Behaving Oddly- Hereditary Spherocytosis

Hereditary Spherocytosis (HS) is the commonest disorder of red cell membrane leading to haemolysis (incidence~500/million). It is inherited in an autosomal dominant manner in 75% of cases and recessively in the remaining quarter. Subjects with recessive inheritance tend to have more aggressive disease and present earlier in life, as with many other disorders with recessive inheritance.

HS has many odd, non-intuitive features. Newborn with HS do not have anaemia, but are diagnosed due to prolonged jaundice. Adults present with anaemia, jaundice and with bilirubin containing gallstones. Thus, recurrent gallstones with jaundice may not necessarily presage primary gallstone disease, but may indicate underlying HS. Anaemia may be mild or absent because of compensation by the marrow, thus leading to difficulty in diagnosis. The diagnosis is usually made by noticing spherocytes on the blood film. It is thought that because of defective anchoring of the red cell membrane to the underlying cytoskeleton, bits of the membrane are gradually lost, reducing the surface area to volume ratio, causing spherocytosis.

Oddly, subjects with HS may develop obstructive jaundice due to CBD stones in addition to underlying indirect hyperbilirubinaemia. When this happens, red cell survival is paradoxically increased. This is thought to be due to increase in the cholesterol content of the red cell membrane which increases its distensibility and thus reduces the propensity for lysis while passing through the splenic sinusoids.

As stated above, mild HS may not be associated with anaemia due to compensation by the bone marrow. The reticulocyte count will be raised in such subjects, but may return towards normal after splenectomy. An important clue to the diagnosis of HS, particularly in infants, is the presence of a high mean corpuscler haemoglobin concentration (MCHC). Normally, MCHC would be expected to be around 31. In HS, MCHC is 36 or higher. A combination of high MCHC (>35) and a high RDW (15 or higher), is virtually diagnostic of HS. I know of no other condition that lends itself to diagnosis by looking at the MCHC alone.

Subjects with HS have more porous red cell membrane. While this allows Na to enter freely, thus increasing the risk of osmotic haemolysis (exploited in the osmotic fragility test, now supplanted by more sensitive and specific tests such as EMA), it also leads to a leakage of K ions when blood is cooled after being drawn. This often leads to the finding of pseudohyperkalaemia in such subjects.

Other odd things happen in these subjects. While splenectomy imparts a lifelong higher risk of arterial and venous clots in HS, due to higher leucocyte, red cell, platelet and fibrinogen levels, non-splenectomised subjects with HS actually have a lower cardiovascular risk than their relatives. It is thought that higher serum bilirubin and lower serum cholesterol protects such subjects against cardiovascular events.

Euler's Number, e

e is perhaps the most celebrated, most feted number in Maths this side of pi. First described by Euler, it is difficult to define as it's an irrational number, i.e. without an integeric value and unexpressable as a fraction. It is best denoted as the limit of (1+1/n)^n, when n tends to infinity. The mathematical value of e is approximately 2.71828..

You do not begin to appreciate the importance of e until you consider the following problems, provided as examples:

1. A bank offers you 5.6% interest rate, compounded for 5 years. How much would you get back after 5 years?
2. UK has a population of 63 million, Germany has 78 million. UK is growing at the rate of 2 new subjects per 1000 per year, Germany at roughly half that. How many years would it take for UK's population to catch up with that of Germany's?
3. A food item will spoil at a critical bacterial mass of 5 million per gram of foodstaff. If initially, there were only 10,000 bacteria, and if the number of bacteria grow from 10,000 to 20,000 in 1 minute and to 250,000 in the next 10 minutes, how long would it take for the food to spoil?

You need e to solve all the problems. Essentially, if an entity is growing continuously, at a given rate, you can use e to calculate its growth. e is the natural base log (log 10 being the "common" log). OTOH, the natural log of a number when the result is expressed as an exponent of e is called "ln" (l for Lima, n for November, using phonetics.

A simple way to remember this is that e^kt gives you the value of an entity growing at the rate of "k" after a certain time period "t", while ln x gives you the exponent- i.e. the power by which the entity has grown. If the rate of growth is known, ln x will let you find the time taken for the entity to grow from y to x.

Things will become clearer with the above examples.

1. A bank offers you 5.6% interest rate, compounded for 5 years. How much would you get back after 5 years if you invested £5000?

You might think the answer is 5000*(1.056)^5, but you would be wrong. The answer of 6565 worked this way is less than what you'd actually get.

The answer is 5000*e^(0.056*5)= approximately 6616. This is how compound interest is calculated.

On the other hand, you might wonder how long it would take for your money to double at this rate of 5.6%? If time for doubling is denoted as t, you get e^(0.056t)= 2. However, you know that ln 2 = 0.693 from a scientific calculator. Hence ln 2 = 0.693 = 0.056t or t = 12.375 years.

Since interest rates are expressed as percentages, you can simply multiply both numerator and denominator by 100 and get t=69.3/5.6= 12.375 years.

In practice, 69.3 is rounded off to 72 to allow us to take advantage of the fact that 72 is divisible by many small integers (the usual duration of a fixed deposit). However, this gives us an approximate result. Thus 72/5.6 in the previous example would give 12.85 years. This is called the rule of 72.

Similarly, you can calculate the trebling time for your deposit at the same rate of interest. Thus, e^0.056t = 3 . ln 3 = 1.10 approximately. Thus for trebling, you can use the rule of 110. Your money will treble in 110/5.6 = 19.64 years.


2. UK has a population of 63 million, Germany has 78 million. UK is growing at the rate of 2 new subjects per 1000 per year, Germany at roughly half that. How many years would it take for UK's population to catch up with that of Germany's?

In this case, if the projected period for equalisation of numbers is t, you can write the equation as:

63 million*e^(0.002t)= 78 million* (e^0.001t)

Or e^.002t/e^.001t = 78/63.

Here, it is useful to know that since e is a logrithmic function, e^x/e^y = e^(x-y).

Thus, in the above problem, e^(.002t-.001t) = 78/63.

Or e^.001t = 1.238

now, ln 1.238= 0.213 = .001t

or t = .213/.001 = 213 years.


3. A food item will spoil at a critical bacterial mass of 5 million per gram of foodstaff. If initially, there were only 10,000 bacteria, and if the number of bacteria grow from 10,000 to 20,000 in 1 minute and to 250,000 in the next 10 minutes, how long would it take for the food to spoil?

Here, after 1 minute, 10,000* e^(k.1), where k is the rate at which bacteria are multiplying.

Or 10,000*e^k= 20,000- This is equation 1.

After a further 10 minutes, 20,000*e^[k.(1+10)]= 250,000- This is equation 2.

Using logrithmic notation, e^[k(1+10)] can also be written as e^k * e^10k

Dividing equation 2 by equation 1, we get:

(20,000*e^k*e^10k = 250,000)/(10,000*e^k = 20,000)

You get 2e^10k = 12.5

or, e^10k = 6.25.

ln 6.25 = 1.83 = 10k, or k = .183

How long will it take to attain the critical mass of 5 million bacteria per gramme? (call this T)

10000*e^(.183T) = 5 million

or e^.183T = 500

ln 500 = 6.21 = .183 T,

or T= 6.21/.183 = 33.93 minutes.

The Nyquist Limit

Doppler echocardiography is often used to assess stenotic or regurgitant valves. Two types of doppler signals are used- pulsed wave doppler (PWD) and continuous wave doppler (CWD). Both serve different purposes and can be viewed as complementary.

PWD is used as a localising tool. It accurately detects that a systolic murmur is, for example, a consequence of aortic stenosis rather than mitral regurgitation. By producing a spectral image, PWD demonstrates a direction of flow towards or away from the tranducer. A spectral wave of aortic stenosis would, for example, be directed away from a transducer placed at the apex. In the case of PWD, a single transducer does both the sending and receiving.

Echocardiography relies on the shift in ultrasound frequency caused by red cells flowing towards or away from the transducer. This is called doppler shift and is given by F= 2Fo.v.cos theta/c, where Fo is the transmitted frequency, v denotes velocity of blood flow, theta is the angle between the transducer and plane of flow and c is the velocity of ultrasound waves in the medium in use, in this case, blood. When the transducer is parallel to the direction of flow, theta is 0, and cos theta is 1. Thus F= 2Fo. v/c.

Note that the doppler shift, i.e, the detected change in frequency is proportional to twice the emitted frequency. This illustrates an important limitation of PWD called "Nyquist limit". The Nyquist limit is always half the sampling frequency. That is to say that the maximum frequency accurately detectable with a sampling frequency of f is f/2. If emitted frequency is more than the Nyquist limit for the sampling frequency, than a phenomenon called "aliasing" occurs, where the recorded spectral wave is cut off at its peak and appears on the other side of the baseline (mimicking combined stenosis and regurgitation in the case of pure stenosis, for example), thus giving a distorted image. One way of reducing aliasing is by reducing the "sample volume", i.e. by placing the transducer as close to the valve being examined as possible. Thus, the ultrasound waves have to travel a shorter distance, thus raising the frequency at which sampling occurs, and thus the Nyquist limit.

CWD overcomes this shortcoming by using 2 transducers- one to transmit, and one to receive. There is thus no Nyquist limit. CWD is thus used to measure high velocity flows, such as through a severely stenotic valve (velocity being a function of Doppler shift in the above equation). Using the modified Bernoulli equation, one can estimate the pressure change across a defective heart valve. Thus Delta P (change in pressure)= 4 V^2. For example, if blood is flowing through a stenotic aortic valve at 4m/s, the pressure differential across the valve is 64 mm Hg.

The limitation of CWD is that while it can measure, it cannot localise. Thus, it is likely to confuse AS with MR if the jets happen to be in range. This distinction is only achievable by PWD, which samples a limited frame. In practice therefore, one should localise the jet with PWD, taking care to avoid aliasing and then measure the velocity and thus delta P with CWD.

Immunohistochemistry as a Diagnostic Tool in Cancer

Unfortunately, cancer has often spread before it is diagnosed. In such cases, it can be a diagnostic challenge to determine the site of the primary neoplasm. In some cases, an extensive search for the primary may prove fruitless, leading to a diagnosis of carcinoma with unknown primary or CUP. It is in such cases that immunohistochemistry comes into its own.

Immunohistochemistry (IHC) is the application of a large panel of monoclonal antibodies directed to a plethora of tissue specific antigens to the putative cancer tissue, followed by the use of immunofluorescence to identify stained areas.

Every tissue has its own signature antigens. Because such antigens will often be present in other tissues, they can be rather non-specific and no one antigen should be relied upon while looking at a biopsy sample. Rather, pathologists rely on a panel of antigens while performing IHC.

Carcinomas, i.e cancers of epithelial origin will demonstrate cytokeratins (CK). CK 8 and 18, and CK 1-3 are universally present in all carcinomas and are initially looked for to differentiate carcinomas from tumours of mesenchymal origin, i.e. sarcomas.

Sarcomas themselves will always have a protein called vimentin. Unfortunately, vimentin can also be produced by the occasional carcinoma.

The most useful cytokeratins are CK7 and CK20. Presence or absence of one or both of these cytokeratins is initially employed to narrow down the possible range of neoplasms.

Thus CK7+ and CK20+ will include transitional cell carcinoma, ovarian mucinous carcinoma and pancreatic carcinoma. CK7+ but CK20- will identify breast, lung, thyroid, gallbladder and cholangiocarcinoma. CK20+ and CK7- comprises gastrointestinal, particularly colon carcinoma and Merkel cell carcinoma. CK7- and CK20- narrows the field down to prostate, renal, adrenal and hepatocellular carcinomas.

Here I will give examples of some of the commonest clinical challenges faced by pathologists and how they are resolved.

1.Tumour deposits in pleura. Primary may be lung, breast, thyroid or pleura itself. Stain for TTF1, CK5/6, WT-1, calretinin, CK7, CK20, CEA, PAX 8, gross cystic disease fluid protein 15 (GCDFP15), mammaglobin, ER, PR.

Thus, TTF-1 positive- lung or thyroid. Thyroid will demonstrate thyroglobulin and PAX 8, lung will not. If lung, stain for BER-EP4, BG8 and MOC-31. All three are seen only with adenocarcinomas.

TTF1 negative- GCDFP15+, mammaglobin+, CEA positive, ER+, PR+ means breast cancer.

2. Deposits in axillary nodes. Cytokeratin negative, but positive for Melan A, HMB 45, S100. Diagnosis is metastatic melanoma.

3. Liver mass- primary or secondary? CK7-, CK20-, Hep-par 1+, alpha-fetoprotein positive, CEA (p), in situ hybridisation positive for albumin-primary hepatocellular Ca.

4. Liver deposit- CK20+, CK7-, CEA+, CDX2+, villin+ denotes metastatic colon cancer.

5. Peritoneal deposits- CD10+, RCA+, CK7-, CK20-, PAX2+ will mean this is a Clear cell carcinoma of the Kidney.

6. Lymphadenopathy- CD5+, Cyclin D1 positive, CD10-, CD23-, CD19, 20, 22 and 79A+ (pan B cell antigen) denotes Mantle cell lymphoma.

7. Adrenal deposits- CK7-, CK20-, simple CK (1, 8, 18+), EMA-, Melan A+, S100+, AdCB4P means adrenocortical carcinoma

8. Liver deposits- CK5/6+, CK7+, CK20+, uroplakin+, thrombomodulin+ denotes TCC of urothelial tract.

9. Pleural tumour- CK5/6+, CK7+, CK20-, TTF1-, BER-EP4-,WT-1+, calretinin+ denotes mesothelioma.

10. Vulval tumour- CK7+, CK20-, GCDFP15+, CEA+, ER+, HER2- is consistent with Pagets disease of vulval appendage- usually sweat duct.

The Pathobiology of "Autosomal Dominant"

Have you ever wondered why certain traits are autosomal dominant and others are autosomal recessive?

In most cases, the explanation is fairly straightforward. Diseases, such as Huntington's or myotonic dustrophy, which are transmitted in an autosomal dominant fashion are mostly due to the toxic effect of the mutant protein encoded by the abnormal gene. This protein builds up inside the endoplasmic reticulum and interferes with the synthesis of other vital proteins, which then leads to the abnormal phenotype.

With autosomal recessive inheritance, it is usually an useful function that is lost. Both alleles encoding an essential protein are lost, and thus the product can no longer be synthesised, for e.g. the CFTR protein in cystic fibrosis.

There are notable exceptions to this general principle. Sometimes, the product of one normal allele is insufficient to perform the requisite function of the putative gene. This is called haploinsufficiency. Thus, the condition is transmitted as autosomal dominant, with a dose-response gradient between those heterozygous and homozygous for the abnormal allele. Examples of haploinsufficiency contributing to autosomal dominant transmission are dyskeratosis congenita, Williams syndrome and autosomal dominant retinitis pigmentosa. Marfans and Ehlers Danlos syndromes are also inherited in a similar way.

A different, but nonetheless fascinating mechanism of autosomal dominant inheritance is illustrated by an autoinflammatory condition called TRAPS (TNF Receptor Associated Periodic Syndrome). In this syndrome, the 55 kDa TNF receptor on the cell surface is abnormal, and does not shed on binding to the pro-inflammatory cytokine TNF alpha. Thus prolonged signalling through TNF alpha leads to an augmented inflammatory response, with excessive production of NF-kappa B. Further, it is thought that the usual neutralising effect of circulating "soluble" (non-membrane bound) TNF receptor on TNF alpha is reduced.

The TNF alpha receptor is homotrimeric, i.e. it is composed of three similar units. Most subjects with TRAPS have only 1 abnormal allele, usually due to a missense mutation, while the other allele is normal. However, assuming that even one mutated component of the trimer would lead to abnormal receptor function, one can see that even with one normal allele, 7 out of 8 receptors would be abnormal (the odds are only 1 in 2^3 that in a given trimeric receptor, all 3 components would be from the normal allele). Thus, the condition would transmit as autosomal dominant.

The Problem of Isolated Uveitis

One of the most common reasons for referral to the Rheumatologist from the Ophthalmologist is the young subject with recurrent or troublesome episodes of uveitis, often in association with a positive ANA or other features suggestive of an autoimmune aetiology such as a raised ACE. The query is whether such subjects have an underlying systemic disease contributing to their uveitis. Certain features can help narrow down the D/D.

1. HLA B27 associated spondarthritis presents with acute unilateral anterior uveitis that improves within 3 months but often recurs in the other eye. Therefore simultaneous or closely spaced occurrence of uveitis in both eyes is not characteristic of this condition, even if arthralgias are present. Prognosis is excellent.
2. The uveitis associated with IBD is often more chronic, posterior to the lens, and bilateral and more common in women. In subjects with IBD, the uveitis often presents prior to bowel symptoms (10/17 in one series).
3. Anterior uveitis is associated with the presence of deposits on the back of the cornea, called keratic precipitates (KP). In the case of sarcoidosis, these KPs are often large and greasy, therefore fine keratic precipitates make sarcoid unlikely.
4. The uveitis of sarcoid is often chronic, bilateral, posterior as well as anterior and unaccompanied by systemic features.
5. Syphilitic uveitis may not be accompanied by systemic features, In Asians, always rule out tuberculosis.
6. In subjects above the age of 45 with chronic posterior uveitis, rule out lymphoma, particularly DLBCL type of NHL. Higher risk in HIV positive cases. Thus, lymphoma is an uveitis mimic.
7. In children with JIA, uveitis is more common in those with oligoarticular arthritis and positive ANA. Polyarticular involvement and absence of ANA are less commonly associated with arthritis.
8. Behcet's disease often causes a panuveitis, is often associated with retinal vasculitis, is silent, and thus can lead to blindness. It's important to be aware that while anterior uveitis presents with pain, redness, photophobia, headache or brow pain and constricted pupils, posterior uveitis presents with a white eye, is silent, and can only be picked up initially by the presence of vitreous cells on slit lamp examination. Being silent, it can lead to blindness, and therefore needs a high index of suspicion. This is also true for children with JIA.
9. Multiple sclerosis can be associated with pars planitis (intermediate uveitis).
10. TINU or Tubulointerstitial Nephritis with Uveitis is a rare condition that combines uveitis with interstitial nephritis. It can be sen in subjects with Sjogren's syndrome or sarcoid.
11. Other autoimmune conditions that present less commonly with uveitis are SLE and GPA (Wegener's).

All In The Family

A 29 year old man presents with recurrent gout, in association with a very high uric acid level at over 800 umol/l. His blood tests show impaired renal function, with a creatinine of 180umol/l and an e-gfr of 28 ml/min. His father has also had gout from an early age with similarly high serum uric acid levels, but normal renal function. However, a brother had to have dialysis starting at age 36, leading to transplantation. This brother also suffered from gout.

The patient's only dietary risk factor for gout was a fondness for Coca-cola. He did not drink much alcohol and consumed little red meat, shellfish, prawn or offal.

What is the diagnosis?

The Genius of Adolf Eugen Fick

Fick was a 19th century German physiologist. In 1870, he worked out that cardiac output could be measured from a subject's oxygen consumption, if the concentration of oxygen entering and leaving a given organ were known.

Thus, cardiac output= Oxygen consumption/Arterial O2 content-Venous O2 content

It is easy to calculate the arterial or venous oxygen content if you know the O2 saturation (SaO2) in arterial or venous blood, as almost all the O2 is carried by haemoglobin. Each gram of Hb carries 1.34 ml of O2.

Thus, Arterial oxygen content= 1.34*Hb concentration*SaO2

As cardiac output is expressed in l/min and Hb in g/dl, to make matters uniform, you have to multiply the denominator by 10.

Thus, cardiac output (l/min)= O2 consumption (l/min)/[10*1.34*Hb(g/dl)*SaO2]-[10*1.34*Hb(g/dl)*SvO2]

In practice, it is rarely necessary to determine the absolute cardiac output, but Fick's principle has been put to good use to determine the degree of left to right shunt by calculating the respective blood flow to pulmonary (Qp) and systemic circulation (Qs)in conditions such as ASD & VSD. In significant left to right shunt, Qp>Qs. If Qp is more than twice Qs, most authorities would recommend percutaneous closure of ASD or VSD with an Amplatzer device or a surgical closure if the shunt is very large, as with some VSDs.

It is easy to determine O2 saturation during cardiac catheterisation consecutively in the superior venna cava, right atrium, inferior vena cava, right ventricle and pulmonary artery. Pulmonary venous oxygen content is considered to be the same as the oxygen saturation of arterial blood measured during pulse oximetry, provided there is no right to left shunt. The latter can be detected by a drop off in O2 saturation while moving into the putative chamber, e.g decline in SO2 while moving from right atrium to right ventricle, suggests a right to left shunt from a VSD, while the same thing happening while moving down from the SVC to the RA indicates a right to left shunt via an ASD.

As the SVC normally has 3 times the flow as in the IVC, the systemic mixed venous O2 content is calculated as (3*SVC SO2+IVC SO2)/4.

Oxygen consumption can be be calculated by using a special spirometer with the subject rebreathing air and using a CO2 absorber.

Using the Fick principle, Qp/Qs can be written thus:

Qp/Qs= [Oxygen consumption/ 10*1.34*Hb*(Pulmonary venous SO2-Pulmonary arterial SO2)] divided by,
[Oxygen consumption/10*1.34*Hb*(Systemic arterial SO2-Systemic mixed venous SO2)]

or simply, Qp/Qs= Systemic arterial SO2-Systemic mixed venous SO2/Pulmonary venous SO2-Pulmonary arterial SO2

In practice, an alternative and non-invasive way to measure the pulmonary:systemic flow ratio in left to right shunts is to use Doppler Echo to find the area of the aortic and pulmonary outflow tracts and multiply them with their respective velocity time integral.

If each circle has a radius of 5 cm, calculate the area of the shaded portion

A 65-Year Old Lady with Muscle Weakness and High CK

The lady presented with a 4-week history of increasing muscle weakness, resulting in a fall down the stairs on the day of admission. There was very little muscle pain.

Her background history included hyperetension & hypercholestrolaemia, and she was taking amlodipine 10 mg OD, bendroflumethiazide 2.5 mg OD and simvastatin 80 mg OD. She had been on these medications for ~2 years.

On admission, she had grade 4/5 muscle weakness proximally in both upper and lower limbs, without fasciculations or UMN signs. Cranial nerves were not involved.

Tests showed normal renal function, electrolytes & FBC. CK was elevated at 14000 U/l. Urine dipstick showed 4+ blood, but RBC within normal limits on microscopy.

ANA, ENA, dsDNA, Ig were all negative or within normal limits. ESR & CRP were not raised. MR scan of thigh muscles showed diffuse oedema and enhancement on T2 weighted images.

Thoughts?

T-cell Receptor Rearrangement- unravelled

Cells of the adaptive immune system, i.e. B- and T-cells are amazingly diverse. They have to be to deal with myriad antigens that the body is exposed to. This diversity is achieved by rearrangement of the receptors on B and T cells during development. While I was aware of this, there was one unanswered question that had always puzzled me...until today. I'll share this shortly.

It is common knowledge that B cell "receptors" are comprised of immunoglobulins, which consist of heavy and light chains. Both have invariant constant (C) regions and variable regions. It is obviously the latter that contribute to their diversity and specificity. In the heavy chains, the variable regions are further comprised of V, D, and J regions ( stand for variable, diversity and joining). The light chain variable chains lack a D region and only consist of V & J regions. Each V, D or J domain has numerous alleles- up to 60, 70 or even more. Thus there are a high number of combinations possible, remembering that a given Ig chain will contain only one each of V, D or J. (Figure 1). These regions are flanked by signal sequences that allow their recognition in order that recombination may occur. These recognition sites undergo further changes -mutations- on exposure to antigens, a process called somatic hypermutation, resulting in increased affinity of the antibody for its antigen- affinity maturation.

Now we come to the reason for this post. T cells, instead of having immunoglobulins on their cell surface, have dimeric receptor chains- either a combination of alpha and beta or a combination of gamma and delta. alpha-beta T cells make up ~95% of all T cells, while the gamma-delta T cells are in a minority. It was therefore puzzling for me to note that in haematological malignancies such as leukaemias and lymphomas, it was usual to test for rearrangement of receptor chains in gamma chains, which are present in only 5% of T cells, rather than in alpha or beta chains, which make up the other 95%.

Here's why. Using standard methods such as Southern blotting, or increasingly now, PCR, you need a much smaller number of probes if were looking for clonality in T-gamma chains, than if you employed T-alpha or T-beta chains. Firstly, T-gamma chains only have V & J region and lack a D region, unlike T-alpha or T-beta. More importantly, the T-gamma chain has far less recombination-capable segments than either T-alpha or T-beta chains. For example, T-alpha has 70 V segments and at least 61 J segments spread over a large area (thus increasing the difficulty of Southern blotting). On the other hand the T-gamma chain has only 14 V segments and 5 J segments, and is therefore much easier to probe.

BTW, the same enzymes- RAG 1 and RAG 2, are responsible for splicing and rejoining of the various V, D and J regions in both B and T cells, which is the basis of severe combined immunodeficiency when there is a problem with RAG. However, unlike B cells, somatic hypermutation and affinity maturation does not occur in T cells. Nevertheless, further diversity is introduced by an enzyme called Terminal deoxynucleotidyl transferase (TdT) which randomly makes changes in the junctions between V, D and J chains. As the enzyme TdT is only present in immature lymphocytes, its presence or absence can inform the stage in lymphocyte ontogeny at which a lymphoid neoplasm arose.

Figure 1. Germline organisation of the T cell receptor genes

The Lady With Microcytic Anaemia...Revisited

Shonkus, I wanted to revist this case we discussed some time ago, as I learnt something that could throw further light on the diagnosis. To begin with, here's the discussion so far. You are in bold and I am in italics.

Tnx....!!.Hope Ur in Gr8 spirits...FJS reported to me c/o Cough 2 mths, & occasional SOB with Fatigue.She is a housewife nd apparently looks healthy. Took t/t in 2010 for GERD & All.Rhinitis provided by then Physicain Dr Jagdish MD(Int.Med).Reports 2010 depict a normal AEC, ESR as 34mm/1st hr. Hb% was not done at tht time. on 18th instant upon detecting Pallor Inv.were ordered. Hb% was 7.5gm/dl, ESR 50 mm/1st hr, AEC 250, TLC 14,300/cmm, N-73%,RBC count 4.59mill./cmm, RBC indices :MCV 58.6 um3 (82-93),MCH 16.3pg (28-32), MCHC 27.9 g/l(32-36), RBS 95mg/dl, Throat C/s indicate Normal Flora. CXR PA Normal. She was surprised nd her husband attending to her complained of her irregular dietary habits. Upon , gentle prodding she gave a further h/o Bleeding PR since last yr on & off few episodes which she attributed to Piles,one episode 15 days back which stood controlled. Asked for, Thallasemia Profile, a stool Examination, Endoscopy, including proctosigmoido & Colonoscopy at a higher center. Ur discussion on this is solicited....Tnx....



SRL report is in hand FJS f/37 : Hb% 7.7g/dl , Hematocrit 27.4 , RBC 4.45mil/muL, MCV 61.6 fl, MCH 17.2pg, MCHC 27.9g/dl , Red Cell distribution width 21.4% , Platelet count 429thou/muL , Mean platelet vol 9.2 fl ,WBC count 13thou/muL , N 69% , RBC- Marked anisocytosis, mild poikilicytosis, microcytic hypochromic, with elliptocytes & ovalocytes. Serum Iron 97mic.gm/dl , TIBC 435 micgm/dl , % saturation 22 . Hb Variant Analysis : HbA 95.6% , Hb A2 2% , Hb F 0.3% , HbS,D,C 0%. Unknown Unidentified peak 2.1 (0.00 - 2.00 )....yes platelets here are high with raised total count....
Whenever I deal with RBC Indices the tall fair complexioned bespectaled ,well dressed Dr SB Pandey Asso.Prof Patho. who used to churn out these with monotonous regularity with a pronunciation as bland as it could be......nd believe me it seems as it is just yestday only I left the class...!!!....Golam case will post eve.....



The lady with the anaemia doesn't look as straightforward as it looks. While the tests would on the face of it, suggest iron deficiency, notice that while the TIBC is raised, the serum iron is in the normal range and that transferrin saturation is only slightly low, discordant with the severity of anaemia. Yet, it is almost certain that she has a degree of iron deficiency, as such marked anisocytosis & high RDW is not seen in pure thal trait.

However, notice the extremely low MCV and the very low haematocrit, and the relatively normal RBC count despite the severe anaemia. These are all features of thal minor. Yet, HbA2 is not raised. How do you reconcile this?

I believe this lady has a combination of iron deficiency anaemia and delta-beta thalassemia trait. Since delta chains are a component of HbA2, the latter has failed to rise. Thal traits are quite common among Asians.

I'd investigate the iron deficiency anaemia exactly as you have, but be aware that the response to iron therapy may not be as impressive as you'd otherwise expect.

To start from where we left off, I am convinced because of the reasons described above that this lady has a combination of thal trait and iron deficiency anaemia. But this time, I'd like to provide a bit more information, and perhaps a rider.

Firstly, I have learnt that iron deficiency anaemia (IDA) can calse false normalisation of the HbA2 in subjects with thal trait. When you treat the IDA, the HbA2 may rise above the upper limit of normal in these subjects. Although this lady's HbA2 is far below the threshold of around 3.2% considered diagnostic of beta thal trait, I'd repeat her Hb and HbA2 after a three week course of iron, say FeSO4 200 mg tds. The Hb should rise by at least 2 g/l in pure IDA, which I expect not to happen in this case because of concurrent thal trait. However, I'd expect the HbA2 to rise considerably- above 3.2%, confirming coincident beta thal trait besides her IDA.

There is however, one other possibility. The HbA2 would not be expected to rise in alpa-thal traits because the alpha globin chain is a component of HbA2. Alpha thal is coded by 4 genes, i.e. 2 paired genes on chromosome 16. If one or two genes are deleted, the patient hardly has any anaemia, but may have slight microcytosis. However, if 3 genes are deleted, the patient can have severe microcytic anaemia resembling beta thal.

Here's where her ethnicity comes in. Although alpha thal traits are well documented in Asians, particularly the alpha 1 trait or cis-deletion (--/alpha, alpha), beta thal trait is far more common in this population. In fact 8% of Bangladeshis carry the beta thal trait (4% among Pakistanis and 3.5% among indians, compared with only 0.1% in white British). On balance therefore, I feel this lady has a combination of IDA and either beta thal trait or beta-delta thal trait. You should get the answer if you repeat her HbA2 levels after a month on iron replacement therapy.

A Girl with High ACE

A 10-year-old girl was referred to the Paediatric Surgery clinic because of recurrent epigastric and left hypochondrial pain every three months, lasting three to five days. The abdominal discomfort was associated with episodes of nausea. The patient had also had occasional nose bleeds.
At birth she had aspiration of meconium but had been otherwise well until the age of 10. Her father had been diagnosed with ulcerative colitis and her father’s sister had coeliac disease. An ultrasound scan of the abdomen showed a mildly enlarged spleen and a right ovarian follicle. Given her family history, she underwent colonoscopy with biopsy which was inconclusive.

At the age of 19, she was seen in the Gastroenterology clinic and had a repeat abdominal ultrasound scan and additional blood tests. Her scan confirmed a large spleen and the blood tests showed an elevated angiotensin converting enzyme (ACE) level (207 U/L). In view of her splenomegaly, she was referred to the Haematology clinic where she was screened for sarcoid, rheumatoid, haematologic and autoimmune diseases. However, further investigations were normal and no diagnosis could be reached. ACE level was again raised at 250 U/l.

She continued to be afflicted by recurrent episodes of abdominal pain. Therefore she was seen again in the Gastroenterology clinic and was further investigated with oesophago-gastroduodenoscopy with biopsy, ultrasound scan of the liver and portal vein, and abdominal magnetic resonance scan. The enlarged spleen was again evident but no other abnormality was found.

During one of her last clinic consultations, she mentioned that she had been on holiday to the Dominican Republic in 2003 and recalled being bitten by an insect. Following this, she had felt ill for several months with episodes of abdominal pain associated with fever, diarrhoea, nose bleeds and some joint swelling.

She was referred to the Infectious Disease clinic where she was tested for pathogens which could have been encountered during her holiday overseas. These included Epstein–Barr virus, Cytomegalovirus, Toxocara, Leishmania and Schistosomiasis. All tests were negative.
At this time she reported some mild joint swelling and discomfort affecting her fingers. A Rheumatology opinion was sought.

What do you think was the diagnosis?

A Young Man with a Rash & High CRP

Some time ago, I was referred a 21-year old Caucasian man who had had a red rash on his legs a couple of months ago. Over the same period, he had been to the Emergency Department twice with pain and redness in his right eye, diagnosed as acute uveitis. The GP wondered if he had vasculitis.

In his other history, he had lost a stone in weight (6.3 kg) in 6 months without really trying. Over the last 12 months, he had had recurrent pilonidal sinus related infections in his bottom, diagnosed and treated with antibiotics by his GP. There was no history of Raynaud's, oral or genital ulcers, sicca symptoms or psoriasis. There had been no joint pains. He did not smoke but drank lager at weekends.

His GP checked his blood and documented: Hb 10.8 g/dl, MCV 82, WBC 11, Neutrophils 9, ESR 120, CRP 85, urine 300 RBC (normally <45), no proteinuria, normal U&Es & LFTs. RF negative, ANA weak positive in a nucleolar pattern, ENA, dsDNA, ANCA negative.

Examination showed no rash or synovitis, normal systems and an apparently healthy looking man.

I made the diagnosis, but only after calling him at home to ask him a question I had omitted in clinic.

Thoughts?

A Sick Lady With An Abnormal Xray

Forty-five year old female, life-long non-smoker presents with with dyspnoea, fever, non productive cough, wheeze, night sweats and weight loss over several weeks. This is her X-ray. What's the diagnosis?

Picture, courtesy UpToDate.

The Acromegaly That Wasn't

One of the most challenging cases I have ever been asked about was regarding a 31 year old Caucasian man. When he walked into the Rheumatology clinic, referred by his GP with arthralgias and a raised, fluctuating CK, even the junior doctors felt he was a straightforward case of acromegaly. He had the classical facies, a large tongue that he felt was "growing" and tingling in his hands. CK fluctuated between 270 and 2000, yet muscle power, EMG and muscle biopsy were normal, as were numerous tests for metabolic myopathy.

He also reported more than one episode of near loss of consciousness. He saw the neurologists with this, who felt this was syncope. A tilt table test showed a sharp drop in BP while upright, associated with tachycardia.

Baseline IGF-1 and timed levels of Growth Hormone after stimulation with glucose were normal. MRI of pituitary revealed a microadenoma.

ECG was not done.

Echo was reported as normal. However, when you looked at the detail, the LV size was the upper limit of notmal, the left atrium was dilated and the E:A ratio at the mitral inlet was at the upper limit of normal at 1.48.

Nerve conduction studies showed no evidence of entrapment neuropathy at the wrist. Autoimmune screen, including every conceivable autoantibody in the book, was negative. Inflammatory parameters were normal.

Can you suggest a diagnosis?

ECG Problem

What's the Diagnosis?

A Young Man With Hypercalcaemia

A 32 year old man is referred by his GP with hypercalcaemia. Corrected serum calcium is 2.75 mmol/l (11 mg/dl). Serum PTH is raised at 105 pg/ml (normally up to~70). Serum phosphate is normal. Serum 25(OH)D levels are at the upper limit of normal. Chest Xray is normal, as is a myeloma screen. Alkaline phosphatase is not raised. Renal function is normal, with an egfr of >90 ml/min.

What's the likely diagnosis?

Trivial Signs?

I recently saw a middle aged man with a swelling at the base of his left middle finger. Just that. Nothing else. He didn't even have much pain. His GP had sent him to Rheumatology because he couldn't explain the symptoms.

He looked well. The left middle MCP was swollen. All other joints were normal. He had had lots of bloods in primary care, including FBC, U&Es, LFTs, ESR, CRP, Rheumatoid factor- all normal. A plain X-ray had revealed an odd looking cyst in the head of the 3rd metacarpal, but all else was normal.

There was no history of trauma.

Thoughts?

Hypokalaemia & Hypertension

A relatively common problem in clinical practice is the young subject with hypertension in association with hypokalaemia and metabolic alkalosis. Physicians should resist the temptation of labeling this subset as "Essential Hypertension". A definitive diagnosis can be achieved with a bit of perseverance.

As medicine evolves, new paradigms displace old ones. Primary hyperaldosteronism was thought to be exceedingly rare. Not so. It is now recognised that 10-15% of subjects with hypertension have primary hyperaldosteronism. This is particularly true in young subjects, subjects with a strong family history of hypertension and those with hypokalaemia.

Yet, too much reliability has been placed on the presence of hypokalaemia as a marker of this condition. In fact, less than 50% of subjects with primary hyperaldosteronism have hypokalaemia. The rest are normokalaemic. Therefore it is important to have a high index of suspicion in young subjects, those with a strong family history of early onset hypertension, those with family history of stroke before age 40, and those with refractory hypertension, i.e. subjects whose BP remains uncontrolled despite three anti-hypertensives, including a diuretic.

The assessment should begin by measuring the ratio of plasma aldosterone to plasma renin activity (PRA). A ratio of >30 with an absolute concentration of aldosterone of>415 pmol/l and in particular, a completely suppressed PRA is virtually diagnostic of primary hyperaldosteronism.

The most common cause of this condition is idiopathic adrenal hyperplasia (IHA), causing bilateral hyperplasia of the adrenals, accounting for 65% of cases. Around 30% of cases are due to unilateral adrenal adenomas. The other 5% comprises 3 rare disorders- hereditary hyperaldosteronism types 1,2 and 3.

Type 1 hereditary hyperaldosteronism is a fascinating condition. Also called glucocorticoid remediable aldosteronism (GRA), this rare autosomal dominant condition is due to the fusion of two genes- CYB11B1 and CYB11B2, whereby the enzyme aldosterone synthase comes under the control of ACTH. Thus, aldosterone is made in the zona fasciculata rather than zona glomerulosa and is suppressible by steroid therapy. Half of these subjects have a normal serum potassium.

Type 2 is simply an inherited predisposition to adrenal adenoma or hyperplasia.

What if the plasma renin and aldosterone are both raised? Think of renovascular hypertension, renin secreting tumours, coarctation of aorta and subjects on diuretics.

Then there are those whose plasma renin and aldosterone are both suppressed. These subjects have a non-aldosterone related cause for their hypertension such as hypercortisolism due to Cushing's syndrome, Syndrome of Apparent Mineralocorticoid Excess (SAME), or excessive liquorice intake, certain causes of congenital adrenal hyperplasia such as 11-beta hydroxylase deficiency, deoxycorticosterone secreting tumours and Liddle's syndrome.

Two inherited conditions deserve special mention. SAME is a rare autosomal recessive condition due to inherited deficiency of 11-beta hydroxysteroid dehydrogenase deficiency. This is a renal enzyme that converts cortisol to inactive cortisone. When this enzyme is deficient or inhibited by liquorice, or overwhelmed by excessive production of cortisol, as in Cushing's syndrome (particularly due to ectopic ACTH production from small cell or other tumours), cortisol is freed up to act on the mineralocorticoid receptors and mimic the action of aldosterone. These patients tend to have a very low serum potassium.

The other inherited disorder with suppressed aldosterone and renin levels is Liddle's syndrome. This is an autosomal dominant condition where epithelial sodium channels responsible for reabsorption of tubular sodium at the expense of potassium remain constitutively open due to inability to remove the beta or gamma subunits of these channels. The removal of these channels from the tubular epithelium depends on an uniquitin ligase called Nedd, which is defective in Liddle's patients. Intuitively, direct potassium channel blockers such as amiloride and triamterene work well in the treatment of hypertension in Liddle's syndrome, while aldosterone antagonists such such as spironolactone do not. It's worth remembering that as in GRA, over half of subjects with Liddle's syndrome have normal serum potassium.

In subjects with high aldosterone:PRA ratio, the next test is salt loading to see if the serum aldosterone levels are suppressible. This can be done as an inpatient after infusing IV saline, or as an outpatient, after loading for 3 days with oral salt tablets. Subjects with primary aldosteronism will not suppress their aldosterone levels after salt loading.

Such subjects should then have a CT scan to pick up an adrenal adenoma putatively responsible for the excess aldosterone. If the CT does not show an adenoma, and irrespective of the CT results in subjects over the age of 40, most authorities would recommend that patients should have adrenal vein sampling for aldosterone:cortisol ratio to distinguish functional adenoma from IHA. Subjects under 40 with an obvious adenoma on CT should be directly considered for surgical adrenalectomy. Conversely, those with IHA would benefit from medical management with spironolactone.

Acknowledgement: uptodate.com