If you’ve ever had blood drawn as part of a routine checkup, or for donation, you probably recall the procedure being very quick and simple. Today, it is routine to collect blood from people, to separate the blood into its various components, to store those components, and then to infuse them into other people. "Packed red blood cells," "platelets," and "fresh frozen plasma" are terms that you’d hear all day long if you were to volunteer on a medical ward. Along with saline, blood products are among the most common agents infused into patients. Each day, transfusion saves many lives, and one can hardly imagine modern medicine without it.
But it’s one of the most dangerous things that you can do to someone, if you don’t know what you’re doing.
Early blood transfusions
In 1628, William Harvey, an English physician, discovered how blood moves through vessels in the body, that it circulates through arteries and veins, and within just a few years scientists were attempting transfusions. Their rationale was simple and still makes sense today. If somebody is ill, his or her blood could be deficient in something. By giving patients blood from someone else, the deficient component will be replaced and they can get better. By extension, if the patient has a hemorrhage, the deficiency is the quantity of blood itself, so transfusion should also be helpful in this type of patient. It made perfect sense in the 17th century, given the assumption by anatomists of the time that all blood was the same.
All blood certainly looked the same and in 1665, another English physician, Richard Lower, was able to keep dogs alive with blood transfused from other dogs. In the years that followed, Lower and other researchers even succeeded in transfusing small amounts of blood between different animals, including from lambs to humans. But most transfusion attempts had fatal consequences. Sometimes the dogs, lambs, or humans died of a high fever. Other times, death followed other reactions that the researchers could not understand.
For two and a half centuries, doctors experimented occasionally with transfusion and continued finding that small amounts of transfused blood sometimes did not harm the recipient and other times was fatal. In rare cases humans could receive blood even from a non-human animal and live, while others would die after receiving blood from another human. Transfusion was like playing Russian roulette, so it was attempted only in desperation.
In 1881, for instance, the sister of William Stewart Halsted, a 29 year-old New York City surgeon, developed a severe hemorrhage after giving birth. She would have died except that Halstead drew his own blood and injected it immediately into his sister’s vein. The transfusion saved her life because she and her brother had compatible blood types, although he did not know about blood compatibility at the time. Halsted got lucky with his sister, but science was only years away from unraveling a mystery that would make transfusion safe.
That research happened at the turn of the 20th century, in connection with work on a phenomenon called hemagglutination. This is a clumping of blood cells that researchers were observing in the blood of victims of mismatched experimental transfusions, and it happens because all blood is not the same. Blood has thousands of different components and slight differences in some of them can spell failure if blood or a blood product is given that is inappropriate for the recipient. On the other hand, all blood is similar in its basic components.
Whole blood, plasma, and serum
If you know anyone who is diabetic, you may have heard something about that person’s blood sugar, or blood glucose. Glucose is a type of sugar (see our module Energy Metabolism I: An introduction). It’s the main source of energy in cells, and since its concentration in the blood should not be too high, nor too low, diabetics check their glucose levels frequently. Usually, they do this with a device that requires only a drop of blood. It’s called “whole blood,” because an individual needs only to prick his or her finger to release a drop. Nothing is separated out of the blood sample, so the machine reads the concentration of glucose in blood the way it exists within the body. For other blood tests, though, you may have heard your doctor or nurse mention plasma or serum levels. On routine exams, they tell you about your serum cholesterol or your serum triglycerides. On other occasions they may mention tests for plasma levels of certain chemicals, or you may have heard of somebody either donating plasma or receiving it.
In addition to water with numerous dissolved compounds such as glucose, blood contains cells. Physicians commonly talk about the blood cells collectively as a solid or cellular component of blood, because they can be easily separated from the liquid component. The liquid component is mostly water, but two different “versions” of this liquid can be prepared, depending on how the separation is performed.
The term plasma refers to everything in the blood without the cells. It is obtained by drawing a blood sample into a tube that has an agent that slows clotting, then spinning the tube in a centrifuge. During spinning, everything in the tube becomes many times heavier than its normal weight under Earth's gravity. Since blood cells, cell fragments, and very large molecules are denser than water, as they get heavier they move toward the bottom of the tube much faster than they would without spinning. What’s left on top is the plasma. (See Figure 1 for a diagram.) The percentage of whole blood volume that is packed cells is called the hematocrit and its value usually correlates with how well a person is making and maintaining hemoglobin and red blood cells (more on that below).
Plasma includes not just water, but also numerous agents called clotting factors that are involved in forming blood clots. In contrast to plasma, serum lacks many of the clotting factors. Serum is obtained by drawing a blood sample into a tube that is not treated to prevent clotting, but rather designed to encourage clotting. The sample is allowed to sit while it clots over time, thereby consuming most of the clotting factors. Then, the sample is centrifuged and liquid that ends up on the top of the tube, called serum, is free of most clotting factors. Thus, plasma minus clotting factors equals serum (Figure 2).
In laboratory medicine, the decision on whether to use whole blood, plasma, or serum for a certain test often involves a tradeoff of various advantages and drawbacks of each. Serum takes longer than plasma to prepare, for instance, and this can be a problem during emergencies or when measuring the concentration of a blood chemical that changes rapidly over time. If clotting factors are what you’re trying to measure (in a patient with hemophilia, a disorder where the blood doesn't clot normally, for instance) then you must use plasma, not serum, because the latter lacks clotting factors.
There are several settings when serum is preferable, for example the need to measure antibodies in a patient’s blood. The term serology, though its literal meaning is the study of serum, often refers to the diagnostic assessment of serum for antibodies.
The discovery of blood types
The age of serology began in Austria, at the University of Vienna, where physician-researcher, Karl Landsteiner worked in forensic anatomy (Figure 3). In 1900, Landsteiner noticed that blood cell clumping, or agglutination, following mixing of blood samples from different patients released toxins into the blood sample. He started mixing blood from patients, not just with whole blood samples, but also with serum from other individuals. He observed that when mixed with serum from a different person, blood cells would either clump or not clump, and that the clumps could be either small or large. Because the clumping had to be the result of the cells reacting with something in the serum, Landsteiner wondered if perhaps blood might indeed differ between individuals, an idea that went against the common thinking of his era.
Landsteiner set up a series of experiments using blood from just six volunteers (himself included), but mixing different blood components in various combinations and careful repetition of each mixing experiment turned those six volunteers into one of the greatest medical discoveries of the early 20th century. Based on his findings, Landsteiner proposed that there were three types or groups of blood that differed by the presence of factors in the serum that today we call antigens. Blood could be mixed between two people, without agglutination, he said, so long as the people were of the same type. He named the three types "A," "B," and "C" (the latter was eventually changed to "O").
It was a watershed study that ushered in an era of experimental blood transfusions in hospital settings, leading to the first ABO-matched transfusion, carried out at New York’s Mount Sinai Hospital in 1907. Transfusion could become routine only after physicians gained some understanding of the complexity of serum and blood cells. Such an understanding began when Landsteiner defined his blood groups and began systematic experiments aimed at honing in on the causes of hemagglutination. That research would quickly enable a revolution in medicine and especially in surgery.
The cellular components that are separated from plasma or serum include various types of cells and cell fragments. The main types are red blood cells (also called erythrocytes), white blood cells (also called leukocytes), and platelets (also called thrombocytes).
Platelets are actually cell fragments because they are pieces of precursor cells called megakaryocytes that break up during maturation. What is left are tiny pieces, generally 2 microns across, called platelets. Essentially, a platelet is a package enclosed by a membrane. Inside and on the surface of the platelet are various clotting factors and other proteins important to the stopping and prevention of bleeding. Clotting factors are also present in the cells that line blood vessels and, as noted earlier, clotting factors are also dissolved in the blood itself, outside of cells. The clotting factors from all three of these sources come together to stop the bleeding whenever a blood vessel breaks.
When a blood vessel wall is damaged, a certain protein called fibrin is exposed that sticks to platelets. This attracts platelets to the injured vessel wall. Not only do platelets stick to the damaged area, but they also become sticky to other platelets. The result is called a platelet plug, which results in hemostasis, or the stopping of bleeding (Figure 4). The most obvious example of this is the scab that forms on your skin when you skin an elbow or knee.
Red blood cells
As cells go, red blood cells (RBCs) are very small - 6-8 microns in diameter. Mature RBCs are filled entirely with the protein hemoglobin and their job is to transport oxygen in the blood. A person with an abnormally low number of RBCs, or a low concentration of hemoglobin in the blood or in RBCs, is said to have anemia. There are many different kinds of anemia, which can result either from reduced production of RBCs or accelerated destruction of RBCs.
Regardless of the condition that causes it, anemia can vary in its effects from very mild to very severe. When the number of RBCs or the amount of hemoglobin is just slightly below normal a person may feel totally normal, or may feel fatigue only with strenuous activity. But as the anemia worsens, a person will feel very sick and appear pale. With fewer RBCs and/or less hemoglobin, their hematocrit will be below normal and their muscles fatigue easily, because less oxygen is delivered to the muscle cells. To compensate for the decreased oxygen-carrying ability of the blood, the heart beats faster in order to move more blood, but the decreased ability to carry oxygen also can affect the heart itself.
RBCs are particularly relevant to Landsteiner’s work on transfusion research, since they comprise most of the cellular component of blood and account for much of what early transfusions provided to recipients. Transfusions in those early years consisted of whole blood, though today RBCs are stored and infused as packed red blood cells (PRBCs) in most transfusions.
White blood cells
White blood cells (WBCs) have their name because they are more of a clear color and are not red since they do not contain hemoglobin. They are bigger than RBCs and are part of the immune system. WBCs are classified into two groups: granulocytes and agranulocytes. Each group consists of different subtypes (see Figure 5) and their numbers and proportions are what physicians want to see when they order a complete blood cell count with differential, (often abbreviated as “CBC w/diff”).
Granulocytes are WBCs that show granules, little dots in their cytoplasm, when viewed under a microscope. They are thus called "granular." The dots are secretory vesicles filled with various enzymes and other compounds that vary among three types of granulocytes that exist in blood:
Neutrophils are the most abundant type of WBC, accounting for 40-70 percent of all WBCs. They are much bigger than RBCs and also very short-lived. While RBCs live in the blood an average of 120 days, a typical neutrophil lives only for 6-10 hours. The function of neutrophils is to eat up bacteria and damaged tissue. They do this by releasing from their granules enzymes that break down the bacteria and cytokines, which amplify the antibacterial response, partly by telling the body to manufacture still more neutrophils. On account of this function, neutrophils are produced more rapidly than usual when the body is fighting a bacterial infection and the number of neutrophils in the blood can rise quickly and dramatically. Because they are so short-lived, the neutrophil count also drops quickly when the infection is brought under control. Thus, because neutrophils are the most abundant WBC and because they are so short-lived, the neutrophil count is a very good indicator for determining whether a patient has an infection.
The other two types of granulocytes are called basophils and eosinophils. The function of basophils is to escalate the body’s inflammatory reaction and have been implicated in allergies. Their granules contain an anticoagulant called heparin and special compounds called histamines, which cause blood vessels to dilate (become wider). Eosinophils fight multicellular parasites such as hookworms and tapeworms and so their granules contain enzymes that are particularly effective against these organisms.
Agranulocytes are WBCs that do not show granules when viewed under a microscope, and they come in two subtypes. (Hence, they are "agranular.")
Monocytes account for 2-10 percent of WBCs, making them the third-most abundant WBC after neutrophils and lymphocytes. Monocytes circulate in the bloodstream and then move into other tissues when an infection is detected. When they arrive at the infection site, they transform into another type of cell, usually a macrophage, and begin to engulf and digest bacteria, dead or dying cells, and other infectious material. Some monocytes migrate into bones where they transform into special bone cells called osteoclasts, whose function is to degrade calcified parts of the bone. This is important in the bone remodeling process by which the bone changes its shape in response to stress and exercise, but it also happens in certain bone diseases, such as osteoporosis.
Lymphocytes are the second most abundant type of WBCs, accounting for 20-50% of the WBC count. They are subdivided into B-lymphocytes and T-lymphocytes (aka, B-cells and T-cells), each of which is yet further divided into various subtypes. The role of B-cells is to produce antibodies, which attach to agents that the body’s immune system considers foreign. This helps to defend the body against infection. However, it can also lead to problems when antibodies are made against an individual’s own tissue, or against something else that benefits the individual, such as a tissue or organ transplant. T-cells are involved in cell-mediated immunity, fighting against infections from viruses and bacteria, and may help the body attack cancer.
Rejection of blood
A good example of B-cells making antibodies against foreign tissue is the reaction of blood transfusion recipients to donor blood of a different type. Prior to the late 19th century, nobody had a clue as to why a transfusion would succeed or fail because, as noted earlier, they assumed all blood to be identical. But with improvements in the microscope and in the dyes used to stain cells, this view started to change. In the years prior to Landsteiner’s discovery, pathologists could see that RBCs were not always exactly the same. Sometimes RBCs would look slightly bigger or smaller than usual, or would stain darker or lighter. They wondered whether these observable differences might have something to do transfusion outcomes, but they had not devised a way to test the idea.
Even with the hand-cranked centrifuges available in those days, Landsteiner could separate the cells from the liquid in blood fairly easily. That produced plasma, and after separating it from the cells, Landsteiner could keep the cells alive for short periods by suspending them in saline (salt water). He found that mixing cells with plasma would cause clotting, even when the plasma and cells were from the same volunteer. However, if a blood sample was left to clot prior to centrifugation, the resulting liquid extract did not cause clotting of fresh RBCs from the same volunteer. That’s because the liquid extract was serum; it lacked clotting factors because the clotting factors had been consumed before Landsteiner had separated out the liquid.
Landsteiner did not know about the clotting factors, but he could deduce that serum and plasma must be different, and this led him to ask a question: What would happen if serum from one volunteer were mixed with saline-suspended RBCs from other volunteers? As happens often in science, a simple question would prove to be the key, since it was a question that Landsteiner was equipped to answer. He needed only to take blood from himself and five other volunteers, extract several samples of serum and blood cells, mix the samples in various combinations, and observe the mixtures both with the naked eye and a microscope.
Certain donor serum samples mixed with blood cells from other donors resulted in no hemagglutination. However, serum from those same samples would agglutinate cells from the other donors. Landsteiner also found that some of the volunteer samples could be mixed with one another with no agglutination. (See Figure 6 for a visual chart of the results.) The presence or absence of hemagglutination sorted the six subjects into three categories that Landsteiner called blood groups.
Further testing showed that one group differed more from the other two, more than those two differed from one another. When exposed to blood cells from the two other groups together, serum from one group of donors would form very big clumps, whereas serum from the other two groups would form only small clumps when exposed to cells from the two other groups together. To explain the results, Landsteiner reasoned that the cells from the volunteers differed in chemical agents present on the cell's surface. He called these chemical agents "haptens." Today they are called antigens and we know that they’re present not just on RBCs, but also on the membranes of all our cells.
Landsteiner named the smaller clumping groups A and B and reasoned that the serum from each must be reacting to the presence of just one hapten that was not its own. When it came to the third group, however, which he first called C (later changed to O), Landsteiner reasoned that their serum must be reacting to the presence of two foreign haptens, thereby resulting in stronger hemagglutination. Group C donors, he suggested, must have no haptens on their RBCs. Thus, when serum from a type A donor is mixed with B and C cells, it reacts only to the cells of type B donors, whose RBCs have a type B hapten. Similarly, he said that group B serum reacted to cells from group A donors, because those cells possessed type A haptens. In contrast, he proposed, for type C individuals haptens A and B were both foreign, so their sera reacted more strongly.
Using multiple samples from blood drawn over several weeks from all six volunteers, Landsteiner repeated the experiments and found that grouping pattern always came out the same. Once that was certain, he published the findings with his proposal that the success or failure of transfusion depended on the A/B haptens, but a study of just six subjects did not provide enough confidence for anyone to attempt a transfusion in humans based on the experimental results. More data were needed, and Landsteiner knew it.
He had two of his trainees/assistants recruit twenty-two additional blood donors and repeat the process that they had used on the original six. It’s fortunate that they did, because in 1902 analysis of the results from the expanded study led the team to define a fourth blood type. They called it "AB" since it consisted of people whose RBCs had both antigens; their cells would agglutinate if mixed with any serum but their own type, but their serum would not cause agglutination in cells of any type (Figure 7).
After a few more years of testing samples from an increasing number of volunteers, Landsteiner and a growing association of colleagues were confident that all humans must fit into the A, B, O, or AB group, and this is what led to the successful 1907 transfusion at Mount Sinai in New York. Leading the Mount Sinai team was Landsteiner’s colleague, Reuben Ottenberg. Like Landsteiner, Ottenberg hailed from Vienna and both were at the beginning of careers that would last a half-century and enable medicine and surgery to advance more in a few decades than it had in all the previous ages of human civilization.
Antibodies: The key to blood compatibility
As for why the various sera reacted this way to Landsteiner’s "haptens," scientists eventually worked out that the reason was antibodies. Also known as immunoglobulins, antibodies are proteins produced by a type of B-lymphocyte called plasma cells. While some antibodies circulate attached to the surface of the cells that make them, other antibodies detach and float freely in the blood. Thus, they are present in serum.
A person in blood group A does not make antibodies against antigen A, but they do make antibodies against antigen B, and thus against RBCs from group B donors. With blood group B, the scenario is the opposite; they make antibodies against antigen A and thus against RBCs from group A donors. People in blood group O (what Landsteiner called group C) make antibodies against both A and B antigens, because both antigens are foreign to them, while people in group AB do not make antibodies against either antigen (Figure 8).
Landsteiner’s discovery of the ABO groups eliminated the Russian roulette quality that had characterized blood transfusion over the centuries – up to and including Halsted’s courageous but lucky experience in transfusing his hemorrhaging sister. After Ottenberg’s transfusion milestone in 1907, surgeons knew that they could replace lost blood without killing the blood recipient. This allowed them to develop a multitude of new operations that otherwise would have been impossible. Medicine changed profoundly and Landsteiner would be awarded the Nobel Prize in Physiology and Medicine in 1930.
This was not the end of the story, however, either for Landsteiner or his colleague Ottenberg. For the bulk of the population, ABO grouping alone worked well enough, but by the 1930s the understanding of blood was growing still more complex. One reason for this was another surface antigen on RBCs that also could come into play when blood mixed. It’s called the Rh factor and both Landsteiner and Ottenberg would be central to its discovery. Another reason was that scientists would also come to understand the genetic basis underlying the existence of the RBC antigens.
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