Clinical Hematology
Sickle-cell disease
Sickle-cell disease is an inherited mutation that
produces a hemoglobinopathy: the glutamate at the number 6 amino acid in
the 146-amino acid b chain of hemoglobin (Hb) is
replaced by valine. This mutation results
from a single base-pair substitution in the gene encoding for the b chain.
The resulting Hb in sickle-cell patients is denoted HbS. In sickle-cell
disease, when the HbS encounters
regions where there is a low O2 tension (like venous blood arising
from an exercising muscle), the HbS
polymerizes, forming long strands within the RBC that distorts its shape (see
figure, right). The distorted RBCs lose
there normal flexibility and pile up, blocking blood flow through capillaries
and small vessels. One can enter a
positive feedback situation whereby the local ischemia (reduced blood flow)
causes further drops in the O2 levels, which causes still further
sickling, and the patient is said to be in sickle-cell
crisis. Patients also suffer from
severe hemolytic anemia—a reduction
of the number of circulating RBCs due to hemolysis of the fragile sickle
cells. Most patients with sickle-cell
disease have a reduced life span—especially in poor third-world countries where
the disorder is most prevalent.
Sickle-cell disease is found only in
individuals who are homozygous for the mutation—i.e., they have two copies of
the mutation, one inherited from their father and one from their mother. Individuals who are heterozygous for the
trait (i.e., have only one copy of the mutation) do not exhibit symptoms, since
they have adequate levels of HbA
(normal hemoglobin). But, individuals
exhibiting the sickle-cell trait can pass the trait on to their offspring.
In the African-American population, the incidence of
sickle-cell trait (heterozygous) is ~8% in the general population. In Equatorial Africa, the incidence of the
trait can reach levels >50% in local populations. This high incidence posed a mystery for many years. Why would such a seemingly lethal gene
remain at such high incidence in a general population? If the individuals who contract the disorder
die before they reproduce, then should not the mutation die with them since it
would not have been passed on to their offspring? The mystery was solved when epidemiologists looked also at the
incidence of malaria in the population.
Malaria is a mosquito borne parasite (Plasmodium falciparum) that invades
RBCs. Persons who have the sickle-cell
trait have an unusually high resistance to infection by the parasites: the HbS
in their RBCs interferes with the ability of the parasite to reproduce. Thus, in tropical regions of the world where
malaria is endemic, the benefit of surviving untreated malaria outweighs the
detriment of the chance of producing offspring that have sickle-cell
disease. Of course, in the United
States, the trait serves no benefit to the individual: malaria is rare in the U.S., and effective
drug treatments are readily available (e.g., quinine and its derivatives). Thus, the relatively high incidence of the
trait in African Americans is a remnant of a trait that greatly benefited their
ancestors.
An aside (for your interest only)… a popular summer
libation evolved from a prophylactic treatment. British soldiers who were supervising construction of the Suez
Canal were issued daily doses of quinine to guard against malaria
infection. Quinine is an extremely
bitter substance, and as such, the soldiers found it more palatable to dissolve
the tablets in bottled soda water sweetened with sugar. Somebody had the bright idea to add gin,
also issued in daily allotments, and voilŕ: the Gin and Tonic is invented!
Pernicious anemia
Pernicious anemia is caused by a
lack of sufficient quantities of vitamin B12. As discussed in lecture, the disorder is rare: one needs only small dietary quantities of
the vitamin on a daily basis, and the liver stores up to a two-year supply of
the vitamin. Thus only strict
vegetarians must take care to acquire the vitamin by taking oral supplements.
Prior to the advent of effective
anti-ulcer drugs, for example H2-type antihistamines like cimetidine
(Tagamet®), one of the sole effective treatments of ulcers was the surgical
removal of the stomach (gastrectomy); note that the stomach secretes into its
lumen vast quantities of HCl, which combined with other factors can cause
gastric (stomach) or duodenal (intestinal) ulcers. It was observed that virtually all gastrectomy patients developed
pernicious anemia typically six months to two years after surgery. Thus, gastroenterologists postulated that
the stomach must secrete some intrinsic factor that is necessary for the
absorption of dietary vitamin B12, and that factor was later
identified and is termed intrinsic
factor.
Intrinsic factor is secreted into the stomach lumen by
gastric parietal cells, the same cells that make the HCl. Intrinsic factor tightly binds to B12
and subsequently serves two functions:
it prevents the denaturation and destruction of the vitamin by
intestinal digestive processes, and the bound form of the vitamin specifically
binds to a site on the mucosal cells of the ileum (the lowest portion of the
small intestines). Once bound to the
mucosal cells, a specific transport process takes up the vitamin (combined with
the intrinsic factor) into the mucosal cells, and the vitamin is subsequently
released into the interstitial fluid where it can then enter the blood.
Gastrectomies as a treatment for ulcers are rarely
today. However, in patients suffering
from gastric secretory diseases, or patients who must undergo gastric resection
(e.g., for stomach cancer) or intestinal resection of the ileum (e.g.,
intestinal cancer), or patients with intestinal mucosal disease (e.g., Crohn’s
disease), pernicious anemia will develop unless the patient receives 1000 mg per month of vitamin B12 via
injections.
Thrombocytopenia
Recall that platelets (thrombocytes)
are cell fragments released from megakaryocytes that rupture in the bone
marrow. The existence of sufficient
quantities of circulating platelets is absolutely essential to stopping bleeding for a period of time sufficient for
wound healing. Thrombocytopenia is the term that describes a low blood platelet
count (defined as < 150,000 per mL, with normal values
typically ~250,000 per mL). Patients suffering from severe
thrombocytopenia (< 50,000 per mL) present with excessive
nose and gum bleeding, easy bruising (n.b., a bruise is a hemorrhage under the
skin), blood in the feces, petechiae (pinpoint skin hemorrhages), and in women,
excessive uterine bleeding during menstrual periods. Causes of thrombocytopenia include increased uptake of platelets
by the spleen (frequently associated with autoimmune disorders), decreased
production rate of platelets (most commonly caused by chemotherapy and/or
radiation therapy in cancer patients), and increased destruction of platelets
(e.g., due to autoimmune disease, drug toxicity to platelets). In severe cases of thrombocytopenia, the
only immediate recourse is to transfuse the patient with platelets derived from
pooled donated blood. Note that in
severe cases, even a simple dental procedure can produce bleeding that is life
threatening. Finally, the common pain
reliever aspirin inhibits the function of circulating platelets (see below),
but it does not decrease the platelet count, so patients who are using aspirin
can exhibit increased bleeding times.
Aspirin hinders platelet-plug formation
A number of hormones and paracrines
are derived from membrane phospholipids
(see figure, right). Thromboxanes,
prostacyclins and prostaglandins are derived from arachidonic acid, where the first step in their synthesis is
catalyzed by an enzyme called cyclooxygenase. Aspirin irreversibly inhibits
cyclooxygenase, and since the products are involved in triggering inflammation,
this explains the antiinflammatory action of aspirin.
Since Tx-A2 is an important player in the
formation of platelet plugs, platelets previously exposed to aspirin are less
capable of aggregating, and thus platelet-plug formation is hindered. Thus people who have recently taken aspirin
can exhibit increased bleeding times.
And, since platelets are also important regulators of clot formation,
clot formation is hindered as well.
Note that aspirin has a beneficial affect in individuals prone to the
formation of thromboemboli—clots
that spontaneously form in the blood stream that can occlude small arteries
causing heart attacks, strokes, etc.
The amount of aspirin needed to afford this benefit amounts to one
“baby” aspirin per day.
Since prostacyclin is also derived from a
cyclooxygenase-catalyzed reaction, then shouldn’t an undamaged vessel be
actually prone to platelet adhesion and aggregation? Recall that prostacyclin release from healthy endothelial cells
inhibits platelet adhesion and aggregation.
The solution of this apparent paradox comes from the fact that
endothelial cells are complete live cells, whereas platelets are merely cell
fragments. True, aspirin will inhibit
endothelial-cell cyclooxygenase, but after the aspirin dose declines, the cell
will manufacture new functional cyclooxygenase. Circulating platelets, on the other hand, do not have the
capability to manufacture new enzyme, so once the cyclooxygenase is inhibited,
it is inhibited for the life of the platelet.
Platelets have a life time in the blood of
approximately 10 days. When you donate
a unit of blood, the blood-drive personnel ask if you have consumed aspirin at
any time during the past 10 days. If
your answer is negative, then the platelets in your donated blood will be separated
out for donation to patients suffering from thrombocytopenia. If your answer is affirmative, then the
platelets in your donated blood will be discarded.
An aside regarding the marketing of drugs (for your interest only)…
Aspirin originally was the trade name of acetylsalicylic acid. It was marketed as a pain reliever by the Bayer Co. starting in 1899. Aspirin gained such wide use that people stopped referring to it as “Aspirin brand of acetylsalicylic acid,” preferring instead to call it simply aspirin. When this occurs, a product loses its trade-name status and becomes generic: other companies can compete by manufacturing their versions of aspirin, and the loss of name recognition hurts the original manufacturer’s profits. Furthermore, other pain relievers came onto the market: Tylenol® brand of acetaminophen, Motrin® brand of ibuprofen, etc. And, these newer products, as pain relievers, had advantages over aspirin: acetaminophen did not upset the stomach (an adverse side effect of aspirin), ibuprofen was more potent, etc.
In response, Bayer launched a cleaver advertising
campaign to promote the continued use of their product. You may recall slogans
like: “If stranded on a desert island,
nine out of ten doctors would prefer Bayer aspirin over Tylenol.” I am sure that this is a true statement, but
it’s a bit deceptive! Solely as a pain reliever, aspirin is not as effective as other drugs. But, look at the other clinical uses of
aspirin: its prevents thromboembolisms,
it reduces fever, it is an effective antiinflammatory drug—and it’s even
effective in treating gout and rheumatic fever, and topically for treating
warts and corns. The other drugs share
some, but not all, of these effective uses of aspirin. And, aspirin is one of the safest drugs on
the market! Overdosing is generally not
fatal, albeit the individual will suffer from terrible dyspepsia and
uncomfortable tinnitus (ringing in the ears).
Consuming an entire bottle of acetaminophen, on the other hand, will
destroy the liver—the only effective treatment being a liver transplant.
So, if you
run the risk of becoming stranded on a desert island, and if you could only pack one
drug, aspirin would be the one of choice!
But, it wouldn’t have to be Bayer brand—any brand of aspirin would do
just fine.
Anticoagulation: the prevention of clot formation
A number of diseases can result in
the pathological formation of clots, including abnormal blood flow in arteries
(i.e., arteries narrowed by plaque), turbulent flow around valves in the veins
and heart, consequences of diabetes and cancer, as well as chemical insults
(e.g., smoking). When an abnormal clot
forms, it has the potential of breaking away from the vessel surface,
travelling downstream, and occluding vessels—i.e., it becomes a
thromboembolis—thereby causing heart attacks, strokes, or ischemia in other
organ systems. Indeed, two-thirds of
the US population can expect a significant thromboembolic episode sometime
during their lifetime, and >40 % will die from one (mostly due to heart
attacks). Thus, anticoagulants are an
important class of drugs to be given prophylactically to individuals prone to
thrombus formation.
In addition, in the laboratory, many
tests require samples of unclotted blood.
Simply drawing blood into a glass tube will initiate the clotting
cascade by activating the intrinsic pathway (i.e., contact activation of factor
XII with the glass surface. For that
reason, many test tubes (vacutaners) are pre-manufactured containing an
anticoagulant.
There are two general classes of
anticoagulants. In vitro anticoagulants—in
vitro means “in glass”—are substances that can prevent coagulation after
the blood is removed from the body (e.g., in a test tube). They work by preventing the activation of
existing factors in the blood. These
include Ca++ chelators like EDTA
(ethylene diamine tetraacetate) and citrate, that bind Ca++ and
remove it from solution. Reducing the
Ca++ concentration prevents activation of the vitamin-K-dependent
factors. Calcium chelators cannot be
used in the body, since reducing extracellular Ca++ levels to a
level sufficient to prevent coagulation would have dire consequences: the heart would cease to function (recall,
electrical activity and excitation-contraction coupling), synapses would cease
to function (recall, the inward flow of Ca++ that triggers synaptic
release), etc.
Another in
vitro anticoagulant is heparin. Heparin is a mucopolysaccharide that is
normally made by mast cells (type of
transformed leukocyte), and is found in particularly high concentrations in
lung tissue; presumably its function in the lungs is to maintain the blood in a
fluid state preventing blockage of small pulmonary vessels and
capillaries. Heparin prevents
coagulation (either in a test tube or in the body) principally by preventing
activation of thrombin, but it also (to a lesser extent) prevents the
activation of other factors. Heparin
works in glass test tubes, but it can also be used clinically in patients. A problem, however, with the clinical use of
heparin is that it cannot be given orally—it is degraded by digestive enzymes
prior to absorption. Thus, it must be
given intravenously, and this can be problematic. Namely, the injection site itself is an injury with a tendency to
bleed; injecting the heparin prevents clotting at the injection site, and the
patient usually develops a bruise (blood oozing out of the vein under the
skin). Thus other drugs that can be
administered orally are usually used for long-term treatment of patients prone
to thromboembolisms.
The other class of anticoagulants
are in vivo anticoagulants—in vivo means “in life.” These types of anticoagulands are
ineffective in preventing clotting involving existing clotting factors
(i.e., the anticoagulants are not effective in vitro). Rather, these
factors act by inhibiting the production of new clotting factors by the liver,
and this is why they only work in vivo. An advantage to using these drugs is that
they can be given orally, thereby avoiding the unpleasant bruising when administering
heparin intrevenously. Thus, these
drugs are clinically referred to as oral
anticoagulants.
The oral anticoagulants were first
discovered in the 1920’s, when investigators discovered the cause of “sweet
clover disease” in cattle. The disease was
characterized by severe bleeding in cattle fed partially fermented sweet-clover
hay. The offending compound in the hay
was coumarin (Dicumarol®). Subsequently, additional derivatives were
manufactured, notably warfaran
(Coumadin®), which was originally developed as a rat poison. Coumarin and warfaran inhibit the conversion
of vitamin K to its different forms as it participates as a cofactor in the
synthesis of the vitamin-K-dependent factors.
Namely, it prevents the synthesis of g‑carboxyglutamate (Gla)
from glutamate (Glu), and thus patients receiving this type of anticoagulant
therapy have lower levels of functional factors.
It is important to emphasize that coumarin and
warfarin have no effect on previously synthesized functional factors already circulating
in the plasma! Finally, apart from the
benefit of being able to administer these drugs orally, there is another
benefit: patients who overdose on the
drug can be effectively treated by administering an antidote, namely large
doses of vitamin K.
Hemophilia
Hemophilia is a sex-linked inherited
disorder resulting in increased bleeding, notably internal bleeding into
joints. In an untreated individual,
even the most minor injury can result in life-threatening bleeding. Sex-linked inherited disorders mean that the
offending gene resides on the X chromosome.
Since males inherit only one copy of the X chromosome (XY genotype),
they invariably exhibit the disease. Heterogeneous
females (XX genotype) do not exhibit the disease, but they can be carriers of
the trait if one of their X chromosomes has the defective gene. A female will only exhibit symptoms if her
father is a male hemophiliac, and her mother is a carrier.
This pattern of inheritance is most strikingly
observed in the pedigree of the Royal families of Europe, starting with the
parents of Queen Victoria, as seen on the next page (purposely printed in
landscape).
The previous figure shows
carrier females as half-filled circles, and males with the disease as filled
squares. It is generally believed that
the hemophilia in these families started with a spontaneous mutation in one of
Queen Victoria’s eggs, since there is no evidence of the disease in any of
Victoria’s prior ancestors. [This notion
has been questioned by some scandal craving “academics” who postulated that
Victoria might have been born out of wedlock—but this is hard to fathom since
(a) her illegitamate father would have had to have had the disease, or (b) she
would have had to have been substituted (switched) with the biological
offspring of her father, King Edward.]
Through subsequent intermarriages, the trait was passed on to the
Spanish royal family (right), the Hessian and Prussian royal families (above
box, left), and to the Russian royal family (above box, right). It escaped being passed on to the German
royal family (left), and the current British royal family (left of box).
Although the disease caused much grief in a number
of the families, probably the most historically important ramification of the
disease occurred in the Russian royal family:
Crown Prince Alexis, the only male offspring of Tsar Nicholas II and
Tsarina Alexandra, suffered from hemophilia.
A number of historians (clearly interested in hematology) have argued
that since Nicholas was preoccupied with ongoing wars, Alexandra became more
influential in directing domestic affairs and affairs of state. She doted over her son and surrounded
herself with half-wit advisors (e.g., Rasputin) who became politically
active. Thus, important governmental reforms
where not undertaken that might have delayed, or prevented, the upcoming
revolution.
There are a number of different
coagulopathies that produce hemophilia, but the two most common are hemophilia types A and B. Type A is caused by a defect in factor VIII,
a cofactor in the intrinsic pathway that is required for activation of the
common pathway by IXa. Type B is caused
by a defect in factor IX, again a factor in the intrinsic pathway. Note that type B hemophilia is sometimes
called Christmas disease, based on
the old (archaic) name of IX, the “Christmas factor.” Although most untreated sufferers of hemophilia die at a young
age, it is important to note that hemophiliacs can form normal clots,
since clotting can still be initiated by the extrinsic pathway. This is why hemophiliacs suffer mostly from
internal (e.g., joint) injuries, where vessels break, but the breakage does not
involve physical lysis of the endothelial cells.
Both types of hemophilia can effectively be treated
by simply administering periodic injections of the clotting factors (either
VIII or IX). Until recently, these
factors were obtained from donated blood.
For mainly economic reasons, the manufacture of the factors first
involved pooling literally hundreds to thousands of liters of donated blood
plasma, and subsequently processing the pooled plasma so as to isolate and
purify the different factors.
Unfortunately, the HIV virus co-purifies with the proteins, so even if
only one of the units if donated plasma contains the virus, this will
contaminate the entire lot of purified factors. Thus in the early 1980’s, the vast majority of hemophiliacs
requiring periodic injections of clotting factors became HIV positive, and a
large number developed AIDS.
The US administration at the time can share much of
the blame for this disaster, since even though significant epidemiological
evidence existed that AIDS was a blood-borne disease, much of this evidence
compiled by the CDC was suppressed, pending proof that the nation’s
blood supply was indeed contaminated.
After all, proof that AIDS was a viral diesase had not yet been
established! And, more criminal was the
fact that even when the blood supply was implicated in transmission of the
disease, agencies with a financial interest in existing supplies of (possibly
contaminated) blood products were slow to remove them from the market. This caused truly unnecessary transmission
of HIV, and as a result, a number of people responsible ended up in jail.
Today it is safe to receive clotting factors for the
treatment of hemophilia. Changes in the
blood collection procedure (e.g., discarding donations from high-risk
individuals), changes in the way the products are produced (e.g., small pools
of plasma rather than large ones), the ability to assay for HIV and effective
procedures for deactivating the virus have all led to the production of safe
clotting factors. And more recently,
recombinant forms of the factors have become available, thus avoiding altogether
having to aquire the factors from donated blood.
And now for something
completely different: an aside
regarding Russian aristocracy (for your interest only)…
Shortly after the execution of Tsar Nicholas II and
his family, a young woman turned up in Germany claiming to be the Grand Duchess
Anastasia. She claimed to have been
spared execution by a sympathetic guard who spirited her out of Russia; she
came to Germany seeking other surviving Romanov family members who were outside
of Russia prior to the revolution. This
caused a “problem” for the surviving Romanov’s: if she could prove that she was indeed Anastasia, then she was
the rightful heiress to a sizable Romanov fortune invested outside of Russia.
“Anastasia” proved very convincing,
and she rapidly became an international celebrity (the drama even resulted in a
full length film staring Yul Brenner and Ingred Bergman). Although she succeeded in convincing some
family members of her identity, other family members disputed her claim and financed
their own investigations to discredit her.
She subsequently lost several legal battles where the courts ruled that
she had not provided adequate proof that she was indeed who she said she
was. She changed her name to Anna
Anderson, and immigrated to the United States where she married a retired
history professor and became Anna Anderson Manahan. She never shared in the Romanov fortune, and died in 1984 still
claiming all along that she was indeed Anastasia.
The remains of the executed Romanov
family members were discovered in 1979, but were kept secret until 1991 when
Boris Yeltson (who had just been elected President of Russia after the breakup
of the Soviet Union) granted permission for exhumation. Forensic analyses proved inconclusive: the remains did not account for all the
executed family members, and although one group claimed that Anastasia’s bones
were part of the remains, two other groups disputed that assertion. This once again sparked renewed interest in
Anna Anderson’s claim.
In 1995, DNA finger printing
technology had sufficiently progressed that it was possible to prove
conclusively whether or not Anna Anderson Manahan was truly Anastasia. All that was needed were suitable sources of
her DNA and that of a family relation.
Surviving friends of Anna Anderson, anxious to vindicate her claim,
tracked down an intestinal biopsy specimen stored in a hospital pathology
labortory (she had had a minor surgical procedure in 1979). All that was needed was the DNA of a known
relative of Anastasia.
The DNA from a known relative of
Anastasia came from an unusual source.
If you look carefully at the above pedigree of the royal families of
Europe, you will see that Anastasia has direct maternal lineage to Queen
Victoria. Mitochondrial DNA is
inherited solely from the mother, thus Anastasia’s mitochondrial DNA must have
been a close copy of that of Victoria’s.
You will also see in the pedigree that in the British royal family,
Prince Philip (husband of Queen Elizabeth II) also has direct maternal lineage
to Victoria as well. Prince Philip
gratiously agreed to donate a blood sample for the DNA comparison.
Well, to make a long story
short: Anna’s mitochondrial DNA did not
match that of Prince Philip!
Furthermore, Anna Anderson’s DNA did match that from a living
relative of a Polish woman who had been identified in 1936 as being Anna’s true
mother; the woman was discovered by private investigators hired by the Romanov
family members to discredit Anna.
Although by all objective accounts these data prove scientifically that
Anna Anderson was a fraud, Anna’s surviving friends discount the DNA
evidence: they still believe to this
day that Anna Anderson was Anastasia, the youngest daughter of Nicholas and
Alexandra, who miraculously escaped execution by the Bolsheviks, but was
wrongfully denied here title and inheritance.