocytes)
than normal RBCs and look darker. Megaloblastic anaemia can also have hypersegmentation
of neutrophils, presence of anisocytosis, poikilocytosis, Howell Jolly bodies and Cabot’s rings.
The reticulocyte count remains close to normal but increases quickly following treatment with
vitamin B12 and/or folic acid. This also indicates the eectiveness of the treatment.
Note Pernicious anaemia is a chronic macrocytic anaemia characterized by achlorhydria
(absence of free hydrochloric acid in stomach). It is caused by the lack of intrinsic factor
in the stomach. Intrinsic factor helps in the absorption of vitamin В (extrinsic factor). Thus
the aetiology of megaloblastic anaemia and pernicious anaemia are dierent although the
laboratory ndings are similar.
Normocytic Normochromic Anaemia
In this type of anaemia, the red cells are of normal size and colour. This may be associated with
normal reticulocyte count or increased reticulocyte count (reticulocytosis). The laboratory
ndings of normochromic normocytic anaemia, low reticulocyte count and pancytopaenia
(i.e., decrease of all blood cells—RBC, WBC and platelets) suggests aplastic anaemia. On
the other hand, increased reticulocyte count, with normochromic normocytic red cells
and decreased myeloid: erythroid ratio in the bone marrow smear indicates increased
erythropoiesis as seen in haemolytic or haemorrhagic conditions.
Haemolytic Anaemias
There are a number of causes of haemolytic anaemias. In most cases abnormal red cell
morphology will be noted. Increased reticulocyte count, presence of nucleated RBCs (NRBC),
various types of inclusion bodies, poikilocytosis and increased bilirubin concentration are
some of the common ndings.
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Medical Laboratory Technology: Volume 1
Haemoglobinopathy
These are congenital defects of haemoglobin leading to haemolytic anaemias and/or poor
haemoglobin function. Sickle cell anaemia is recognized by the presence of sickle cells in
circulation. The sickle cell screening test helps to make the initial diagnosis, which is
conrmed by haemoglobin electrophoresis. In case of thalassemia, haemoglobin maturation
is disturbed. Hb F (foetal haemoglobin) is found in the foetus, but the synthesis of Hb F
gradually decreases and is replaced by the synthesis of Hb A in the rst year of life. In case
of thalassemia major, Hb F is markedly elevated. In thalassemia minor, Hb A2 is increased
with or without an increase in Hb F. The largest fraction, however, is the normal Hb A.
Haemoglobinopathies are best diagnosed in the laboratory by haemoglobin electrophoresis.
Hereditary Spherocytosis
Abnormal morphology of red cells and increased fragility of red cells are the routine
laboratory ndings of this disorder.
Drug-induced Anaemia
This is recognized from the Heinz body formation in red cells and conrmed by the G-6-PD
assays.
LeukAemiAs
Leukaemias are malignant disorders of the white blood cells (WBCs). Based on their course
they are broadly classied as acute and chronic. Untreated acute leukaemias have a short
survival period whereas patients with chronic leukaemias might live for several years.
Leukaemias are further classied on the basis of the cell of origin (myelocytic or lymphocytic).
Though incomplete, a simple classication of leukaemias is as follows:
• Acute myelocytic leukaemia
• Acute lymphoblastic leukaemia
• Chronic myelocytic leukaemia
• Chronic lymphocytic leukaemia
Unlike leukocytosis, which is a transient increase of the white cells and is a benign condition,
leukaemias are cancerous conditions which originate in the bone marrow and eventually spread
to the circulation and can also inltrate other organs and tissues in the body. The white cell count
may be markedly and consistently elevated. All leukaemias if untreated are eventually fatal.
Acute Myelocytic Leukaemia (AML)
This is also called acute granulocytic leukaemia (AGL). Acute myelocytic leukaemia is
characterized by the presence of large number of myeloblasts in the bone marrow and their
appearance in the peripheral blood. Myeloblasts in the peripheral blood might vary from a
few to as much as 95%. Many variants of AML have been described depending on the type
of cell predominating in the peripheral blood. These could be myeloblasts, promyelocytes,
monoblasts or blasts of both myeloid and erythroid series. In classical AML, myeloblasts
contain Aüer rods which take up eosinophilic stain with Romanowsky staining. Their
detection can be markedly improved by a modied peroxidase staining when they are
described as Phi bodies. Their presence in myeloblasts is diagnostic of AML. When blasts
are not easily detected in the peripheral blood smear (aleukaemic leukaemia) the diagnosis
may be missed and can only be made on bone marrow examination. At this stage most
Interpretation of Laboratory Findings in Haematology
patients show pancytopenia, i.e., a decrease in RBC, WBC and platelet counts. In cases where
myeloblasts are not well dierentiated, many cytochemical stains and immuno-logic markers
can be used to dene the exact type of leukaemia.
335
Chronic Myelocytic Leukaemia (CML)
Chronic myelocytic leukaemia (CML) is also called chronic granulocytic leukaemia (CGL). It
is characterized by the presence of a marked increase in the cells of the myelocytic series. The
predominant cells are neutrophilic myelocytes and metamyelocytes. But there is an increase
in both less mature and more mature cells of the neutrophilic series. The total leukocyte count
is generally higher than 50000/µL and may be as high as 200000/µL. There is an increase in the
cells of the eosinophilic and basophilic series as well. Thrombocytosis is common. A marked
reduction in neutrophilic alkaline phosphatase activity and detection of Philadelphia-1
(Ph-1) chromosome by chromosomal analysis conrms the diagnosis of CML and dierentiates
it from benign conditions of leucocytosis in which immature cells of neutrophilic series might
appear (leukaemoid reaction).
Acute Lymphoblastic Leukaemia (ALL)
It is a malignancy of the cells of lymphocytic series and it is characterized by the presence
of increased number of lymphoblasts in the bone marrow and also in the peripheral blood.
In many children, it can be cured by intensive and sustained treatment. It is critical to
dierentiate AML from ALL as the treatment regimen for each is entirely dierent. In case
of diculty in diagnosis, several special cytochemical stains and immunological markers
can be used to make a denitive diagnosis. Acute lymphoblastic leukaemia is the most
common form of leukaemia in children than other leukaemic conditions.
Chronic Lymphocytic Leukaemia (CLL)
CLL is characterized by a marked increase of mature lymphocytes (>15000 cells/µL). The
cell count may go very high and 80–90% of the cells look like immature lymphocytes. This
occurs more commonly in older adults. There are two types of CLL—a slow growing type
that patients may survive for many years without any treatment, and a fast growing type that
requires treatment.
Lymphomas
Lymphomas are malignant (cancerous) conditions of the cells of the lymphocytic series. In
lymphomas, the disease is initially localized to the lymph nodes but can eventually spread to
involve the bone marrow or the peripheral blood. Blood and bone marrow picture vary from
normal to presence of marked changes, including pancytopaenia.
Multiple Myeloma
It is a malignant condition of the plasma cells. The bone marrow aspirate shows a marked
increase in the number of mature and immature plasma cells, which might constitute as
many as 90% or more of the cells of the bone marrow. Rarely the plasma cells appear in the
peripheral blood when the condition is described as plasma cell leukaemia. Peripheral blood
smear shows marked Rouleaux formation; therefore, the erythrocyte sedimentation rate
(ESR) is markedly elevated. There is a marked increase in the abnormal globulin (monoclonal
gammopathy) in the plasma. This is recognized by the presence of an abnormal protein in
serum electrophoresis and in the urine called Bence Jones protein.
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Medical Laboratory Technology: Volume 1
misceLLAneOus DisOrDers
Polycythaemia Vera
This is a malignant condition of the red cells with increased red cell count, high haematocrit
and haemoglobin values, increase in WBC and platelet counts and a decrease in ESR.
Secondary polycythaemia is far more common than polycythaemia vera.
Infectious Mononucleosis
It is an acute infectious disease caused by the Epstein Barr virus. It is associated with an
increased number of lymphocytes and the presence of atypical lymphocytes. It is more
prevalent in children and young adults and is classied under benign lymphoproliferative
disorders. The serological test, known as the heterophil antibody test (HAT), conrms the
diagnosis.
Genetic Anomalies in White Cells
Various types of genetic anomalies are recognized in the laboratory by the abnormal
morphology of white cells. Two of these are: Alder–Reilly anomaly and Pelger–Hüet anomaly.
The Alder–Reilly anomaly is characterized by the presence of large, coarse granules in the
cytoplasm of leucocytes from birth; patients are often aicted with a deformity of bones and
joints. Pelger–Hüet anomaly aects the segmentation of the nucleus in polymorphonuclear
cells (neutrophils). The nucleus appear stunted, coarse and lumpy, and rounded or bilobed
(hyposegmented). These abnormal neutrophils appear to maintain the functional abilities of
normal granulocytes. Pseudo–Pelger–Hüet anomaly is seen in many conditions including
granulocytic leukaemias.
Acquired Abnormalities of White Cells
Acquired abnormalities of white cells might occur under dierent conditions and may
be normalized when the pathologic condition disappears. These abnormalities include
the presence of toxic granulation, Döhle bodies and cytoplasmic vacuolization in WBCs.
Causes for these abnormalities are aributed to infections, drug poisoning and burns.
Hypersegmentation occurs in case of megaloblastic anaemia.
Interpretation of Laboratory Findings in Haematology
337
review QuestiOns
1. What are the common ndings of anaemias? From the available laboratory reports how
would you dierentiate between iron deciency anaemia and pernicious anaemia?
2. Which laboratory ndings will indicate the following clinical conditions?
Aplastic anaemia, Macrocytic anaemia, Microcytic hypochromic anaemia, Leukocytosis,
Neutrophilia, and Eosinophilia
3. What are the laboratory ndings of haemolytic anaemias?
4. What is the clinical signicance of Aüer bodies? What laboratory ndings will
dierentiate between AML and CML, and between ALL and CLL?
5. If the dierential count indicates the presence of a high number of plasma cells, what
would be the most likely clinical diagnosis? What would be the laboratory ndings for
serum proteins?
6. List the laboratory ndings that will guide the physician towards the diagnosis of: (1)
polycythaemia vera (2) Infectious mononucleosis.
7. What laboratory ndings indicate the occurrence of Pelger–Hüet anomaly and Alder–
Reilly anomaly? Are these disorders inherited?
8. What are the clinical conditions that may cause the following laboratory observations?
Toxic granulation, Basophilic stippling, Hypersegmentation, Sickling of red cells, Target
cell formation, Presence of basket cells (smudge cells), Hypochromasia, Heinz body
formation, Presence of NRBC, Presence of Howell Jolly Bodies, Presence of haemoglobin
crystals within and outside red cells.