Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5543_Библиотеки_им_академика_М_И_Перельмана.pdf
288
Medical Laboratory Technology: Volume 1
• Immature Granulocytes: Immature granulocytes, originating in the bone marrow, are
passed into the circulation under severe bacterial infection. They are 12–18 μM in size,
nucleus without lobes, with chromatin varying in colour from dark red to purple. The
cytoplasm is pale blue or pink in colour with many large mauve or dark red granules.
Toxic granulations may be seen in which granules are very large and darkly stained. If
immature polymorphonuclear neutrophils are seen in the blood smear, as ‘band form’
or ‘stab cells’, they must be reported as a percentage of leukocytes.
• Blast Cells: Presence of other immature cells without granules and with nucleoli (lym-
phoblasts) must be reported.
• Toxic Granulation: In many severe bacterial infections neutrophils contain prominent
granules, which take up an intensely purple black colour in the cytoplasm. These may
also appear in some hereditary disorders (Chediak Higashi).
• Vacuoles: In conditions such as severe infections, burns, chemical poisoning, malignan-
cy and others, cytotoxic injury may result in the occurrence of vacuoles in the cytoplasm
and nucleus.
• Döhle Bodies: These are small round or oval discrete light blue-stained inclusion bodies
(1–5 μM) that occasionally appear in the cytoplasm of neutrophils in severe infection,
burns and on exposure to toxic agents.
• Hypersegmentation: When more than ve lobes are seen in segmented neutrophils,
which are normally 2–3 lobed, they are classied as hypersegmented neutrophils (technically called shift to the right, indicating increased maturity) that may be due to an
inherited disorder or may occur in megaloblastic leukaemia.
• Hyposegmentation: This anomaly is characterized by failure of normal lobe develop-
ment (less than 3) in neutrophils. The nucleus may take up a dumbbell shape or may
appear as a band neutrophil. It occurs in acute myelocytic leukaemia, severe infections,
toxic states and hereditary disorders (Peiger Hüet anomaly). Non-hereditary dumb-
bell-shaped neutrophils are also known as pseudo Peiger Hüet anomaly.
• Aüer Bodies: These cytoplasmic inclusions are found in myeloblasts using the Roma-
nowsky stains in patients with acute myelogenous leukaemia. Their detection can be
markedly improved by a modied peroxidase staining technique, when they are referred to as Phi bodies. They are needle-like in shape with intense red or purple red
colour, usually found as a single inclusion.
• Smudge Cells: These are degenerated lymphocytes that look like baskets (often called
basket cells) with no cellular wall. Their presence may be associated with chronic lymphocytic leukaemias. The presence of a few smudge cells in a smear is considered normal and is probably due to faulty technique.
• Atypical Lymphocytes: These abnormal lymphocytes are often visible in the circula-
tion with a viral infection, especially in case of infectious mononucleosis (glandular
fever), whooping cough and measles. They are also found in tuberculosis, severe malaria and the acquired immunodeciency syndrome (AIDS). These are of variable size
(12–18 μM), usually irregular in shape, with round or irregular nuclei. The nucleus is
round or irregular and lies to one side of the cell. Nucleoli may also be seen. The cytoplasm is often darker blue in colour and forms a dark edge. It does not contain granules.
• Lymphoblasts: These are the most immature of all leukocytes. These are visible in the
circulation of leukemic patients. The cells are 15–25 μm in size with large, round, pale,
mauve-coloured nuclei that contain 1–5 nucleoli. The cytoplasm is dark blue, with a
clear unstained area around the nucleus. It does not contain granules.
• Megakaryocytes: These are the parent cells of thrombocytes and found in the bone
marrow. They are seen in the circulation only under abnormal conditions. They are
Routine Haematological Tests
Platelet count/500 RBC RBC count/ L
/L
500
289
relatively large (60–100 μM) with irregular, greatly lobulated but dense nuclei and the
cytoplasm contains numerous ne granules, mostly dark red, and thrombocytes. The
cell wall is not clearly dened.
Estimation of Platelet Count
For a quantitative estimate of the platelet count, the following procedure may be followed. A
normal smear with a normal RBC count of 5 × 106 RBC/μL or cu. mm and a normal platelet
count of 3 × 105 platelets/μL or cu.mm should show 8 to 20 platelets per oil-immersion eld.
A beer estimate would be to determine the number of platelets per 500 RBC and apply the
following formula:
Platelet count
Note This method is inaccurate and is employed only to double check the platelet count
done directly with Neubauer counting chamber (haemocytometry).
Other Stains for Blood Smears
There are four stains used in staining thin blood lms. These are grouped under Romanowsky
stain, which essentially contains azure В and eosin dyes. The Romanowsky stains most widely
used include:
• Leishman stain, which was described earlier.
• May-Grünwald stain, which is used with the Giemsa stain.
• Giemsa stain, which can be used alone or together with May-Grünwald or Jenner
stain.
• Field stains A and B, which are prepared in water unlike the above-mentioned stains,
which are made up in methanol. Field stains are used for both thin and thick blood
lms.
The Romanowski stains prepared in methanol can be used to x thin lms before being di-
luted on the slide to stain lms. Beer results are obtained by xing rst with methanol, and
then staining with pre- prepared diluted stains, as described earlier.
Principle of Romanowski-Giemsa stain
Normal blood smears are stained by the standardized azure B-eosin Y Romanowsky procedure.
Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and
eosinophil granules. Azure В very rapidly gives rise to blue stained chromatin, neutrophil
specic granules, platelets and ribosome-rich cytoplasm; also to violet basophil granules.
Subsequently the azure В in certain structures combines with eosin to give purple azure
B-eosin complexes, leaving other structures with their initial colours. The selectivity of
complex formation is controlled by rate of entry of eosin into azure В stained structures. Only
faster staining structures (i.e., chromatin, neutrophil specic granules, and platelets) permit
formation of the purple complex in the standard method.
Reagents
May-Grünwald stain
May-Grünwald powder 5 g
Methanol (q.s.) 1000 mL
Rinse out and clean a 1-L volumetric ask with methanol. Add a few clean dry glass beads.
Add the staining powder and methanol. Mix well to dissolve the stain. Make to the nal volume of 1 litre (q.s., quantum sucit). Label the bole and write the date.
Medical Laboratory Technology: Volume 1
Note The stain is improved by keeping for 1–2 weeks, mixing at intervals. It is important to
prevent moisture from entering the stain during its preparation and storage.
Giemsa stain
Powdered Giemsa stain 0.75 g
Methanol (CH3OH) 65 mL
Glycerol (C3H8O3) 35 mL
Put the ingredients in a bole containing glass beads and shake. Shake the bole three
times a day for four consecutive days. Filter into a staining bole. Label the bole and write
the date. May-Grünwald and Giemsa are stable for ve years. The boles must be kept closed.
The advised storage temperature is 18–30°C. Used solutions and solutions that are past their
shelf-life must be disposed of, according to local disposal guidelines.
Field stain
Field stain В powder 4.8 g
Distilled water, heated to 80°C (q.s.) 600 mL
Mix until dissolved. When cool, lter into a 1000 mL bole. Label the bole and write the date.
Storage Upon receipt, store at 2–30°C in dark. Product should not be used if there are any
signs of deterioration or if the expiration date has passed. Do not expose to excessive heat or
moisture. Product is light sensitive so protect from light. The expiration date applies to the
product in its intact packaging when stored as directed. This is commonly 180 days.
Procedures
A. May—Grünwald and Giemsa stains (Figure 10.22)
1. Fix the blood lm with methanol for 2-3 min.
2. Dilute May-Griinwald stain 1:2 using equal volumes of stain and buered water. For
example, mix 10-mL stain and 10-mL water.
Giemsa staining: (a and b) Make 1:24 dilution of Giemsa stain before use, (c) Flood the
blood smear with freshly diluted Giemsa stain, (d) Allow the stain to stay on the smear
for the desired time, (e) Wash the stain o with buered water, (f) Dry the stained smear
on a rack
Routine Haematological Tests
3. Dilute Giemsa stain 1 in 10 using one volume of stain and nine volumes of buered
water. Mix gently. (For example, 2-mL stain and 18-mL water).
Note Prepare only enough stain for 1 day’s use, as the diluted stains do not keep
well. Prepare the Giemsa mixture slowly and carefully. Shaking causes the stain to
precipitate.
4. Cover the slide with diluted May-Grünwald stain for 5 min.
5. Tip the stain o and replace with diluted Giemsa stain for 10 min.
Important: The staining time may need to be adjusted, especially when a new batch
of stain is received or the stain has been stored for a long time.
6. Wash the stain o in a stream of buered water. Do not tip the stain o as this leaves
a deposit of stain on the lm.
7. Leave clean water on the slide for 2–3 min to dierentiate the lm. The time for
dierentiation depends on the stain and pH of the water used. The pH should be
between 6.8 and 7.0.
8. Tip the water o and place the slide in a draining rack to dry.
B. Field Stain
The Field stain (Figure 10.23) is rapid and convenient. It was originally introduced for the thick
lms for malarial parasites. With some modications it can be used for the rapid screening
of blood smears.
1. Fix the thin blood lm with ethanol for 2–3 min.
2. Dip the slide into Field stain В and count up to ve. Drain and wash the slide in the
rst container of tap water.
3. Drain and dip the slide into Field stain A and count up to 10. Drain and wash the slide
well in the second container of tap water.
4. Examine the colour of the lm. It should appear mauve, neither too blue nor too pink.
If the lm is not satisfactory, return the slide either to the Field stain A or to the Field
stain В for a few more seconds, as needed.
Field staining (rapidprocedure): (a) Dip the smear ve times in stain A, (b) Take out, and
(c) Wash in distilled water, (d) Repeat the same with stain B (d, e, f), (g) Finally, dry the
stained smear on a rack
Additional information
• Deposits caused by May-Grünwald stain or neutral water can be seen with the naked
eye in the liquid on the slide. Drain o the stain. Rinse the slide twice in methanol. Dry
and re-stain using fresh or ltered May-Grünwald stain.
• Deposits of Giemsa stain can be seen with the naked eye or under the microscope. Rinse
with methanol, but wash o immediately with neutral water. Dry the slide and repeat
the staining procedure from the beginning.
292
• Too much blue in the lm (basophilic staining): Prepare a solution of 1% boric acid in
95% ethanol. Rinse the slide twice in this preparation. Wash at once in neutral water.
Dry and examine under the microscope. Basophilic staining can usually be prevented
by using buered water at more acid pH and, if necessary, altering the dierentiation
time. Poor staining may also be caused by impurities in the dyes; therefore, the use of a
standardized stain is recommended.
• Clearing of stain on the lm is done with water, not methanol.
Medical Laboratory Technology: Volume 1
automateD systems in Haematology
Introduction of automation in the routine tests performed in haematology has considerably
improved the accuracy of results and also the eciency of the laboratory.
There are two major techniques currently used for automated cell counting: (1) Electrical
Impedance Cell Counting, and (2) Light-Scaer Cell Counting. Many new instruments use a
combination of the two.
Electrical Impedance Cell Counting
This electrometrie method of cell counting, rst devised by the Coulter Counter (Hialeah,
Florida, USA) is still the most popular one. For example, the Coulter Model S reports seven
parameters: Hb, Hct, RBC count, WBC count, MCV, MCH and MCHC.
Blood cells are suspended in an electrolyte solution (Isoton) and made to ow from an out-
er chamber into an inner chamber through an orice of 100 μm diameter (Figure 10.24a); an
electrode is placed in each chamber to sense the electric current owing through the orice.
When a cell (poor conductor of electricity) passes through the orice, it imparts resistance to
the electrical conductivity between the two chambers. The intermient resistance imposed
when each cell ows through the orice, is recorded as a voltage pulse that corresponds to the
counting of the cell. In addition, the degree of resistance, which is proportional to the volume
of the cell, gives the machine the capability of measuring the size of the cell. Red cells and
white cells are counted separately by diluting the blood in dierent diluents (Figure 10.24b).
The white cell diluent is Drabkin solution, which, in addition to the WBC count, reports Hb
concentration. Of all the indices, MCV is actually measured from the average amplitude of
voltage pulse in the region of red cell size (6 to 9 μm). Haematocrit value is calculated from
MCV and RBC count (MCV × RBC in millions divided by 10). Other indices are calculated
(MCH and MCHC) in the same way as described earlier. With the improvement in technolo-
gy, recent models are reporting platelet count, lymphocyte count and average population of
dierent sizes of cells.
Other automated systems, based on electrometric counting have come to the Indian market
like the Celloscope (Lars Ljungberg & Co.), Toa micro-cell counter (Toa Electric Co., Ltd.),
Particle Counter (Erma Optical Works Ltd.) and several others.
Diluents for Red Blood Cell Counting
It is always advisable to use the diluent suggested by the manufacturer. This prolongs the life
of the machine. Unfortunately, this may prove to be expensive. Hence, alternative diluents can
be tried out with minimal risk. For example, Eagle’s solution can be used in place of Isoton.
Phosphate-buered physiological saline (0.85% NaCl), adjusted to pH 7.4, is acceptable. Use
a combination of KH2PO4 and Na2HPO4 (0.1% solution of each) and adjust the pH before the
nal volume of NaCl solution is made. The diluents must be particle-free, hence use distilled
water and good quality reagents; lter if necessary through Millipore lters.
Routine Haematological Tests
293
Principles of electrometrie blood cell counting (Coulter model): (a) Essential components
of ow of cells, (b) Process of reporting seven parameters of complete blood count, CBC
Correction of leukocyte count of particle counter
The white cell diluting uid of the particle counter, for example, Coulter Counter needs to be
corrected for specimens with a high number of NRBCs (nucleated red blood cells), because
the diluent does not haemolyse the red cells and they are counted by the counter as WBCs.
The formula for the correction is as follows (the same formula as described under manual
counting):
294
100 + 8
Corrected WBC count =
Medical Laboratory Technology: Volume 1
Uncorrected WBC count
100 + NRBC per
100 WBCs
100
Example If Coulter counter reports 6000 WBC/μL (uncorrected) and the NRBC per 100
WBC is 8, report the corrected value by the following calculation:
Corrected WBC =
6000
100 55 10
3
.
Light-Scatter Cell Counting
In this method, a laser or a tungsten-halogen lamp is pointed towards the blood stream,
which passes through a channel that is so narrow that cells can only pass through one by
one (Figure 10.25). This is achieved through a hydrodynamically focused ow, called sheath
ow. When the cell passes through the light beam the light is scaered or reected into a
Automated dierential counter. As the white cells pass in a single row under a light path,
the size and nature of the cells are recognized by two detectors through light scattering
technology. The results are compared with automated dierential count, using blood
smears.
* WBC is determined from the study of blood smear (dierential count).
Routine Haematological Tests
particular direction, which depends mainly on the size of the cell but also on the shape and
the refractive index. A photo-detector is placed at the angle at which the light is scaered.
The use of lasers is preferred due to the focused light beam that they produce. The progress
in laser technology, based on semiconductors, makes this also the more price ecient light
source. Calibration of these instruments has to be performed using human blood.
295
Automated differential count
In order to achieve dierential counts, it is necessary that the instrument can distinguish the
dierent cell types. This can be done in both the two techniques of cell counting described
above. Each manufacture uses dierent mechanisms to achieve this task.
The basic principles that are used are:
• For the electrical impedance based instruments such as the Beckman Coulter Ac*T
series the cells are subjected to special reagents which shrink the cytoplasm of each
type of white blood cell (WBC) to a dierent degree. This allows classifying the cell
by size.
• Instruments like the Hemalog D from Technicon Corp. use chemicals that inuences or
stain the cells in specic ways and the cell can thus be distinguished when illuminated
by a laser beam either by their scaering properties or the colour of the light that they
emit.
• The light-scaered method can be rened such that the intensity of the scaered light
is measured in various angles. The ratio of intensities from the dierent detectors
characterizes the cell. This method has the advantage that cells do not have to be
modied.
• With the availability of the digital camera and computing power, the computer can
also imitate the manual methods. In these instruments, a blood smear is recorded and
compared to the images of cells in the computer.
• For research instruments, other principles are exploited as well and may nd their way
into industrial labs in the future. Among them are advanced methods to stain or ‘tag’
the cells in dierent ways using special stains. The particular cell can then be identied
by a characteristic wavelength, which can be excited to produce light emission of a
particular colour. Other methods use UV light that produces dierent light emission
spectra for dierent cells.
Just as in manual cell counting, advanced instruments for automated cell dierential
often use a combination of the principles described above. This allows for beer accuracy and
precision.
Current Progress in Automation
The CBC count and leukocyte dierential count (LDC) are among the most frequently
requested clinical laboratory tests. These analyses are now highly automated. Correct
interpretation of results, however, requires extensive knowledge of the analytic performance
of the instruments and the clinical signicance of the results they provide.
During the last 2 decades, automated blood cell counters have undergone a formidable
technological evolution owing to the introduction of new physical principles for cellular
analysis and the progressive evolution of computer software. In addition to the traditional
parameters of the complete blood count (CBC) and leukocyte dierential count (LDC), the
more complete analysers are able to provide much more information, both quantitative,
such as the extended dierential count (EDC). The new technology is now able to recognize
cells that are normally absent from peripheral blood such as blasts, atypical lymphocytes,
296
immature granulocytes, and nucleated RBCs (NRBCs). For some consolidated parameters,
such as WBC and RBC counts, haemoglobin concentration, or mean corpuscular volume
(MCV), analytic performance is generally excellent. For others, in particular, certain
components of the LDC and reticulocyte or platelet counts, especially at low concentrations,
performance is less satisfactory.
The traditional microscopic method based on the count of 100 cells has 3 types of error:
• statistical error
• distributional error owing to unequal distribution of cells in the smear
• errors in identifying cells related to the subjective interpretation of the examiner.
The most important error is statistical because it is invariably related to the total number of
cells analysed. This method, therefore, suers from imprecision, poor accuracy, and reduced
clinical sensitivity. The automated counters performing LDCs analyse thousands of cells per
sample and can produce morphologic and quantitative ags, which have signicantly reduced error and allow for reliable absolute counts at low and high concentrations. Expressing
WBC populations in absolute values has many uses, from noting the increase in lymphocytes
in lymphoproliferative diseases or viral infections, to the increase in eosinophils in parasitosis
and allergic diseases, to the increase in neutrophils seen in infections and acute inammation.
The absolute count is even more useful for monitoring neutropenia during chemotherapy or
after bone marrow transplantation. In case of monocytes, only an absolute count can discern
monocytopenia and study its causes or associations (e.g., marrow aplasia, hairy cell leukaemia, HIV infection, megaloblastic anaemia).
Medical Laboratory Technology: Volume 1
Flow cytometry
Flow cytometers (FC) are automated instruments that quantitate properties of single cells, one
cell at a time. They can measure cell size, cell granularity, the amounts of cell components like
total DNA and m-RNA. Typically, up to three to six properties or components are quantitated
in a single sample, cell by cell, for about 10,000 cells, in less than one minute (not counting
time to prepare the sample, which might be an hour or more). Simply measuring cell size and
granularity is sucient to distinguish the major categories of leukocytes in peripheral blood.
This is the basis for clinical instruments that do automated complete blood counts (CBC).
Adding uorescent probes to the cells enables quantitation of specic structures (“ow
cytouorometry”). The most common use of ow cytouorometry is for total DNA per cell
in biopsy specimens from tumors, for clinical cancer diagnosis and prognosis. Another
major use is quantitation of T-lymphocytes in blood to determine when an HIV infection has
resulted in AIDS, and the degree to which anti-HIV drugs are working.
Flow cytometers take in a suspension of monodisperse (single, unclumped) cells and run
them one at a time (single le) past a laser beam. As each cell passes through the laser beam,
scaered and uorescent light are quantitated.
Flow cytouorometry (FC) can be contrasted with uorescence microscopy (FM). FC can
quantitate total amounts of a component per cell for a large number of cells (typically 10,000,
up to 100,000 easily). FC can sort thousands of living cells according to their uorescent
properties, while FM cannot.
retiCuloCyte Count
Reticulocytes are juvenile red cells that pass into the blood stream from the bone marrow.
Reticulocytes stay in circulation for about 24 h and mature into erythrocytes. Reticulocytes
have ribosomal and cytoplasmic remnants, which pick up supravital stain. Following this
process of supravital staining, a blood smear is made and the number of reticulocytes is
counted against the number of red cells observed in the blood smear.
Routine Haematological Tests
/L
100
297
Clinical significance
The number of reticulocytes (abbreviated as retics) in the blood circulation indicates the
degree of bone marrow activity. When the marrow is very active (e.g., in haemolytic anaemia
or acute blood loss), their number increases. This is known as reticulocytosis. In case of
aplastic anaemia (poor activity of bone marrow), the reticulocyte count is decreased.
Normal value
Infants: 2–6% of RBCs (8–110 × 103/μL of whole blood)
Adults (and children): 1–2% of red cells (100–300 × 103/μL of whole blood)
Specimen
EDTA-anticoagulated blood is commonly used but other anticoagulants do not interfere.
Heparinized capillary blood can also be used. A blood specimen collected within 2–3 h should
be used; older blood specimens show artefacts.
Principle
Reticulocytes cannot be clearly dierentiated in the blood smear with Leishman stain during
the dierential count. They appear light blue and polychromie but may go unnoticed. Hence,
for the enumeration of reticulocytes, special supravital staining is needed. In this procedure,
the stain (new methylene blue or brilliant cresyl blue) enters the cell in the living condition
(supravital staining) and then the blood smear is made. This is unlike the dierential count
procedure, where the smear is made before staining.
New methylene blue is the preferred stain over brilliant cresyl blue stain. It stains deeply
the lamentous net-like structure (reticulum) present in the cytoplasm of reticulocytes and
they are more readily identied. The most immature reticulocytes are those which are bigger
in size and have a heavy reticulum inside. As the reticulocytes mature in the circulation, the
reticular structure is reduced to a few dots or short strands, and ultimately erythrocytes form
without any trace of reticular structure.
Unlike haemocytometry, a direct absolute count of reticulocytes is not possible. A relative
count is taken against the number of red cells and expressed as a percentage of red cells. This
relative value can be converted to an absolute value by the following equation:
Absolute count of reticulocytesof blood =
/L
Equipment and supplies
• Microscope
• Microscope slide
• Glass spreader
• Test tubes—small (5-mL) and large (15-mL)
• Test tube rack
• Funnel
• Filter paper
• Two Pasteur pipees
• Hand tally counter (Figure 10.26)
• Grease-free slides
Reagent
• Sodium chloride solution (0.85%) in water
Sodium chloride (NaCl) 8.5 g
Water (q.s.) 1000 mL
Reticulocyte
((%) RBC count