Why I cannot find dendritic cells in blood smear?

According to many sources including Wikipedia, there are haematopoietic stem cell derived dendritic cells in the blood.

  • figure 1 - haematopoietic cell lines - ref


  • figure 2 - blood smear cell lines - ref

Why is that?





Further, from Kuby's Immunology:



When I was young, I was wondering why the size of chronic lymphocytic leukemia cells described in Western countries is always smaller than that of our Japanese patients. Do we have a different disease? But, I found later that this difference is caused by the methods how blood smears are dried [1].

How to dry blood smears?

After blood smears are made on microscope slides, they are left and dried naturally without using a fan before staining (natural-air drying), or they are dried immediately in an airstream by blowing air onto the smears (forced-air drying). As far as I know, in most or many hospitals in Western countries, they use the natural-air drying method, although the forced-air drying method is sometimes recommended in the English literature. In all the hospitals in Japan, blood smears are dried immediately by a fan. Natural-air drying is not done routinely. The natural-air drying method causes the smears to dry slowly, and cells shrink, particularly when there is high humidity. But, I cannot find any articles to describe how cells shrink on different humidity or on different temperature.

According to many pictures presented from Western countries, it is sometimes difficult to see fine intracellular structures on naturally dried smears. The forced-air drying method, on the other hand, causes the smears to dry more rapidly, and cells shrink less. It is then easier to see fine intracellular structures. However, it is difficult to see hair-like surface projections on hairy cells by forced-air drying, making the diagnosis of hairy cell leukemia (HCL) difficult. By photos, I will show a morphological difference in HCL cells made by these two methods, and discuss advantages and disadvantages of these methods.

Example 1: HCL- HCL is characterized by the cells having hairlike surface projections along the cellular outlines. Five different types of HCL have been described (2), and they differ clinically, morphologically, phenotypically, and/or genetically. Fig. 1 shows HCL cells of a patient, who was diagnosed to have splenic diffuse red pulp small B-cell lymphoma by splenectomy. The blood smear made by forced-air drying (Figure 1A) showed a large agranular lymphocyte, or sunny-side up lymphocyte, with broad agranular cytoplasm and unclear hair-like projections. A smear naturally dried (Figure 1B) showed a smaller-sized cell with clear hairy projections on the cell surface, characteristic of HCL. If we did not make a naturally dried smear, we could not make the diagnosis of HCL, because it is usually difficult to see hair-like projections on the slides made by forced-air drying, except for on the head of the smear, where the cells are dense and dry slowly.

Figure 1A: Peripheral blood smears of a patient with splenic diffuse red pulp small B-cell lymphoma. In this a blood smear was made by forced-air drying.

Figure 1B: By Natural Drying.

Until about 20 years ago, I did not know that hair-like projections do appear when naturally dried smears are made, but not when forced-air dried slides are made. A unique, noteworthy variant of HCL, called HCL Japanese variant, was proposed some 30 years ago by Japanese investigators, but hair-like projections were not found in blood smears of their article (3), probably because blood smears were made by forced-air drying. When large lymphocytes lacking azurophilic granules in the cytoplasm are found on the smears made by forced-air drying, I strongly recommend making naturally dried smears in addition to make forced-air dried smears in Japan. The incidence of HCL is lower in Japan than in Western countries, and this may be partly explained by the method to prepare blood smears.

Example 2: large granular lymphocyte leukemia (LGLL): Large granular lymphocytes (LGL) are large lymphocytes with azurophilic granules in their cytoplasm. Normal LGL are either NK cells or T cells, and LGLL includes three different disorders T-cell LGLL (T-LGLL), chronic lymphoproliferative disorder of NK cells, and aggressive NK-cell leukemia. T-LGLL is most common, and exhibits indolent clinical course (4). I want to focus whether normal LGL and T-LGLL cells are really large in size. What is the definition of large lymphocytes? The definition differs remarkably among investigators. The FAB classification defines B lymphocytes large when they are more than twice the size of RBC (5). Others define lymphocytes large simply by cell diameter (6,7). When the definition of FAB classification is applied to T-LGLL cells obtained from our eight patients, they were not large when blood smears were made by forced-air drying. When smears were made by natural-air drying in our three patients and three normal individuals, the size of RBC did not change significantly, but that of T-LGLL cells and normal LGL became smaller than the smears made by forced-air dying. Accordingly, the ratio of T-LGLL cells to RBC became further lower (manuscript, in preparation).

Understanding mRNA

You‘re given the shot in the arm. The cells within the vicinity absorb the mRNA encapsulated in an liposome wrapper known as transfection.

The infected cell machinery uses the mRNA, like it does with any internally generated mRNA, to manufacture the so-called protein spike that is the hallmark of the SARS Cov2 virus.

The cell then exports the protein spike to its cell wall exposing it to the outside world.

Immune cells spot the foreign body and mount a response to neutralize it by generating antibodies to attach to it.

Once attached, the antibodies have marked the cell for destruction by killer T-cells and the immune system in doing so learns to neutralize the threat throughout the body.

The mRNA in one or more liposomes goes into one cell. The commandeered cell makes lots of spike proteins. The immune system detects foreign spike protein, cleans up the mess, and the cell dies.

One cell, one liposome, lots of spike proteins. Not every cell in the body is affected.

So 500 liposomes transfect 500 cells. Because the dose is in the muscle tissue most of the transfections occur at the point of the injection. Some cells even get more than one liposome attached. So 500 liposomes might only get into 450 cells, for example.

Other remote cells are indirectly affected -but no mRNA from the injection is involved.

There are lymph nodes in your armpit. A help! call goes out from the immune system's cells that got awakened by the spike protein arrival. That call is answered in part of the immune system that lives in your armpit. It then creates new, additional special immune cells. These guys can get into really complicated biochemical "cascades".

Biochemical cascade, also known as a signaling cascade or signaling pathway, is a series of chemical reactions that occur within a biological cell when initiated by a stimulus.

Vaccine mRNA getting inside the lymph node cells does not really happen. And is not part of the hugely complex stuff in your armpit.

One difference between virus and vaccine I'd like to bring out more.

A virus infects a cell and makes it produce more virus, which infects other cells and so on and so on. The immune system learns about the virus and confers immunity post-infection. Well, if the person survives.

The mRNA infects a cell and makes it produce the spike protein on the Covid virus. And then that's it. The immune system learns about the protein and thereby the virus and confers immunity post-vaccination.

The spike protein does not get into the bloodstream.

Here's a decent text description of how mRNA vaccines work that goes into a bit more detail than others:

I'll also add a bit more explanation to clarify some issues in the thread:

The mRNA vaccines consists of an mRNA molecule encapsulated in a lipid nanoparticle shell. The vaccine gets injected intramuscularly into the arm. While the lipid nanoparticle shell does help it get into cells, it does not get into all different types of cells equally. There is a type of immune cell called dendritic cells (DCs) whose job it is to surveil the body for potential pathogens, take them up into the cell (via endocytosis), break the pathogen up into small fragments (antigens), and present those antigens to other immune cells (like T-cells and B-cells) to active those cells and start the adaptive immune response.

Because of their role in recognizing foreign particles, the DCs are the main type of cell taking up the mRNA vaccines. Inside the DCs, the mRNA causes the cell to produce the spike protein, some of which goes to the surface of the cell and some of which gets cut up and presented on antigen-presenting MHC molecules. The process of taking up the mRNA (likely through specific lipids in the nanoparticle shell) helps activate the DCs, which causes them to migrate to lymph nodes so that they can present their antigens to immature T-cells and B-cells. Interactions between the DCs and these immune cells will activate the T-cells and B-cells to cause them to replicate and spread throughout the body to activate the cellular and humoral arms of the adaptive immune response.

So, it is neither the mRNA nor the spike protein that gets spread throughout the body. Rather, the mRNA likely gets take up by DCs (which is the important part) as well as some cells in the local area of injection and maybe some other cells scattered throughout the body (if some, say, does get into the blood stream). However, it is the uptake of the mRNA by the DCs and their downstream activation of other immune cells that allow the replication of the T- and B-cells and for these immune cells to spread throughout the bloodstream and provide body-wide protection against the SARS-CoV-2 virus.

Another good reference for the science behind mRNA vaccines (though focused on speaking to researchers working in the area):

It would be helpful if you provided a link to where you read this in order for us to clarify.

It would be helpful if you could provide link(s) to the study(s).

Excellent TW, a lot was covered here. I want to say how much I appreciate the rapid responses on the forum it's so helpful. We're not out of the woods yet. I have no background in this material, just a natural interest in science and so I try to master what I can and, particularly, what's hot at the moment. I've always loved microbiology but I wasn't strong mathematically, so I didn't think I'd survive a science career. I taught AP French for 18 years and I'm retired now. I do remember a lot of the basics so I'm not lost. Let's continue.
Picking up where the liposome blanket gets the mRNA into the cytoplasm. Clearly the liposome blanket disintegrates and ultimately becomes part of the cell membrane. This exposes the mRNA to the local ribosomes but also to RNAse then, which goes to work ripping it apart? or can the ribosomes connect with the mRNA while it is still encased? Do any of the adjuvants in the vaccine neutralize the RNAse to maximize spike protein production time?

Are the current mRNA structures self-amplifying?

Also, mRNA is generic then in the sense that it doesn't have to have any genetic connection to your specific DNA, just a single strand of an RNA sequence coding the protein you want? And the poly A tail is also generic as in it works on everyone for all ribosomes?

In a Hopkins/Bloomberg study/report entitled Technologies to Address Global Catastrophic Biological Risks ( sounds awful) on pg. 47 they talk about using cytomegalovirus as a vector for mass inoculation of vaccine material. I'm assuming this would spread via air and breath droplets or could it be engineered for simple skin to skin contact? I can't imagine how they could get exogenetic mRNA to stay active in saliva or breath droplets and I guess it would just enter through epithelial cells. I realize they're working on it and don't have it, it's just fascinating.

One last thing. On pg. 51 of the same report it sounds as if the positive mRNA is replicated by a viral replicase which creates first a neg mRNA copy from which a duplicate positive is mass produced. Is this mRNA copying going on while the ribosomes are making the protein or is this something else? Check out the article when you can. I understand the "ase" suffix indicates an enzyme, is this RNAse they're talking about? I do wish scientific vocab was a bit more consistent. Thanks team.

Oh, is it possible to print this thread? I'm taking notes but I'd love to have a hard copy. I've attached the Bloomberg report.

Multiple Types Of White Blood Cells Made Directly From Embryonic And Adult Stem Cells




The likely most immediate benefit is cells that can be used for safety screening of new drugs, says study leader Igor Slukvin, an assistant professor in the university's Department of Pathology and Laboratory Medicine.

"Toxicity to the blood-forming system is a key limit on drug development, so these cells could be used for safety testing in any drug development," says Slukvin, who performs research at the National Primate Research Center in Madison.



Eventually they found a recipe that would cause the cells to move through a process of progressive specialization into a variety of adult cells. Slukvin's study was published in the Journal of Clinical Investigation.

The result included osteoclasts, cells that play a role in osteoporosis, and eosinophils, which are involved in allergy and asthma. Other adult cells included dendritic and Langerhans cells, which direct other immune cells to attack infections, and neutrophils, the most common type of white blood cell.

"While we now can make almost all types of blood cells from embryonic and adult pluripotent stem cells, the next major challenge is to produce blood stem cells (called hematopoetic stem cells) that might be used in a bone marrow transplant," Slukvin says.

This life-saving procedure can replace the entire blood-forming system in a patient with blood cancer, but more than one-third of patients cannot find a well-matched bone marrow donor and thus risk graft-versus-host disease, a sometimes-fatal attack on the patient by the transferred immune system.

Compatibility problems should disappear if the blood-forming stem cells are based on the patient's own cells, Slukvin says. "Eventually, we want to make therapeutic cells that could be used instead of bone marrow transplants."

In the interim, Slukvin expects the new technique to produce cells that model a variety of medical conditions.

"We can take cells from patients with a disease of the blood system and explore the cause and treatment of that specific disease. We can generate blood cells which are normal or abnormal, and study the mechanisms and treatments of blood cancers," he says.

Scientists now suspect that many cancers have their own stem cells, a long-lived malefactor that spawns cells that form the bulk of the tumor.

"Cancer has these stem cells, and we need to target them for treatment. But when patients come to the clinic, they already have cancer, so the malignant transformation already started," says Slukvin. "By reprogramming blood cancer cells to pluripotent stem cells and differentiating these cells back to blood, we hope to generate cancer stem cells in a dish that would be a good model for studying how these cells formed, to figure out what external factors make them go bad. This could be a crucial step in treating or preventing cancer."

Story Source:

Materials provided by University of Wisconsin-Madison. Note: Content may be edited for style and length.

What culture conditions should a researcher use to generate macrophages and DCs? How do the in vitro-generated and in vivo cells compare?

A.M.M. Like others, we use the simple and conventional methods of growing bone marrow cells in either granulocyte–macrophage colony-stimulating factor (GM-CSF) or CSF1 (also known as M-CSF) to obtain mouse DCs and macrophages, respectively. These methods have the advantage of generating reasonably homogeneous and reliable numbers of cells which can be manipulated in vitro.

However, the cells obtained are probably not representative of their counterparts in tissues. Their early stage of differentiation and homogeneity are unlikely to be replicated in vivo. More importantly, they have not been exposed to any of the environmental factors known to regulate myeloid cell differentiation and function in tissues. For example, DCs and macrophages in the intestine show many unusual properties compared with their equivalents in other tissues, blood or bone marrow and these are known to be conditioned by local factors 29,30,48 . However, the mediators involved are still being identified and no in vitro conditions have yet been found that replicate the full characteristics of the populations in situ. The same issues apply to other tissues, and so real advances will only be made using cells isolated from the organs themselves.

F.G. DCs obtained in culture from monocytes or bone marrow precursor cells exist in two functionally and phenotypically distinct states, immature and mature. Fundamental progress in understanding the cell biology of antigen presentation and the cellular mechanisms that allow DCs to initiate immunity or promote tolerance has been made using these cells 49 .

Macrophages can also be obtained from monocytes or bone marrow precursors following culture with CSF1. This experimental model has allowed investigators to identify cytokines and bacterial products that control their effector functions. M1 macrophages, the prototypical activating stimuli of which are IFNγ and lipopolysaccharide (LPS), exhibit potent microbicidal properties, whereas M2 macrophages support TH2-type effector functions and may play a role in resolving inflammation through endocytic clearance and trophic factor synthesis 50 .

However, the heterogeneity of the mononuclear phagocyte system is poorly recapitulated by these in vitro models. In other words, in vitro-generated macrophages and DCs, albeit useful to study the cell biology of phagocytosis and antigen presentation, for example, do not represent a model to study the specialized functions of the diverse cell types that are present in vivo, or the regulation of their development and functions by the tissue microenvironement. As an example, many tissue macrophages and DC subsets do not derive from blood monocytes, and some may not even derive from the bone marrow 1,4 . In vivo studies are required to analyse the functions of the mononuclear phagocyte system.

D.A.H. Individual colonies produced in vitro from bone marrow in GM-CSF contained classical DCs as well as granulocytes and macrophages 51 . Bone marrow contains a common progenitor (the macrophage and DC progenitor (MDP) 52 ), and a so-called common DC progenitor (CDP) 15 . CDPs are probably identical to high-proliferative potential colony-forming cells (HPP-CFC) that require more than one factor to produce colonies 19 . There are at least seven cytokines that one should consider in studying myeloid APC differentiation CSF1, GM-CSF, granulocyte colony-stimulating factor (G-CSF), IL-3, FMS-related tyrosine kinase 3 ligand (FLT3L), IL-4 and IFNγ.

A dogma has emerged that cells grown in GM-CSF are DCs, and those grown in CSF1 are macrophages 19 , but cells grown in both of these conditions are phagocytes and have been studied as functionally distinct macrophages 53 . Both cell types are in vitro artefacts in vivo, progenitors do not encounter any of these factors in isolation, and there are no obvious in vivo counterparts to the in vitro-derived cells. HPP-CFCs require a combination of IL-3, GM-CSF, CSF1 and IFNγ for optimal proliferation to differentiate into macrophages 19 . Although DCs were originally thought to be CSF1-independent, this is clearly not the case 54 and antibody against CSF1R is now used for the purification of DC progenitors 15 .

FLT3, the receptor for FLT3L, is a marker for haematopoietic stem cells and common myeloid progenitors, and it is retained on classical DCs in the spleen. FLT3L can expand myeloid APC populations in vitro and in vivo 15 but, like CSF1, probably acts on the HPP-CFC in combination with other factors.

G.J.R. DCs and macrophages derived in culture show functional properties generally consistent with their counterparts in vivo. However, they are only approximations of cells that exist in vivo. They are useful for cell biological studies, for migration studies to some extent and in some cases they hold valuable promise as agents of cell-based immune therapies. Cultured DCs fuelled the growth of the DC field in the mid-1990s, so they are historically important.

Now, the DC field has moved beyond this phase and makes use of cultured DCs in combination with in vivo models. Generally, DCs cultured in GM-CSF are thought to be counterparts of inflammation-derived DCs, but this needs to be formally shown.

I have less experience with cultured macrophages. Perhaps they are over-used in studies of M1 and M2 polarization states, and peritoneal macrophages isolated by peritoneal lavage may too often be thought to model macrophages from any anatomic site.

However, overall, I think the field appropriately uses cultured DCs and macrophages without over-extending the interpretation of the data generated from them.

S.G. The ability to generate large numbers of DCs from mouse bone marrow and from human blood monocytes with the aid of growth-factor cocktails has proved irresistible to investigators for obvious reasons for example, the direct isolation of these cells from blood and tissues is tedious and often results in poor yields and the introduction of artefacts.

Less well appreciated is the rapid 'acculturation' of macrophages that occurs ex vivo. Kupffer cells, for example, do not express CD11b in situ, but rapidly acquire this complement receptor in cell culture. Even modifying culture vessels with media and growth factors cannot prevent this artefact of isolation. Alveolar macrophages are round and loosely adherent in vivo, but are profoundly altered in morphology by adherence to tissue culture plastic, as are all macrophages. The use of undefined, non-physiological supplements such as fetal bovine serum is particularly egregious, and defined media should be used wherever possible.

In situ analysis is therefore mandatory, but DCs and macrophages in the tissue can be difficult to access, quantify or test functionally. Therefore an integrated approach, with appropriate awareness of artefact, is required. My own fantasy is to be able to recreate the full panoply of macrophage phenotypes observed in vivo, from embryonic stem cells and other haematopoietic stem cells, entirely in vitro. This will require knowing their in situ phenotype in detail and elucidation of the cellular and extracellular environment in different organs, such as the liver, gut, lung, brain and uterus.

COA - Practice Questions

These fine suspensory ligaments are composed of numerous fibrils arising from the surface of the ciliary body and inserting into the lens equator.

The cones are used during daylight to allow detailed vision and color perception. They predominate in the macular area and receive visual images, partially analyze them, and submit this modified information to the brain.

Loss of accommodation is due to a gradual hardening of the lens substance, beginning with the nucleus, so that it is more resistant to changes in shape.

There are approximately 125 million rods present in the extramacular area of the retina. These rods function best in dim light and are responsible for what is called scotopic vision.

It is the adjustment after we enter a dark movie theater that permits us to walk up the aisles with some degree of accuracy. Pilots during World War II soon learned they had to become dark adapted for bombing missions at night.

A prism is a wedge-shaped piece of glass that bends light toward its base because of a change in the direction of light waves. It creates this alteration of direction because of the change in the index of refraction between light traveling in air and light traveling in glass. When light emerges from the prism, it undergoes another change in direction toward the base.

Chromatic aberration is seen naturally in rainbows. White light penetrates a suspended droplet of water and is broken up into its spectral components.

Clinically, chromatic aberration is seen in patients who have corneal edema. The most common occurrences are in persons with severe or acute glaucoma or those wearing ill-fitting contact lenses. The liberated edema fluid breaks up the intact bundle of white light, and patients complain of seeing halos around lights.

All the colors, whether they are reds, blues, greens, or oranges, have exactly the same speed of light, which is 186,000 miles per second.

A wavelength is the distance from the top of one wave to the top of the next, whereas the frequency is the number of waves passing in 1 second. Red light may have a longer wavelength than blue but this indicates only its vertical vibration. All colors, white included, travel at the same speed.

Placido's disc uses the cornea as a mirror. The disc is used to detect keratoconus. The cone-shaped deformity of the cornea is reflected in the distortion of the annular rings, which appear irregular and oblong on the cornea.

If light strikes any optical surface at an angle greater than the critical angle, the light, instead of passing through that surface, will be totally reflected.

This principle is well established. Some ophthalmoscopes are based on total reflection by virtue of light striking a prism. In fiberoptic bundles the light is inside the bundle and cannot escape because the angle of incident light exceeds the critical angle and the outer coat of the light has a low refractive index. The light emerging from this fiberoptic bundle is compressed, intense, and very high in illumination. It is a pure light because none of it escapes or is broken down to its spectral components.

Large frames that contain strong prescriptions, that is -5.00 diopters or more, frequently slide down the nose with reading. The effect of gravity drops the lens, and the vertex distance of the lens to the eye changed. With plus lenses it increases the prescription with minus lenses it does the reverse. Also, unwanted base-up prism is added with plus lenses, with the opposite, or base-down, occurring with minus lenses.

The optical center and the mechanical center do not coincide. The optical center of a lens is that place of the lens that does not contain unwanted prism. It is the point detected on the lensmeter where the rays of light come into focus. The optical center should be in line with the eye. The mechanical center is the geographic center, and in a perfectly round lens it coincides with the optical center. The perfectly round center, not the mechanical center, concerns us.

Spherical aberration: The image from a spherical lens is never a single point because the central and paraxial rays form more concentrated images than those rays that pass through the periphery of the lens.

-The degree of spherical aberration depends on:
1) the aperture of the system it is reduced by closing down the size of the aperture
2) the precise form of the lens used the error can be reduced, making the curvature of the anterior surface greater than the curvature of the posterior surface
3) the curvature of the lens the fault can be diminished by making the peripheral curves less sloped these are called aplanatic surfaces

Astigmatism of oblique pencils: If light rays strike a lens at an angle instead of perpendicular to it, the image will be distorted in a form similar to that produced by a cylindric lens. Some light rays will strike the lens early and some later. The extra distance has to be traveled by the later rays to strike the lens. Moreover, a flatter section of the lens will be astigmatic, sharp in one direction and fuzzy in the other. If the light rays strike a lens perpendicularly, this type of lens distortion does not occur.

Concave mirrors magnify the image of the object only if it is placed within the focal point of the mirror. Cosmetic mirrors always are concave, and the face has to be placed close to the mirror to have its image enlarged and in focus.

Why I cannot find dendritic cells in blood smear? - Biology

You, the student
Dr Holly Ressetar . . . . .Room 4047 . . (293-1687) . . and Dept No. 293-2212 for all
Dr Gregory Konat. . . . . .Room 4016 . . (293-0594)
Dr William Beresford . . . . . Room 4003 . . (293-0589) Home phone 292-0083

Required Textbook:
Histology. A Text and Atlas by M.H. Ross, E.J. Reith, and L.J. Romrell, 4th OR 3rd edition, Williams & Wilkins, Baltimore, 2003.

Other resources: "Histology Lab Assistant" On the Anatomy SBLC computers Should be available as an icon

Histology Full-text by Beresford at & Histology Powerpoints at the same site . an older version that you can only print out as one huge file. The local HSC version has separate chapters.
The 703 Course site on SOLE
A good online Atlas is at http://129.241.42/pathology/nlm_histology
Other Atlases are listed in the Introduction to Histology Fulltext

The 35 mm slides, projected in the lecture to orient you for the lab, will be put in the lab a day or so after the lecture, along with a slide projector.

The Histo component is primarily a task-based independent exercise for you - to be competent in visual microscopic diagnosis, and in knowing the functional and some clinical correlates of what you recognize. The textbook was chosen thus for laboratory helpfulness, but also for relative brevity, and because one of the authors is on the USMLE Anatomy sub-committee constructing Step I of the licensing examination. Using the book for review next year will be easier if you are already familiar with it. We expect you not 'to have learned it', but to have consulted it intelligently and extracted enough ideas and information to do well on the exams.

Clinical correlations Our number of lectures relative to other basic sciences is few, so that we weave clinical significance into the presentations and exam questions instead of having clinicians visit.

  1. describe some of the routine techniques used for the microscopic preparation of cells and tissues and explain the effects of these techniques on histological appearance
  2. identify the structural components of cells at the light and electron microscopic levels
  3. interpret the activities and properties of living cells based on the observation of fixed specimens
  4. recognize examples of the basic tissue types for later application in organology and pathology
  5. identify selected organs and their parts by examining stained sections with the light microscope and by examining electron micrographs in textbooks, and
  6. correlate structure with function in all cells, tissues and organs studied (eye and ear will be studied later in Neurobioogy)

HS 303 Histology Laboratory: The basic agenda

2 Then, we'll go to the lab, 4023, to find our assigned places with a key to the microscope locker and the drawer with the slide sets. (With the key is the number to unlock your Gross Anatomy locker, out in the hallway. Gross directions will be given this afternoon.)

3 The individual slide sets will be checked against an inventory (not all slides are present) and the completed inventory sheets handed in. [Another version of the inventory is at the back of this Lab Guide.]

4 Using a well-stained slide, we'll try using the microscope and getting binocular fusion of the images (not everyone will the first time).

5 We shall also try the microscope on some slides showing the less usual methods of tissue preparation, and we'll get some first impressions of how tissues and cells look with various staining methods. What one stains for is what one sees, and the picture is always a very partial one. The figures specified in the text-atlas give one an initial idea of what to expect. The standing assignment is to read in advance the chapter matching the lecture topic, at the least, to have read the text going with the plates. Other textbooks can substitute for Ross & Romrell.

6 Also on Monday, we shall start thinking about cells as the fundamental component of organ systems, and taking a preliminary look at them in some slides. However, the electron microscope (EM) is far better than the light microscope for seeing cell structure, so that here the `lab' exercise extends beyond lab time, being mostly one of your looking at cytology pictures in the textbook and atlases, and using the histology computer program (Histology Lab Assistant) in the SBLC.

7 On Thursday, we'll start the first tissue - Epithelium, and the lab will involve searching designated slides for examples of what has been shown as 35-mm projected slides in the lecture. Read the `Epithelium' chapter and PowerPoints beforehand.

8 The point of a lab is to tackle messy reality rather than looking at pretty pre-packaged views. [You know the theory of cardiovascular and respiratory systems, but can you confidently do CPR?] Mixed in with, or at the end of, most lists of things to find in a lab session, are comments on how the material fails to connect with the theory. Skim through these comments before going systematically down the list. By the end of the Module, you will be competent in the skills of microscopy and intelligently reading slides, will appreciate the significance of cell and tissue structure for normal function and pathology, and should know enough to pass the boards.

9 The assessments and exams will have typical written questions - you will be provided with examples and can get them from previous years. The histology lab exam involves your going around microscopes and EM figures set up in the lab in numbered sequence, with just over a minute per station. Answer the question on the card. The kinds of question we ask are at the end of the Neural Tissues section Lab exam format. Frame your lab thinking, from the start, in terms of questions by reformatting the neural examples for each tissue.

10 After the first exam, life gets easier. (a) We are on organ systems, whose components and physiology you know. (b) The light microscope is operating at its most favorable, showing how tissues and special cells are organized to make up an organ. (c) We have left behind the initially difficult mixture of three levels of analysis - cytology, tissues, & organs - to concentrate on organs.


Take only your book-bag (& computer, if you have it with you) into 4023. Locate your place - labeled in alphabetical order from the far end of the lab. The last nine in the Class alphabetical order will find their places on the side benches. You will have sitting places in the lab, but this will be sorted out in the course of the morning.

Find the small key at your place and attach it to your key ring or something visible, when dropped in the snow. (Note: your key number does not match your microscope number.)

Use the key gently to open the top drawer at your place & take out the two small boxes of slides. Place them near the middle of the bench. Do not balance your slide box in the open drawer or leave it close to the edge of the bench. [The sound of a slide box hitting the floor is painful and expensive.]

Open the locker, and carefully lift out the microscope. Find the microscope-use directions (next two pages). Follow them with any well-stained slide. Ask us for help. [We will not issue oil for the X100 oil lens] Switch off the scope. Complete the receipt form.

Note the convention on slide numbering below. Check your slide boxes against the inventory. Leave the completed inventory form at your place.

Put your scope & slides away carefully. Lock the drawer & locker. About half of you share with a dental student. Please be considerate throughout the semester. Meaning, among other things: always put the slides back in the correct slots in the boxes do not leave personal items - half-used suckers, b-c pills, toothbrushes, costly textbooks, etc - in the drawer or locker and, if needed, only good-humored notes to your unseen colleague, please.

Students in places 59-94 have the older (better?) Leitz microscopes. These have an extra flip-up lens at the top of the condenser under the stage. This lens should be switched OUT for the X4 low-power objective, but very definitely switched INTO use for all other objectives. After a while you'll get the hang of this. Whatever `scope you have, try out one of the other kind between now and the first exam. The exam will be set up using both kinds, and for SOME stations you can use all the `scope controls


You can use your access cards to get into the lab at any times, except Tuesday, Wednesday & Tuesday and Thursday 2-4, when the Dental students have class, or at their exam times. When our exams come around, we'll work to avoid any conflicting review times

A Simple squamous epithelium
For now, just classify this as such, regardless of whether a particular example is endothelium, mesothelium, or epithelium

1 Problems of finding it in the H-1 kidney slide are: (i) that this thin plastic section has the corpuscles widely spaced (because it is thin) and the stain is weak, so that there is little contrast between the corpuscles and the surrounding tubules (ii) the corpuscle is dominated by the irregular, tangled glomerulus, which may reach right out to touch the capsule, therefore finding the epithelium of the enclosing capsule takes perseverance.

2 Slide A-5 of silver-stained mesentery outlining mesothelial cells has a lot of gold or brown precipitate (junk) to confuse the picture. Also, the cell outlines are often incompletely stained, and the two epithelial layers are at different depths of focus.

3 The heart (B-7) is itself a vessel lined by endothelium. However, its inner surface is irregular and extends in as valves. Any simple squamous epithelium will appear only as a thin line of flat, dark nuclei at a free surface with occasional hints of cytoplasm, if the epithelium has stayed intact. The small vessels at the heart's outer surface present another problem: most are cut in cross- section, and because the lining endothelial cells run lengthwise with the vessel, their nuclei appear only as dots at the inner margin.

B Stratified squamous epithelium, non-keratinized
Where the uterine cervix protrudes some way into the vagina (L-10), the epithelium is mostly the protective stratified squamous (non-cornified) variety. Hunting far and wide along this epithelium should eventually reveal a region of simple columnar ep. well inside the uterus. (There may be stretches of stratified columnar ep., as well.) The picture here and in many other organs is visually complicated because tubular glands, also lined by simple columnar epithelium, extend down from the surface epithelium, with which we are primarily concerned. This situation arises in the stomach (E-5) and colon, where there is not that much surface epithelium, but most of the epithelial tissue is actually glandular below the surface lining the organ. The glandular epithelium is less clearly a simple columnar ep. since the epithelium is forced into the tubular shape of the little gland.

C Pseudostratified columnar epithelia
The airway epithelium has several cell types, and an apparent multilayering based on the nuclei, whereas the epithelium of the ductus deferens and epididymis has two layers of nuclei and only two cell types - tall columnar and short basal. Both sites have features that suggest the location in the body (pale mucus-secreting goblet cells and cilia - airway lining sperm in the male lumen), and from seeing these features one can infer indirectly that the situation is 'pseudo' rather than truly stratified. Slide G-9 of lung has little if any sign of bronchi: it was cut too peripheral to the main airway. Slide G-6 of trachea has a ragged epithelium, heavily infiltrated with lymphocytes - the small, dark nuclei, but the BM shows up as a pale gray band just below the epithelium. A general problem with epithelia at first is to see the epithelial cells, but to ignore the many connective tissue cells of the underlying lamina propria. In places the pseudostratified columnar epithelium of the airway undergoes metaplasia (conversion) into a more stratified squamous epithelium. This you may encounter as scattered islands of abnormality in the epithelium.
In the testis slide, do not confuse the tails of maturing sperm sticking out into the lumen of the tubules in the testis itself with the stereocilia of the nearby epididymal epithelium. (Stereocilia are inappropriately named long microvilli, with no microtubules and no ability to beat.)

D Cilia and microvilli
In LM, cilia should be long and spiky, and individually distinct as you use the fine focusing. Microvilli, when densely packed as a brush/striated border, are about one third the height of cilia, are not seen individually, and stain PAS+. Brush borders, terminal bars, prickle cells etc. need oil immersion objectives and good specimens to be seen convincingly.

E Keratinized stratified squamous epithelium
The keratin tends to break up into tatty layers, when processed and cut for LM histology. In 'thick skin' (F-1) note that roughly half the thickness of the epithelium is living cellular tissue (next to the connective-tissue dermis), whereas the outer half is a dead, but still cellular keratin layer. You need now only know the epithelial type, not thick-thin differences, nor the several layers in the epithelium except for the keratin and living layers.

F Stratified cuboidal epithelium
This epithelium is recognized from the two concentric rings of round nuclei. The sweat gland ducts are small, dark structures, mostly far down in the dermis away from the surface epithelium of the skin. Some regions of the duct system in salivary glands have strat. cuboid ep. However, along the total length of the system, simple and stratified cuboidal and columnar epithelia, and transition forms, can be seen, depending on the luck of the section. Go by the rule - that at a particular site what is there is what is there - and start to rely on your own judgment.

G Transitional epithelium (Urothelium)
The plastic-imbedded bladder slide (H-7) has some dark cells flattened on the surface of the epithelium. These do not look like typical surface umbrella cells, but may be dead or dying cells, cast of from the bladder or upstream in the urinary system that have stuck to the surface. 1 With large, small and medium, there are too many terms to choose from. After the experience of having looked at a lot of vessels, the wide range of terms for size is useful, but at first it isn't. For the lab exam, know: elastic artery, muscular artery, arteriole, capillary, vein, large vein/vena cava, and have a try at venule for structures like capillaries but several diameters wider. Think very small for capillaries in LM.

2 The classification of layers/tunics gets pushed beyond good sense. Theory intrudes on the light- microscopic lab. reality. Thus, much of the heart is lined by a very thin endocardium which it is not convincing to subdivide likewise the intima of an arteriole is negligible in LM. Also the layers can vary in thickness, e.g., the endocardium or be mixed up as far as the cells are concerned, as in the vena cava where fibroblasts and VSMCs do not respect the others' territory, and produce three tunics with combinations of c.t. and muscle. You should be able to cope with pointer questions on the layering in elastic and muscular arteries, and the heart.

3 Vessels within a class vary quite a bit. The veins within organs, e.g., the heart and ovary, can get quite large without acquiring smooth muscle, whereas small veins in the limbs already have a little muscle in a media. If in doubt about a vessel, cruise around to compare veins and arteries in the same region.

4 Lymphatics are a distraction, but can usually be disregarded because they squash flat. They should have large lumens, very thin walls, valves, but no RBCs. Leave until lymph node, where there have to be lymph vessels going to and from the node (cut them off at the pass!)

5 In large vessels one sees more detail than is comfortable, if the theory has been kept simple. In the outer media of the aorta there are some muscle bundles that run 'longitudinally' (actually in a spiral with a long pitch) in the intima of the vena cava, an internal elastic lamina can be seen etc.

6 The comparison slide of vein with muscular artery (we don't speak of muscular veins) is stained red for elastic so that the inner line of the artery is the lamina elastica interna. There is more elastic at the start of the adventitia , giving a reasonable impression of an external elastic layer. Between the two is the media in which a few elastic fibers can be seen. As in most vessels of this size, the intima is negligible in light microscopy, leaving only a media and adventitia to be distinguished.

7 For the venule-vein distinction, use for guidance any accompanying artery or arteriole, allowing for the vein or venule to have 2X or 3X wider lumen. On the exam you will be given guidance, e.g., by asking for a one-word answer, that we are not asking for small-medium-large distinctions.

8 In the VASA VASORUM vessels in the adventitia of the aorta, the veins and venules have quite a lot of elastic in them. A guide to both is that any muscle does not form a continuous layer, which it should for arteriole.

9 In the OVARY, there are many arteries and arterioles. The veins are flattened, may have blood cells in them, but are to be distinguished by the endothelial cell nuclei lined up, lining a space in the connective tissue. They need work to be found.

10 CAPILLARIES are very small, so think an order of magnitude down. For cross-sections expect a red RBC around which curves a fine line including one, maybe two, curved endothelial cell nuclei. That's as exciting as it gets.

11 The ADVENTITIA of any tube will blend off into the CT of surrounding structures. Under medium power, one can usually get an idea of where to stop calling the CT adventitia. The same problem arises for periosteum and endosteum. HOW TO APPROACH A KIDNEY Cautiously, because although the individual uriniferous tubule makes sense, when a million of them are packed together at different levels, fixed imperfectly, and stained in an unbalanced way (plus, maybe, a little pathology), the images can become difficult to interpret.

Look at all the kidney slides against a white background and using the eyepiece, reversed, as a low-power magnifier. Questions: Is it a unilobar animal kidney? If it is human multilobar kidney, do you have more than one lobe present and how small is the piece, e.g., does it go out to the capsule? How far towards the hilus does it go? Where are the cortex and medulla? Is the medulla intact? How is the section stained - H. & E. , PAS, a trichrome? See below for how this information can help.
If the kidney is unilobar, or you have only part of one lobe of a multilobar one, you will not see renal columns. If the apex of the pyramid is cut off, you are missing the papilla and with it the papillary ducts. Some structures, e.g., the renal sinus and its calyces, are best appreciated by the gross anatomist or pathologist doing a conventional dissection, and working with a complete kidney. With PAS staining, the individuality of the nephrons and collecting tubules shows up because the basal laminae are revealed, and the staining shows up the microvillous border of the proximal tubules.

To start the microscopy, use the piece of human kidney stained with H. & E. Go to the cortex and search along to a place where the renal corpuscles and surrounding labyrinth are distinct from the parallel elements of the medullary rays. The corpuscles contain a capsular space, usually visible, and the enclosing capsule lined by the parietal epithelium, seen only as flattened nuclei of the simple squamous epithelial cells. In the glomerulus all you can say is that the many nuclei have to belong to podocytes (the visceral epithelium), endothelial cells and, towards the vascular pole, mesangial cells. The cells cannot be reliably distinguished in these specimens with only high-dry mag., but with TEM (& SEM) it's easy.
The task of telling afferent from efferent arterioles is not for novices. There is a difference in caliber, but one needs to see both arterioles at the same time. The most one should try to say is that the vessel fastened to a macula densa is likely to be an afferent arteriole.

To build confidence one can work through the structures that give no trouble, before tackling anything harder. The majority of the tubules cut in cross & oblique section around the glomeruli have to be prox. convoluted tubules. Next, mixed in with these will be a few distal convol. tubule profiles, with wider lumens, cells staining less red, no brush border and more nuclei per cross-section. Scan quickly over a number of corpuscles until you find a JGA with a macula densa. The other side of the distal tubule to its macula densa will be typical distal tubule. Thus, in the cortical labyrinth you have reliably identifiable prox. & distal tubules to go back and check with, when you are later looking for straight proxs. and distals in medullary rays and the outer medulla.

The third easy tubule is the collecting duct. Find the cortico-medullary junction and cruise down and back through the medulla with the idea of assigning one chunk to outer zone, the other to inner. In the inner medulla, find a collecting duct - pale cells, distinct cell outlines, some luminal bulging, & a significant lumen. Go down into the papillary region, if it is present, and trace from the surface inwards the papillary ducts. (In the large slide, the epithelium of these ducts is grotesquely flattened from excess pressure and all the tubules are distended - artefact).

The outer medulla is the place to hunt among the many profiles for thin segments. The difficulty here is that they are small and mixed in with a lot of other things. The epithelium is simple squamous, but definitely more visible than endothelium of capillaries. While you are in this region of outer medulla look for straight parts of prox. and distal tubules. (You may need to go back to the cortex for a check on prox.-distal differences.) One problem is that the tubules of your chosen part of the medulla may be cut at an unfavorable angle if so, move around. You will not see loops (Henle's) of nephrons, only the components - straight distals & proxs. & thin segments. Now, you can try for the collecting ducts in the medullary rays, where they will be sparse and central, so that the wider cuts through rays are better bets and for the connecting tubules (CTs) coming to rays from the labyrinth. CTs have a lower, paler cuboidal ep. than distal tubules. They are an emaciated version of the collecting ducts seen deep in the medulla: if you cannot find them & 'ray' collecting ducts, never mind. If you have a dark serous gland with no islets but a lot of ducts, search over it very carefully for any mucous secretory units (they do look different from fat cells under high power) in order to distinguish parotid from submandibular.

DUCTS are named for postion, function and appearance so: interlobular/excretory versus intralobular/secretory/striated (some of our slides are cut to miss interlobular ducts). "Striated" for the basal mitochondria and infoldings for ion transport so that the secretory duct can alter the secretion, in contrast to the excretory or drain-pipe kind.

LABIAL, BUCCAL, etc. minor salivary glands. The ID requires using two or three magnification powers: to be sure of the gland (in newborns they look serous, because mucus has not yet built up), and to see that the organ is lip, cheek, palate, or tongue. [Our only palate is a fetal sagittal-face section.] Tongue is easy from the papillae. Cheek has more adipose tissue than lip, and lacks a red margin. The glands will be on the mucosal side below the thick strat squamous epithelium.

Let's get serious about SEROUS.

SEROUS: 1. applied to glands and certain glandular cells 2. signifies major protein secretion occurring but not of mucous glycoprotein 3. examples - pancreatic acinar cells, gastric chief cells, parotid secretory cells

SEROUS: a. relating to serous body cavities - pleural, pericardial, peritoneal b. mobile tubes or theirenlargements in these cavities have an external layer - the t. serosa c. Tunica serosa and serous membrane are both covered by simple squamous epithelium (mesothelium) to allow lubricated movement (serous fluid lubricates) d. inflammation, interruption of the mesothelia, & fusion of the two connective tissues --> adhesions, restricting movement, and impairing function GLANDS & SECRETIONS: vital for epithelial function and protection, digestion, metabolism, excretion, & control.
Secretions are useful materials made and released by cells. Glands comprise assemblies of secreting epithelial cells. Mesenchymally derived cells secrete many substances - cytokines, prostaglandins, synovial fluid, antibodies, etc., but such cells are not considered to be glandular. Nerve cells also secrete materials, but are viewed in the first instance as neural, not glandular, entities.

Questions for a gland: What is the product(s)? What are its functions? Where is the product released? How much is needed? What is the construction of the gland? Diffuse? Aggregated? Shape? Complexity? How are the gland components controlled? How are epithelia, myoepithelium, smooth muscle, vessels involved? What are the cellular & molecular biologies? Protein & peptide transcription, glycosylation - sites & enzymes, compartment targetting & intracellular traffic, precursor forms & storage, release & destruction mechanisms? Which cell types form tumors? What markers show this?

How far away from the secreting cells does the secretory procduct act or otherwise be useful? And what is the fate of the secreting cell? The notorious CRINE FAMILY spreads its influence via secretions thus: autocrine - leaning on the family at home and on oneself paracrine - leaning on the neighborhood endocrine - down the pike to put it to another city. Meanwhile, in the family's exocrine factory (which quite respectably makes laundry detergent) the employee can hand over the product with no injury - merocrine/eccrine secretion while parting with an arm and a leg - apocrine secretion or become engorged with the product and die as the way to deliver it - holocrine secretion. [A cytogenic mode produces gametes, although 'holocrine' is also applied to gamete formation.]

DETERMINANTS OF GLAND STRUCTURE - What sort of secretion how much is needed & where it is to be released & used. Hormones are potent & little is needed, are used at a distance, & need not be modified in a duct. Therefore, to construct an endocrine gland, clumps or cords of epithelial cells face wide capillaries with leaky or fenestrated endothelium on a support of reticular fibers. If intracellular storage is not enough, follicles form, e.g. in thyroid, parathyroid, and pituitary glands. Epithelial cytology reflects the hormone's chemistry: variables being granule size & density, ER, lysosomes, PAS reactivity (indicating glycoproteins), etc.

EXOCRINE GLANDULAR TYPES based firstly on morphology, secondly on the product(s) 1 Diffuse 'unicellular' gland - mucus-secreting goblet cells in an epithelium 2 Secretory sheet - all surface cells secrete mucus, e.g., stomach lining 3 For concentration on the job of secretion, to reduce bulk, & to let the lining epithelium get on with its work, exocrine glands become separated from, but attached by a duct to, an epithelium.

Simple: a single or no duct Compound - branching duct system (& gland is usually large) Each of these major classes is subdivided according to the shape(s) of the secretory units: simple straight tubule, simple coiled tubule, simple branched tubule, & simple acinar/alveolar versus compound tubular, compound acinar, & compound tubulo-acinar/alveolar. Alveoli can be greatly dilated, e.g. active breast, prostate, or have a lumen barely visible in LM. 4 Product classes - Serous cells produce and store proteins mucous cells make bulk glycoproteins (mucins), which often push the nucleus to the base of the cell. (Many so-called serous cells include glycoproteins among their products, thus certain salivary glands have mucous tubules with crescents of serous (seromucous) cells capping the ends.) Another product is lipid, but few cells make only lipids.

DUCT TERMS are based on position and function, which overlap but do not coincide exactly. Within a lobule are intralobular and intercalated ducts, & the intralobular duct may or may not be secretory (actively altering the secretion). Interlobular and interlobar ducts are usually excretory. Secretory duct cells possess basal infoldings, some microvilli, & many mitochondria (whose enzymes stain red with eosin) for pumping ions to specify electrolyte composition and water content.

Lastly, a word from a Histologist

Medicine and surgery treat people. People are made up of 200 or so types of cell organized in various organ systems. Effective treatment now means knowing these cells, their normal roles, and their weaknesses. For example, pernicious anemia results from an autoimmune attack on the proton pump of gastric parietal cells an attack which also knocks out the cells' contribution to iron absorption. The HIV virus gains a foothold by misusing a normal, surface glycoprotein of a subset of lymphocytes. Such knowledge should quite soon provide effective preventive, as well as curative, remedies for these two fatal diseases.

Histology is the subject that introduces you to the happy family of cells that makes up you, and the less happy ones of your patients. The subject has two faces: a practical, well circumscribed, lab-exercise goal - to recognize and describe the light- and electron-microscopic features of cells, tissues and organs the second face is the open-ended and ever expanding, detailed knowledge about cells, cellular activites and biochemistry, and their relations to medical problems. The limitless aspect gives your curiosity free rein, but it need not worry you as a taker of exams. What you will be expected to 'know' is limited to the contents of one atlas-text, and what you hear in 30-odd lectures.

We would like you to leave us: (1) feeling confident with light-microscopic slides and a few electron micrographs, of the kind that you will meet in histopathology, rounds and conferences (2) with some appreciation for the potential power of the microscope in medicine (3) and, for some of you, a lasting curiosity about how events at the cellular level will hamper your ability to treat mental disturbance, vascular blockage, persistent cough, diabetes, glue ear, and other such everyday problems of medical practice. [For some of you? Curiosity, like affection, is hard to direct.]

Antibody tests




Did You Know.






To evaluate the performance of the RUV-III method on Nanostring gene expression data, we examined 4 studies comprising one in-house and three different published datasets that had technical replicate samples. The details of each study are given separately below.

Example 1: Lung cancer study

Our in-house Nanostring dataset was part of a study of the expression of DNA repair genes in lung adenocarcinoma (LUAD). The data was generated using 15 nCounter cartridges, with the majority of the normal samples being run in a 15th cartridge a year after the rest of the experiment ( Supplementary Figure S1a ). For more details on this study, we refer to the supplementary file and Supplementary Figure S1 .

We began our analysis with an examination of the Average Plot (See ‘Materials and Methods’ section) of the raw nCounter data ( Supplementary Figure S2 ). The spiked-in controls were roughly constant across the cartridges, though occasionally showing substantial variability. The average counts of the HK genes and the library sizes show a marked decline in the 15th cartridge while the averages of the POS and NEG spiked-in controls are fairly stable across all cartridges. This inconsistency poses a challenge for the nCounter normalization.

In Figure 1A, we present three RLE plots (See ‘Materials and Methods’ section), the first for the unnormalized data. For the nCounter normalized data, we applied a standard option: mean+2sd of the NEG transcripts for background correction, and geometric means for both the POS and the HK transcripts. In the third, the data were normalized by RUV-III using the 500 genes with lowest variance as the negative control set, all technical replicates samples ( Supplementary Figure S1b ) and k = 6 (See ‘Materials and Methods’ section). In the RLE plot of the raw data (Figure 1A) we see substantial differences within and across cartridges, with samples from cartridge 15 standing out. This remains the case with nCounter normalized data, whereas the RUV-III normalization yields much more uniform RLE plots, centred around zero (Figure 1A).

Comparing the performance of nCounter and RUV-III normalization methods. (A) The RLE boxplots of unnormalized, nCounter normalized and RUV-III normalized datasets. Ideal RLE distributions would be centered around zero and similar to each other. The boxplots of unnormalized data clearly show substantial variation within and between batches. (B) Log ratios of genes of all technical replicate samples in unnormalized, nCounter normalized and RUV-III normalized datasets. RUV-III normalization led to smaller log ratios change of genes between technical replicate samples. (C) Scatter plots of ERCC1 and RRM1 gene expression obtained from nCounter normalized data (r = −0.47) and RUV-III normalized data (r = 0.31) and two publicly available datasets including Hou et al.’s ( 25) microarray gene expression data (r = 0.40) and the TCGA lung adenocarcinoma RNA-SeqV2 (r = 0.0). The nCounter normalization shows very different pattern compared to that of the RUV-III normalized data and the publicly available datasets.


We evaluated the similarity of technical replicate samples by producing TRA plots (See ‘Materials and Methods’ section and Figure 1B). These show that RUV-III normalization leads to a considerable reduction in the differences between technical replicate samples.

To assess the effectiveness of the different normalizations, we attempted to recapitulate some established biology in our data. A known biological signal in this context is that the expression of the genes RRM1 and ERCC1 should be moderately positively correlated ( 14–17). In Figure 1C, we present four scatter plots of the expression levels of these two genes. The nCounter normalized data scatter plot clearly deviates from the other three, including the RUV-III normalized data, two of which recapitulate the finding of ( 14).

We also examined all 84 different nCounter normalization options, displaying in Supplementary Figure S3 the medians of the RLE plots for each sample, the TRA plot and the correlation between RRM1 and ERCC1 for all 84 options, and for RUV-III. Repeating all 84 normalization options on the lung dataset using a new set of reference genes obtained by the geNorm ( 18) method led to results similar to those obtained using the initial set of reference genes (data not shown). In all the assessments we have presented, the RUV-III normalization markedly improves upon the nCounter normalizations.

Example 2: Inflammatory Bowel Disease (IBD) study

The study by Peloquin et al. ( 19) of Crohn's disease (CD) and ulcerative colitis (UC) included samples from three tissues (colon, rectum and terminal ileum) under each of five different conditions: CD-uninflamed, CD-inflamed, UC-uninflamed, UC-inflamed and healthy. The cartridges were run in three batches (CodeSets) numbered 2, 3 and 4, see ( Supplementary Figure S4a ) and the supplementary file for fuller details.

First, we examined the Average and RLE plots of the unnormalized data ( Supplementary Figure S5a and b ). Both show noticeable batch differences. The RLE plots of the data normalized by Peloquin et al. show that there is still noticeable variation within and between batches ( Supplementary Figure S5a ), and this is strongly supported by the batch-colored PCA plot (Figure 2A). There one of the two clusters is dominated by samples from batch 3, consistent with a batch difference rather than the tissue difference we would expect to see biologically. Interestingly, samples from batch 3 do not stand out if we make our PC2 against PC1 plot with the correlation matrix ( 20). However, batch 3 does stand out in PC3 against PC1 plot using the correlation matrix ( Supplementary Figure S6 ).

Comparing the performance of different normalization methods on Nanostring data for the inflammatory bowel disease study. (A) Scatter plots of first two principal components (log scale and centered) for Peloquin normalized Nanostring data colored by reagent lots (left) and by different tissues (right). The second principal component captures all samples of batch 3, which clearly demonstrates that batch effects remain. (B) Same as a, for RUV-III normalized Nanostring data colored by reagents lots (left) and by different tissues (right). The different tissues cluster as expected biologically. (C) Differential expression analysis of terminal ileum individuals with inflamed Crohn's disease between pairs of Nanostring batches.


We then normalized the data by RUV-III using all genes as negative controls, all technical replicates ( Supplementary Figure S4b ) and the maximum value of K (See ‘Materials and Methods’ section). The RUV-III normalization led to RLE plots that were more uniform and centered around zero, and also to the separation of ileum from colorectal tissues in PCA plots (Figure 2B). This is now consistent with what is known about the biology of these tissues ( 21).

In order to determine whether the batch differences visible in Figure 2A are of any consequence, we performed a differential expression analysis for each disease state between pairs of batches, comparing the data normalized by Peloquin et al. with that normalized by RUV-III. The results (Figure 2C) are displayed in the form of volcano plots with -log10(P-value) plotted vertically against the log(fold-change) horizontally for each gene counted. More such plots can be found in Supplementary Figure S6 . Ideally, we should see little evidence of differential expression in these plots, whereas we see a lot in the data normalized by Peloquin et al., far more than in the RUV-III normalized data.

Noble et al. ( 22)reported microarray gene expression data profiling colon tissue in some of the same states studied in Peloquin et al. This enabled us to compare the two normalization results using an orthogonal platform.

We note that the batch effects remaining in the data normalized by Peloquin et al. (Figure 3A) can influence downstream analyses. For example, these data suggest that the CCDC101 gene is upregulated in UC uninflamed colon compared to normal colon tissue, whereas we do not see this in either the RUV-III normalized data or the Noble et al. microarray data (Figure 3B). In Supplementary Figure S8 we see similar results for six other genes. The residual batch effect in the data normalized by Peloquin et al. clearly matters, and it is not present in the RUV-III normalized data.

Impact of batch effects on differential expression analysis. (A) Expression pattern of CCDC101 gene across different batches of reagents. The Peloquin normalized Nanostring data showed a substantial difference between batch 3 and other batches whereas RUV-III largely removes this unwanted variation. (B) The expression pattern of the CCDC101 gene across different the disease states of colon samples. The gene is apparently upregulated in colon samples in the uninflamed ulcerative colitis state compared to healthy individuals in the Peloquin normalized data, whereas there is no evidence of differentially expression in the RUV-III normalized data or the Noble et al. microarray data.


Example 3: Human T-cell activation

For a discussion of the study by Ye et al. ( 23) of CD4 + T-cell conditions including activated 4 h, activated 48 h, INFβ 4 h, Th17 48 h and unstimulated T cells for 4 h, we refer to the supplementary file. The Nanostring data consisted of 1788 assays generated over 4 months in 2012 and 2013 ( Supplementary Figure S9a ). We found only 46 technical duplicates samples, distributed across Nanostring cartridges in 2013 only ( Supplementary Figure S9b ).

The Average Plot ( Supplementary Figure S10 ) of the unnormalized data show a clear downward shift in expression values from 2013 compared to 2012 (hereafter called time effects) this is most clear in the positive spike-in controls. The RLE plots of the same data show that the majority of samples with 48 h conditions were shifted up compared to those with the 4 h conditions ( Supplementary Figure S11a ), and the individual expression patterns of HK ( Supplementary Figure S12a ) and gender genes (data not shown) confirm this.

The pattern observed in the raw data is strikingly reversed in the data normalized by Ye et al. ( Supplementary Figure S11a ), where samples under the 48 h conditions now have generally lower values compared to those with 4 h conditions, and this too is reflected in the housekeeping gene expression patterns ( Supplementary Figure S12b ). This is likely the result of using HK genes for normalization across cartridges. We retrieved the authors’ normalized gene expression microarray data from GEO, and produced RLE plots for the 236 genes used in their nCounter assays ( Supplementary Figure S11b ). These RLE plots are very different from those of the Nanostring data normalized by Ye et al.

Our earlier strategy of using all genes as negative controls and all technical replicates failed to produce a reasonable RLE plot, so we used the nine (from 236) genes with lowest variance in the microarray data as a set of negative controls and all technical replicates to normalize the Nanostring data using RUV-III (k = 1). The RUV-III normalization largely removed the technical variation between the T-cell conditions, but the time effects remained in the data due to not having technical replicate across 2012 and 2013. Enlarging the number of negative control genes from 9 to 100 produced little change ( Supplementary Figure S11a ).

Next, we carried out a differential expression analysis between pairs of conditions to examine the concordance between log fold-changes found using the data normalized by Ye et al. and the RUV-III normalized data and the corresponding results from the microarray data. The result for one of 10 pairwise comparisons is seen in Figure 4A while those for the other 9 are in the supplementary file ( Supplementary Figure S13 ). This was repeated for the 100 least variable genes ( Supplementary Figure S14 ). In 17/20 comparisons, the RUV-III normalized data give results closer to those from the microarray data.

Differential expression analysis between activated 48 h and unstimulated 4 h conditions and the expression pattern of SOCS1 gene. (A) Scatter plots of log fold changes obtained by differential expression analysis between activated 48 h and unstimulated conditions for Ye-normalized data and RUV-III normalized data, each compared to the corresponding comparison from Ye et al. microarray data. The dotted red line is the 45° line. (B) Boxplot of log fold change (Ye)—log fold change (microarray) and log fold change (RUV-III)—log fold change (microarray). (C) Expression pattern of SOCS1 gene across all the T cell conditions in Ye-normalized data, RUV-III normalized data and Ye et al. microarray data.


We also display several genes whose expression levels differ across the conditions, and compare the patterns of differences seen in Ye-normalized, RUV-III normalized and the microarray data (Figure 4B and Supplementary Figure S15 ). In all of these comparisons, the RUV-III normalized data showed better agreement with the microarray data than did the Ye-normalized data.

Example 4: Dendritic cell study

The final dataset we examined is from the study by Lee et al. ( 24) of four different CD14 + T cell conditions, unstimulated for 0 h (UNS 0h), Escherichia coli bacterial lipopolysaccharide for 5 h (LPS 5 h), influenza virus for 10 h (FLU 10 h) and interferon beta (INFβ) for 6.5 h. We refer to the supplementary file for fuller details, particularly Supplementary Figure S16b for sample collection times and Supplementary Figure S17 for the TRL plot.

We began our analysis with average and RLE plots of the raw and Lee-normalized data ( Supplementary Figure S18a and 19 ), and we saw that the UNS samples really stood out. Not only were they very spread out in time ( Supplementary Figure S16b ), we noticed that they consisted of 165 that were assayed under the UNS condition alone (U), 276 that were assayed under all four conditions (ULVI), with smaller numbers for another four types ( Supplementary Figure S16a ). We were led to define 11 sample usage types, see supplementary file for a full explanation of this term, and Supplementary Figure S16a for their numbers.

In Figure 5A we present PCA plots of the Lee-normalized data stratified by condition and colored by gender (row 1) and by usage type (row 2), while Supplementary Figure S20b has the same data with the initial RUV-III normalization.

Scatter plots of first two principal components of Lee-normalized data and RUV-III normalized data with all technical replicates and 10 pseudo-replicate pairs. (A) Principal component analysis of each condition colored by gender (first row) and by usage type (second row). Ideally, PCA plots within condition should have two clear clusters corresponding to males and females, preferably defined by PC1. (B) Same as A, for RUV-III normalized data using all technical replicate samples and 10 pairs of pseudo technical replicate across U and ULVI types of the UNS condition.


A differential expression analysis comparing the U-samples with the rest of the samples showed striking heterogeneity of Lee-normalized gene expression data under the UNS condition ( Supplementary Figure S21a ). By definition, there cannot be replicate pairs spanning the U and the ULVI sample usage types, so we created 10 carefully matched pseudo-replicate pairs that did so (See ‘Materials and Methods’ section). We then used RUV-III with all technical replicates and the 10 pseudo-replicate pairs, the 340 least variable genes as negative controls and k = 10. The resulting RLE plot is in Supplementary Figure S19 and the corresponding PCA plots are in rows 3 and 4 of Figure 5.

When we normalized using RUV-III with the pseudo-replicates spanning the U and ULVI sample usage types, the resulting PCA plots split into two clusters on PC1 corresponding to gender for three of the four conditions (Figure 5B). It seems likely that there is heterogeneity under the LPS 5 h condition which could be removed by creating suitable pseudo-replicates that would lead to a gender split on PC1 for that condition too, but we did not explore that. These clusterings, the RLE plots in Supplementary Figure S19 and the greatly reduced gene expression heterogeneity evident in Supplementary Figure S21 demonstrate the value of using pseudo-replicates with RUV-III.

Breakthrough therapy gives hope for new leukaemia treatment

Clinical trials of the potent new anti-cancer drug venetoclax showed it was effective in killing cancer cells in people with advanced forms of chronic lymphocytic leukaemia (CLL) when conventional treatment options had been exhausted.

Seventy-nine per cent of those involved in the trial had promising responses to the breakthrough therapy – including twenty per cent who went into a complete remission.

A small number of patients had such a profound response that even very sensitive tests were unable to detect any remaining leukaemia in their bodies.

CLL is one of the most common forms of leukaemia, with around 1000 people diagnosed with the cancer in Australia every year.

The results from the trials conducted at The Royal Melbourne Hospital and the Peter MacCallum Cancer Centre, in collaboration with the Walter and Eliza Hall Institute, as well as trial sites in the US, were published in the New England Journal of Medicine.

Professor Andrew Roberts, a clinical haematologist at The Royal Melbourne Hospital and head of clinical translation at the Walter and Eliza Hall Institute, said most trial patients responded positively to the therapy, showing substantial reductions in the number of leukaemia cells in their body.

“Many patients have maintained this response more than a year after their treatment began, and some patients remain in remission more than four years on,” Professor Roberts said.

“This is a very exciting result for a group of people who often had no other treatment options available.”

The drug has been granted Priority Review status by the US Federal Drug Agency (FDA) for treating some types of CLL. The designation is granted to medicines that the FDA has determined to have the potential to provide significant improvements in the treatment, prevention or diagnosis of a disease.

Venetoclax was developed based on a landmark discovery made in the 1980s by Walter and Eliza Hall Institute scientists that a protein called BCL-2 promoted cancer cell survival. Venetoclax was co-developed for clinical use by US pharmaceutical companies AbbVie and Genentech, a member of the Roche group, and was discovered by AbbVie scientists as part of a joint research collaboration that involved Walter and Eliza Hall Institute scientists.

Professor Roberts said the drug works very specifically by overcoming the action of BCL-2.

“High levels of BCL-2 protect the leukaemia cells from dying, so the leukaemia can grow and become resistant to standard treatments. Venetoclax selectively targets the interaction responsible for keeping the leukaemia cells alive and, in many cases, we’ve seen the cancerous cells simply melt away,” Professor Roberts said.

“The fact that a targeted drug, given on its own, can produce such a profound reduction in the leukaemia burden in the patient, to the point we cannot find the leukaemia even with our best tests, underscores what a powerful strategy targeting the BCL-2 gene is,” said Professor John Seymour, Chair of the Haematology Service at Peter MacCallum Cancer Center.

“These results set the foundation for building towards the dream of cure for CLL.”

Phase 2 and phase 3 studies are currently being undertaken to test venetoclax across a range of blood cancers globally, including at many sites in Australia.

Victorian institutions involved in this study, The Royal Melbourne Hospital (Melbourne Health), Walter and Eliza Hall Institute and Peter MacCallum Cancer Centre, are three of the ten organisations that make up the new Victorian Comprehensive Cancer Centre, a powerful partnership committed to cancer research, treatment and care.

“The clinical trials of venetoclax have been led by researchers within the VCCC, and demonstrate the power of this initiative in enabling world-first research to happen in Victoria, and bringing better treatments to Victorians with cancer,” said Professor Roberts, who is also the inaugural Metcalf Chair of Leukaemia Research at the University of Melbourne.

The trial was funded by AbbVie and Genentech (a member of the Roche Group). The researchers were also supported by the National Health and Medical Research Council, the Leukaemia & Lymphoma Society (US), the Webster Bequest, Cancer Council Victoria, the Australian Cancer Research Foundation, the Victorian Cancer Agency and the Victorian Government Operational Infrastructure Support Program.


Although viruses may elicit an acquired immune response, we still face big challenges in combating viral infections, since intracellular agents may develop different strategies to escape from the host immunity. Moreover, viral infections usually cause chronic diseases causing difficulties in the fight against them. Regarding COVID-19, little is known about humoral and cell-mediated immunity, since this is a very recent pandemic. Although a ton of information is being generated, it is still difficult to have a clear picture of host immunity, concerning the role of antibodies not only for diagnosis but also for individual’s protection. Cellular immunity seems to be preserved and many vaccines are being produced rapidly. Some crucial questions are still not fully answered: how durable is immunity and protection after infection with SARS-CoV-2? Will the primary immune response protect in subsequent reinfections? Is there a cross-reaction of SARS-CoV-2 with other antibodies? What is the mutation rate of this virus?

Antiviral agents have been employed and, while vaccines are still being produced, natural products should be assayed in an attempt to discover new antiviral drugs. As mentioned above, propolis antiviral action has been evaluated in vitro, while few in vivo assays and clinical trials have been carried out to explore its potential to stimulate host immunity to fight against viruses. Specific to COVID-19, the effects of propolis should be firstly investigated directly on the virus in vitro or on infected individuals alone or in combination with antiviral drugs, due to its immunomodulatory and anti-inflammatory action. Bachevski et al. [232] also recommended propolis as a prophylactic product for high-risk groups, such as individuals in close contact with infected patients.

The search for therapeutic targets may be useful to find out how propolis can help to control COVID-19 and bioinformatics approaches can add new insights. Using a homology-based structural model of TMPRSS2 and molecular docking, Kumar et al. [235] investigated the binding potential of CAPE, Withaferin-A (Wi-A) and Withanone (Wi-N) to TPMRSS2. Despite their binding affinity towards TMPRSS2, Wi-A and Wi-N seemed to be more efficient in binding and interacting with this protease. Maruta and He [236] reported another approach for virus control: the discovery of PAK1 blockers. Among mammalian kinases called PAKs (RAC/CDC42-activated kinases), PAK1 is the major “pathogenic” kinase and its abnormal activation is responsible for some diseases including viral infection. PAK1 may also suppress both T and B cells, affecting antibody production. CAPE found in propolis were shown to inhibit RAC, which activates PAK1. These authors also mentioned a large-scale clinical trial using propolis for COVID-19 patients in the Netherlands ( Thus, we recommend the investigation of propolis effect against SARS-CoV-2 replication in vitro. A possible inhibitory action of propolis in ACE activity could be investigated in vivo as well.

Researchers have tested the use of propolis for different purposes and the number of bioproducts containing propolis (nanoparticles, mouthwashes, gels, chewing gums, ointments) and patents have increased, [21] indicating its promising therapeutic applications. Since propolis is nontoxic and practically without side effects, patients should ask for the medical recommendation to include propolis in combination with the antiviral agents or with the new vaccines when available. Previous research from our group revealed that propolis might exert a synergistic action in combination with some antibiotics. [22, 237, 238] Thus, we strongly suggest the evaluation of propolis effectiveness in combination with different antiviral drugs to obtain a new treatment for viral diseases. In this context, Altindis et al. [239] investigated in vitro the effects of propolis and olive leaf extract (OLE) alone or in combination with acyclovir regarding their antiviral activity against HSV-1. This combination exerted an efficient antiviral effect and caused no CPE, suggesting that reduced doses and side effects of acyclovir could be achieved by a simultaneous administration of propolis and OLE. Propolis could be used for COVID-19 prevention (prophylactic effect) or even in combination with the recommended treatment of this disease (therapeutic effect) due to its biological properties. We also recommend the evaluation of propolis administration simultaneously with vaccines, due to its adjuvant properties, to enhance the individuals’ immune response. The addition of propolis to antivirals and vaccines should be investigated in clinical trials aiming to use lower doses of the drugs and reduce side effects moreover, the use of capsules with standardized extraction and defined concentrations of propolis should be considered. Propolis antiviral and immunomodulatory activity and proposals for anti-SARS-CoV-2 approaches are shown in Figure 3 .

Propolis antiviral and immunomodulatory activity and proposals for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) approaches. Propolis and its constituents can induce (1) pro-inflammatory cytokine production, (2) reactive oxygen species (ROS) generation by innate immune cells and (3) interferons (IFN) type I production by virus-infected cells in the onset of viral infection. (4) Antibody production (humoral immunity) and (5) cell-mediated immunity may be enhanced by propolis. Propolis may be investigated in combination with (6) vaccines and (7) antivirals. Propolis anti-inflammatory action may help to control (8) the cytokine storm. (9) p21-activated kinases (PAKs) and (10) angiotensin-converting enzyme (ACE) inhibition. (11) transmembrane protease serine 2 (TMPRSS2) interaction with propolis constituents can be considered in the control of SARS-CoV-2.

Last but not least, this review was designed in an attempt to positively contribute to this pandemic scenario, to inspire new research that may help with different aspects of COVID-19, controlling the virus and benefiting people who are infected or not.

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