chemistry – Page 5 – Lablogatory

Osmolality

Just like molarity, normality and mass units such as mg/dL, osmolality is a way of expressing concentration. In fact, osmolality is the concentration, of dissolved ions/particles of all types in a solution. It is expressed as milliosmoles per kilogram of water (mOsm/kg of H₂O) and it takes into account everything dissolved in that solution, regardless of size or charge. In a solution of serum, plasma or urine, that’s a LOT of dissolved particles!

The most commonly used osmometers measure this combined concentration by means of a technique called freezing point depression. Any time you dissolve a solute in a solution, you lower the solution’s freezing point below that of the pure solvent. Thus ocean water freezes at a lower temperature than pure water. Serum, which is a water-based solution, also has a lower freezing point than pure water. Measuring how much the temperature is lowered is an accurate means of measuring the number of particles present.

On the other end of the spectrum, dissolved particles also change the temperature at which a solution becomes a vapor. There have been osmometers on the market that measure osmolality by vapor pressure measurements. These osmometers have the drawback of not being capable of detecting volatile solutes which may be present in the solution, as they would boil off before the serum reached its boiling point. This would include such compounds as ethanol, methanol, isopropanol and ethylene glycol. A freezing point osmometer detects and includes these solutes in its measurement.

Osmolality can also be estimated from the concentrations of the major solutes present. This is referred to as a calculated osmolality. Many main chemistry analyzers currently in use will give a calculated osmolality, or the LIS can be programmed to calculate it. There are multiple formulas in use for calculating osmolality, but they all use the concentrations of the major solutes contributing to osmolality, sodium (and chloride), glucose and urea nitrogen (BUN). In the US the formula for a calculated osmolality is some version of:

(2 X Na⁺ in mEq/L) + (glucose in mg/dL ÷ 18) + (BUN in mg/dL ÷ 2.8)

Two times the sodium accounts for the chloride also. If you are using SI units, this formula is simply 2 times the sodium + glucose (in mmol/L) + BUN (in mmol/L).

A calculated osmolality cannot be used to replace a measured osmolality. Since the calculated osmolality uses only sodium, glucose and urea nitrogen, this method will give falsely low osmolalities whenever there is a significant excess of solutes other than these three. The presence of the volatile solutes mentioned earlier will not be detected. In addition, more common solute excesses such as severe lactic acidosis or ketoacidosis will also cause an elevated osmolality that would be missed with a calculated value. The primary clinical utility of a calculated osmolality is in conjunction with a measured osmolality. Subtracting the calculated from the measured value can tell you whether there is an “osmolal gap” present. The difference in the two values is caused by solutes other than the three included in the calculation, and may indicate the presence of an agent that should be followed up on, like alcohol or ethylene glycol.

Measuring urine osmolality is a good way to determine the ability of the kidneys to retain water and concentrate the urine.

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

But How does it Work?

There’s an old saying that goes like this: if you understand it, it’s obsolete. Sadly, in this day and age of rapidly advancing technology, this saying is truer than ever. I say “sadly” because what this means for us in the laboratory is that we are becoming less and less likely to be able to troubleshoot and repair our own instruments. This is another thing I sometimes miss about bygone laboratory medicine. Taking instruments apart used to be fun.

Many instruments now are considered “black boxes” by clinical laboratory scientists. They may not understand the principles behind how the instrument works, and even if they do know, they are not inclined or encouraged to attempt to fix it if it stops working. In the early days of laboratory medicine, we could repair most of the instruments we used in the laboratory. Now we can repair almost none of them. Instruments have become so sophisticated, with so many bells, whistles and extras, that even if you know the basics of how the instrument works, being able to fix it when it goes down is no longer a possibility.

For example, most big main chemistry analyzers work on the basis of two principles: some type of photometry and ion-selective electrodes (ISE). Knowing that information used to make it possible to troubleshoot and do some repairs on the photometer system, as well as replace ISEs. Troubleshooting and repair was a matter of checking the functioning of your optics and cleaning as necessary, replacing tubing and replacing electrodes and fluids for the ISE part. Medical technologists were much more likely to repair systems themselves than to call Service in. That ability is rapidly becoming a lost art however.

Modern instruments are much more than a photometer and a set of ISEs. The sheer volume of working parts in current instrumentation is orders of magnitude higher than in old instruments, and most of those parts are robotics in the instrument rather than analytical components. With more sophistication and technical abilities though, come more things that can go wrong. And these are things that cannot be fixed by the clinical laboratory scientist working the instrument.

Of course, for every lost ability is a gained ability. While local troubleshooting may not be possible, many of these major instruments are now connected to the internet. Troubleshooting can be done remotely by the people who do have the knowledge to service them. Honestly, I probably do not want to return to the days of fixing my own instruments in the case of the big chemistry analyzers, but I still do enjoy troubleshooting my mass spectrometers. And it was nice to know I could fix things.

Bilirubin

Occasionally I get a question about exactly what forms of bilirubin our assays are measuring, or what a direct bilirubin (DBili) measures versus a total bilirubin (TBili). This post is a short discussion on bilirubin.

Bilirubin is the degradation product of heme, which is the oxygen carrying group found in hemoglobin. Every hemoglobin molecule has four heme groups. Each of those heme groups will degrade into a bilirubin, so anything causing red blood cell destruction or increased turnover will generally result in increased bilirubin levels. This bilirubin is called unconjugated bilirubin because nothing is bound to it. Unconjugated bilirubin is extremely water insoluble and it is carried to the liver, mostly by albumin. In the liver, an enzyme called UDP-glucuronyltransferase (UDP-GT) adds glucuronic acid molecules to the bilirubin. This bilirubin is now called conjugated bilirubin, and it is water soluble.

There are four basic forms of bilirubin found in blood, unconjugated, mono-conjugated (one glucuronic acid added), di-conjugated (2 glucuronic acids added) and protein bound. They are also referred to as alpha, beta, gamma and delta bilirubins, respectively. How much of each form is measured depends on the assay used to measure it.

Most of the currently available wet chemistry assays for bilirubin use diazotized salts of sulfanilic acid to react with bilirubin and form a colored compound. The initial reaction occurs with the conjugated, water soluble forms, and is referred to as the “direct-reacting” or “direct” bilirubin. Then an accelerant is added to the assay, and the rest of the bilirubin reacts, giving you a total bilirubin. Therefore DBili is a measure of most of the conjugated forms but usually also includes any protein-bound forms that may be present. Unconjugated bilirubin is part of the total, but is not measured directly by diazo reactions.

A few assays give a direct spectrophotometric measurement of the conjugated and unconjugated forms themselves, specifically the dry slide technology available on Ortho Diagnostics instruments. Other than these few assays, to directly measure the unconjugated bilirubin requires an HPLC method which measures all the various forms separately. Transcutaneous bilirubin instruments give a measure of total bilirubin.

In disease states, monitoring the TBili concentration is important, however knowing whether the bilirubin present is conjugated or unconjugated will give you an idea of what the underlying problem may be. Elevated unconjugated bilirubin (high total with low direct) suggests increased hemolysis, or inability of the liver to conjugate bilirubin. Neonatal jaundice is usually caused by unconjugated bilirubin due mainly to immature liver enzymes, ie not enough UDP-GT to conjugate all the bilirubin present. Additionally, high unconjugated bilirubin will move into the tissues because of its water insolubility, and can cause brain damage if the concentration is high enough for long enough, a condition known as kernicterus. Elevated conjugated bilirubin (high total and high direct) suggests conditions causing inability of the liver to properly drain bilirubin into the bile (cholestasis). Prolonged high unconjugated bilirubin is a more serious condition than prolonged high conjugated bilirubin, because conjugated bilirubin can be excreted in urine and tends not to accumulate in the tissues.

The ABCs of Vitamin D

Vitamin D is produced from 7-dehydrocholesterol in the skin when the skin is UV-irradiated by sunlight. In humans, vitamin D3 or cholecalciferol is specifically produced. Plants produce predominately vitamin D2 or ergocalciferol. While human bodies can utilize vitamin D2, they preferentially use D3 and the rest of this post will be talking about D3.

Vitamin D is actually more of a steroid hormone than a vitamin. Unlike vitamins, Vitamin D is produced in the body, and like hormones, it is produced in skin cells and acts on cells at sites distant from its site of production. The primary functions of vitamin D include actions to increase blood calcium levels. Low blood calcium levels cause a release of parathyroid hormone (PTH), which in turn activates vitamin D. Vitamin D then actively increases calcium absorption from the intestine and helps mobilize calcium from the bone. Vitamin D has been studied and associated with health benefits ranging from decreasing the risks of getting various types of cancer to lowering the risks of heart attacks and type 1 diabetes, causing it’s measurement to become an almost routine part of most physicals and resulting in a large testing volume in the lab.

In the body, Vitamin D exists in multiple forms. The vitamin D produced in the skin is hydroxylated in the liver to give 25-hydroxy-vitamin D (25-OH-D), the main circulating form of Vitamin D. This form is not biologically active. When the correct physiological signals are received however (low calcium and high PTH), another hydroxyl group is added to 25-OH-D in the kidneys, to form the biologically active form, 1,25-dihydroxy-vitamin D (1,25-diOH-D). 1,25-diOH-D is present in very low concentrations.

25-OH-D is the form measured when assessing a person’s overall vitamin D status. It is in the greatest concentration in the body and has a half-life of 2 to 3 weeks. It can be measured by a variety of immunoassays as well as by tandem mass spectrometry. Unfortunately not all assays measure the same forms of 25-OH-D, and thus values can differ significantly depending on the assay used to measure them. This is a major problem because vitamin D has health-based reference intervals, not population based. This means that studies have determined that 25-OH-D concentrations below 30 µg/dL suggest vitamin D deficiency. So all assays use this 30 µg/dL cut-off, even though all assays don’t measure the same amount of vitamin D in samples. Cholesterol is another example of an analyte with health-based reference intervals. We say a person’s cholesterol should not exceed 200 mg/dL, rather than establishing the population-based reference intervals for cholesterol for our population.

1,25-diOH-D is much more difficult to measure because it occurs in much lower concentrations, with a half-life in the body of 4 to 6 hours. It is generally only measured when renal function is impaired, or to check for diseases involving vitamin D metabolism. It is often ordered in error when the healthcare provider actually wants to know the patients overall vitamin D status, the 25-OH-D concentration. Assays for measuring 1,25-diOH-D include radioimmunoassays and extraction followed by liquid chromatography-tandem mass spectrometry. This testing is usually performed in reference labs.

Making Solutions

It often seems to me that the art (or science) of making solutions is becoming a lost one. In this current day and age when most of our solutions come in a pre-made form and only require mixing, or at most, thawing and mixing, I believe we’re losing the ability to make solutions ourselves.

This thought came to me when I overheard a comment in a hallway about a shortage of physiological saline. It was back-ordered and we’d be in dire straits soon if we didn’t get any in. And I wondered: if we have solid sodium chloride in the laboratory and we have water, how can we have a shortage of physiological saline? And physiological saline is incredibly easy because you don’t even need to know the molecular weight of sodium chloride. If you want 0.9% physiological saline, that 0.9 grams of NaCl in 100 ml of water.

The same is true for any other easily made-up solution. We’re so used to having them pre-made for us, that we’re forgetting everything we learned in school about how to make solutions. Of course, being able to make solutions from scratch does presuppose that the lab still has chemicals, a balance and a pure water source. My lab does, but that’s because we run a lot of laboratory developed tests (LDT). Most laboratories may no longer keep chemicals, and even if they do, using a home-made reagent turns your assay into a LDT. Plus so many pre-made reagents have proprietary formulas that making them up from scratch is not possible. But for simple reagents like physiological saline, that perhaps is being used to perform dilutions or wash cells, I find it kind of sad that we rely on “store bought” reagents so much that we never consider making them ourselves. In that respect, I guess I’m something of a lab dinosaur.

Don’t get me wrong. I’m totally in favor of making our lab lives as easy as possible and pre-made solutions are one of the wonderful things that do that for us. In addition, if you buy pre-made reagents, you remove one variable that can affect results – was the reagent made up correctly, using the correct chemicals. On the other hand, I believe it’s also a good idea to know how to make up a solution if you should need to do so.

It’s a little comforting to know that this loss of ability may not be confined to the lab. I heard a pharmacist talk about a shortage of total parenteral nutrition (TPN) solution, which I suspect at one time every pharmacist knew how to make up from scratch.

Dried Blood Spots – Sample Extraordinaire

When you mention dried blood spot samples (DBS), most people think of newborn screening. That’s natural because the most common usage for DBS is newborn screening. However, DBS samples are actually one of the most versatile, stable and easily stored samples that it’s possible to collect from a human. And did I mention useful?

To create a DBS sample, whole blood, often from a finger or heelstick, is spotted onto a very specific weight of filter paper. The weight and type of filter paper is important so that all DBS are created equally, and so that differences in testing are not introduced due to filter paper differences. Enough blood is used to thoroughly saturate the paper (in most case roughly 50 uL will saturate the marked dot) and then the blood spot is allowed to dry completely.

DBS are ideal samples for population based testing. The list of positive attributes is long. They are easily obtainable (fingerstick). They use very little blood (50-60 uL). Once dried they are not subject to the sample degradation effects that plague liquid samples. They are simple to transport with no possibility of spilling or breaking. They store easily, taking up very little space, and studies suggest that once dried, the sample is stable for years, whether at room temperature, refrigerated or frozen.

In addition to these obvious benefits, a truly remarkable number of analytes can be measured from one or two 6 mm punches out of a dried blood spot, with a punch containing roughly 10 uL of blood. Protein enzymes are generally stable in a dried blood spot, allowing enzyme activity to be measured from DBS. Viruses such as HIV can be measured in DBS. Even RNA and DNA is stable in these spots, as evidenced by the PCR assays that are being performed using them. These assays include such things are Cystic Fibrosis (CF) mutation testing and screening for severe combined immunodeficiency (SCID).

Besides the PCR testing for CF and SCID, the newborn screen itself uses the DBS sample to measure some or all of the following: either T4 or TSH for hypo- or hyperthyroidism, hemoglobin variants for sickle cell anemia, 17-hydroxyprogesterone for congenital adrenal hyperplasia, immunoreactive trypsinogen for CF, amino acids and acylcarnitines for amino acid, fatty acid and organic acid disorders, and an enzyme for galactosemia. Each one of these assays is performed using the single punch from a DBS. The DBS is an almost overlooked sample type. However it has the potential to be used for a huge variety of testing.

Autoantibodies in Diabetes Testing

In order to diagnose diabetes, technologists measure glucose or hemoglobin A1C concentrations or perform an oral glucose tolerance test. That being the case, what are autoantibody tests ordered on patients with diabetes or pre-diabetes, or those suspected of having diabetes?

Diabetes is classified as type 1, type 2, gestational and “other specific types”, with about 40 different types of diabetes known. Type 1 and type 2 account for the majority of the diabetes cases, with type 1 accounting for roughly 10% of all diabetes and type 2 accounting for roughly 90%. Type 1 diabetes (sometimes referred to as autoimmune diabetes) is caused by the autoimmune destruction of the pancreatic beta cells. This results in an absolute insulin deficiency and requires insulin to treat. Type 2 diabetes is associated with obesity and insulin resistance and is usually treated initially with diet, exercise and weight loss. Several drugs are currently available to treat hyperglycemia in type 2 diabetes if behavioral modifications do not succeed in lowering glucose levels.

The diagnosis of diabetes is made based on the glucose level and/or hgbA1c concentration, however, not all cases are clear-cut or follow the classic presentations or indications to allow classification as type 1 or 2. Yet determining whether diabetes is type 1 or 2 is important since the treatments are different. Also, persons with type 1 diabetes are prone to other autoimmune disorders, such as autoimmune thyroid disease. In cases that are less than clear-cut, measuring autoantibodies can provide useful information. The main autoantibodies, along with their utilities include:

ICA – Islet cell cytoplasmic autoantibodies are rare in the general population but are present in 70-80% of new-onset type 1 diabetics. They are also present before the onset of type 1 diabetes, so the presence of ICA in a non-diabetic is indicative of a markedly increased risk of type 1 diabetes. If ICA are detected in a type 2 diabetic, it suggests the slowly progressive autoimmune destruction of the beta cells, and that the person actually has a form of diabetes known as latent autoimmune diabetes of adulthood (LADA). Although present in 70-80% of newly diagnosed type 1 diabetics, ICA decline to a frequency of about 5 – 10 % by 10 years post diagnosis.

GAD65/GAD67/GADA – these are the 65 KD and 67KD forms of glutamic acid decarboxylase autoantibodies. GADA like ICA are present in roughly 70-80% of new-diagnosis type 1 diabetes. They also are indicative of progression to type 1 diabetes if found in non-diabetics or type 2 diabetics. However GADA are also found in up to 3% of the general population, so ICA are more specific for type 1 diabetes.

IA2 or IAA – insulin autoantibodies are the least common type of autoantibody present at onset of type 1 diabetes, only occurring in 50-60% of children, and occurring uncommonly in adults with new onset type 1 diabetes. They are also the least disease-specific of the autoantibodies. In addition assays to measure IA2 do not distinguish between antibodies to insulin that may be produced against insulin being administered, versus insulin autoantibodies.

IA-2A – insulinoma-associated-2 autoantibodes are present in roughly 60% of new-onset type 1 diabetes and generally more prevalent then IAA.

ZnT8A – This is the newest autoantibody discovered and it is raised against the transporter that moves zinc from the cytoplasm to the insulin-containing secretory granules in the beta cells. ZnT8A are present in 60-70 % of new onset type 1 under the age of 20, and about 40% after 20 years old. They are also present in 14% of cases which are negative for GADA, IAA and IA-2A. They are also common in LADA patients so can be useful in that diagnosis.

The autoantibodies are especially useful when the diagnosis of the type of diabetes is unclear, and when there is some suspicion that a person with type 2 diabetes may in fact have autoimmune diabetes. Because none of these autoantibodies are present in greater than 80% of type 1 diabetics, measuring several of them is sometimes necessary in order to sort out the diagnosis.

Newborn Screening for Severe Combined Immunodeficiency

The most recent disorder that has been recommended for addition to all US newborn screening (NBS) programs is severe combined immunodeficiency (SCID). SCID is actually a group of at least 14 primary immunodeficiencies which affect the individual’s immune system, making it impossible for them to adequately fight off infections. A baby with SCID who does not receive treatment rarely survives the first year of life, being unable to clear repeated and massive infections. Everyone probably remembers the story of the “Bubble Boy” who survived 12 years in a completely sterile environment. The “Bubble Boy” had SCID.

Screening newborns for this disorder would seem like a no-brainer; however, because SCID is actually many different primary immunodeficiencies, finding a screening test that would pick up all or the majority of them has been problematic. In recent years, people began looking at one of the hallmarks of all SCID, a lack or very low number of functional T-cells.

During maturation in the thymus, T-cells undergo gene rearrangement, and during this process small extra-chromosomal circles of DNA are created as the segments of DNA are clipped out of the gene. These small DNA circles are call T-cell receptor excision circles, or TREC. In 1998, Douek et al (1) developed a PCR assay to quantify TREC as a measure of thymic function. When that assay was published, researchers began wondering whether there would be any TREC produced in a disorder like SCID with no functional T-cells. Very quickly the TREC PCR assay was adapted to measure the presence of TREC in dried blood spots, and several papers showed that in SCID individuals, essentially no TREC are produced. Thus the PCR assay for TREC became a viable screening test for SCID in newborns.

Currently 18 States are either already screening for SCID or are in the process of adding SCID screening to their NBS. There is a ways to go before this screening becomes part of all programs in the US, however, given the morbidity and mortality associated with the disease and the availability of a test, it’s hopefully only a matter of time.

1. Douek DC, McFarland RD, Keiser PH, Gage EA, Massey JM, Haynes BF, et al. Changes in thymic function with age and during the treatment of HIV infection. 1998. Nature. 396:690-695.

Pseudohyponatremia: Is This Sodium Really Low?

Periodically I get a call from a clinician saying, “What’s wrong with your sodiums?” In general, this call is triggered by a sodium <125 mmol/L. My first response question is always: What are the child’s protein and/or lipid levels?

At issue here is the type of ion-selective electrode (ISE) used to measure the electrolytes. There are two basic types, indirect and direct, and knowing which one your chemistry analyzer uses is important. Direct ISEs are exactly that. They measure the ion activity in the sample directly, in whatever fluid volume is present in the sample, and are basically not affected by other constituents in the sample. The activity is then converted to concentration and a result is produced. Indirect ISE’s do not do a direct measurement. They dilute the sample first and measure the concentration of electrolytes in the diluted sample. This usually works well, but becomes problematic when the sample happens to have a high concentration of proteins or lipids. The reason for that is this: systems using indirect ISE measurement assume that the sample is all water. In reality, normal plasma/serum is roughly 93% water with 7% solids present (proteins and lipids). If the sample being analyzed has less than 93% water, for example when either protein or lipid makes up more than 7% of the volume, the resulting measurement will be falsely low, as you can see from this table. A normal, 7% solids sample that an indirect ISE measurement would give you a value of 135 mmol/L; if the solids are 20%, that sample will give you a value of 116 mmol/L.

True concentration	% water	Direct ISE measurement	Indirect ISE measurement
145 mmol/L	93	145 mmol/L	135 mmol/L	(145/0.93L)
145 mmol/L	80	145 mmol/L	116 mmol/L	(145/0.8 L)

This is called pseudohyponatremia. The sodium is not really low; it’s perfectly normal. The instrument is giving you a falsely low value. The vast majority of wet chemistry analyzers measure electrolytes by indirect ISE. Only a few big chemistry analyzers measure electrolytes using direct ISEs, and those usually have a correction factor so that the directly measured results are more in line with the big majority of indirect ISE measurements.

What can you do about falsely low sodiums caused by hyperproteinemia or hyperlipidemia? If it’s related to lipids, you may be able to clarify the sample by centrifugation or chemicals and get a real result. Alternatively, blood gas analyzers and some POC analyzers, like the i-STAT, measure electrolytes by direct ISE. If you have or can get a whole blood sample, you can use these analyzers to give you a real result. Otherwise you may be explaining pseudohyponatremia to a concerned physician.

On the Lab Medicine Website

In case you’ve missed it, here is the table of contents for the current issue of Lab Medicine. New articles are uploaded regularly, so be sure to check back often.

Theoretical knowledge helps troubleshoot wonky results, but unfortunately that knowledge is easy to forget if it’s not used every day. If you’ve worked the chemistry bench long enough to have forgotten some of theory behind the analytes, check out this series of articles to refresh your memory.

In the latest edition of our podcast series, Dr. Alex Thurman walks listeners through diagnosing a new acute leukemia in the middle of the night.