MEDICINE AND SURGERY "F"
Course of LABORATORY MEDICINE
Prenatal and neonatal clinical analyses
PRENATAL CLINICAL ANALYSES
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Prenatal clinical analyses are most often concerned with genetic disorders of the foetus. In principle several fluids of foetal origin can be obtained and submitted to chemical, biochemical and microbiological analysis, e.g. amniotic fluid and blood. This however is technically difficult and of limited diagnostic value since these fluids equilibrate with maternal blood, thus eventual pathological alterations may be concealed.
Obtaining samples of foetal tissues or fluids always entails some risk of abortion (e.g. in the case of amniocentesis estimates range between 0.05% and 0.5%; in villocentesis between 0.5 and 1%), thus the indications must be critically evaluated. Risk factors that constitute strong indications are:
- maternal age > 35 years
- history of miscarriages or neonatal deaths
- exposure to theratogenic agents or infectious diseases during pregnancy
- hereditary diseases in the paternal or maternal lineages
- abnormal nuchal translucency or ultrasound finding
- positive tri-test. The triple test is carried out on maternal blood (starting from the 14th week of gestation) and relies on the measurement of the serum concentrations of (i) chorionic gonadotropin, (ii) alpha fetoprotein, and (iii) non conjugated estriol. The tri-test is positive if (i) is elevated while (ii) and (iii) are decreased; positivity is associated to an increased risk of Down syndrome of the foetus (predictive value = 60%)
PREPARATION OF SAMPLES
Foetal cells can be obtained from the amniotic fluid (amniocentesis), from the chorionic villi (villocentesis, chorionic villus sampling) or from foetal blood (cordocentesis).
is possible from the 16th to the 22nd week of gestation, and is effected using a specifically designed syringe, under echographic control.
(biopsy of the chorionic villi of the placenta) is possible starting from the 10th-12th week of gestation and is not used after the 15th because at that time amniocentesis (which has the same diagnostic indications less risk) becomes possible.
(percutaneous umbilical cord blood sampling, PUBS) cannot be performed before the 17th week of gestation and entails a 1-2% risk of miscarriage.
STUDY OF THE CHROMOSOMES
The foetal cells obtained by amniocentesis or villocentesis are cultured in artificial media and, when confluent, they are treated with colchicine to block all mitoses in the metaphase. The samples are then stained using quinacrine or Giemsa, and observed under a microscope. Metaphase chromosomes are well formed and easy to visualize; dedicated computer softwares for image analysis are available.
Pathological conditions that may be diagnosed with this method include aneuploidies (alterations in the number of the chromosomes and/or structural anomalies):
1. Alterations in the number of the sex chromosomes
1a. Monosomy: kariotype X0 (Turner syndrome)
1b. Sexual trisomies: kariotypes XXX, XXY (Klinefelter syndrome) and XYY
2. Alterations in the number of the chromosomes
Trisomies: 21 (Down syndrome); 18 (Edwards syndrome); 13 (Patau syndrome)
3. Translocations (exchanges of genetic material between different chromosomes)
3a. Balanced translocations
3b. Unbalanced translocations
4. Partial deletions or partial duplications
5. Somatic mosaicism
(some but not all of the cells present any of the aneuploidies listed above)
STUDY OF SELECTED GENES
In the presence of familiarity for hereditary diseases, selected genes can be amplified using the
Polymerase Chain Reaction
method and sequenced, to screen for mutations.
Polymerase Chain Reaction
is carried out by adding to the genetic material to be tested the RNA primers corresponding to the beginning of the gene of interest (of bothDNA strands), a heat-resistant DNA polymerase, and an excess of deoxyribonucleotides tri-phosphate. A thermal cycle is then started in which the mixture is heated to promoted the dissociation of the complementary DNA strands, and cooled to allow the DNA polymerase to synthesize the segment (gene) that follows the chosen primer. After a chosen number of cycles the DNA segment (gene) of interest has been greatly amplified and can be submitted to the detection of its nucleotide sequence (sequencing).
Examples of hereditary genetic diseases that can be diagnosed by selective amplification and sequencing include:
Frequency at birth
Polycistic kidney disease
Sickle cell anemia
Hemoglobin beta subunits
Hemoglobin beta chain
Chloride membrane transporter CFTR
Coagulation factor VIII
All the mitochondria of the foetus (or of the adult) cells come from the ovum, i.e. they are of maternal origin. Several genetic diseases due to mitochondrial defects are known and are maternally inherited. They can be diagnosed by studying the mitocondrion DNA extracted from any cell (including those obatined by amniocentesis, or villocentesis). The most common mitochondrial diseases are myopathies and neuropathies.
OTHER DISEASES THAT CAN BE DIAGNOSED IN THE FOETUS
Foetal infections may be diagnosed from the presence of bacteria in the (normally sterile) amniotic fluid. They are severe, life-threatening conditions and should be promptly treated with antibiotics administered to the mother or directly in the amnios.
Lung maturity can be estimated by the lecithin/sphingomyelin ratio (normal value >2:1) and the surfactant/albumin ratio (normal value >55) in the amniotic fluid.
Rh incompatibility and risk of foetal erythroblastosis.
PERINATAL CLINICAL ANALYSES
Several clinical analysis are routinely carried out on neonatal blood and urine. Technically these are not different from the equivalent analyses one could carry out on the adult; but some diseases must be diagnosed immediately after birth because they require prompt therapy. Examples of these include:
Phenylketonuria and other metabolic defects
The reason why diagnosis is so urgent is that the foetus suffering of any of these conditions is normal at birth, because the metabolism of the mother (if she is healthy) compensates for the defect. After birth the disease becomes evident and usually begins to produce organ damage (most often in the brain).
Questions and exercises:
1) At which gestational age the amniocentesis is indicated:
11th - 15th week
16th - 22nd week
after the 18th week
2) The diseases that can be diagnosed from the fetal cells obtained by the procedures described in question 1 are:
Monogenic hereditary diseases, chromosomal abnormalities, mitochondrial diseases
Monogenic hereditary diseases, chromosomal abnormalities, skeletal deformities
Mitochondrial diseases, chromosomal abnormalities, hydrocephalus
3) The prenatal diagnosis of inherited enzymatic defects is achieved by
PCR followed by gene sequencing
Detection of abnormal concentrations of the substrate of the affected enzyme
4) To select the gene to be amplified by PCR one needs:
the appropriate DNA primers
the appropriate gene promoter
the appropriate RNA primers
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Thank you Professor (lecture on bilirubin and jaundice).
The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?
This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.
Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.
(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.
(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.
Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!
In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).
is it possible to take gulonolactone oxidase to synthesize vitamin C
instead of vitamin C supplement?
Bellelli: no, this approach does not work. The main reason is that
the biosynthesis of vitamin C, as almost all other metabolic processes, occurs intracellularly.
If you administer the enzyme it will at most reach the extracellular fluid but will not be
transported inside the cells to any significant extent. Besides, there are other problems
in this type of therapy (e.g. the enzyme if administered orally, may be degraded by digestive
proteases; if administered parenterally, may cause the immune system to react against a
non-self protein). In theory one could think of a genetic modification of the inactive human
gene of gulonolactone oxidase, but the risk and cost of this intervention would not be
justified. In addition to these considerations, except for cases of shipwreckage or
other catastrophes, a proper diet or administration of tablets of vitamin C is effective,
risk-free and unexpensive, thus no alternative therapy is reasonable. However, I express my
congratulations for your search on the biosynthesis pathway of ascorbic acid.
Resorption and not reabsorption would lead to hypercalcemia ie bone matrix being broken down.
Bellelli: I am not sure to interpret your question correctly. Resorption indicates destruction of the bone matrix and release of calcium and
phosphate in the blood, thus it causes an increase of calcemia. Reabsorption usually means active transport of calcium from the renal tubuli to the blood, thus
it prevents calcium loss. It prevents hypocalcemia, and thus complement bone resorption. To avoid confusion it is better use the terms "bone resorption" and "
renal reabsorption of calcium". If you have a defect in renal reabsorption, parthyroid hormone will be released to maintain a normal calcium level by means of
bone resorption; the drawback is osteoporosis.
In Reed and Frost model: I haven't understood what is the relationship
between K and R reproductive index. Thank you Professor!
Bellelli: in the Reed and Frost model K is the theoretical upper limit of
. R the reproductive index is the ratio (new cases)/(old cases) measured after
one serial generation time. R
is the value of R one measures at the beginning
of the epidemics, when in principle all the population is susceptible.
What is the link between nucleotide metabolism and immunodeficiencies and mental retardation?
Bellelli: the links may be quite complex, but the principal ones are as follows:
1) the immune response requires a replication burst of granulocytes and lymphocytes, which in turn requires
a sudden increase of nucleotide production, necessary for DNA replication. Defects of nucleotide metabolism
impair this phase of the immune defense. Notice that the mechanism is similar to the one responsible of
anemia which requires a sustained biosynthesis of nucleotides at a constant rate, rather than in a burst.
2) Mental retardation is mainly due to the accumulation of nulceotide precursors in the brain of the
newborn, due to the incompletely competent blood-brain barrier.
How can ornithine transaminase defects cause hyperammonemia? Is it due to the accumulation
of ornithine that blocks the urea cycle or for other reasons?
Bellelli: ornithine transaminase is required for the reversible interconversion of ornithine
and proline, and thus participates to both the biosynthesis and degradation of ornithine. The enzyme is
synthesized in the cytoplasm and imported in the mitochondrion. Depending on the metabolic conditions
the deficiency of this enzyme may cause both excess (when degradation would be necessary) or defect
(when biosynthesis would be necessary) of ornithine; in the latter case, the urea cycle slows down. Thus
there is the paradoxical condition in which alternation may occur between episodes of hyperammonemia
and of hyperornithinemia.
When we use the Berthelot's reaction to measure BUN do we also have to
measure the concentration of free ammonia before adding urease?
Bellelli: yes, in principle you should. Berthelot's reaction detects ammonia,
thus one should take two identical volumes of serum, use one to measure free ammonia,
the other to add urease and measure free ammonia plus ammonia released by urea. BUN is
obtained by difference. However, free ammonia in our blood is so much lower than urea that
you may omit the first sample, if you only want to measure BUN.
Why do we have abnormal electrolytes in hematological neoplasia e.g.
Bellelli: I do not have a good explanation for this effect, which may have
multiple causes. However, you should consider two factors: (i) acute leukemias cause a massive
proliferation of leukocytes (or lymphocytes depending on the cell type affected) with a very
shortened lifetime; thus you observe an excess death rate of the neoplastic cells. The dying
cells release in the bloodstream their content, which has an electrolyte composition different
from that of plasma: the cell cytoplasm is rich in K and poor in Na, thus causing hyperkalemia.
(ii) the kidney may be affected by the accumulation of neoplastic white cells or their lytic products.
Gaussian curve: If it is bimodal is it more likely to be a "certain diagnosis" than if it is
unimodal or does it only show the distinguishment from health?
Bellelli an obviously bimodal Gaussian curve indicates that the disease is clearly
separated from health: usually it is a matter of how precise and clear-cut is the definition of the disease.
For example tuberculosis is the disease caused by M. tuberculosis, thus if the culture of the sputum is
positive for this bacterium you have a "certain" diagnosis (caution: the patient may suffer of two diseases,
e.g. tuberculosis and COPD diagnosis of the first does not exclude the second). However, in order to have
a "certain" diagnosis it is not enough that distribution of the parameter is bimodal, it is also required that the
patient's parameter is out of the range of the healthy condition: this is because a distribution can be
bimodal even though it is composed by two Gaussians that present a large overlap, and the patient's
parameter may fall in the overlapping region. Thus, in order to obtain a "certain" diagnosis you need to
consider not only the distribution of the parameter(s) but also the patient's values and the extent of the
Prof can you please elaborate a bit more on the interhuman variability and its difference
with the interpopulation variability please?
Bellelli: every individual is a unique combination of different alleles of the same genes;
this is the source of interindividual variability. Every population is a group of individuals who intermarry and
share the same gene pool (better: allele pool). Every allele in a population has its own frequency. Two
population may differ because of the diffferent frequencies of the same alleles; in some cases one
population may completely lack some alleles. The number and frequencies of alleles of each gene
determine the variance. If you take two populations and calculate the cumulative interindividual variance
of the population the number you obtain is the sum of two contributions: the interindividual variance within each population, plus the interpopulation variance
between the means of the allele frequencies. For example, there are human population in which the frequency of blood group B is close to 0% and other populati
ons in which it is 30% or more.
Prof can you please explain again the graph you have showed us in class about thromboplastin?
(Y axis=abs X axis= time)
Bellelli: the graph that I crudely sketched in class represented the signal
of the instrument (an absorbance spectrophotometer) used to record the turbidity of the
sample (turbidimetry). The plasma is more or less transparent, before coagulation starts.
When calcium and the tissue factor (or collagen) are added. thrombin is activated and begins
digesting fibrinogen to fibrin; then fibrin aggregates. The macroscopic fibrin aggregates cause
the sample to become turbid, which means it scatters the incident light. The instrument reads
this as a decrease of transmitted light (i.re an increase of the apparent absorbance) and the
time profile of the signal presents an initial lag phase, which is called the protrombin or
thromboplastin time depending on the component which was added to start coagulation
(tissue factor or collagen).
Prof can you please explain the concept you have described in class about
the simultaneous hypercoagulation and hemorrhagic syndrome? How can this occur?
Bellelli: The condition you describe is observed only in the Disseminated
Intravascular Coagulation syndrome. Suppose that the patient experiences an episode of
acute pancreatitis: tripsin and chymotripsin are reabsorbed in the blood and proteolytically
activate coagulation causing an extensive consumption of fibrinogen and other coagulation
factors. Tripsin and chymotripsin also damage the vessel walls and may cause internal
hemorrages, but at that point the consumption of fibrinogen may have been so massive that
not enough is left to form the clot where the vessel has been damaged, causing an internal
hemorrage. Pancreatitis is a very severe, potentially lethal condition, and DIC is only one of
the reasons of its severity.
You said that certain drugs (ethanol, cocaine, cannabis, opiates...) cause a
necessity of higher and higher dosage, for two reasons: the enzyme in the liver is inducible and
the receptors in the brain are expressed less and less. So, first, I am not sure I got it right, and
second I did not understand how expressing less receptors leads to a necessity of higher
Bellelli: You got it correctly, but the detailed mechanism of resistance may
vary among different substances, and not all drugs cause adaptation.
The reason why reducing the number of receptors may require an increased dosage of the drug
is as follows: suppose that a certain cell has 10,000 receptors for a drug. When bound to its
agonist/effector, each receptor produces an intracellular second messenger. Suppose that in
order for the cell to respond 1,000 receptors must be activated. The concentration of the
effector required is thus the concentration that produces 10% saturation. You can easily
calculate that this concentration is approximately 1/10 of the equilibrium dissociation constant
of the receptor-effector complex (its Kd), the law being
Fraction bound = [X] / ([X]+Kd)
where [X] is the concentration of the free drug.
After repeated administration, the subject becomes adapted to the drug, and his/her cells
express less receptors, say 5,000. The cell response will in any case require that 1,000
receptors are bound to the effector and activated, but this now represents 20% of the total
receptors, instead of 10%. The drug concentration required is now 1/4 of the Kd.
Continuing administration of the drug further reduces the cell receptors, but the absolute
number of activated receptors required to start the response is constant; thus the fewer
receptors on the cell membrane, the higher the fraction of activated receptors required.
Why does hyperosmolarity happen in type 2 diabetes and not in type 1?
Bellelli: Hyperosmolarity can occur also in type 1 diabetes, albeit
infrequently. The approximate formula for plasma osmolarity is reported in the lecture on
osmolarity = 2 x (Na
) + BUN/2.8 + glucose/18
this is expressed in the usual clinical laboratory units (mEq/L for electrolytes, g/dL for non-
electrolytes). The normal values are:
osmolarity = 2 x (135 + 5) + 15/2.8 + 100/18 = 280 + 5.4 + 5.6 = 291 mOsmol/L
Let's imagine a diabetic patient having normal values for electrolytes and BUN, and glycemia=400 mg/dL:
osmolarity = 280 + 5.4 + 22.4 = 307.8 mOsmol/L
The hyperosmolarity in diabetes is mainly due to hyperglycemia, even though other factors
may contribute (e.g. diabetic nefropathy); however the contribution of glucose to osmolarity is
relatively small. As a consequence in order to observe hyperosmolarity the hyperglycemia
should be extremely high; this is more often observed in type 2 than in type 1 diabetes, for
several reasons, the most relevant of which is that in type 1 diabetes all cells are starved of
glucose, and the global reserve of glycogen in the body is impoverished: there is too much
glucose in the blood and too few everywhere else, thus reducing, but not abolishing, the risk of
extreme hyperglycemia. Usually in type 2 diabetes the glycogen reserve in the organism is not
impoverished, thus the risk of extreme hyperglycemia is higher.
Hemostasis and Thrombosis lecture: I don't understand why is sodium citrate
added to the serum solution to measure the prothrombin time.
Bellelli: in order to measure PT or PTT you want to be able to start the
coagulation process at an arbitrary time zero, and measure the increase in turbidity of the
serum sample. To do so you need (i) to prevent spontaneous coagulation with an anticoagulant;
and (ii) to be able to overcome the anticoagulant at your will. Citrate (or oxaloacetate; or EDTA)
has the required characteristics: it chelates calcium, and in this way it prevents coagulation;
but you can revert its effect at your will by adding CaCl
in excess to the amount
of citrate. You cannot obtain the same effect with other anticoagulants (e.g. heparin) whose
action cannot be easily overcome.
Dear professor I cannot do the self evaluation test because it says the the
time has expired It is not possible because I havent even started them
Bellelli: this is due to the fact that the program registers your name and
matricola number from previous attempts. I shall fix this bug. Meanwhile try to use a fake
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