During breakfast the next morning, Dr. Kirk suggested that we move our discussion into Holmes’ laboratory. The three of us grabbed our cups and trooped, once again, into the perpetually cluttered laboratory.

“I want to run through several blood testing procedures,” explained Kirk over his shoulder, “to demonstrate the limitations and differences between the testing methods used in this investigation.”

Returning to the bench we had cleared earlier, Dr. Kirk unpacked a variety of containers, spray bottles, pipettes, two vials and a square of white cloth from his leather bag.

“Strictly speaking,” began Dr. Kirk, “there is no such thing as a test for blood. Because blood is a highly complex and somewhat variable mixture, any test must be directed toward identifying one or more of its regular constituents. The standard blood tests used by the police in the Sheppard investigation were sensitive but nonspecific; that is, they were able to detect the presence of hemoglobin, but they could not prove its source or identity. Although the hemoglobin of blood varies between species, all varieties of hemoglobin are detected by the same tests.”  

Setting the two vials (one labeled X and the other Y) on the laboratory bench, Dr. Kirk explained that each vial contained blood – one from canine and the other from human sources — but he did not tell us which vial contained blood from which species.

Dr. Kirk proceeded to use two separate pipettes to remove blood from each vial and placed four drops of blood from each onto a cloth square. He folded his arms with a look of satisfaction while Holmes and I stared expectantly at two rows of four blood stains on a piece of cloth.

“Now we begin the experiment,” Kirk declared.

First, he tested one spot from each row with a benzidine reaction. We saw that both tested spots developed the same blue-green color.

Next, he sprayed two spots, one from each row, with luminol. He doused all candles and lanterns in the room, and as we expected, a bluish-white luminescence glowed from each spot.

Demonstrations of the phenolphthalein test and the leuco-Malachite green test gave the same results – both tested blood spots, whether dog or human — reacted in the same manner. 

Dr. Kirk, obviously enjoying his professorial role, removed two smaller, capped bottles from his kit.

“As we discussed earlier, Cleveland investigators found blood throughout the Sheppard house, but nonspecific tests like those we just conducted cannot distinguish between human and animal blood. When detectives used a precipitin test, they discovered that only five spots of blood were human in origin: two on the basement stairs and three more on the stairs descending from the second floor.”

Holmes and I knew that a German biologist invented the precipitin test in 1901 when he injected protein from a chicken egg into a rabbit and then mixed serum from the rabbit with egg white. As a result, the egg proteins separated from the liquid to form a cloudy substance known as a precipitin or antibody. In the same manner, when rabbits are injected with a small quantity of human blood, they develop anti-human antibodies. Investigators can add a drop of the rabbit anti-human serum to suspect blood, and the blood will agglutinate (clump) or precipitate its protein if it is of human origin. The test is highly sensitive and will work even on small traces of blood.

We watched as Dr. Kirk uncapped the two small bottles, identifying the liquid within as anti-human rabbit serum. He used pipettes to remove blood from vials X and Y, once again, but this time he placed each sample into separate bottles of anti-human rabbit serum, and the results were markedly different.

“As you can see,” said Kirk, stepping back to reveal the two bottles, “blood from the vial marked X has formed a cloudy precipitin, while the other has not. The blood in vial X is human in origin.

Without giving us a chance to comment, Dr. Kirk continued his demonstration.

“The next investigative step was to ascertain, as far as possible, from whom the blood came. We did not have the benefit of DNA profiling back then,[1] but exclusion of a suspected individual was possible in 1954 based on blood grouping.”

Holmes bolted upright at the mention of DNA.

“And what, exactly, is DNA?”

I knew Holmes was loath to admit ignorance on this one blood-related subject, so I took a risk and explained based on my extensive reading of futuristic materials. I tried to be modest.

“My dear fellow, DNA — or deoxyribonucleic acid — is the molecule that carries genetic instructions for the development of all known living organisms. And DNA profiling can help identify individuals based on their unique genetic code, or their DNA.”

 I smiled. Holmes relaxed, and Dr. Kirk continued.

“Forensic serology, as you know, deals with blood and other body fluids such as saliva and semen. It is important not only for narrowing suspicion on the guilty but also in showing a suspect’s innocence.”

Holmes waved his hand in agreement.

“Forgive me for lecturing,” apologized Kirk. “I know you are both well-informed on the subject of blood.”

“Indeed, we are, Dr. Kirk,” Holmes replied with a reassuring smile. “And that is why we find this discussion extremely fascinating. Please continue with every detail.” I nodded in agreement.

“Well then . . . Again, as you know, all members of the human race can be divided into a number of categories by the blood groups alone. Several different kinds of blood group factors are detectable, each of which is independent of the others, and form the basis of several blood grouping systems. The ABO and the Rh systems are used most frequently; the MNS system is less well-known. A person’s blood group never changes throughout life; nor can it be faked or altered in any way.

“When blood is dry, the ABO is far more valuable than any other blood grouping system. It divides the human race into four groups: Group A individuals have “A” antigens in their blood; Group B have “B” antigens; Group AB individuals contain both “A” and “B” antigens, and Group O individuals contain neither. The most common Group O includes a little over 40 percent of all humans. The A group is almost as frequent as O. The B group is found in 10 to 15 percent of the population, and AB is found in only about 5 percent.

“Both the A and B factors are detectable, even in very old, dry blood, but when the blood has been dried, the methods for detecting either factor are quite different from that used for fresh blood, and far more difficult. In fluid blood samples, of course, blood type is determined according to which antibodies cause red cells to agglutinate. In dried bloodstains red cells have ruptured, so direct agglutination tests are no longer feasible.

“Of all the persons who were concerned with the blood in the Sheppard case, only Mary Cowan, Coroner Gerber’s laboratory analyst, showed an understanding of the techniques of grouping dry blood.

“Miss Cowan found that Marilyn Sheppard belonged to the most common group O, and she demonstrated the presence of the extremely common M factor in Marilyn’s blood.

“Dr. Sam’s blood was never grouped by anyone prior to the trial, but I obtained blood from him in the Cleveland jail and determined that his blood was group A.”

“And what did you find when typing blood at the crime scene?” I asked with a glance at Holmes.

Dr. Kirk paused a moment to remove his glasses once again. He took a minute to close his eyes and rub the bridge of his nose. When he opened his eyes a moment later, he looked exultant. “I found important differences in the blood deposited in the murder room . . . somebody, other than Marilyn or Sam, shed blood in that room the night of the murder.” 

This statement took my breath away, and I could not speak. Holmes remained impassive as he queried Dr. Kirk in a cool tone.

“You are suggesting, I assume, that the murderer incurred an open and bleeding wound during the course of his or her attack.”

“More than merely suggesting, Mr. Holmes, I am stating this as fact, based on the story told by the blood itself!”

Holmes stared at Kirk with narrowed eyes, considering the validity of his statement I am certain. I was more than ready to depart the frigid laboratory, but Holmes suddenly turned and dragged three chairs from behind a large screen. Flapping away years of dust with an old cloth, he took a seat and invited us to join him as he crossed his legs and calmly continued to question Dr. Kirk.

“Dr. Kirk, exactly what kinds of differences did you find in the blood deposited in the murder room?”

“Significant differences in agglutination and solubility,” answered Kirk. “Blood removed from the mattress, unquestionably the blood of the victim, was grouped and found to be devoid of A and B factors, thus ABO typing revealed it was Group O.”

“So, you agreed with Mary Cowan on that point,” I inserted.

“Yes. Furthermore, the blood from the mattress was readily soluble in distilled water, and agglutination after treatment with anti-serum and cells was immediate as compared with controls of anti-serum and cells alone. I then tested blood removed from a 1/2-inch-in-diameter spot deposited on the wardrobe door, and it showed the same results. Agglutination was rapid – less than one minute — and solubility of the blood was excellent.”

“Most likely from the victim as well,” Holmes said.

“Right again. I then tested a very large, 1-inch-diameter, unique spot of blood found on the wardrobe door. Grouping of this blood was performed simultaneously with the same sera and cells and in an identical manner as carried out with the previously mentioned samples. But this time, several differences were immediately apparent.”

“Ah ha!” Holmes exclaimed. “We know from our own work that all blood shed by a person at one time will have the same composition. And if two persons shed blood at the same time, any differences between the two deposits will prove their separate origins.”

“Yes, now the blood from the very large spot was significantly less soluble than the smaller spot on the same door and control samples from the mattress. In running the agglutination tests on the uniquely large spot, in every instance and with tests for both A and B factors, agglutination was much slower – taking up to 20 minutes — and less certain than the controls. Delayed agglutination clearly indicated that the large blood spot also was Group O, but its behavior was so different as to be striking.

“It must be recognized that storage of blood on the mattress was not identical with storage on the wardrobe door, but Marilyn’s blood from both sources gave the same reactions, thus demonstrating that storage conditions could not explain the marked differences between the two spots on the door. I consider these differences to constitute confirmatory evidence that the blood of the 1-inch-diameter spot had a different individual origin from most of the blood in the bedroom.

“I further subjected samples of blood from the murder room to paper electrophoresis – where a solution of dry blood is allowed to flow down a wet filter paper curtain as a direct current flows across the curtain. Proteins separate and follow distinct paths, creating patterns that differ with blood from different people, or when the length of storage time is greatly different. Results showed that the pattern created by the unique blood spot was markedly different from the pattern associated with the sample of Marilyn’s blood deposited at the same time from the same wardrobe door.”

Holmes was about to comment further, but I had new information and was impatient to sally forth.

“Here’s where things get really interesting,” I said, grabbing my notes once again. “In 1997 Dr. Mohammad Tahir, an expert DNA forensic scientist, tested bone, tooth, and fingernail samples from the exhumed body of Dr. Sam Sheppard (Dr. Sheppard died in 1970) and several strands of Mrs. Sheppard’s hair. He established their DNA profiles and tested them against 1954 evidence obtained from the coroner’s office, including the blood stain on the bed sheet, two vaginal swabs, the one blood stain on Sam Sheppard’s trousers, a wood chip from the basement stairs, a blood stain found in the porch and the largest (unique) blood spot on the wardrobe door.”

Taking a breath, I added, “Dr. Tahir also had a saliva sample from Richard Eberling – the Sheppard’s window washer in the fifties, later imprisoned for murdering his benefactress. Richard Eberling did odd jobs for Mrs. Sheppard and was familiar with the basement entrance through the lakeside of the house. He told police – without their asking – that his blood had dripped through the house when he cut his finger while changing screens in the bedroom.”

“And that is what forensic psychologists call a ‘gratuitous offering,’” inserted Dr. Kirk. “When a suspect volunteers an explanation without having been asked, it usually indicates an effort to cover something up. Be that as it may, our work is based upon the indisputable physical evidence connected with this case.”

“Indeed, but Dr. Tahir’s DNA testing confirmed your conclusion, Dr. Kirk, that a third person was in the Sheppard home the night Marilyn Sheppard was killed. His DNA testing determined that the bloodstains found on the wood chip from the basement stairs, the porch and – most significantly — the one blood stain on Sheppard’s trousers contained DNA that did not match Marilyn’s nor Sam’s DNA.’

“Most interesting,” said Holmes. “That finding can most logically be explained if the murderer had a fresh wound that dripped blood onto the unconscious Sam Sheppard’s trousers.”

I paused to savor Holmes’ engagement, yet worried about his completely unreadable expression as I referred once again to my notes.

“Although no sperm had been reported in 1954, Tahir examined vaginal swabs from the victim’s autopsy, and he found two sperm heads. Through DNA testing, he excluded Dr. Sam Sheppard as a donor of the DNA found in that sperm! He wrote that Richard Eberling ‘could not be excluded’ as a DNA contributor to the sperm fraction, as well as to the blood on the stairs and the stain on Sam’s trousers.” 

Dr. Kirk appeared cautiously elated with this new information and responded as the exacting forensic scientist he was.

“Discovery of sperm not belonging to Sam Sheppard definitely confirms that this murder began as a sexual attack and supports Sheppard’s story of an intruder,” he said. “However, although Richard Eberling must be considered a prime suspect, thousands of other men would, likewise, not be excluded from that DNA profile.”

“Including, I assume, Mayor Spencer Houk,” said Holmes with a nod, “but what did Dr. Tahir’s testing reveal about the unique blood spot on the wardrobe door — the one showing slow agglutination?”

          “Dr. Tahir did test that spot and reported that it contained a mixture of DNA, and I quote, ‘consistent with having originated from Marilyn Sheppard and Richard Eberling.’”

          “Ahh, but my dear Watson,” purred Holmes, “We know from Dr. Kirk’s earlier testing, that the very large blood spot on the wardrobe door was type O, and the medical records I have read indicated Richard Eberling’s blood to be type A.”

          Admittedly, I was a bit taken aback with that unexpected comment, but I held a finger up in protest as I pulled out my copy of A Mockery of Justice and turned to a well-marked page.

          “There seems to be some confusion on that point Holmes. According to the investigation carried out for this book, Mr. Eberling maintained his blood type was O, but later records indicated type A.”

“Let me point out, however,” jumped in Kirk, “the results of DNA testing conducted by an expert in 1997 simply compound what we already know. While there may be some confusion regarding Eberling, we know for certain that Dr. Sam Sheppard was of blood type A and had no open wounds. 

“Furthermore, the spots of human blood found outside of the murder room certainly did not come from a blood-dripping weapon as ascertained by the prosecution. My experiments proved conclusively that no weapon, however bloody, will continuously drip blood.

“No. Marilyn fought for her life, and she bit down hard on the person who killed her. Be assured, whoever murdered Marilyn Sheppard beat her unmercifully with his left hand and had a nasty, bleeding wound somewhere on his or her hand or arm.”

Holmes began pacing up and down the aisle between laboratory benches, hands clasped behind his back, obviously deep in thought. I was soon to be the focus of his famously deductive reasoning.

“Watson, as I remember your notes on the 2000 civil trial, the State’s DNA analyst disagreed with Dr. Tahir’s conclusions. In reviewing Dr. Tahir’s DNA tests on the unique blood spot and that one blood spot on Dr. Sheppard’s trousers, the State analyst testified that the DNA could not be matched to Mrs. Sheppard, Dr. Sheppard nor Richard Eberling.”

“Yes,” I replied. “The State concluded that this indicated contamination and sought to disregard all DNA evidence.”

“As expected,” smiled Holmes enigmatically. “However, what if that conclusion – rather than indicating contamination – simply confirmed the third alternative?”

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[1] DNA profiling was developed in 1985 by scientist Sir Alec Jeffreys in Great Britain; positive identification of an individual from blood has yet to be achieved.