RH Negative Blood: Monkey Blood, Blue Blood, Pure Blood, True Blood, or Ordinary Blood? (2023)

RH Negative Blood: Monkey Blood, Blue Blood, Pure Blood, True Blood, or Ordinary Blood?

For simplicity, RhD is used for Rh positive blood and Rhd is used for Rh negative blood in this series of blood articles. Readers are reminded to use RH Negative Blood Dictionary if needed.

In the beginning long before individual blood types (ABO) were identified, humans believed everyone had the same kind of blood. After all, you can’t look at blood and fathom out differences other than cherry red color from spurting arteries and a darker hue from veins. There are old documents that describe crude attempts at blood transfusion because of this observation.

Karl Landsteiner, an Austrian scientist by birth, published a short note in 1900 in medical circles on the agglutination (clumping) of red blood cells he’d observed of some people by the serum (the clear fluid that separates naturally from red blood cells when left standing in blood tubes) of others. He observed this in the lab as he worked. We now call the clumping he noticed antibody action. Landsteiner would have never published this note unless he’d done hundreds of tests first in the lab. In 1901 Landsteiner wrote another article, a landmark and revolutionary report, and in it he identified three separate blood groups he discovered in humans—types A, B, and O. In this paper, he published the steps he used, so that other interested scientists could repeat his results, and they did. TypeAB was discovered a year later in 1902 by Landsteiner lab colleagues, Alfred von Decastello and Andriano Sturli.

Landsteiner detailed the background of his amazing discovery in his 1930 Nobel lecture, and it was reprinted in Science 1931.(1) His background was in chemistry, but he notes the normal, ordinary chemical methods didn’t help him establish results, but the use of serological reagents did. These reagents opened the door to protein chemistry in plants and animals. He observed that proteins in plants and animals were different, and each species had individual and specific proteins. He describes how observing the protein reactions made him wonder if human blood contained the same individual differences. To find the answer, he’d have to make serological reagents for human blood testing like he did for plants and animals. He’d completed experiments on all types of animals: goats, rabbits, guinea pigs, cattle, sheep, chickens, chimpanzees, and Rhesus monkeys.

In Landsteiner’s time period the clumping, that many had observed besides him, was assumed to be caused by diseased blood. However, he believed it was due to individual differences or factors within the blood and not caused by disease. He called those factors, X Factors in his writings. To prove his theory that clumping wasn’t caused by disease, he chose healthy men as subjects—himself and five other men in his lab. (2)

It’s important to understand the procedure he used because he was pioneering all the blood work that followed and teaching his approach to his many assistants.

This was the procedure as related to Zimmerman by Levine and Wiener: (3) He drew blood in tubes and let the specimen stand until it separated into two components. The heavier red blood cells turned into a clot at the bottom of the tube and the other component a clear serum floated to the top. We now call the clear serum, plasma. Plasma helps the red blood cells flow throughout the bloodstream.

Next, Landsteiner mixed one man’s red blood cells with another man’s clear serum. He observed and documented the interactions. From Owen 2000 (2) Lansteiner listed his name and the other five colleagues on a chart with the results. From it, I learned Landsteiner had Type O blood.

Landsteiner observed two antagonistic reactions and concluded a factor or antigen was at play. He named the antigens (blood proteins) A and B, alphabetically. The third component he isolated he named zero, but wrote it as O, and it got misinterpreted as alphabet O. He used zero because that blood group didn’t react to the antigens A or B.(3) Zimmerman. He called antibodies that destroyed A antigens anti-A antibody and antibodies that destroyed B antigens anti-B antibodies. He found antibodies in a small fraction of the clear serum that we now call gamma globulins. Now, picture a blood tube of separated red cells from the clear serum and read Landsteiner’s Rules listed below:

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Blood group A red cells carry the A antigen and A serum (clear fluid) contains anti-B antibody.

Blood group B red cells carry the B antigen and B serum (clear fluid) contains anti-A antibody.

Blood group O red cells carry no antigens and O serum (clear fluid) contains both anti-A and anti-B antibodies.

Blood groupAB red cells carry the A and B antigens and AB serum (clear fluid) has no antibodies.

A current rendering of Lansteiner’s Rules from Owens 2000 (2):

A – B – = AB

aa B – = B

A – bb = A

aa bb = O

In this manner, by testing large numbers of people for blood group the proportions of blood types emerged and provided a way to make transfusions safe. However, the truth was, the majority of his peers ignored his revolutionary discovery in 1901 because most didn’t believe it.

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Disappointed, Landsteiner left blood group work and continued other research projects until World War I intruded. Phillip Levine (a doctor and Landsteiner research associate) wrote that Landsteiner lost his lab facilities, faced personal deprivations and frustrations, as well as starvation during that bleak time. (3) Landsteiner came to America in 1923 and resumed his blood group work with Doctor Phillip Levine at the Rockefeller Institute.

In America twenty-two-years later, he again pursued the antigens on red blood cells. He believed there were more antigens than just A and B (ones he discovered back in 1901) in human blood. To find more antigens, he settled on using rabbits and not humans for his tests.

Note to Readers: Landsteiner is the genius and the leader in the lab. He had the ideas, and the devoted lab assistants did the tedious work to prove them, just as he once did. Also, back then he wasn’t looking for antigens, he called them X factors. If you read the old studies the word “factor” is used instead of the more current term “antigen.” For continuity, I use Landsteiner’s name in procedures being done by his doctor assistants.

Landsteiner’s goal was to make serum markers or reagents for testing to find new blood factors. Here’s the procedure he used described by Wiener to Zimmerman 1973: He injected (.5cc or 10 drops) group A human red blood cells into live rabbits. This immunized the rabbit to the human A antigen, and forced the rabbits to make anti-A antibody. This process also immunized the rabbit against weaker antigens or factors that might also be on the red blood cell. Later, he’d withdraw the blood from the rabbit, isolated the anti-body containing serum, and then mixed it with blood group A human red blood cells (in the lab). Like a magnet the rabbit’s anti-A antibody would attach to the human red blood cells. Then the group A cells with anti-A antibody attached could be removed from the rabbit’s serum. Now, with the anti-A gone, the rabbit’s serum could be challenged with another batch of human group A red cells. If the serum reacted with some but not all, then clearly those that did react carried some unknown factor, the X Factor.

As you can see, the work wasn’t difficult, but it had to be slow and tedious. Using the “test serums” developed from the rabbit work, Lansteiner and Levine, announced in 1927 the discovery of three new human red blood cell factors, which they named M, N, and P. (4) This discovery helped Landsteiner move closer to his goal, which was subdividing mankind into groups and individualities based on blood types. After this joint discovery, Levine left Landsteiner’s lab because his Rockefeller contract ended.

Note to Readers: Levine and Wiener were Landsteiner disciples and they started a slugfest over Rh discovery that muddied the waters for decades. Both men worked closely with him, and when Levine left the lab he had to agree to Landsteiner’s rule to abandon all blood work research. This is a gentlemen’s agreement, not a signed contract. Levine honored his promise for almost ten years until called in to consult on a horrific transfusion case at Bellevue hospital in 1937. For more on this blood feud, see the next article in this series titled Blood Feud.

As Landsteiner’s blood work progressed, man’s close biological kinship to animals was being established. Researchers needed lab animals and monkeys were expensive. Writing in Science 1931,(1) he wrote that “isoagglutionation isn’t limited to human blood, it did occur in many animals especially apes, but results were never as consistent as seen in man. Only the highest anthropoid apes—their blood cells being indeed distinguishable from those of man, but have blood group characteristics which are shown to coincide with those of man.” In fact, his blood research proved that the higher up an ape was on the phylogenetic tree the closer its blood profile became to man.

By 1935 Alexander Wiener worked in the serological laboratory at the chief medical examiner’s office in New York City. Wiener was interested in studying the evolution of agglutinogens M and N in apes and monkeys. (5) Wiener has a free and vast amount of published papers on his ape/ monkey experiments for those interested online. Wiener, working alone, used the same techniques Levine and Landsteiner used to identify the M, N, and P factors, Wiener took anti-M antibodies and anti-N antibodies and proved beyond a shadow of doubt that monkeys had the same M factor in their blood as humans do.(3) This is the same M factor shared by RhD and once Rhd blood was discovered, with that blood group as well. This should silence, I hope, the “we have pure blood” groups. To really drive the point home, check out this online article, read the tables in the mid section.

Landsteiner suggests to Wiener they next make a new “test serum” injecting cells from related test animals to see if the resulting anti-sera displayed human characteristics. This is the first time two animals are used in the same test. They chose to inject rhesus monkey blood into guinea pigs and rabbits because the rhesus monkey was higher up the phylogenetic tree than sheep. The antisera from the rabbits caused a reaction.

This was the successful procedure: They injected rabbits with .5cc (10 drops) of rhesus blood and the rabbit made anti M serum. They strained out the M cells with the anti-M antibodies stuck to them and discarded it. They took the remaining serum, freed from the anti-M antibody, and mixed it with a new batch of type M human. This new rabbit serum hit a home run by reacting with most but not all human blood. The new “rabbit test serum” had detected an unidentified antigen. It goes without saying here the scientists were testing human blood for “rhesus-like” factors and found one.

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Note to Readers: What is being used here is rabbit serum (clear fluid) infused with antibodies against rhesus monkey blood, or to put it another way they’re using anti-rhesus serum the rabbit made in self defense. Rhesus blood isn’t used directly, it’s rabbit serum filled with antibodies against rhesus blood. This is why Landsteiner is careful to call it “rhesus like” in their published papers. Later, they got the same test result using guinea pigs. They injected the rabbit with 10 drops of guinea pig blood and used the same procedure described above. “Guinea like” produced the exact result. While both rhesus like and guinea like produced the same test result, the rhesus like reaction was stronger.

Next, using the same procedures mentioned earlier the men removed the known antigens, A, B, M, N, P and retested the new “rabbit test serum.” Again the anti-rhesus serum and the anti-guinea serum clumped the majority (85%), but not the minority 15%.(6) Using this method, they proved that human red blood cells carried a new and unidentified factor or antigen. The antigen-antibody reaction they discovered was weak in comparison to A&B antigen reactions. They calculated there were only ten thousand sites for this new antigen on a red blood cell as compared to the millions of sites for A & B antigens.(3)

They had no idea this discovery might connect to a human disease at the time. Don’t forget: Landsteiner, Levine, and Wiener don’t see or treat patients, they’re lab workers.

Landsteiner’s & Wiener’s goal was to find all the antigens on red blood cells to make transfusions safer, and enhance the use of their test serums for use in blood tests to solve legal paternity disputes. While they were impressed with the new test reagent, the two weren’t impressed enough to publish a paper on their discovery until January 1940. (6) At that time publishing was their “life blood,” so they definitely weren’t impressed.

Note to Readers: Landsteiner had retired in 1939, but Rockefeller let him use a lab until his death in 1943. He died in the lab.

Despite retirement in 1939 Landsteiner still searched for his X factors, and Wiener looked for human blood similarities in his ape work. Of the two men, Wiener had more familiarity with apes and monkeys. Wiener named the discovery the Rh factor in honor of the Rhesus blood donor. The Rh factor gave Landsteiner et all, one more tool to use in establishing blood profiles to characterize human blood. For years, both Landsteiner and Wiener referred to their discovery as the monkey-rabbit-human experiment.(7)

Landsteiner wrote in his Rockefeller report April 13, 1940: “In collaboration with Wiener it has now been found that such {rabbit anti-rhesus sera} may also contain antibodies reacting with an unknown antigen, tentatively designated as Rh, present in human blood in about 80% of individuals.” (3)

The poor rabbit did all the work and doesn’t get to share in the fame and glory, does it? What so many people tend to forget is that a new and important blood test was discovered.

In conclusion, Landsteiner, in his own words, called the name “Rh” tentative. (1) He was well aware of the evolution corollary and wanted to avoid his blood test being connected to it for obvious reasons still in existance today. I hope the reader understands it could have been called X factor or KL factor just as easily as it was called Rh factor. Wiener had a “gift of words” and he understood that naming something gave it importance. In fact, the name was changed in 1982. (See Dictionary for explanation-LW) and their discovery Rh factor is now called LW after them. I will explain later that when non-scientific people use Rh factor, most are referring to the tests done by Landsteiner and Wiener because of the rhesus monkey, but doctors are referring to a another blood system that absolutely does not have one thing in common with a rhesus monkey! This is another reason you’re confused. I will explain why in the next article: Blood Feud.

I think it’s clear that Landsteiner considered changing the name Rh factor because the monkey connection was misleading, but died before doing so. But please note even he called it rabbit anti-rhesus sera in his annual report. For the record, I hope I’ve changed some minds about the so-called proof that Rhd blood types have nothing in common with monkeys. Wiener proved 78 years ago, that humans shared M antigens with monkeys. Both RhD and Rhd. Owen states clearly that both RhD and Rhd tested positive using the guinea pig antibody test described above.

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Finally, scientists report that no matter how potent they make the dose human antibodies from all blood types do not clump Rhesus monkey cells.(2) I think it’s time to put the monkey business myths to bed, don’t you?


1. Landsteiner, K. 1931 Individual differences in human blood blood. Science 73:403-409.

2.Owen, R. 2000 Karl Landsteiner and the First Human Marker Locus. Genetics 155:995-998.

3.Zimmerman, D 1973 Rh: The Intimate History of a Disease and Its Conquest. New York, Macmillan Publishing Co., Inc.

4. Landsteiner, K., Levine, P. A new agglutinable factor differentiating individual human bloods. Proc Soc Exp Biol Med. 1927: 24:600-602

5. Schwarz, H. 2003 Historical Review: Karl Landsteiner and His Major Contributions to Haematology. British Journal of Haematology 121:556-565.

6. Landsteiner, K and A.S. Wiener, 1940 An agglutinable factor in human blood recognized by immune sera for Rhesus blood. Proc. Soc. Exp. Biol. Med. NY 48 223-224.

7. Wiener, A. 1969 Solutions of the Rh-Hr Nomenclature Problem. New York Journal of Experimental Medicine 69:2915-2935.


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