LABORATORY
MEDICINE: HISTORY AND MODERN ASPECTS
LABORATORY TESTS IN HEMATOLOGY – I
LABORATORY TESTS IN HEMATOLOGY – II
Clinical
Laboratory diagnostics(laboratory
medicine) is the medical
discipline devoted to obtain, explore and employ knowledge about using various
techniques for the analysis of body fluids composition and properties of cells
and tissues, and interpretation of the results in relation to health and
disease.
It should be stressed that laboratory diagnostics or
laboratory medicine is both the clinical discipline and the separate medical
science. These two fields of laboratory diagnostics are tightly bound as in the
case of other clinical sciences. Laboratory tests are used in various stages of
the diagnostic process in all fields of clinical medicine, being along with
imaging studies, electrophysiological and other procedures the main source of
information on the health status of the patient. It is estimated that laboratory
results can be the basis of 60 % -70 % of medical decisions. In addition to
routine diagnostics in symptomatic patients, laboratory tests are used for
screening, treatment monitoring and medical jurisprudence. Thus, laboratory
diagnostics generating around 10 % of all healthcare costs is crucial for the
healthcare decision making process, contributing to improved outcomes and cost
savings
Clinical
laboratory (also
called medical laboratory) is a facility that provides controlled conditions in
which tests are done on clinical specimens in order to acquire information
about the health of an individual (or patient) for the purpose of diagnosis,
treatment, and
prevention of disease or
medical research.
The clinical
laboratory consists of four major divisions (or departments).
1. Medical
Microbiology and Parasitological Laboratory: This laboratory
deals with the study of human pathogens. Pathogens are biological agents that
cause diseases to their hosts. They include microorganisms (bacteria, viruses
and fungi) and parasites (e.g. intestinal worms, lice and malaria parasites) of
medical importance. In bigger health centresor research institutes, medical microbiology and
parasitological laboratory is usually split into sub-unit like bacteriology, parasitology
and virology laboratories.
2. Haematology: This laboratory is
involved in the performance of relevant tests (on blood) in the diagnosis of
blood diseases, (e.g. Anaemia, Haemoglobinopathies, Leukaemiaetc) and blood transfusion services e.g. blood
group, blood cross matching.
3. Clinical
Biochemistry Laboratory or Clinical Chemistry : This
division of laboratory is concerned with
the performance of quantitative and qualitative tests on clinicalspecimens to
investigate the state of various body chemistries. Such clinical specimens
include body fluids (e.g. whole blood, plasma, serum, urine, sweat,
cerebrospinal fluid) and occasionally faeces, tissue, hair e.t.c.
4. Histopathology
Laboratory: This is
the laboratory where tissues (or cells) are processed for microscopic
examination in order to investigate or study disease manifestations on the
tissue (or cells), structure, for diagnostic purposes e.g. Cancer diagnosis. In
the laboratory, tissue samples are processed onto glass slides from which
effects of diseases on the histological architecture of tissues can be
microscopically examined and hence diagnostic inferences are made.
Universal
Precautionary Measure in Clinical Laboratory
Garner (1997) defined Universal Basic Precaution as
the prevention of transmission of blood pathogens through strict respect of
rules concerning care and nursing. Gerberding et
al.,(1995) also defined universal precaution as the routine use of
appropriate barrier and techniques to reduce the likelihood of exposure to
blood, other body fluid and tissue that may contain blood borne pathogens.
Universal basic precautions assume that all clinical
specimens contain infectious agents and should therefore be handled as such.
This approach eliminates the need to identify infected patients or specimen
from Human Immunodeficiency Virus (HIV) or other blood borne pathogen infected
patients.
The followings are
the laboratory universal safety precautions.
1. Universal
precautions should apply to blood and all body fluid containing visible blood,
semen, vaginal secretions, tissues, cerebrospinal fluid, peritoneal fluid,
pericardial fluid, synovial fluid and amniotic fluid.
2. Laboratory
workers should use protective barriers appropriate for the laboratory procedure
and the type and extent of exposure expected. All persons processing blood
should wear gloves and laboratory coats; and these should be removed before
leaving the
laboratory.
Biological safety barriers should be used wherever necessary.
3. Hands should be
washed immediately when contaminated with blood or other body fluids, after
removing gloves and after completing laboratory activities.
4. Use of needles
and syringes should be minimised. They should be used in situations in which there
is no alternative. If used, needles should not be recapped or bent or broken by
hand. After use, needles and other sharp instruments should be placed in a ‘sharpsafe’
puncture-resistant container for disposal.
5. Specimens of
blood should be placed in strong-leak-proof containers during transport.
6. Mouth pipetting
must not be performed in the laboratory. Mechanical devices should be used.
7. Contaminated
materials used in the laboratory should be decontaminated appropriately before
reprocessing or disposal.
8. Laboratory work
surfaces should be cleaned and decontaminated with appropriate disinfectant
after a blood or body fluid spill and at the end of day’s work.
History of
laboratory medicine
Three distinct
periods in the history of medicine are associated with three different places
and, therefore, different methods of determining diagnosis: From the middle
ages to the 18th century, bedside medicine was prevalent; then between 1794 and
1848 came hospital medicine; and from that time forward, laboratory medicine
has served as medicine’s lodestar. The laboratory’s contribution to modern
medicine has only recently been recognized by historians as something more than
the addition of another resource to medical science and is now being
appreciated
as the seat of
medicine, where clinicians account for what they observe in their patients.
The first medical diagnoses made by humans were based
on what ancient physicians could observe with their eyes and ears, which
sometimes also included the examination of human specimens.
The ancient Greeks attributed all disease to disorders
of bodily fluids called humors, and during the late medieval period, doctors
routinely performed uroscopy. Later, the microscope revealed not only the
cellular structure of human tissue, but also the organisms that cause disease.
More sophisticated diagnostic
tools and
techniques—such as the thermometer for measuring temperature and the stethoscope
for measuring heart rate—were not in widespread use until the end of the 19th
century. The clinical laboratory would not become a standard fixture of
medicine until the beginning of the 20th century. This four-part article
reviews the history and development of diagnostic methods from ancient to
modern times, as well as the evolution of the clinical laboratory from the late
19th century to the present.
Ancient diagnostic methods
In ancient
treating the middle and
lower classes included divination through ritual sacrifice to predict the
outcome of illness. Usually a sheep would be killed before the statue of a god.
Its liver was examined for malformations or peculiarities; the shape of the
lobes and the orientation of the common duct were then used to predict the fate
of the patient.
Ancient physicians
also began the practice of examining patient specimens. The oldest known test
on body fluids was done on urine in ancient times (before 400 BC). Urine was
poured on the ground and observed to see whether it attracted
insects. If it
did, patients were diagnosed with boils. The ancient Greeks also saw the value
in examining body fluids to predict disease. At around 300 BC, Hippocrates
promoted the use of the mind and senses as diagnostic tools, a principle that
played a large part in his reputation as the “Father of Medicine.” The central
Hippocratic doctrine of humoral pathology
attributed all disease to disorders of fluids of the body. To obtain a clear
picture of disease, Hippocrates
advocated a diagnostic
protocol that included tasting the patient’s urine, listening to the lungs, and
observing skin color and other outward appearances. Beyond that, the physician
was to “understand the patient as an individual.” Hippocrates related the
appearance of bubbles on the surface of urine specimens to kidney disease and
chronic illness. He also related certain urine sediments and blood and pus in
urine to disease. The first description of hematuria, or
the presence of blood in urine, by Rufus of Ephesus surfaced at around AD 50
and was attributed to the failure of kidneys to function properly in filtering
the blood. Later (c. AD 180), Galen (AD 131–201), who is recognized as the
founder of experimental physiology, created a
system of pathology that
combined Hippocrates’ humoral theories
with the Pythagorean theory, which held that the four elements (earth, air,
fire and water), corresponded to various combinations of the physiologic
qualities of dry, cold, hot
and moist. These
combinations of physiologic characteristics corresponded roughly to the four
humors of the human body: hot + moist = blood; hot + dry = yellow bile; cold +
moist = phlegm; and cold + dry = black bile.
Galen was known
for explaining everything in light of his theory and for having an explanation
for everything.
He also described
diabetes as “diarrhea of urine” and noted the normal relationship between fluid
intake and urine volume. His unwavering belief in his own infallibility
appealed to complacency and reverence for authority. That dogmatism essentially
brought innovation and discovery in European medicine to a standstill for
nearly 14 centuries. Anything relating to anatomy, physiology and disease was
simply referred back to Galen as the final authority from whom there could be
no appeal.
Middle Ages
In medieval
practiced, and
the urine flask became the emblem of medieval medicine. By AD 900, Isaac Judaeus, a Jewish
physician and philosopher, had devised guidelines for the use of urine as a
diagnostic aid; and under the Jerusalem Code of 1090, failure to examine the
urine exposed a physician to public beatings. Patients carried their urine to
physicians in decorative flasks cradled in wicker baskets and, because urine
could be shipped, diagnosis at long distance was common. The first book
detailing the color, density, quality and sediment found in urine was written
around this time, as
well. By around AD 1300, uroscopy became
so widespread that it was at the point of near universality in European
medicine.
Medieval medicine
also included interpretation of dreams in its diagnostic repertoire. Repeated
dreams of floods indicated “an excess of humors that required evacuation,” and
dreams of flight signified “excessive evaporation of humors.”
Seventeenth
century
The medical advances of the 17th century consisted
mostly of descriptive works of bodily structure and function that laid the
groundwork for diagnostic and therapeutic discoveries that followed. The status
of medicine was helped along by the introduction of the scientific society in
The invention of the microscope opened the door to the
invisible world just as Galileo’s telescope had revealed a vast astronomy. The
earliest microscopist was a Jesuit priest, Athanasius Kircher (1602–1680) of
Kircher’s writings included
an observation that the blood of patients with the plague contained “worms;”
however, what he thought to be organisms were probably pus cells and red blood
corpuscles because he could not have observed
Bacillus pestis with a
32-power microscope. Robert
Hooke (1635–1703) later used the microscope to document the existence of
“little boxes,” or cells, in vegetables and inspired the works of later histologists;
but some of the greatest contributions
to medical science
came from Italian microscopist, Marcello Malpighi (1628–1694). Malpighi, who
is described as the founder of histology, served as physician to Pope Innocent
XII and was famous for his investigations of the embryology of the chick and
the histology and physiology of the glands and viscera. His
work in embryology
describes the minutiae of the aortic arches, the head fold, the neural groove,
and the cerebral and optic vesicles.
Uroscopy was still in
widespread use and had gained popularity as a method to diagnose “chlorosis,” or love-sick young women, and sometimes to test
for chastity. Other methods of urinalysis had their roots in the 17th century,
as well.
The gravimetric analysis of urine was introduced by
the Belgian mystic, Jean Baptiste van Helmont (1577–1644).
Van Helmont weighed
a number of 24-hour specimens, but was unable to draw any valuable conclusions
from his measurements. It was not until the late 17th century—when Frederik Dekkers of
that urine that
contained protein would form a precipitate when boiled with acetic acid—that
urinalysis became more scientific and more valuable. The best qualitative
analysis of urine at the time was pioneered by Thomas Willis
(1621–1675), an English physician and
proponent of chemistry. He was
the first to notice the characteristic sweet taste of diabetic urine, which
established the principle for the differential diagnosis of diabetes mellitus
and diabetes insipidus.
Experiments with blood transfusion were also getting
underway with the help of a physiologist in
as indicators of
health status.
18-th century
The 18th century is regarded as the “Golden Age” of
the successful practitioner, as well as the successful quack. Use of phrenology
(the study of the shape of the skull to predict mental faculties and
character), magnets, and various powders and potions for treatment of illness
were a few of the more popular scams. The advancement of medicine during this
time was more theoretical than practical. Internal medicine was improved by new
textbooks that cataloged and described many new forms of disease, as well as by
the introduction of new drugs, such as digitalis and opium. The state of
hospitals in the 18th century, however, was alarming by today’s standards.
Recovery from surgical operations was rare because of septicemia. The concept
of antisepsis had not yet been discovered, and hospitals were notorious for
filth and disease well into the 19th century. One notable event that is a
forerunner to the modern practice of laboratory measurement of prothrombin time, plasmathromboplastin time and other coagulation tests, was
the discovery of the
cause of coagulation. An English physiologist, William Hewson (1739–1774) of Hexham,
from the corpuscles and
skimmed off the surface. Hewson found
that plasma contains an insoluble substance that can be precipitated and
removed from plasma at a temperature slightly higher than 50°C. Hewson deduced that coagulation
was the formation in the
plasma of a substance he called “coagulable lymph,” which is now known as
fibrinogen. A later discovery that fibrinogen is a plasma protein and that in
coagulation it is converted into fibrin attests to the importance
of Hewson’s work.
The clinical diagnostic methods of percussion,
temperature, heart rate and blood pressure measurements were further refined,
and there were some remarkable attempts to employ precision instruments in
diagnosis. Leopold Auenbrugger (1722–1809)
was the first to use percussion of the chest in diagnosis in 1754 in
of heart and lung
diseases using Auenbrugger’s chestthumping technique. Corvisart’s translation of Auenbrugger’s treatise on percussion, “New Invention
to Detect by Percussion Hidden Diseases in the Chest,” popularized the practice
of thumping on a
patient’s chest. The resulting sounds are different when the lungs contain
lesions or fluids than in healthy people. This observation was validated by
postmortem examination.
James Currie (1756–1805), a Scot, was the first to use
cold baths in treatment of typhoid fever; and by monitoring the patient’s
temperature using a thermometer, he was able to adjust the temperature and frequency
of the baths to treat individual patients. It took another hundred years,
however, before thermometry became a recognized feature in clinical diagnosis.
Additional advances in urinalysis occurred with J.W. Tichy’s observations of sediments in the urine of febrile patients (1774); Matthew Dobson’s proof that thesweetness of the urine and blood serum in diabetes is caused by sugar (1776); and the development of the yeast test for sugar in diabetic urine by Francis Home (1780).
19-th century
1893 T. W. Richards invents the nephelometer;
Hermann M. Biggs establishes Diagnostic Laboratory in
1895 Franz Ziehl and
Friedrich Neelsen introduce their modification of the
acid-fast stain for tuberculosis;
William Roentgen discovers X-rays; William
Pepper Laboratory is established at the
1896 S. Riva-Rocci invents the sphygmomanometer; C. W.
Purdy publishes Practical Urinalysis and Urinary Diagnosis; Ferdinand Widal develops the
agglutination test for
identification of the typhoid bacillus; in
laboratories existed in
1897 The first commercial clinical laboratory
established in
Clinical Research Association, receives
specimens by mail.
20-th century
1899 American
Society for Microbiology is founded.
1902 The DuBoscq visual colorimeter is first introduced
into clinical laboratories.
1903 Ayer
Clinical Laboratory is established at
1904 Christian
Bohr discovers the reciprocal relationship between pH and oxygen content of
hemoglobin (Bohr effect); M. Beijerinck obtains
the first pure culture of the sulfur-oxidizing bacterium Thiobacillus thioparus;
the first ultraviolet
lamps and the first
practical photoelectric cell are invented.
1905 H.J. Bechtold discovers immunodiffusion.
1906 American
Hospital Association is formed from the Association of Hospital Superintendents
of the
1908 Todd
and Sanford publish the first edition of
Diagnosis by Laboratory Methods.
Venipuncture is in widespread use by 1920.
1911 Oskar Heimstadt invents the fluorescence microscope.
1912
1913 D.D.
van Slyke is appointed chemist at Rockefeller
Hospital Laboratory; American Association of Immunologists is founded.
1916 K.M.G. Siegbahn
develops X-ray spectroscopy. P.A. Kohler develops the colorimeter–nephelometer.
1918
1920 First clinical
laboratory method for serum phosphorus is established; the use of venipuncture
for diagnostic testing becomes widespread; Victor Meyers establishes the
University of Iowa center for training clinical chemists, primarily for
hospital positions; Conference of Public Health Laboratories is founded.
1921 First
clinical laboratory method for serum magnesium is introduced; The Denver
Society of Clinical Pathologists, precursor of the American Society of
Clinical Pathologists, is founded in
1922 ASCP is
founded in
1925 American
Type Culture Collection is founded.
1926 Arne
Tiselius develops moving boundary electrophoresis of proteins; Theodor Svedberg
determines the molecular weight of hemoglobin by ultracentrifugation; ASCP appoints
a “Committee on the Registration of Laboratory Technicians” to define and
classify medical technicians.
1929 Otto Folin introduces the use of the light filter
in colorimetry;
R. Gabreus develops the erythrocyte sedimentation
rate as an index of severity of disease;
M. Knoll and E. Ruska invent the electron microscope;
ASCP establishes its Board of Registry for certifying medical technologists;
Mayo Clinic has 21 laboratories by this date.
1930 Kay
develops the first clinical laboratory method for alkaline phosphatase, thus
beginning clinical enzymology; refractometry is first used in clinical
labs for the determination of protein in urine; ASCP issues its first medical technologist certification to P.H. Adams for Ft. Wayne, IN; Beckman
Instruments is founded.
1932 Cherry and Crandall develop the clinical laboratory method for serum lipase activity; American Society of Clinical Laboratory Technicians, precursor of
the American Society for Medical Technology, is founded.
1934 Commercial development of the electron microscope takes place.
1935 Beckman Instruments Co. introduces the first
pH meter; ASCP Board of Registry first requires a college degree for medical technologist certification.
1937 First hospitalbased blood bank is established at Cook
County Hospital, Chicago, IL;
ASCP and its Board of Registry officially oppose state licensure of medical technologists.
1938 Somogyi develops 2 major clinical laboratory methods for serum and urine amylase activity; Gutman develops the first assay for acid phosphatase.
1939 Conway and Cook develop the first clinical laboratory method for blood ammonia; American Medical Technologists is founded.
1940 Visual colorimeters begin to be replaced by photoelectric colorimeters in clinical labs; RCA demonstrates the first commercial electron microscope.
1943 Penicillin is successfully used in therapy.
1944 William Sunderman applies refractometry of proteins in the clinical lab.
1945 S. Borgstrom develops the whole blood clotting time test; itemized charges for hospital services are begun.
1946 The Vacutainer evacuated serum collection tube is introduced by Becton Dickinson Co.; Arne Tiselius separates proteins by chromatography; College of
American Pathologists is founded.
1947 Edwin Land develops the Polaroid camera; American Association of Blood Banks is founded.
1948 American Association of Clinical Chemistry is founded.
1950 R.S. Yalow and S. Berson develop radioimmunoassay; Levey and
Jennings adapt the Shewhart QC
chart to use in clinical laboratories;
Histochemical Society is
founded.
1954 Kuby develops
the clinical lab method for serum creatine phosphokinase
activity; A. Walsh develops the atomic absorption spectrometer.
1955 Wroblewski and LaDue develop the clinical laboratory method
for serum lactate dehydrogenase; Karmen develops
the clinical laboratory method for aspartate aminotransferase; Leonard Skegges develops the concept of “continuous
flow dialysis” in connection with treatment of renal disease; Severo Ochoa
synthesizes RNA.
1956 Wroblewski and LaDue develop the method for serum alanine
aminotransferase activity called “serum glutamic- pyruvic transaminase” and
recognize its greater
specificity for liver disease compared with that of aspartate aminotransferase;
J. Edwards proposes prenatal screening for genetic disease.
1957 Van Handel and Zilversmit develop
a direct chemical method for the determination of triglycerides.
1959 The first clinical laboratory chemical analyzer,
the singlechannel “Auto-Analyzer,” is introduced by Technicon Corp.; Technicon first applies
flame photometry to
automated methods.
1960 Methods
for serum creatine phosphokinase isoenzymes are
developed; the first method
for gamma-glutamyl transferase in
serum is developed;
Perkin-Elmer Corp. introduces atomic absorption
spectrometry
for the determination of
calcium and magnesium; the laser is developed; Feichtmeier invents the mechanical pipettor (Auto Dilator).
1961 Becton
Dickinson Co. introduces disposable hypodermic syringe and needle.
1962 Siegelman develops
a method for glutamic dehydrogenase;
IBM introduces disk storage for computers;
International Society for Clinical
Laboratory Technology is founded.
1965 Scanning
electron microscope is developed; the
Medicare and Medicaid (Titles 18 and 19 of
the Social Security Amendments).
1966 Medicare/Medicaid
officially goes into effect.
serum of patients with
testicular teratocarcinoma; MetPath Laboratories is founded;
1968 The
first random-access analyzer is introduced by DuPont (the ACA); the 1% Medicare
allowance for unidentified costs is reduced to zero;
The use of laboratory tests:
Laboratory investigations
are involved in every branch of clinical medicine.
The results of
laboratory tests may be of use in:
1. Diagnosis and in
the monitoring of treatment.
2. Screening for
disease or in assesing the prognosis.
3. Reseach into the biochemical
basis of disease
4. Clinical trials of
new drugs
Laboratory investigations hold the key for the diagnosis and prognosis
of diabetes mellitus, jaundice, myocardial infarction, gout, pancreatitis,
rickets, cancers, acid-base imbalance etc. Successful medical practice is
unimaginable without the service of clinical laboratory.
In
general, laboratory tests can be broadly divided into two groups:
In 1. Discretionary
or selective requesting, the tests are carried
out on the basis of an individual patient's clinical situation. The case for
discretionary requesting has been put admirably (Asher, 1954):
1. Why
do I request this test?
2. What
will I look for in the result?
3. If I find what I am looking for, will it affect my diagnosis?
4. How will this investigation affect my management of the patient?
5. Will this investigation ultimately benefit the patient?
In contrast, 2. Screening
tests are
used to search for disease without there being any necessary clinical
indication that disease is present.
The situations in which discretionary test requests are undertaken are
listed in Table 1.2
Table 1.2 Test selection for
the purposes of discretionary testing
Category |
Example |
To confirm a diagnosis |
Plasma
(free T4) and (thyroid-stimulating hormone, TSH) in suspected
hyperthyroidism |
To aid differential diagnosis |
To
distinguish between different forms of jaundice |
To refine a diagnosis |
Use
of ACTH to localize Cushing's syndrome |
To
asses the severity of disease |
Plasma
(creatinine) or (urea) in renal disease |
To monitor progress |
Plasma
(glucose) to follow of patients with diabetes mellitus |
To
detect complications or side effects |
ALT
measurements in patients treated with hepatotoxic drug |
To monitor therapy |
Plasma
drug concentration in patients treated with antiepileptic drugs |
Screening may take two
forms: 1. Well-population screening in which typically a spectrum of tests
is carried out on individuals from an apparently healthy population in an
attempt to detect presymptomatic or
early disease. The value of well-population screening has been called into
question and certainly should only be initiated under certain specific circumstances
which are listed in Table 1.3.
Table 1.3 Requirements for well-population screening
The disease is common or life-threatening
The tests are sensitive and specific
The tests are readily applied and acceptable to the population to be
screened
Clinical, laboratory and other facilities are available for follow-up
Economics of screening have been clarified and the implications accepted
2. Case-finding screening programmes
perform appropriate tests on a population sample known to be at high risk of a
particular disease.
These are inherently more selective and yield a higher proportion of
useful results (Table 1.4).
Table 1.4 Examples of tests used in case-finding programmes.
Programmes to detect diseases in |
Chemical investigations |
Neonates: |
|
PKA (phenylketonuria) |
Serum
[phenylalanine] |
Hypothyroidism |
Serum [TSH] and/or [thyroxine] |
Adolescents and young adults: |
|
Substance abuse |
Drug screen |
Pregnancy: |
|
Diabetes mellitus in the
mother |
Plasma and urine
[glucose] |
Open neural tube defect (NTD) in the foetus |
Maternal serum [a-fetoprotein] |
Industry: |
|
Industrial exposure tolead |
Blood [lead] |
Industrial exposure to pesticides |
Plasma cholinesterase activity |
Malnutrition |
Plasma [albumin]
and/or [pre-albumin] |
Thyroid dysfunction |
Plasma [TSH] and/or [thyroxine] |
ADVANTAGES
OF SCREENING
First, an uncommon or unexpected disease may be found and created (Table
1.5). Second, the early requesting of a battery of tests might be expected to
expedite management of the patient. Most studies have not shown this to be so.
Table 1.5 Advantages of screening in identifying unexpected test results
Disease |
Unexpected abnormal test results |
Hyperparathyroidism |
Raised plasma calcium |
Hypothyroidism |
Raised
plasma TSH and/or a low T4 |
Diabetes mellitus |
High random plasma glucose |
Renal tract disease |
Raised
plasma creatinine or urea |
Liver disease |
Increased plasma ALT, AST |
DISADVANTAGES
OF SCREENING
It is easy to miss significant abnormalities
in the 'flood' of data
coming from the laboratory, even when the abnormalities are
'flagged' in some way. Most of the abnormalities detected will be of little or
no significance, yet may need additional time-consuming
and often expensive tests to clarify their importance (or lack of it).
In other instances, to simplify requesting, a wide range of tests are
routinely requested on all patients in a particular category, for example,
admission screening on all those admitted through the Accident and Emergency
(A&E) Department. Mention should also be made of batteries of tests which
are generally requested on a discretionary basis but where the test group
collectively provides information about an organ system (e.g. tests for liver
disease) or a physiological state (e.g. water and electrolyte status). Many
laboratories analyse and report these functional or organ-related groups. For
example, a 'liver function test' group might consist of plasma bilirubin,
alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT)
and albumin measurements.
Clinical biochemical tests comprise over ⅓
of all hospital laboratory investigations.
Core biochemistry: Most biochemistry
laboratories provide the "core analyses", commonly requested tests
which are of value in many patients, on a frequent basis.
Core biochemical tests:
1. Sodium, potassium, chloride and
bicarbonate
2. Urea and creatinine
3. Calcium and phosphate
4. Total protein and albumin
5. Bilirubin and alkaline phosphatase
6. Alanine aminotransferase (ALT) and
Aspartate aminotransferase (AST)
7. Glucose
8. Amylase
Specialized tests:
Not every laboratory is equiped to carry out all possible biochemistry
requests.
Large departments may act as reference centres where less commonly asked for tests are performed.
Specialized tests:
1. Hormones
2. Specific proteins
3. Trace elements
4. Vitamins
5. Drugs
6. Lipids and lipoproteins
7. DNA analyses
The emergency lab:
All clinical biochemistry laboratories
provide facilities for urgent tests. An urgent test is designated as one on
which the clinician is likely to take immediate action. The main reason for
asking for an analysis to be performed on an urgent basis is that immediate
treatment depends on the result.
Emergency tests:
1. Urea and electrolytes
2. Blood gases
3. Amylase
4. Glucose
5. Salicylate
6. Paracetamol
7. Calcium
Specimen collection:
The biological
fluids employed in the clinical
biochemistry laboratory include blood,
urine, saliva, sputum, faeces,
tissue and cells, cerebrospinal
fluid,peritoneal fluid, synovial fluid, pleural fluid, stones.
Among these, blood (directly or in the form
of plasma or serum) is frequently used for the investigations in the clinical
biochemistry laboratory.
Identification of
patients and specimens
The
correct patient must be appropriately identified on the specimen and request
form, as follows:
1. Patient
identification data (PID). This
usually comprises name plus unique number.
2. Test
request information. This
includes relevant clinical details (including any risk of infection hazard),
the tests to be performed and where the report is to be sent.
3. Collection
of specimens. In the correct tube and the appropriate preservative.
4. Matching
of specimens to requests. Each
specimen must be easily and unequivocally matched to the corresponding request
for investigations.
Table 1.1 Some commoner
causes of errors arising from use of the laboratory.
Error |
Consequence |
Crossover of addressograph labels between patients |
This can lead to two patients each with the other's set of results. Labels
between patients. Where the patient is assigned a completely wrong set of
results, it is important to investigate the problem in case there is a second
patient with a corresponding wrong set of results |
Timing
error |
There are many examples where timing is important
but not considered. Sending in a blood sample too early after the
administration of a drug can lead to misleadingly high values in therapeutic
monitoring. Interpretation of some tests (e.g. cortisol) is critically
dependent on the time of day when the blood was sampled |
Sample collection tubeerror |
For some tests the nature of the collection tube is
critical which is why the Biochemistry Laboratory specifies this detail. For example,
using a plasma tube with lithium-heparin as the anticoagulant invalidates
this sample tube for measurement of a therapeutic lithium level! Serum
electrophoresis requires a serum sample; otherwise, the fibrinogen interferes
with the detection of any monoclonal bands. Topping up a biochemistry tube
with a haematology (potassium-ethylenediamine tetraaceticacid
(EDTA) sample) will lead to high potassium and low calcium values in the
biochemistry sample |
Sample taken from close to the site of an intravenous
infusion |
The blood sample will be diluted so that all the
tests will be correspondingly site of an intravenous (IV)
infusion low with the exception of those
tests which might be affected by the composition of the infusion fluid itself.
For example, using normal saline as the infusing fluid would lead to a
lowering of all test results but with sodium and chloride results which are
likely to be raised |
Analytical error |
Although comparatively rare, these do inevitably
happen from time to time and any result which is unexpected should lead the
requesting clinician to discuss the matter further with the Laboratory.
Transcription errors within the Laboratory are increasingly less common
because of the electronic download of results to the Laboratory computer as a
source of the printout or results on the VDU. Most errors generated within
the Laboratory occur at the Reception as a result of mislabelling of samples
within the Laboratory |
COLLECTION OF
BLOOD:
Venous blood is most commonly
used for a majority of biochemical investigations. It can be drawn from any
prominent vein (usually from a vein on the front of the elbow).
Capillary
blood (<0.2
ml) obtained from a finger or thumb, is less frequently employed.
Arterial
blood (usually drawn under local anesthesia)
is used for blood gas determinations.
Precautions
for blood collection
: Use of
sterile (preferably disposable) needles and syringes, cleaning of patients skin,
blood collection in clean and dry vials/tubes are some of the important
precautions.
Biochemical
investigations can be performed on 4
types of blood specimens – whole
blood, plasma, serum and red blood cells. The selection of the specimen depends
on the parameter to be estimated.
1. Whole blood (usually mixed with an anticoagulant)
is used for the estimation of hemoglobin, carboxyhemoglobin, pH, glucose, urea, non-protein nitrogen,
pyruvate, lactate, ammonia etc. (Note : for glucose determination, plasma is prefered in recent years).
2. Plasma, obtained by
centrifuging the whole blood collected with an anticoagulant, is employed for
the parameters—fibrinogen, glucose, bicarbonate, chloride, ascorbic acid etc.
3. Serum is the supernatant
fluid that can be collected after centrifuging the clotted blood. It is the
most frequently used specimen in the clinical biochemistry laboratory. The
parameters estimated in serum include proteins (albumin/globulins), creatinine,
bilirubin, cholesterol, uric acid, electroylets (Na+,
K+, Cl-),
enzymes (ALT, AST, LDH, CK, ALP, ACP, amylase, lipase) and vitamins.
4. Red blood cells are employed for
the determination of abnormal hemoglobins, glucose 6-phosphate dehydrogenase, pyruvate
kinase etc.
Collection
and preservation of blood specimens
Lack of
thought before collecting specimens or carelessness in collection may adversely
affect the interpretation or impair the validity of the tests carried out on the
specimens. Some factors to consider include the following:
1. Diet Dietary
constituents may alter the concentrations of analytes in
blood significantly (e.g. plasma [glucose] and [triglyceride] are affected by
carbohydrate and fat-containing meals, respectively).
2. Drugs Many drugs
influence the chemical composition of blood. Such effects of drug treatment,
for example, antiepileptic drugs, have to be taken into account when
interpreting test results. Details of relevant drug treatment must be given
when requesting chemical analyses, especially when toxicological
investigations are to be performed.
3. Diurnal
variation. The
concentrations of many substances in blood vary considerably at different times
of day (e.g. cortisol). Specimens for these analyses must be collected at the
times specified by the laboratory, as there may be no reference ranges relating
to their concentrations in blood at other times
Care
when collection blood specimens
The
posture of the patient, the choice of skin-cleansing agent and the selection of
a suitable vien (or other source) are the principal
factors to consider before proceeding to collect each
specimen:
1. The
skin must be clean over
the site for collecting the blood specimen. However, it must be remembered
that alconol and methylated spirits can cause
haemolysis, and that their use is clearly to be avoided if blood [ethanol] is
to be determined.
2. Limbs
into which intravenous infusions are
being given must not be selected as the site of venepuncture unless particular
care is taken. The needle or cannula must first be thoroughly flushed out with
blood to avoid dilution of the specimen with infusion fluid.
3. Venepuncture
technique should be standardised as
far as possible to enable closer comparison of successive results on patients.
4. Venous
blood specimens should be obtained with minimal stasis Prolonged stasis
can markedly raise the concentrations of plasma proteins and other
non-diffusible substances (e.g. protein-bound substances). It is advisable to release
the tourniquet before withdrawing the sample of blood.
5. Posture
should be standardised if possible When a patient's
posture changes from lying to standing, there may be an increase of as much as
13% in the concentration of plasma proteins or protein-bound constituents, due
to redistribution of fluid in the extracellular space.
6. Haemolysis
should be avoided, since
it renders specimens unsuitable for plasma K+, magnesium and many protein and enzyme
activity measurements.
7. Infection hazard High-risk specimens require special care in collection,
and this danger must be clearly indicated on the request form.
Vacutainers used
for blood collection and storage
Care of
blood specimens after collection
Blood
specimens should be transported to the laboratory as soon as possible after collection.
Special arrangements are needed for some specimens (e.g. for acid-base
measurements, or unstable hormones) because of their lack of stability. Most
other analytes are stable for at least 3 h in whole
blood, or longer if plasma or serum is first separated from the cells. As a
rule, whole blood specimens
for chemical analysis must not be stored in a refrigerator, since ionic pumps that maintain
electrolyte gradients across the cell membrane are inactive at low temperatures. Conversely,
separated serum or plasma is best refrigerated, to minimize chemical changes or
bacterial growth.
Several changes occur in whole blood
specimens following collection. The commoner and more important changes that
occur prior to the separation of plasma or serum from the
cells are:
1. Glucose is
converted to lactate: this process is inhibited by fluoride;
2. Several
substances pass through the erythrocyte membrane, or may be added in
significant amounts to plasma as a result of red cell destruction insufficient
to cause detectable haemolysis. Examples include K+ and lactate dehydrogenase;
3. Loss of CO2 occurs,
since the Pco2, of blood is much higher than in air;
4. Plasma [phosphate] increases due to hydrolysis of organic ester
phosphates in the red cells;
5.
Labile plasma enzymes lose their activity.
ANTICOAGULANTS
Certain biochemical tests require unclotted blood. Serum from coagulated blood is
the specimen of choice for many assay systems.
Heparin (inhibits the convension prothrmobin to thrombin) is the most widely used
anticoagulant for clinical chemical analysis. Heparin is an ideal
anticoagulant, since it does not cause any change in blood composition.
However, other anticoagulants are prefered to
heparin, due to the cost factor.
Ethylene diamine tetra
acetic acid (EDTA) is a chelating
agent, and is particularly useful for hematological examination because it preserves
cellular components of the BLOOD. It chelates with calcium and blocks coagulation.
EDTA is employed to collect blood for hematological examinations It
may affect some of the clinical chemistry tests.
Sodium fluoride is usually used as
a preservative for blood glucose by inhibiting the enzyme systems involved in
the glycolysis. Without an antiglycolytic agent,
the blood glucose concentration decreases about 10 mg/dl per hour and false
results may be obtained. Fluoride is also anticoagulant. It should not be used for enzyme assays, as
well as when the test involves enzymatic analysis.
Citrate is widely used for
coagulation studies.
Oxalate inhibits blood
coagulation by forming insoluble complexes with calcium ions. Potassium oxalate
may be used at a concentration of 1 -2 mg/ml blood. At concentration of > 3 mg/ml, oxalate may cause hemolysis.
Potassium or sodium oxalate : These compounds
precipitate calcium and inhibit blood coagulation. Being more soluble,
potassium oxalate (5-10 mg per 5 ml blood) is prefered.
Potasium oxalate
and sodium fluoride
: These anticoagulants
are employed for collecting blood to estimate glucose. Further sodium fluoride
inhibits glycolysis and preserves bfood glucose
concentration.
Ammonium
oxalate and potassium oxalate : A mixture of these two compounds in the
ratio
3 : 2 is used
for blood collection to carry out certain hematological tests.
HEMOLOYSIS
The rupture or lysis of RBC,
releasing the cellular constituents interferes with the laboratory
investigations. Therefore, utmost care should be taken to avoid hemolysiswhen
plasma or serum
are used for biochemical
tests. Use of dry syringes, needles and containers, allowing slow flow of blood
into syringe are among the important precautions to avoid hemolysis.
PRESERVATION
OF BLOOD SPECIMENS
Plasma or serum should be separated within 2 hours after blood
collection. It is ideal and advisable to analyse blood, plasma or serum,
immediately after the specimen collection. This however, may not be always
possible. In such a case, the samples (usually plasma/serum) can be stored at 4°C until
analysed. For enzyme analysis, thesample are preserved at -
Sampling Errors:
1. Blood sampling
technique. Difficulty in
obtaining a blood specimen may lead to haemolysis with
consequent release of potassium and other red cell constituents.
Results for these will be falsely elevated.
2. Prolonged stasis
during venepuncture. Plasma water diffuses into the
interstitial space and the serum or plasma sample obtained will be
concentrated. Proteins and protein-bound components of plasma such calcium or thyroxine will be falsely elevated.
3. Insufficient
specimen. Each biochemical
analysis requires a certain volume of specimen to enable the test to be carried out.
4. Errors in timing. The biggest source of error in the
measurement of any analyte in a
24-hour urine specimen is in the collection of an accurately timed volume of
urine.
5. Incorrect specimen
container. For many analyses
the blood must be collected into a container with anticoagulant and preservative.
For example, samples for glucose should be collected into a special container
containing fluoride which inhibits glycolysis; otherwisethe time taken to deliver the sample to
the laboratory can affect the result.
6. Inappropriate sampling
site. Blood samples should
not be taking downstream from an intravenous drip.It is
not unheard of for the laboratory to receive a blood glucose request on a
specimen taken from the same arm into which 5% glucose is being infused.
7. Incorect specimen storage. A blood sample stored overnight before
being sent to the laboratory will show falsely high potassium, phosphate and
red cells enzymes such as lactate dehydrogenase, because of leakage into the
extracellular fluid from the cells.
Many hormones show circardian rhythm. For example, ACTH has maximum
peak at early morning, and minimum level at afternoon. Maximum level of growth
hormone is during night and minimum is in the day time. Many reference values are age related; e.g., levels of
urea and cholesterol are more in geriatric patients. Exercise will increase the
level of transaminases and creatinine. Triglyceride level is to be done in fasting
condition. Caffeine (coffee and tea) will increase the levels of free fatty
acid, glycerol, total lipids and glucose. Smoking will increase the levels of
GH, cortisol and triglycerides.
COLLECTION OF
URINE:
An
early morning fasting specimen is generally the most concentrated specimen.
Therefore, this is preferred for microscopic examination and for the detection
of proteins, beta chorionic gonadotropin and other metabolites.
Urine,
containing the metabolic waste products of the body in water is the most
important excretory fluid. For biochemical investigations, urine can be
collected as a single specimen or for 24 hours. Single specimens of urine,
normally collected in the morning, are useful for qualitative tests e.g.,
sugar, proteins. Twenty four hour urine collections (done between 8 AM to 8 AM)
are employed for quantitative estimation of certain urinary constituents e.g.,
proteins, hormones, metabolites.
Depending
on the test, either a random or a complete
timed collection of urine is
needed. The timed collection is obtained as follows:
1. Just before the collection period is
due to start, the patient empties his/her bladder. This urine must be discarded.
2. Thereafter, from the start (e.g. at 8 AM) to the end of the collection
period, all urine passed by
the patient must be added to the container. If this contains preservative, the
specimen must be mixed gently each time more urine is passed and added to the
collection.
3. At the end of the period (e.g. 8 AM the next day, in the case of a
24-h collection), the patient empties his/her bladder. This urine must be included in the collection.
4. The period over which the collection
was made must be recorded and written on the specimen container and the request
form.
For large volumes, an aliquot (e.g. 25 mL)
may be sent to the laboratory, but the complete specimen must first be mixed
and its volume recorded on the container and the request form.
Urine specimens tend to deteriorate unless the correct
preservative is added from the start, or the specimen is refrigerated
throughout the collection period. The changes include:
1. destruction of glucose by bacteria;
2. conversion of
urea to ammonia, by bacteria, with fall in [H+] and precipitation
of phosphates;
3. oxidation of urobilinogen to urobilin and porphobilinogen to porphyrins.
Preservatives for urine : The preservatives are
used (1) to reduce bacterial action; (2) to minimise chemical decomposition,
and (3) to decrease atmospheric oxidation of unstable compounds. The most
satisfactory form of preservation of urine specimen is to refrigerate it during
the collection. Formalin, thymol, chloroform,
toluene, concentrated HCI and glacial acetic acid are the commonly used urine
preservatives.
For the
collection of 24 hr urine samples, preservatives have to be used or else urine
undergoes changes due to bacterial action. Hydrochloric acid, toluene, light
petroleum, thymol,
formalin etc., are among the common preservatives used.
Timed
Urine Specimen
Usually,
urine sample is collected for the 24 hour period. This will minimise the influence
of short-term biological variations and diurnal rhythms. Generally, collection
of urine samples are done from 6 AM to next 6
AM. The bladder should be
emptied when the collection is started (6 AM), and this urine is discarded.
Thereafter all the urine should be collected. The next day urine is
voided at 6 AM and this sample is also collected.
CEREBROSPINAL FLUID:
CSF is
a fluid of the nervous system. It is formed by a process of selective dialysis of
plasma by the choroid plexuses of the ventricles of the brain. The total volume
CSF is 100-200 ml.
Collection
of CSF : CSF is collected
by puncturing the interspace between the 3rd and the 5th number vertabrae,
under asepetic conditions and local anesthesia.
Biochemical investigations on CSF : Protein,
glucose and chloride estimations are usually performed in the clinical
biochemistry laboratory.
The interpretation of results:
Most biochemical analyses are quantitative. Many tests measure the amount
of the analyte in a small volume of the sample
(blood, plasma, serum, urine or some other fluid or tissue). The tests results
are commonly expressed in molar units. A mole of any compound always contains
6* 1023 molecules.
Describing how much of ananalyte is present in moles indicates how many
molecules of the substance are present. Molar units can be converted to mass
units: one mole is the molecular weight of the substance in grams. Results are
reported as concentrations, usually in terms of the number of moles in one litre (mol/l).
Molar units:
Mole |
Abbreviation |
Definition |
Milimole |
mmol |
*10-3 of a mole |
Micromole |
µmol |
*10-6 of a mole |
Nanomole |
nmol |
*10-9 of a mole |
Picomole |
pmol |
*10-12 of a mole |
Femtomole |
fmol |
*10-15 of a mole |
Enzymes are not usually expressed in moles but as enzyme activity in
"units". Large molecules such as proteins are reported as grams or
milligrams. Blood gas results (PCO2 or PO2) are expressed in kilopascals (kPa), the units in which partial pressures are measured.
Biological factors
affecting the interpretation of results:
1. Sex of the
patient. Reference ranges
for some analytes such as serum creatinine are different for men and women.
2. Age of the patient.
There may be different references ranges for neonates, children, adults and the eldery.
3. Effect of diet.The sample may be inappropriate if taken
when the patient is fasting or after a meal.
4. Time when sample
was taken. There may
be variations during the day and night.
5. Stress and
anxiety.
6. Posture of the
patient. Redistribution of fluid may effect the
result.
7. Effects of
exercise. Strenuous exercise can release enzymes from tissues.
8. Medical history.Infection or tissue injury can affect
biochemical values independently of the disease process being investigated.
9. Pregnancy. This
alters some references
ranges.
10. Menstrual cycle. Hormone measurement will vary through the menstrual
cycle.
11. Drug history.
The
clinician may well ask the following questions on receiving a biochemistry
report:
1. Does
the result fit in with what I expected on the basis of the clinical examination
and history of the patient?
2. If the
result is not what I expected, can I explain the discrepancy?
3. How
can the result change my diagnosis or the way I am managing the patient?
4. What
should I do next?
QUALITY CONTROL:
Quality control in clinical biochemistry laboratory
refers to the reliability of investigative service. Any error in the laboratory will
jeopardize the lives of patients. It is therefore utmost important that the
laboratory errors are identified and rectified.
Quality control comprises of four interrelated
factors namely precision, accuracy, specificity and sensitivity.
Precision refers to the
reproducibility of the result when the same sample is analysed on different
occasions (replicate measurements) by the same person. For instance, the precision
is good, if the blood glucose level is 78, 80 and 82 mg/dl on replicates.
Accuracy means the
closeness of the estimated result to the true value e.g., if true blood urea
level is 50 mg/dl, the laboratory reporting 45 mg/dl is more accurate than the
one reporting 35 mg/dl.
Specificity refers to the
ability of the analytical method to specifically determine a particular
parameter e.g., glucose can be specifically estimated by enzymatic glucose
oxidase method.
Sensitivity deals with the
ability of a particular method to detect small amounts of the measured
constituent.
METHODS OF QUALITY CONTROL
1. Internal quality control refers to the
analysis of the same stored sample on different days in a
laboratory, the
results should vary within a narrow range.
2. External quality control deals with the
analysis of a sample received from outside, usually from a national or regional
quality control centre. The results obained arethen
compared.
Accuracy
It is the
closeness of a result to the true value. For example, if one technician
performs a test on a serum which is known to contain 5,0 mmol/L
glucose and obtains a result of 4.9 mmol/L.
A second technician does the same test on the same sample, and gets the
result of 4.5 mmol/L. Then the
value of the first technician is considered as accurate. Values farther away
from the true value are less
accurate than those closer.
Precision
This refers to the reproducibility of the result. If one technician performs
glucose analysis on the same sample on three different occasions and obtains 4 mmol/L, 3.9mmol/L, and 4.1 mmol/L, then the results have been reproduced very well,
and the precision is very good. Precision depends on the technique, the
reagents, as well as on the
technician.
Specificity
Specificity of a reaction denotes that
only one substance will answer that particular test.
For example, in the case of glucose oxidase method, only glucose molecules are
assayed. So it is a very specific method. But if the reducing property of the glucose is utilised for the
assay purpose (e.g., Nelson Somogyi method), then other reducing agents in the blood will interfere in
the reaction, and hence specificity is lowered. Specificity is determined by the
method of the analysis.
Sensitivity |
It indicates that whether the method could
be utilised to test a very dilute solution. For example, biuret method is used
for solutions having a few g of protein/dl. Spectrophotometric method is useful
to detect a few mg of protein/dl, while ELISA method is employed if the
solution has only microgram of protein/dl. Thus ELISA method is most sensitive.
Generally speaking, as the sensitivity is increased, specificity is decreased.
Limit
of Errors Allowable in Laboratory:
In a laboratory, error may not be totally avoided; but should be kept at
a minimum. The limits are denoted by the term, percentage error. The percentage of allowable error in
an assay is given by the
formula:
Difference
between maximum and minimum of normal
range
————————————————— x100
Mean of
the normal range x 4
The percentage error, therefore, will vary from test to test. To take an
example, in the case of blood glucose analysis, the normal range is 70 to 110
mg/dl. If these values are substituted in the formula:
(110
minus 70)
———————— x 100 = 10%
90x4
Now, in the case of blood urea (normal range of 20-40 mg/dl), the
percentage error will be
(40
minus 20)
———————— x 100 =
16%
30-x4
Biochemical testing outside the
laboratory:
The methods for measuring some biological compounds in blood and urine have
become so robust and simple to use tht measurements
can be made away from the laboratory – by the patient's bedside, in the ward,
in the home or even in the shoping centre.
Tests performed away from the laboratory:
The most common blood test outside the
laboratory is the determination of glucose concentration, in a finger stab
sample, at home or in the clinic. Diabetic patients who need to monitor their
blood glucose on a regular basis can do so at home or at work using one of many
commercially available pocket-sized instruments.
A portable bench analyser. This
instrument may be used to monitor patients' glucose and cholesterol, and its frequently used in many outpatient
clinics and in screeningcentres.
Common tests
on blood performed away from the laboratory
Analyte |
Used
when investigating |
A |
Blood
gases |
Acid-base
status |
|
Glucose |
Diabetes
mellitus |
|
Urea |
Renal
disease |
|
Creatinine |
Renal
disease |
|
Bilirubin |
Neonatal jaundise |
|
Therapeutic
drugs |
Compliance
of toxicity |
|
Salicylate |
Detection
of poisoning |
|
Paracetamol |
Detection
of poisoning |
|
Glucose |
Diabetic
monitoring |
B |
Cholesterol |
Coronary
heart disease risk |
|
Alcohol |
Fitness
to drive/confusion, coma |
C |
Common
tests on urine performed away from the laboratory
Analyte |
Used
when investigating |
A |
Ketones |
Diabetic
ketoacidosis |
|
Protein |
Renal
disease |
|
Red
cells/haemoglobin |
Renal
disease |
|
Bilirubin |
Liver
disease and jaundise |
|
Urobilinogen |
Jaundise/haemolysis |
|
pH |
Renal
tubular acidosis |
|
Glucose |
Diabetes
mellitus |
B |
hCG |
Pregnancy
test |
The
tests commonly performed away from the laboratory can be categorized as
follows:
A. Tests performed
in medical or nursing settings.
They
clearly give valuable information and allow the practitioner to reassure the
patient or family, or initiate futher investigations
or treatment.
B. Tests performed
in the home, shopping centre or clinical setting.
They
can give valuable information when properly and appropriately used.
C. Alcohol
tests.
Manual vs automation in clinical laboratory
Automation in
clinical laboratory is a process by which analytical instruments perform many
tests with the least involvement of an analyst. The International Union of Pure
and Applied Chemistry (IUPAC) define automation as “the replacement of human
manipulative effort and facilities in the performance of a given process by
mechanical and instrumental devices that are regulated by feedback of
information so that an apparatus is self-monitoring or selfadjusting”.
Presently no
currently available clinical instrument fully meets this definition,
however the term ‘automation’
is applicable to the individual steps in many analytical processes and modern
instrumentation is improvising with more and more intelligence built into new
generations of laboratory analyzers to soon come up to the IUPAC definition.
Automated instruments
enable laboratories to process a much larger workload without a relative
increase in manpower. Automation in clinical laboratories has evolved from
fixed automation whereby an instrument performs a repetitive task by itself,
and has progressed to programmable automation, which permits it to perform a
variety of different tasks. Intelligent automation has recently been introduced
into a few individual instruments or systems to enable them to self-monitor and
respond appropriately to changing conditions. Instead of
resorting to
manual means automation leads to reduction in variability of results and error
of analysis by doing away with jobs that are repetitive and monotonous for an
individual and that can lead to boredom or casual attitude. However, the
improved reproducibility attained by automation is not necessarily associated
with improved accuracy of test results since accuracy is mainly influenced by
the analytical methods used. The significant improvement in quality of
laboratory tests in recent years is due the combination of well-designed
automated instrumentation with good analytical methods and effective quality
assurance programs. Automation may initially incur high costs for procurement
of the equipments but is
economical in the long run
due to the reduction in the manpower required to perform the tasks.
Automated
analyzers usually include the mechanized versions of basic manual laboratory
techniques and procedures, and several ways have been developed for automating
them. When initially introduced, automation mimicked manual test procedures and
was applied to those tests requested most often. All the individual steps in
the procedure are duplicated. Analytical methods, which are quicker and with
fewer steps as well as modification of existing protocols are being developed
as the manufactures have integrated computer hardware and software into
analyzers to provide automatic process control and data processing
capabilities.
Types
of analyzers
Semi-auto analyzer: Here, the
samples and reagents are mixed and read manually
Batch analyzer: The
reagent mixture is mixed and fed automatically. One reagent is stored in the
machine at a time enabling one batch of a specifc test
to be automatically conducted e.g. RA 100.
Random Access autoanalyzers: These analyzers can store more
than one reagent. Samples are placed in the machine and the computer is
programmed to carry out any number of selected tests on each sample e.g. Hitachi 912.
Figure-1: Semi-Autoanalyzer
Figure-2: Autoanalyzer
Some important techniques employed in the clinical
laboratory analysis
are as follows:
(a) Optical Techniques
(b) Electrochemical Techniques
(c) Chromatography
(d) Electrophoresis
Optical Techniques
Analytical techniques that make use of light spectrum
either of a specific
wavelength or as visible light
spectrum can be collectively referred to as Optical
Techniques. The major optical techniques used in clinical laboratory are
microscopy and spectrophotometry.
Many biochemical quantitative analyses done in
clinical laboratory are based on measurements of radiant energy (light)
emitted, transmitted, absorbed, scattered or reflected when the substance being
measured interact with an incident light, under controlled conditions.
Techniques of measuring such radiant energy (light) are termed spectrophotometric techniques.
Specific spectrophotometric techniques (or instrumentation
design) depend on whether the interaction between the incident wavelength of
light and the substance being measured results into light absorption, (or
transmission) reflection or scattering.
In colorimetric method, (an example of
spectrophotometric), light of a specific wavelength is made to pass through a
solution of which concentration is to be determined. The amount of light
absorbed by the solution is measured (absorbance). A known standard solution of
the substance being measured is treated same way and its absorbance is
measured, the concentration of the test solution is derived by simple
extrapolation. The
specific wavelength of light made to pass through the solution is
dependent on the colour of the test solution. Complementary colour of that of the solution is made use
of: e.g.
Colour of
Solution Complementary Colours of Light
Blue 1. Blue Yellow (e.g. 450 nm)
Bluish-green 2. Bluish-green Red (630 nm)
The basic principle of colorimetric technique is
Beer-Lambert law. Beer-Lambert law states that ‘when a specific wavelength of
light (monochromatic light) passes through a colouredsolution, the amount of light absorbed is directly
proportional to the concentration of the solution (intensity of the colour) and
the length path through the
solution.The biochemical
substance or analyte (e.g. glucose, cholesterol) to be
measured in a clinical specimen (body fluids) is allowed to specifically react with
chemical agent(s) to form a coloured product
(in solution). The absorbance of the coloured solution
formed is measured using spectrophotometer (an instrument used to measure the
amount of light
absorbed or transmitted by
substances in solutions). Since absorbance of a substance in solution is
directly proportional to its concentration, the concentration of the substance
of interest is calculated from its absorbance and the absorbance of a standard
solution can be treated the same way.
Picture of a Spectrophotometer
Picture of an Atomic Emission Flame Photometer
Electrophoretic
Techniques
Electrophoresis is a method of separation of mixtures
based on differential rate of movement of charged particles when subjected to
an electric field at a specific pH. Electrophoretic technique is typically used
in the clinical laboratory for the separation of proteins. It is primarily a
qualitative method of analysis, but it can be adopted for quantitative
analysis.
Principle of Electrophoretic Separation of Proteins
Proteins in serum vary in their iso-electric
points. Iso-electric
point of a protein is the pH at which there is no net charge (zero charge) on
protein particles. At a pH alkaline to its iso-electric point, a protein will carry a net negative
charge and therefore migrates to the anode when a current is passed, whereas at
a pH acidic to its iso-electric point, it will carry a net positive charge and
migrate to the cathode when a current is applied.
Iso-electric point of
serum proteins varies from 4.7(albumen) to 7.3 (gamma globulin).
Hence, at a buffered pH of say 8.6, each protein
fraction will migrate at different rates when subjected to an electric field.
Electrophoretic technique can be used to detect or
identify an abnormal protein present in plasma as a result of disease
conditions. Example is in a disease called multiple myeloma, an abnormal
protein called Bence -Jones
protein can be detected by electrophoretic method. Determination of Haemoglobin genotype of an individual is also done
using this technique.
Chromatographic
Techniques
Chromatography is a method of separation of mixtures
which utilizes differential affinity of the separating molecules substance in
the mixture, for mobile and stationary phases, over which the substances to be
separated are distributed. A mobile phase may be a gas or a liquid (solvent) in
which the substance (mixture) is solubilised, while a stationary phase is either a solid or
a liquid supported (stationed) on a solid matter, over which the mobile phase
carries the mixture. Substances (in the mixture) that have greater affinity for
the mobile phase are separated first, after the order of their affinities for
the mobile
phase constituents of
the mixture that have greater affinities for the stationary phase are separated
much latter during the process. As the mobile phase carries the mixture over
stationary phase (like an effluent), the separated constituent are collected as
different fractions. The different fractions can be identified and also
quantified. Chromatographic technique is typically named after the mobilestationary phase e.g. Gas-liquid chromatography,
or after the working principle e.g. ion-exchange chromatography. Others
include:
· Thin layer chromatography
· Molecular sieve chromatography
· High performance liquid chromatography
Centrifugation
Centrifugation is a process that involves the use of centrifugal
force for the separation of mixtures. In the clinical laboratory setting, the
major use of centrifugation is as follows:
(1) Separation of plasma, serum and red cells from
whole blood, when a particular fraction of the blood is needed for tests.
(2) Acquisition of urine sediment for microbiological
examination
(3) Any laboratory procedure (test) that require
separation of a particular fraction of a suspension.
Many particles or cells in a liquid medium (suspension)
at a given time, will eventually settle at the bottom of a container due to
gravity. However, the length of time required for such separation may be long.
When a suspension is rotated at a certain speed or revolution per minute,
centrifugal force causes the particulates to move away from the axis of
rotation and therefore settles at the bottom of the container as a precipitate.
The remaining solution or liquid is called the supernate or supernatant. The equipment used for
the process is called centrifuge.
There are basically two types of centrifuge used in
clinical laboratory:
1. Bench centrifuge
2. Microhaematocrit centrifuge
Picture of a Microhaematocrit Centrifuge
Picture of a Bench Centrifuge
CLINICAL INTERPRETATION OF LABORATORY TESTS IN
HEMATOLOGY – I
CLINICAL INTERPRETATION OF LABORATORY TESTS IN
HEMATOLOGY – II
Blood constitutes 6 to 8 percent of total body weight. In terms of
volume, women have 4.5 to 5.5 L of blood and men 5 to 6 L. In infants and children, blood volume
is 50 to 75 mL/kg in girls and 52 to 83 mL/kg in boys. The principal functions
of blood are the transport of oxygen, nutrients, and hormones to all tissues
and the removal of metabolic wastes to the organs of excretion. Additional
functions of blood are (1) regulation of temperature by transfer of heat to the
skin for dissipation by radiation and convection, (2) regulation of the pH of
body fluids through the buffer systems and facilitation of excretion of acids
and bases, and (3) defense against infection by transportation of antibodies
and other substances as needed. Blood consists of a fluid portion, called
plasma, and a solid portion that includes red blood cells (erythrocytes), white
blood cells (leukocytes), and platelets (thrombocytes). Plasma makes up 45 to
60 percent of blood volume and is composed of water (90 percent), amino acids,
proteins, carbohydrates, lipids, vitamins, hormones, electrolytes, and cellular
wastes.
Hematology is traditionally limited to the study
of the cellular elements of the blood, the production of these elements, and
the physiological derangements that affect their functions.
Hemopoiesis
Hemopoiesis – the
processes of blood cells formation and development.
There are 2 kinds of hemopoiesis: embrional and postembrional.
Organs of embrional hemopoiesis: 1. In the first
few weeks of gestation the yolk sac is the main site of hemopoiesis.2. Liver. 3. Spleen. 4. Lymphatic nodules. 5.
Thymus. 6. Bone marrow – on 6-th month is central site of hemopoiesis.
Organs of postembrionalhemopoiesis:
1. Bone marrow (vertebrae, ribs, sternum, skull, sacrum and pelvis, proximal
ends of femur). 2. Spleen. 3. Lymphatic nodules.
All blood cells derive from a common stem cell.
Under
the influences of local and humoral factors,
stem cells differentiate into different cell lines. Erythropoiesis and thrombopoiesis proceed independently once the stem
cell stage has been passed, whereas monocytopoiesis and granulocytopoiesis are quite closely “related.” Lymphocytopoiesis is the most independent among the
remaining cell series. Granulocytes, monocytes, and lymphocytes are
collectively called leukocytes (white blood cells), a term that has been
retained since the days before stainingmethods were
available, when the only distinction that could be made was between
erythrocytes (red blood cells) and the rest. All these cells are eukaryotic,
that is, they are made up of a nucleus, sometimes with visible nucleoli,
surrounded by cytoplasm, which may include various kinds of organelles,
granulations, and vacuoles. Despite the common origin of all the cells,
ordinary light microscopy reveals fundamental and characteristic differences in
the nuclear chromatin structure in the different cell series and their various
stages of maturation.
The
developing cells in the granulocyte series (myeloblasts and promyelocytes), for example, show a delicate, fine “net-like” (reticular) structure.
Careful microscopic examination (using fine focus adjustment to view different
depth levels) reveals a detailed nuclear structure that resembles fine or coarse
gravel. With progressive stages of nuclear maturation in this series (myelocytes, metamyelocytes, and band or staff cells), the chromatin
condenses into bands or streaks, giving the nucleus— which at the same time is
adopting a characteristic curved shape—a spotted and striped pattern.
Lymphocytes, on the other hand—particularly in their circulating forms—always
have large, solid-looking nuclei. Like cross-sections through geological slate,
homogeneous, dense chromatin bands alternate with lighter interruptions and
fissures. Each of these cell series contains precursors that can divide (blast
precursors) andmature or almostmature forms
that can no longer divide; the morphological differences between these
correspond not to steps in mitosis, but result from continuous “maturation
processes” of the cell nucleus and cytoplasm. Once this is understood, it
becomes easier not to be too rigid about morphological distinctions between
certain cell stages. The blastic precursors
usually reside in the hematopoietic organs (bone marrow and lymph nodes).
Since, however, a strict blood–bone marrow barrier does not exist (blasts are
kept out of the bloodstream essentially only by their limited plasticity, i.e.,
their inability to cross the diffusion barrier into the bloodstream), it is in
principle possible for any cell type to be found in peripheral blood, and when
cell production is increased, the statistical frequency with which they cross
into the bloodstream will naturally rise as well. Conventionally, cells are sorted
left to right from immature to mature, so an increased level of immature cells
in the bloodstream causes a “left shift” in the composition of a cell
series—although it must be said that only in the precursor stages of granulopoiesis are the cell morphologies sufficiently
distinct for this left shift to show up clearly.
Blood
Cell functions.
Neutrophil granulocytes with segmented nuclei serve mostly to defend against bacteria.
Predominantly outside the vascular system, in “inflamed” tissue, theyphagocytose and lyse bacteria. The blood merely transports the granulocytes to
their site of action.
The function of eosinophilic granulocytes is defense against parasites; they have a direct cytotoxic
action on parasites and their eggs and larvae. They also play a role in the down-regulation of anaphylactic
shock reactions and
autoimmune responses, thus controlling the influence of basophilic cells.
The main function of basophilic
granulocytes and their
tissue-bound equivalents (tissue mast cells) is to regulate circulation through
the release of substances such as histamine, serotonin, and heparin. These
tissue hormones increase
vascular permeability at the
site of various local antigen activity and
thus regulate the influx of the other inflammatory cells.
The main function of monocytes is the defense against bacteria, fungi, viruses, and foreign bodies. Defensive
activities take place mostly outside the
vessels by phagocytosis. Monocytes also break down endogenous cells (e.g., erythrocytes) at the end
of their life cycles, and they are assumed to perform
a similar function in defense against tumors. Outside the bloodstream, monocytes develop into histiocytes;
macrophages in theendothelium of
the body cavities; epithelioid cells;
foreign body macrophages (including Langhans’
giant cells); and many other cells.
Lymphocytes are
divided into two major basic groups according to function. Thymus-dependent
T-lymphocytes, which make up about 70% of lymphocytes, providelocal defense
against antigens fromorganic and
inorganic foreign bodies in the form of delayed-type hypersensitivity, as
classically exemplified by the tuberculin reaction. T-lymphocytes are divided
into helper cells and suppressor cells. The small group of NK (natural killer)
cells, which have a direct cytotoxic function, is closely related to the T-cell
group.
The other group is the bone-marrow-dependent B-lymphocytes or Bcells,
which make up about 20% of lymphocytes. Through their development into
immunoglobulin-secreting plasma cells, B-lymphocytes are responsible for the
entire humoral side of defense against viruses, bacteria, and
allergens. Erythrocytes are the oxygen carriers for all
oxygen-dependent metabolic reactions in the organism. They are the only blood
cells without nuclei, since this allows them to bind and exchange the greatest
number of O2 molecules.
Their physiological biconcave disk shape with a thick rim provides optimal plasticity.
Thrombocytes form
the aggregates that, along with humoral coagulation
factors, close up vascular lesions. During the aggregation process, in addition
to the mechanical function, thrombocytic granules
also release factors that promote coagulation. Thrombocytes
develop from polyploid megakaryocytes in the bone marrow.
They are the enucleated, fragmented cytoplasmic portions of these progenitor
cells.
COMPLETE
BLOOD COUNT
A CBC includes (1) enumeration of the cellular elements of the blood,
(2) evaluation of RBC indices, and (3) determination of cell morphology by
means of stained smears.
INDICATIONS FOR A COMPLETE BLOOD COUNT
Because the CBC provides much information about the overall health of
the individual, it is an essential component of a complete physical
examination,
especially when performed on
admission to a health-care facility or before surgery. Other indications for a
CBC are as follows:
1. Suspected
hematologic disorder, neoplasm, or immunologic abnormality
2. History of
hereditary hematologic abnormality
3. Suspected
infection (local or systemic, acute or chronic)
4. Monitoring
effects of physical or emotional stress
5. Monitoring
desired responses to drug therapy and undesired reactions to drugs that may
cause blood dyscrasias
6. Monitoring
progression of nonhematologic disorders
such as chronic obstructive pulmonary disease, malabsorption syndromes, malignancies, and renal
disease
Taking
Blood Samples
This
means that blood should always be drawn at about the same time of day and after
at least eight hours of fasting, since both circadian rhythm and nutritional
status can affect the findings. If strictly comparable values are required,
there should also be half an hour of bed rest before the sample is drawn, but
this is only practicable in a hospital setting. In other settings (i.e.,
outpatient clinics), bringing portable instruments to the relaxed, seated
patient works well.
A
sample of capillary blood may be taken when there are no further tests that
would require venous access for a larger sample volume. A well perfused
fingertip or an earlobe is ideal; in newborns or young infants, the heel is
also a good site. If the circulation is poor, the blood flow can be increased
by warming the extremity by immersing it in warm water. Without pressure, the
puncture area is swabbed several times with 70% alcohol, and the skin is then
punctured firmly but gently with a sterile disposable lancet. The first droplet
of blood is discarded because it may be contaminated, and the ensuing blood is
drawn into the pipette (see below). Care should be taken not to exert pressure
on the tissue from which the blood is being drawn, because this too can change
the cell composition of the sample.
General blood analysis (normal values)
1. Erythrocytes
(red blood cells) Male - 4-5,1× 1012/L
Female – 3,7-4,7×
1012/L
2. Hemoglobin Male - 130-160 g/L Female – 120-140 g/L
3. Hematocrit Male - 40-48 % Female – 36-42 %
4. Reticulocytes 0,5-1
%
5. Plateletes 180-320 × 109/L
6. ESR
(Erythrocytes sedimentation rate) Male
- 1-10 mm/hour
Female – 2-15 mm/hour
7. Leucocytes 4-9
× 109/L
Neutrophilic band granulocytes
– 1-6 %
Neutrophilic segmented
granulocytes – 45-72 %
Eosinophilic granulocytes – 0,5-5 %
Basophilic granulocytes – 0-1 %
Monocytes – 3-11 %
Lymphocytes – 19-37 %
Erythrocyte Parameters
The quality of erythrocytes is characterized by:
1. MCV (mean
corpuscular volume)
Male - 80-94 mcm3 (Fl) Female – 81-99 mcm3 (Fl)
2. MCH (mean
corpuscular hemoglobin) – 27-31 pg
3. MCHC (mean
corpuscular hemoglobin concentration) – 33-37 % or 20,4-22,9 mmol/L
4. Red Cell Distribution
Width (RDW) - 11,5-14,5 %.
MCV
indicates the volume of the Hgb in each RBC, MCH is the weight of the
Hgb in
each RBC, and MCHC is the proportion of Hgb contained in each RBC. MCHC is a
valuable indicator of Hgb deficiency
and of the oxygen-carrying
capacity of
the individual erythrocyte. A cell of abnormal size, abnormal shape, or both
may contain an inadequate proportion of Hgb. RBC indices are used mainly in
identifying and classifying types of anemias. Anemias are
generally classified according to RBC size and Hgb content. Cell size is indicated by the terms
normocytic, microcytic, and macrocytic. Hemoglobin content is indicated by the terms
normochromic, hypochromic, and hyperchromic.
ERYTHROCYTE
(RBC) COUNT
The
erythrocyte (RBC) count, a component of the CBC, is the determination of the
number of RBCs per cubic millimeter. In international units, this is expressed
as the number of RBCs per liter of blood. The test is less significant by
itself than it is in computing Hgb, Hct, and RBC indices. Many factors influence the level of
circulating erythrocytes. Decreased numbers are seen in disorders involving
impaired erythropoiesis excessive blood cell destruction (e.g., hemolytic
anemia), and blood loss, and in chronic inflammatory diseases. A relative
decrease also may be seen in situations with increased body fluid in the
presence of a normal number of RBCs (e.g., pregnancy). Increases in the RBC
count are most commonly seen in polycythemia vera, chronic pulmonary disease with hypoxia and secondary
polycythemia, and dehydration with hemoconcentration. Excessive exercise,
anxiety, and pain also produce higher RBC
counts.
HEMATOCRIT
Blood
consists of a fluid portion (plasma) and a solid portion that includes RBCs,
WBCs, and platelets. More than 99 percent of the total blood cell mass is
composed of RBCs. The Hct or packed
RBC volume measures the proportion of RBCs in a volume of whole blood and is
expressed as a percentage. Several methods can be used to perform the test. In
the classic method, anticoagulated venous
blood is pipetted into a tube 100
mm long and then centrifuged for
30 minutes so that the plasma and blood cells separate. The volumes of packed
RBCs and plasma are read directly from the millimeter marks along the side of
the tube. In the micromethod, venous or capillary blood is used to fill a
small capillary tube, which is then centrifuged for 4 to 5 minutes. The
proportions of plasma and RBCs are determined by means of a calibrated reading
device. Both techniques allow visual estimation of the volume of WBCs and
platelets. With the newer, automated methods of cell counting, the Hct is calculated indirectly as the
product of the RBC count and mean cell volume. Although this method is
generally quite accurate, certain clinical situations may cause errors in
interpreting the Hct. Abnormalities in RBC size and extremely elevated WBC
counts may produce false Hct values.
Elevated blood glucose and sodium may produce elevated Hct values because of the resultant swelling
of the erythrocyte. Normally, the Hct parallels
the RBC count. Thus, factors influencing the RBC count also affect the results
of the Hct.
HEMOGLOBIN
Hemoglobin
is the main intracellular protein of the RBC. Its primary function is to transport
oxygen to the cells and to remove carbon dioxide from them for excretion by the
lungs. The Hgb molecule consists of two main
components: heme and globin. Heme is composed of the red pigment porphyrin and iron, which is capable of
combining loosely with oxygen. Globin is a protein that consists of
nearly
600
amino acids organized into four polypeptide chains. Each chain of globin is
associated with a heme group.
Each RBC contains approximately 250 million
molecules of hemoglobin,
with some erythrocytes containing more hemoglobin than others. The
oxygen-binding, -carrying, and –releasing capacity of Hgb depends on the ability of the globin
chains to shift position normally during the
oxygenation–deoxygenation process. Structurally
abnormal chains that are unable to shift normally have decreased
oxygen-carrying ability. This decreased oxygen transport capacity is
characteristic of anemia. Hemoglobin also functions as a buffer in the
maintenance of acid–base balance. During transport, carbon dioxide (CO2)
reacts with water (H2O) to form carbonic acid (H2CO3).
This reaction is speeded by carbonic anhydrase, an enzyme contained in RBCs.
The carbonic acid rapidly dissociates to form hydrogen ions (H+) and
bicarbonate ions (HCO3–). The hydrogen ions combine with
the Hgb molecule, thus preventing a buildup of
hydrogen ions in the blood. The bicarbonate ions diffuse into the plasma and
play a role in the bicarbonate buffer system. As bicarbonate ions enter the
bloodstream, chloride ions (Cl_) are
repelled and move back into the erythrocyte. This “chloride shift” maintains
the electrical balance between RBCs and plasma.
Hemoglobin
determinations are of greatest use in the evaluation of anemia, because the
oxygen-carrying capacity of the blood is directly related to the Hgb level rather than to the number of
erythrocytes. To interpret results accurately, the Hgb level must be determined in
combination with the Hct level.
Normally, Hgb and Hct levels
parallel each other and are commonly used together to express the degree of
anemia. The combined values are also useful in evaluating situations involving
blood loss and related treatment. The Hct level is
normally three times the Hgb level. If
erythrocytes are abnormal in shape or size or if Hgb manufacture is defective, the
relationship between Hgb and Hct is disproportionate.
STAINED
RED BLOOD CELL EXAMINATION
The stained RBC examination (RBC morphology) involves examination of RBCs
under a microscope. It is usually performed to compare the actual appearance of
the cells with the calculated values for RBC indices. Cells are examined for
abnormalities in color, size, shape, and contents. The test is performed by
spreading a drop of fresh anticoagulated blood
on a glass slide. The addition of stain to the specimen is used to enhance RBC
characteristics.
Red
Blood Cell Abnormalities Seen on Stained Smear
Descriptive Term |
Observation |
Significance |
Macrocytosis |
Cell diameter > 8 µm MCV > 95 µm3 |
Megaloblastic anemias Severe liver disease Hypothyroidism |
Microcytosis |
Cell diameter < 6 µm MCV < 80 µm3 MCHC< 27 |
Iron-deficiency anemia Thalassemias Anemia of chronic disease |
Hypochromia |
Increased zone
of central pallor |
Diminished Hgb content |
Hyperchromia |
Microcytic, hyperchromic cells Increased bone marrow stores of
iron |
Chronic inflammation Defect in ability to use iron
for Hgb synthesis |
Polychromatophilia |
Presence of red cells not fully hemoglobinized |
Reticulocytosis |
Poikilocytosis |
Variability of cell shape |
Sickle cell disease Microangiopathic hemolysis Leukemias Extramedullaryhematopoiesis Marrow stress of any cause |
Red Blood
Cell Abnormalities Seen on Stained Smear
Descriptive Term |
Observation |
Significance |
Anisocytosis |
Variability of cell size |
Reticulocytosis Transfusing normal blood into
microcytic or macrocytic cell population |
Leptocytosis |
Hypochromic cells with small central zone of Hgb (“target cells”) |
Thalassemias Obstructive jaundice |
Spherocytosis |
Cells with no central pallor, loss of biconcave shape |
Loss of membrane relative to
cell volume Hereditary spherocytosis |
Schistocytosis |
MCHC high |
Accelerated red blood cell
destruction by reticuloendothelial system |
Acanthocytosis |
Presence of cell fragments in circulation |
Increased intravascular
mechanical trauma Microangiopathic hemolysis |
Echinocytosis |
Irregularly spiculated surface Regularly spiculated cell surface |
Irreversibly abnormal membrane
lipid content Liver disease Abetalipoproteinemia Reversible abnormalities of
membrane lipids High plasma-free fatty acids Bile acid abnormalities Effects of barbiturates,
salicylates, and so on |
Stomatocytosis |
Elongated, slitlike zone of central pallor |
Hereditary defect in membrane
sodium metabolism Severe liver disease |
Elliptocytosis |
Oval cells |
Hereditary anomaly, usually harmless |
Types
of Abnormal Red Blood Cell Inclusions and Their Causes
Type |
Causes of inclusion |
Heinz bodies (denatured Hgb) |
Thalassemia G-6-PD deficiency Hemolytic anemias Methemoglobinemia Splenectomy Drugs: analgesics, antimalarials,
antipyretics,nitrofurantoin (Furadantin), nitrofurazone (Furacin), phenylhydrazine, sulfonamides, tolbutamide, vitamin K (large doses) |
Basophilic stippling (residual cytoplasmic RNA) |
Anemia caused by liver disease Lead poisoning Thalassemia |
Howell-Jolly bodies (fragments of residual DNA) |
Splenectomy Intense or abnormal RBC
production resulting from hemolysis or inefficient erythropoiesis |
Cabot’s rings (composition unknown) |
Same as for Howell-Jolly bodies |
Siderotic granules (ironcontaining granules) |
Abnormal iron metabolism Abnormal hemoglobin manufacture |
OSMOTIC
FRAGILITY
The osmotic
fragility test determines the ability of the RCB membrane to resist rupturing
in a hypotonic saline solution. Normal disk-shaped cells can imbibe water and
swell significantly before membrane capacity is exceeded, but spherocytes (RBCs that lack the normal biconcave
shape) and cells with damaged membranes burst in saline solutions only slightly
less concentrated than normal saline. Conversely, in thalassemia, sickle cell disease, and other
disorders. The test is
performed by exposing RBCs to increasingly dilute saline solutions. The
percentage of the solution at which the cells swell and rupture is then noted. Normal
erythrocytes rupture in saline solutions of 0.30 to 0.45 percent. RBC rupture
in solutions of greater than 0.50 percent saline indicates increased fragility.
Lack of rupture in solutions of less than 0.30 percent saline indicates
decreased RBC fragility.
Causes of Altered
Erythrocyte Osmotic Fragility
Decreased Fragility |
Increased Fragility |
Iron-deficiency anemias |
Hereditary spherocytosis |
Hereditary anemias (sickle
cell, hemoglobin C, thalassemias) |
Hemolytic anemias |
Liver diseases |
Autoimmune anemias |
Polycythemia vera |
Burns |
Splenectomy |
Toxins (bacterial, chemical) |
Obstructive jaundice |
Hypotonic infusions |
|
Transfusion with incompatible blood |
|
Mechanical trauma to RBCs (prosthetic heart valves, disseminated intravascular clotting, parasites) |
|
Enzyme
deficiencies (PK kinase, G-6-PD |
ERYTHROCYTE SEDIMENTATION RATE
The erythrocyte sedimentation rate (ESR or sedrate)
measures the rate at which RBCs in anticoagulated blood
settle to the bottom of a calibrated tube. In normal blood, relatively little settling
occurs because the gravitational pull on the RBCs is almost balanced by the
upward force exerted by the plasma. If plasma is extremely viscous or if
cholesterol levels are very high, the upward trend may virtually
neutralize the downward
pull on the RBCs. In contrast, anything that encourages RBCs to aggregate or
stick together increases the rate of settling. Inflammatory and necrotic
processes, for example, cause an alteration in blood proteins that results in
clumping together of RBCs
because of surface attraction. These clumps are called rouleaux. If the proportion of globin to
albumin increases or if fibrinogen 3 levels are especially high, rouleaux formation is enhanced and the sed rate increases.
Causes of Altered Erythrocyte Sedimentation Rates
Increased rate |
Decreased rate |
Pregnancy (uterine and ectopic) |
Polycythemia vera |
Toxemia of pregnancy |
Congestive heart failure |
Collagen disorders (immune
disorders of connective tissue) |
Sickle cell, Hgb C disease |
Inflammatory disorders |
Degenerative joint disease |
Infections |
Cryoglobulinemia |
Acute myocardial infarction |
Drug toxicity (salicylates,
quinine derivatives, adrenal corticosteroids |
Most malignancies |
|
Drugs (oral contraceptives, dextran, penicillamine, methyldopa, procainamide,
theophylline, vitamin A) |
|
Severe anemias |
|
Myeloproliferative disorders |
|
Renal disease (nephritis) |
|
Hepatic cirrhosis |
|
Thyroid disorders |
|
Acute heavy metal poisoning |
|
WHITE BLOOD CELL COUNT
The
WBC count determines the number of leukocytes per cubic millimeter of whole
blood. The counting is performed very rapidly by electronic devices. The WBC may
be performed as part of a CBC, alone, or with differential WBC count. An
elevated WBC count is termed leukocytosis; a decreased count, leukopenia. In addition to the normal
physiological variations in WBC count, many pathological problems may result in
an abnormal WBC count .
Causes of Altered White Blood Cell Differential by
Cell Type
Cell Type |
Increased Levels |
Decreased Levels |
Neutrophils |
Stress (allergies, exercise,
childbirth, surgery) Extremes of temperature Acute hemorrhage or hemolysis Infectious diseases Inflammatory disorders
(rheumatic fever, gout, rheumatoid arthritis,
drug reactions, vasculitis, myositis) Tissue necrosis (burns,
crushing injuries, abscesses Malignancies Metabolic disorders (uremia,eclampsia, diabetic ketoacidosis, thyroid crisis,
Cushing’s syndrome) Drugs (epinephrine, histamine,
lithium, heavy metals, heparin, digitalis, ACTH) Toxins and venoms (turpentine,
benzene) Leukemia (myelocytic) |
Bone marrow depression (viruses,
toxic chemicals, overwhelming infection, Felty’s syndrome, Gaucher’s disease,myelofibrosis, hypersplenism, pernicious anemia, radiation) Anorexia nervosa, starvation,
malnutrition Folic acid deficiency Vitamin B12 deficiency Acromegaly Addison’s disease Thyrotoxicosis Anaphylaxis Disseminated lupus erythematosus Drugs (alcohol, phenylbutazone [Butazolidin], phenacetin,
penicillin, chloramphenicol, streptomycin,
phenytoin [Dilantin], mephenytoin [Mesantoin], phenacemide[Phenurone], tripelennamine [PBZ], aminophylline, quinine, chlorpromazine,
barbiturates,dinitrophenols, sulfonamides,antineoplastics |
Bands |
Infections Antineoplastic drugs Any condition that causes neutrophilia |
None, as bands should be absent
or present only in small numbers |
Basophils |
Leukemia Hodgkin’s disease Polycythemia vera Ulcerative colitis Nephrosis Chronic hypersensitivity states |
None, as normal value is 0–1% |
Eosinophils |
Sickle cell disease Asthma Chorea Hypersensitivity reactions Parasitic infestations Autoimmune diseases Addison’s disease Malignancies Sarcoidosis Chronic inflammatory diseases
and dermatoses Leprosy Hodgkin’s disease Polycythemias Ulcerative colitis Autoallergies Pernicious anemia Splenectomy |
Disseminated lupus erythematosus Acromegaly Elevated steroid levels Stress Infectious mononucleosis Hypersplenism Cushing’s syndrome Congestive heart failure Hyperplastic anemia Hormones (ACTH, thyroxine, epinephrine) |
Monocytes |
Infections (bacterial, viral, mycotic,rickettsial, amebic) Cirrhosis Collagen diseases Ulcerative colitis Regional enteritis Gaucher’s disease Hodgkin’s disease Lymphomas Carcinomas Monocytic leukemia Radiation Polycythemia vera Sarcoidosis Weil’s disease Systemic lupus erythematosus Hemolytic anemias Thrombocytopenic purpura |
Not characteristic of specific disorders |
Lymphocytes |
Infections (bacterial, viral) Lymphosarcoma Ulcerative colitis Banti’s disease Felty’s syndrome Myeloma Lymphomas Addison’s disease Thyrotoxicosis Malnutrition Rickets Waldenström’s macroglobulinemia Lymphocytic leukemia |
Immune deficiency diseases Hodgkin’s disease Rheumatic fever Aplastic anemia Bone marrow failure Gaucher’s disease Hemolytic disease of the
newborn Hypersplenism Thrombocytopenic purpura Transfusion reaction Massive transfusions Pernicious anemia Septicemia Pneumonia Burns Radiation Toxic chemicals (benzene,
bismuth, DDT) Antineoplastic agents Adrenal corticosteroids (high
doses) |