Abnormal Result

Sodium 186mmol/L

Presenting Complaint

The neonate was taken to the Emergency Department due to seizures.

History

Unfortunately the attending clinician at Greys hospital did not have much of the history.

There was no history of diarrhoea according to what she remembered.

Examination

Patient was severely dehydrated clinically.

Later the patient presented with edema, signs and symptoms of nephropathy and biochemical changes in keeping with liver failure.

Laboratory Investigations

Cumulative laboratory history

Urine reducing subs (screen)

Positive ++

Urine reducing substance ID (chromatography):

TLC shows the presence of trace glucose only.

Urine Amino Acid Analysis

Urine organic acids

        Interpretation:
        Urine organic acid analysis by GCMS demonstrates elevation of the liver markers
        4-OH phenyllactate and 4-OH-phenylpyruvate together with lactaturia.
        Succinylacetone, a marker for tyrosinaemia type 1 is absent. These changes
        indicate underlying hepatic dysfunction with lactataemia but are non-specific
        for an IMD per se. Rare forms of lactataemia include defects in pyruvate
        metabolism (gluconeogenic defects such as glycogen storage disease type 1,
        pyruvate dehydrogenase deficiency and thiamine deficiency). In these disorders
        the lactate/pyruvate ratio is normal despite lactataemia. In pyruvate
        dehydrogenase deficiency the CSF/Plasma lactate ratio is typically >2. Please
        note that routine metabolic screening does not exclude galactosaemia. If
        galactosaemia is suspected this should be screened for by measuring red cell
        GALT activity in unspun heparin whole blood or by screening for the common
        African S135L mutation in Black South African patients. As part of our
        gatekeeping policy to limit unnecessary testing, routine metabolic screening at
        Red Cross Children's Hospital consists only of urine organic and amino acid
        analysis. Additional tests must be requested separately based on the working
        differential diagnosis and routine screening results. Relevant legible clinical
        information also aids significantly in interpreting metabolic profiles.

Other Investigations

Urine dipstick:
Blood 3+
Protein: Trace
Glucose (by glucose oxidase) ++/+++ 28 – 55 mmol/L (hence this is most likely the predominant reducing sugar – as suggested by Prof David). This is an interesting finding since the random glucose in the ward the same day was 4.4 mmol/L. Hence two possible theories: likely either severe tubular injury or, when the child had convulsions they gave a dextrose infusion which increased the plasma glucose above the renal threshold.
pH >8.5
Nitrites: Not present
Leucocytes: Not present
Urobilirubin: Trace
Bilirubin: Not present

Final Diagnosis

Severe dehydration with acute kidney injury (pre-renal origin) is the most likely cause of the presentation of seizures.

Take Home Message

Dehydration is a common cause of pre-renal acute kidney injury

My thoughts initially, was that the urine amino acid screening by GCMS demonstrated a heavy generalized aminoaciduria indicative of renal tubulopathy and that cystinosis should be considered as this is the most common inheritable cause of renal tubulopathy in South Africa. However, this was later decided to be changed to rather and “evolving tubulopathy” clinical picture, as shown in the two examples below.

Fanconi syndrome — Generalized proximal tubular dysfunction, referred to as Fanconi syndrome, is characterized by phosphaturia, renal glucosuria (glucosuria with a normal plasma glucose concentration), aminoaciduria, tubular proteinuria, and proximal RTA.

The etiology of Fanconi syndrome includes inherited diseases or acquired causes [Source: Up-to-date]:

Genetic conditions associated with Fanconi syndrome include the following:

• Dent disease (X-linked recessive nephrolithiasis)
• Cystinosis
• Tyrosinemia type 1
• Galactosemia
• Wilson disease
• Lowe oculocerebrorenal syndrome, also referred to as Lowe syndrome
• Hereditary fructose intolerance
• Mitochondrial myopathies

Acquired causes of Fanconi syndrome include:

• Drugs – Medication associated with Fanconi syndrome include
  • aminoglycosides
  • cisplatin
  • ifosfamide
  • valproic acid
  • deferasirox
• Heavy metals
  • lead
  • mercury
  • cadmium

The fact that the urine dipstick was positive for glucose, suggests that either the acute kidney injury is the source of glucosuria or the patient was treated with high dose dextrose, causing the plasma glucose to overwhelm the tubular threshold for glucose transport.

Abnormal Result

PSA: 846.5 ug/L

ALP: 284 U/L (53 – 128)

Presenting Complaint

Painful ”lumps” in groin + constipation

Spine pain

History

Smoker (>45 years)

No other co-morbidities

6/12 history of generalized body pain (mostly spine)

Red Flags (weightloss, night pain not responding to analgesia)

Examination

O/E: Pallor (Hb 8.6), Wasted. Clinically painful bilateral inguinal lymph nodes PR: normal tone, no masses, no blood, prostate smooth

Laboratory Investigations

Other Investigations

Chest X-Ray: Left hilar opacities

X-ray of the limbs: Global lytic lesions involving both proximal femurs

Prostate biopsy

• MACROSCOPY: Specimen consists of two cores, the longest measuring 12mm in length.
• MICROSCOPY Sections show 2 prostatic cores, both infiltrated by a prostatic adenocarcinoma.
• % Ca core 1: 90%
• % Ca core 2: 60%
• Gleason score: 5 + 4
• Grade group: 5
• High grade PIN: Not seen
• Seminal vesicle: Not seen
• Perineural invasion: Present
• Fat (extraprostatic) involvement: Not seen
• PATHOLOGICAL DIAGNOSIS:
• Prostate, needle biopsy: Prostatic acinar adenocarcinoma

Final Diagnosis

Metastatic Prostate Carcinoma with multiple metastases to the bones (thoracic spine and both femurs).

Take Home Message

Prostate-specific antigen (PSA, also known as kallikrein III, seminin, semenogelase, γ-seminoprotein and P-30 antigen) is a 34-kD glycoprotein produced almost exclusively by the prostate gland. It is a serine protease enzyme.

Most PSA in the blood is bound to serum proteins. A small amount is not protein-bound and is called ‘free PSA’. In men with prostate cancer, the ratio of free (unbound) PSA to total PSA is decreased. The risk of cancer increases if the free to total ratio is less than 25%.

The lower the ratio is, the greater the probability of prostate cancer. Measuring the ratio of free to total PSA appears to be particularly promising for eliminating unnecessary biopsies in men with PSA levels between 4 and 10 mg/L.

ALP (alkaline phosphatase) is well known to be a marker of ductal hepatic damage. ALP, being an isozyme, however has its origin from various tissue sources in the body. It is present in the liver, bile duct, kidney, bone, intestinal mucosa and placenta. The majority of ALP in serum is from either skeletal or liver origin. In adults the major form is from liver and in children the major form is from the skeleton.

Blood levels of alkaline phosphatase increase by two to four times during pregnancy. This is a result of additional alkaline phosphatase produced by the placenta.

If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using electrophoresis can confirm the source of the ALP. It would likely in this patient be quite clear that the raised ALP would be due to the excess leakage from the osteolitic lesions from the metastases, but who knows, the patient may have had a beer or five in the preceding 3 weeks leading up to the bloods being drawn. The fact that the other liver enzymes are near-normal, makes alcohol consumption less likely though.

HOSP #	Lab no: SA03948371	WARD	Paediatric Ward
CONSULTANT	Dr. Jody Rusch	DOB/AGE	16 y Female

Abnormal Result

Albumin of 8 g/L

Presenting Complaint

Signs and symptoms of a urinary tract infection made the patient present to a general practitioner.

History

No known chronic medical illness were present upon initial presentation.

No medical treatment was being taken for chronic illnesses.

The patient had reported taking NSAIDS before for pain in the lower abdomen. The exact drug / dose was unknown.

Examination

All clinical findings are unfortunately not available for this patient.

It is known that the patient had been having lower abdominal pains upon presentation (which was not due to pregnancy).

A urinary tract infection was suspected by the initial treating physician. Upon the other finding of edema, investigation towards the cause was investigated.

Typical findings of nephritic syndrome are:

• Fever
• Edema (due to hypoproteinemia)
• High blood pressure (due to activation of the renin-angiotensis-aldosterone system).
• Joint pain
• Muscle pain
• Malar rash
• Foamy urine (proteinuria)

Laboratory Investigations

Albumin 14 g/L

Cholesterol 8.14 mmol/L

Urine Protein:Creatinine ratio: 1.62 g/mmol creat

C3: 0.29 (Low)

C4: 0.07 (Low)

Creatinine 255 – 322 umol/L

Other Investigations

Final Diagnosis

Lupus Nephritis with hypoalbuminemia

Take Home Message

The clinical presentation of this patient is a good example of the findings in patients who initially present with renal failure. The extent of renal failure is often so severe, that when the patient presents with signs and symptoms of renal failure, there are quite significant permanent renal damage already.

Patients with nephrotic syndrome present with significant proteinuria with resultant hypoproteinemia, firstly hypoalbuminemia, followed by the other bigger proteins like gammaglobulins, alpha-1, beta-1 and beta-2 (complement) proteins. Because alpha-2 (macroglobulin) comprises one of the biggest proteins (in molecular size) in the serum, it generally stays in the serum relatively longer than the other leaking proteins.

Because the liver increases its production of proteins to try compensate for the reduction in osmolality, the production of VLDL rises significantly and hence Triglycerides (and cholesterol) rises. Thus cholesterol in this patient measured 8.14 mmol/L.

The pathophysiology of lupus nephritis is that of autoimmunity. Autoantibodies direct themselves against nuclear elements. The characteristics of nephritogenic autoantibodies are antigen specificity directed at the nucleosome. High affinity autoantibodies form intravascular immune complexes, and autoantibodies of certain isotypes activate complement. Hence the C3 and C4 which are low often indicates active lupus disease.

Abnormal Result

Glycerol which is significantly raised on urine organic acid analysis.

Presenting Complaint

Patient is a 3 month old male with signs and symptoms of sepsis.

History

Patient presented with significant failure to thrive.

Laboratory Investigations

Triglycerides : 4.47 mmol/L

Other Investigations

Faecal elastase 81 ug/g stool

Reference range (adults and children > 1 month):

• > 200 ug elastase/g stool: Normal exocrine pancreatic function
• 100-200 ug elastase/g stool: Moderate/mild pancreatic insufficiency
• < 100 ug elastase/g stool: Severe exocrine pancreatic insufficiency

These ranges apply to formed stool samples. Watery stool samples may yield spuriously low elastase results due to dilution, and a formed stool sample should be sent for re-analysis.

Final Diagnosis

Glycerol contamination of the skin – as excluded by the repeat analysis.

Take Home Message

• Glycerol (glycerine) is a common contaminant of urine organic acids due to being present in various skin products / creams. Contamination can be eliminated by thorough cleaning of the perineum with normal saline or doing an “in-out” catheterization procedure for urine collection in neonates. Interestingly glycerol is also one of the main ingredients in KY jelly, a common lubricant use for catheterization.
• High glycerol in serum will present with a falsely high triglyceride level on most routine chemistry analysers due to the inherent enzymatic conversion of triglycerides to glycerol before further steps to measurement.

• Sepsis is more common than inherited metabolic diseases and so is pre-analytical caveats such as glycerol contamination of the perineal skin.

Abnormal Result

• Aldosterone: 2320 pmol/L
• Renin: 76.5 ng/L (Adult reference ranges – Supine: 1.7 – 23.9 ng/L; Upright: 2.7 – 27.7 ng/L)
• Aldosterone:Renin Ratio : 30.3
• The aldosterone:renin ratio (ARR) is a screening test for primary hyperaldosteronism and is most sensitive when both an absolute aldosterone > 350 pmol/L and an ARR > 118 pmol/ng is present.

• Na: 138 mmol/L
• K: 4.5 mmol/L
• Urea 3.7 mmol/L
• Creatinine: 49 umol/L
• Total Calcium: 2.55 mmol/L

Urine dipstick 1+ protein

The urine protein:creatinine ratio was 0.044 g/mmol creat

Presenting Complaint

Patient presented with a 2 week history of blurry vision, intermittent headaches, hot flushes and mild intermittent epistaxis.

History

No known comorbidities

Multiple episodes of otitis media as a child

Presented with hypertension and evidence of target organ damage (retinopathy and left ventricular hypertrophy).

Examination

Vitals: HR 120, Resp. Rate 20, Temp 36.7 deg.C

BP (mmHg)

Right arm 144/92, Left arm 150/90

Right leg 176/74, Left leg 178/107

Gen: Not acutely / chronically ill, JACCOLD neg. Thyroid exam normal.

Funduscopy: Silver wiring, hard exudates, no haemorrhages, no papiloedema

CVS: bounding, peripherals pulses present. JVP raised, Undisplaced apex. Regular HR, no Radial/ femoral delays. Normal S1 & S2 with flowmurmur grade 2.

GIT: soft, non-tender. Ballotable left kidney, non-tender

Resp: Central Trachea, clear viscular breath sounds, no added sounds.

Neurological Exam: GCS 15/15, normal higher functions, no focal neurological signs.

BP control achieved with Doxazocin (increased to 4mg dly) and Atenolol (increased to 50mg dly)

Laboratory Investigations

Other Investigations

ECG: Biatrial enlargement, left ventricular hypertrophy

Chest X-ray: Normal Cardio-thoracic index

Cardiac Ultrasound: Concentric left ventricular hypertrophy with preserved left ventricular ejection fraction. No valvular pathology.

KUB ultrasound: Similar kidney sizes. A mass with a cystic center was noted with no evidence of metastatic disease. Diagnosis suggested to be most likely a pheochromocytoma.

Final Diagnosis

Right-sided phaeochromocytoma

Take Home Message

Aldosterone : Renin ratio cannot be looked at alone. A raised value in either of the Aldosterone and Renin should be investigated further, especially if severely deranged like in this case.

Always investigate hypertension in a child until the cause is found. Hypertension in a child is not normal.

3 consecutive 24 hour urine collection samples are recommended for diagnosis of phaeochromocytoma as some tumours only secrete epinephrines / norepinephrines episodically. In this case however it was not necessary as the case was clear with a markedly raised dU-normetanephrine level.

Screening for pheochromocytoma is an essential part of the workup for secondary hypertension. Urinary vanillyl mandelic acid (VMA) was traditionally used to diagnose phaeochromocytoma. It has a low sensitivity (60-70%). Later, catecholamines measurement in plasma (PCAT) and urine (UCAT) emerged as useful tests. The sensitivity of catecholamines is limited by their episodic release from the tumour cells. The sensitivity ranges from 76-86 % for PCAT and UCAT and the specificity is around 81-99 %.

Metanephrines are methylated metabolites of catecholamines.

Metanephrines are secreted continuously from the tumour cells, independent of the intermittent release of catecholamines. The metanephrines are metabolized by conjugation, primarily in the hepatomesentric organs. Plasma metanephrines (pMN) are measured in the free form (not routinely offered in South Africa) whereas urinary metanephrines (uMN) represent mainly the conjugated form. Hence compared to pMN, uMN is less specific. Studies have shown that plasma free metanephrines have a sensitivity of 96-100 per cent and specificity of 85-100 % superior to that of uMN which has a sensitivity of 93-99.6 per cent and specificity of 71-77 per cent.

Previous methods used colorimetry or spectrophotometry as total MET (metanephrine + normetanephrines) which includes a combined measurement of metanephrine (MN) and normetanephrine (NMN). These methods were superseded by liquid chromatographic assays (LC) that allow individual measurement of MN and NMN.

At Red Cross Hospital Laboratory we use a gas chromatography with mass spectrometry, which is not so widely used for fractionated metanephrines. An isotope dilution method is employed, bringing the method up to internationally recognized standards and the quality assurance of the method at the Red Cross Chemistry lab performs well on the international EQA scheme used.

Abnormal Result

Oral lactose tolerance test:

Presenting Complaint

Patient presented with bloating and abdominal pains.

History

The medical history is not known

Patient reported symptoms of bloating and abdominal pains. Upon further questioning it became known that she had been troubles by these symptoms especially severe after consuming dairy products.

Examination

Not known.

Signs and symptoms often associated with this condition are:

• Abdominal pain and bloating
• Gass / flatulence
• Diarrhoea
• Constipation

Laboratory Investigations

Other Investigations

No other investigations were done.

Final Diagnosis

Patient appears to be lactose intolerant since we expect a rise of >1.7 mmol/L in glucose when lactose is adequately digested and absorbed after a lactose load of 50g.

The following cut-offs are frequently used:

• Glucose rise > 1.7 mmol/L at any of the time points is a normal lactase response
• Glucose rise of 1.1-1.7 is equivocal
• Glucose increases < 1.1 is consistent with lactase deficiency 

Take Home Message

Lactose consists of Glucose and Galactose.

The biochemical handling of oral lactose               

The disaccharide cannot be absorbed and needs to be cleaved before absorbtion by the enterocyte.  This happens by lactase, normaly present on the distal part of the brush border of the enterocyte.  These enterocytes can become damaged by enteritis and loose activity of lactase partially.  Lactase activity also decreases with age, hence elderly do develop partial lactose intolerance. The transporter that carries glucose and galactose into the enterocyte is the sodium-dependent hexose transporter, SGLT-1. As the name indicates, this molecule transports both glucose and sodium ion into the cell and in fact, will not transport either alone.

SGLT1 shouldn’t be confused with a GLUT. 

Inhibition of SGLT1 delays and reduces glucose absorption in the small intestine, thus improving post meal glycemic control. This is beneficial particularly in patients with declining renal function where SGLT2 inhibition is less effective.

SGLT2 is the major transport protein and promotes reabsorption of glucose from the glomerular filtration back into circulation and is responsible for approximately 90% of the kidney’s glucose reabsorption.

SGLT2 inhibitors, also called gliflozins, are a class of medications that alter essential physiology of the nephron; unlike SGLT1 inhibitors that modulate Sodium/Glucose channels in the intestinal mucosa.    

How does lactose intolerance normally present?            

Bloating and abdominal cramps after dairy ingestion. (Bacteria metabolise disaccharides and produce H2, hence don’t light your farts when lactose intolerant.)  It is less pronounced with dairy which has a lot of lactobacilli (live cultures) in it since they can partially digest some of the lactose.  Thus cheese and joghurt doesn’t give such severe Sx as the raw milk products.Good.  For your own education, think about sour milk 

What is the difference between lactose intolerance, milk allergy and galactosaemia?

•         Lactose intolerance is explained above, and can be either primary (defective enzyme) or secondary due to another condition as is the case in enteritis where the enterocytes are regenerating and temporarily expressing less enzyme on the apical brush border.
•         Milk allergy is an abnormal response by the immune system to milk and products containing milk. It’s one of the most common food allergies in children. Cow’s milk is the usual cause of milk allergy, but milk from sheep, goats, buffalo and other mammals also can cause a reaction.  The allergy is most likely to one of the exogenous proteins in animal origin milk. Milk allergy presents like any other allergy with hives and/or urticaria.
•         Galactosemia is the inability to metabolise galactose to glucose for metabolism.

        3 genes can contain a mutation  (GALT, GALK1, and GALE) coding for:

•             Galactose-1-P-uridylTransferase
•             GalactoKinase
•             UDPGalactose Epimerase

HOSP #		WARD	Red Cross Padiatric Hospital
CONSULTANT	George van der Watt / Surita Meldau	DOB/AGE	1y 5m

Abnormal Result

Inorganic phosphate 0.85 L mmol/L (1.00 – 1.95)

Presenting Complaint

It is unknown what this patient’s presenting complaint was.

Common complaints in similar patients are:

1. Photophobia (see slit lamp picture below, which explains why).
2. Bone pain
3. Weakness

History

The items below illustrates the recorded information on the laboratory request forms as they were captured by our lab staff on the respective samples:

      SEPSIS
      Vit D Deficiency
      Vit D Deficiency
      HYPOCALCAEMIA
      ?LOSSES
      ?PTB
      ?PTB
      ?FANCONI
      ?SEPSIS
      ?SEPSIS
      ?SEPSIS
      ? FANCONI SYNDROME
      ? Cystinosis
      Risk factor:
      Contact
      Cough
      Fanconi's ? cystinosis
      Hypocalcaemia
      Pneumonia.
      Pneumonia.
      Fanconi syndrome
      ? Hypoglycaemia
      post iv calcium for hypo
      Sepsis. Cystinosis
      Sepsis. Cystinosis
      Cystinosis
      FANCONIS SYNDROME
      Fanconi's syndrome
      Cystinosis
      Cystinosis
      Sepsis. Fanconi syndrome, hypocalcaemia
      Hypocalceamia
      CYSTINOSIS- HUNGRY BONE D

It can be seen above that the clinicians were noting Fanconi’s syndrome and were querying Cystinosis.

Examination

Data not available. Generally patients with Fanconi’s syndrome present with loss of electrolytes from the proximal and distal renal tubuli:

• Nephropathic cystinosis in untreated children is characterized by renal Fanconi syndrome, poor growth, hypophosphatemic/calcipenic rickets, impaired glomerular function resulting in complete glomerular failure, and accumulation of cystine in almost all cells, leading to cellular dysfunction with tissue and organ impairment. The typical untreated child has short stature, rickets, and photophobia. Failure to thrive is generally noticed after approximately age six months; signs of renal tubular Fanconi syndrome (polyuria, polydipsia, dehydration, and acidosis) appear as early as age six months; corneal crystals can be present before age one year and are always present after age 16 months. Prior to the use of renal transplantation and cystine-depleting therapy, the life span in nephropathic cystinosis was no longer than ten years. With these interventions, affected individuals can survive at least into the mid-forties or fifties with satisfactory quality of life.
• Intermediate cystinosis is characterized by all the typical manifestations of nephropathic cystinosis, but onset is at a later age. Renal glomerular failure occurs in all untreated affected individuals, usually between ages 15 and 25 years.
• The non-nephropathic (ocular) form of cystinosis is characterized clinically only by photophobia resulting from corneal cystine crystal accumulation.

Because the thyroid glands are actively translating and resorbing thyroglobulin, the thyrocytes (thyroid colloid epithelial cells) are prone to accumulation of cysteine within their lysosomes, hence these children often also develop hypothyroidism with a palpable thyroid gland.

Laboratory Investigations

Biochemistry:

Genetic screening

PCR with enzyme digest:

1st PCR with enzyme digest:

Upon the first PCR for a gene screen which was done, there occurred some evaporation in the third tube (lane no. 3 – positive control), upon incubation with the enzyme digest mix. This incubation step is minimum 3 hours at 37 degrees Celcius and do not have the high temperatures associated with the PCR process. Thus the reason for evaporation remains unclear and may have been an incompletely closed lid or a defective PCR tube rim or cap.

It is not expected to see an undigested PCR product (>200bp) as can be seen in the top band in lane 3, hence it is clear that there was incomplete digestion of PCR product in this PCR tube. Even though it is clear that the patient sample ( lane 1) represents a homozygous positive result (lane 2), one cannot authorize results when both the positive and normal control worked well.

Thus the PCR was repeated.

2nd PCR with enzyme digest:

In the second PCR it can be well seen how the Positive control did undergo complete digestion. No evaporation was noted in this run.

Other Investigations

Leucocyte cystine is another investigation which helps make the diagnosis in patients with presumed cystinosis.

Leucocyte cystine report in this patient:

Protein 0.58 g/L

Leucocyte Cystine 1.16 nmol/mg protein

Reference range:

• Normal < 0.1 nmol cystine/mg protein
• Cystinosis > 1.0 nmol cystine/mg protein
• Cystinosis on Rx (target level) < 0.5 nmol cystine/mg protein

Our reporting comment on the Lab Information Systems reads as follows:

Please note that the diagnosis of cystinosis can be confirmed in the majority of South African patients by screening for the common South African Black mutation CTNS-c.971-12G>A which results in an estimated newborn incidence of 1/10 000 in this population. A molecular diagnosis is of value in that siblings of index cases can be screened and identified for early intervention which improves the outcome in this disorder.

Final Diagnosis

Patient is homozygous positive for cystinosis by the common South African mutation, as confirmed on leucocyte cystine as well as on the gene screen.

Take Home Message

** The grey portion at the bottom of the electropherogram – this indicates the movement of the ethidium bromide out of the gel towards the cathode. One should ideally not let a gel run beyond the intended time period as the migration of ethidium bromide “dye front” beyond the smallest band may cause band to “de-stain” and not be visualized well.

The CTNS gene provides instructions for making a protein called cystinosin. This protein is located in the membrane of lysosomes. Proteins digested inside lysosomes are broken down to amino acids. These are then moved out of lysosomes by transport proteins. Cystinosin is a transport protein that specifically moves the amino acid in its dimeric form cystine out of the lysosome.

More than 80 different mutations that are responsible for causing cystinosis have been identified in the CTNS gene. The most common mutation is a deletion of a large part of the CTNS gene (sometimes referred to as the 57-kb deletion), resulting in the complete loss of cystinosin. This deletion is responsible for approximately 50 percent of cystinosis cases in people of European descent. Other mutations result in the production of an abnormally short protein that cannot carry out its normal transport function. Mutations that change very small regions of the CTNS gene may allow the transporter protein to retain some of its usual activity, resulting in a milder form of cystinosis.

The treatment / management of patients with cystinosis includes Cysteamine, a drug which binds cysteine and forms Cysteamine-cysteine (see figure above). This molecule is similar in structure to lysine and can be exported from the lysosomes by a lysine transporter.

Interestingly, South Africa likely has the highest incidence of cystinosis in the world due to a common mutation, G > A mutation in intron 11 of the CTNS gene (c.971-12G > A p.D324AfsX44), likely due to some sort of founder-effect in black and coloured patients: https://link.springer.com/article/10.1007/s00467-014-2980-7 I’m proud of this article as it was published by scientists at our institution.

A useful web site to learn nomenclature of gene variants is HGVS.

Message from Prof David Marais:

Hi S & J
Interesting and I wish we could devote much more effort to solve these cases. Especially since this is the second time this mother has had this sad experience and the next pregnancy may result in the same.

On first principles:

1. This appears autosomal recessive
2. The dysmorphology eliminates many “simpler” inherited errors as homeostasis through the placenta settles imbalances. However, errors involving tissue differentiation, structural components may have dysmorphology. E.g. sterol synthetic defects, mucopolysaccharidoses…It is easy to exclude Smith Lemli Opitz with 1mL serum or plasma even at this stage. However, syndactyly is a very strong feature and hydrops is uncommon but described. Happy to do this if sample is available. Mt abn has been described as well and might explain steatosis though not likely.
3. Microvesicular steatosis in several organs is suggestive of incomplete mobilisation of FA into mitochondria for oxidation or inadequate oxidation in mitochondria. These disorders do not usually result in dysmorphology and it is said renal steatosis is typical. LCHAD deficiency has caused hydrops but not dysmorphism to the best of my knowledge. Wolman’s disease has adrenal calcification but not hypoplasia as far as I know and not typically hydrops and diffuse steatosis – will need to check this again. The steatosis could be secondary to severe metabolic stress.
4. Hydrops fetalis should be taken as a strong clue. The lysosomal disorders can cause these. The list I found in JIMD Reports (2018) Hurler syndrome (MPS-I; OMIM #607014), Morquio-A (MPSIVA; OMIM #253000), Sly syndrome (MPS-VII; OMIM #253220), galactosialidosis (OMIM #256540), sialidosis
   (OMIM #256550), GM1 gangliosidosis (OMIM #230500),
   Gaucher type 2 (OMIM #230900), Niemann-Pick disease
   types A and C (NPD-A and NPC; OMIM #257200, 257220), Farber granulomatosis (OMIM #228000), Wolman disease (OMIM #278000), mucolipidosis II (I-cell disease; OMIM #252500), sialic acid storage disease (ISSD; OMIM #269920), and multiple sulfatase deficiency (OMIM #272200) which have been shown to be associated.

5. Interesting that the spleen is absent and that the adrenals are small. This is hard to explain on any of the metabolic disorders above but I shall have to read more extensively.
6. It may be worth testing the urine or other fluids for sialic acid. Infantile Salla disease is a possibility. (Sialin defect, coarse facies, hydrops fetalis, vacuolated lymphocytes but I was not aware of steatosis.) I can look up if this is practicable with the old fashioned assays and chemicals that we do have. I know I tried to analyse sialic acid in the early 1990s. Will look this up too.

One hopes that the anat path dept keeps samples for work-up. Very important to involve the chem path early if any metabolic disease is suspected as one can do fibroblast biopsy up to a few days in the morgue.

Regards
D

Professor Emeritus AD Marais
Chemical Pathology 6.33 Falmouth Building
University of Cape Town Health Sciences
Anzio Rd, Observatory, 7925
Cape Town, South Africa

Abnormal Result

31 y/o Male

Presenting Complaint

Triglycerides of 78.59 mmol/L

Lipaemia index 3 (value of 1132)

It is likely that the results as set out above was due to a routine follow-up, but unfortunately little clinical information was given by the clinician.

History

The patient is hypertensive and diabetic on treatment since 2018. No other clinical information was given and the drug list was not supplied.

Examination

N/A – No signs and symptoms obtained.

Laboratory Investigations

Other Investigations

We would have loved to do lipid electrophoresis and see better investigations into the cause of the diabetes, but at the time of writing, 14/05/2020, the patient has unfortunately not had the opportunity to follow-up and it can unfortunately not be shown.

In an adult diabetic one would however expect the lipid electrophoresis to be that of a Fredrickson type V.

Hyperlipoproteinemia type V, also known as mixed hyperlipoproteinemia, familial or mixed hyperlipidemia, is very similar to type I, but with high VLDL in addition to chylomicrons.

It is also associated with glucose intolerance and hyperuricemia.

Final Diagnosis

Considering most factors known, and as explained via feedback from Prof. Marais below, diabetes is likely type 2 related to insulin resistance. One should also consider metabolic errors such as glucokinase deficiency causing MODY. Other causes, but unlikely, are endocrine pancreatic insufficiency which could include mitochondrial defects or herbicide-induced diabetes or (post-traumatic) excision of tail of pancreas.  HbA1c shows prolonged exposure to high glucose concentrations: 10.3% and 11.2% the year before.

Take Home Message

The following were my thoughts on causality of the high triglycerides initially:

Increased intake:

• Overeating (unlikely for this high Triglyceride level), that is why one has lipoproteins – to keep the fat in the blood low and store the fat in liver and tissues.
• Excess alcohol consumption, but I’m also not sure (wasn’t sure) if excess alcohol will raise triglycerides this high – it likely may (after prof’s email I think this is very likely the main cause in this patient).

Increased production:

• Kidney failure (nephrotic syndrome) (Prot: Creat ratio will likely exclude this – if borderline, a protein electrophoresis can be done).

Decreased metabolism:

• Some forms of familial hyperlipidemia such as familial combined hyperlipidemia
• Lipoprotein lipase deficiency
• Lysosomal acid lipase deficiency (aka cholesteryl ester storage disease)
• Hypothyroidism (TFT’s normal in this patient though)
• SLE
• Glycogen storage disease type 1  –> NAFLD (non-alchoholic fatty liver disease)

Drugs:

• Isotretinoin, Thiazides,
• Apparently some HIV meds.

How to further test:

Lipid electrophoresis will delineate the Fredericksen Class.

Familial combined hyperlipidemia:

Lipid electrophoresis will show lipoproteinemia type IIB.  

LPL deficiency:

Lab tests show massive accumulation of chylomicrons in the plasma and corresponding severe hypertriglyceridemia. Typically, the plasma in a fasting blood sample appears creamy (plasma lactescence).

The absence of secondary causes of severe hypertriglyceridemia (like e.g. diabetes, alcohol, estrogen-, glucocorticoid-, antidepressant- or isotretinoin-therapy, certain antihypertensive agents, and paraproteinemic disorders) increases the possibility of LPL deficiency.  Also other loss-of-function mutations in genes that regulate catabolism of triglyceride-rich lipoproteins (like e.g. ApoC2, ApoA5, LMF-1, GPIHBP-1 and GPD1) should also be considered.  (remember our case – I won’t mention her name though, patient’s name begins with a K… and ends with …ana).

The diagnosis of familial LPL deficiency is finally confirmed by detection of either homozygous or compound heterozygous pathogenic gene variants in LPL with either low or absent lipoprotein lipase enzyme activity (Jody and I have done this assay with Bharati and Prof once for above patient).

Lysosomal acid lipase deficiency (LAL-D) (aka cholesteryl ester storage disease) – Unlikely – would rather present earlier – the accumulation of fat in the walls of the gut in early onset disease leads to serious digestive problems including malabsorption, the gut fails to absorb nutrients and calories from food. Because of these digestive complications, affected infants usually fail to grow and presents with failure to thrive.  As the disease progresses, it can cause life-threatening liver dysfunction or liver failure). Until 2015, apparently there was no treatment (not sure if this is true though), and very few infants with LAL-D survived beyond the first year of life.

I think the clinical presentation and examination and history is much needed before any further investigations are advised.  

Also, one should appreciate the size difference which is partly responsible for the electrophoretic mobility of lipoproteins on a gel.

Feedback from Prof. David Marais:

Hi Dieter

Thanks for distributing the interesting case information. The patient is at very high risk of developing acute pancreatitis. Hopefully the medical officer will be able to get in touch with the patient and urgently:

• (1) control diabetes mellitus and
• (2) restrict dietary fat intake to 10g/d for a few days whereafter 30-40g/d.
• (3) restrict alcohol intake to preferably zero or certainly <20g/d.
• (4) prescribe fibrate.
• (5) Referral to the lipid clinic – unfortunately may take time owing to shut-down of out-patients clinics in the precautions against corona virus spread.

Such severe hyperTGaemia seen in the neonate, infant or child is most likely due to an error in the lipolytic system and all of these are recessively inherited. LPL deficiency is the commonest but there may also be apoCii, apoAv, GPIHBP1 or LMF1 deficiency.  In adolescent and young adults the same causes apply but also auto-immune LPL inhibition. In these cases all the agarose gel electrophoresis for lipoprotein separation will display a type I pattern. The highest TG conc I have seen in a patient was 695mmol/L and at 6 weeks of age.

In adults the lipoprotein electrophoresis pattern will usually be a type V. Here, there is usually partial lipase deficiency (often polygenic heterozygotes of LPL system components) and a dietary or metabolic stress. Diet containing triglycerides in large amounts and alcohol.  Metabolic stress is mostly diabetes with increased return of NEFA to the liver and export as VLDL. Occasionally, apoE2/2 status with impaired remnant clearance can have a backlogue effect to raise VLDL and chylomicrons. Rarely, in partial lipodystrophies the adipose tissue does not take up NEFA from LPL and the liver puts out more VLDL which competes with chylomicrons for lipolysis. Typically this is associated with diabetes as well. Significant hypothyroidism and renal impairment appear to be excluded as potential secondary causes.

The results indicate long-standing diabetes and hyperlipidaemia. There is likely pseudohyponatraemia. This is because the aqueous part of the aliquot for analysis can be significantly less than the whole volume. The response is to do highspeed or ultracentrifugation so that the lipid can float and the infranatant plasma can be best analysed. Obviously, the whole plasma should be first assayed (in dilution with saline) to be certain of the TG concentration. Alternatively, the lipid volume can be calculated by converting the mmol/L of TG + CE + phospholipid to mass/L and then using the specific gravity of 0.92 g/mL to obtain the volume correction. For practical purposes only the TG and cholesterol values may be used as we do not routinely measure the phosphatidyl choline. Average MW of TG =850da, of CE = 650da, of PL = 750da.  Note that usually 70% of cholesterol is esterified. Cholesterol MW = 387da.

Per L, TG of 79mmol/L is 67g,   CE of 14mmol/L is 9g, total lipid is 85g.

Each g being 1.09mL, makes this 92.4g/L or 9.24g/100mL. This means that the aqueous portion of the aliquot is about 10% too low. This makes the calculated Na+ concentration about 131mmol/L which is still not normal but certainly closer to the reference range.  But the calculation is not highly accurate; partly because PL has not been taken into account; unesterified cholesterol is quantitatively less important.

Diabetes is likely type 2 related to insulin resistance but at this age and especially if dominantly inherited, consider metabolic errors in MODY such as glucokinase deficiency. Unlikely endocrine pancreatic insufficiency which could include mitochondrial defects or herbicide-induced diabetes or (post-traumatic) excision of tail of pancreas.  Not certain if patient is on IV line that could provide lipid (Intralipid in parenteral nutrition) or glucose. Regardless, HbAic shows prolonged exposure to high glucose concentrations.

Regards

D

Professor Emeritus  AD Marais

Chemical Pathology 6.33 Falmouth Building

University of Cape Town Health Sciences

Anzio Rd, Observatory, 7925

Cape Town, South Africa

Abnormal Result

Low growth hormone with an abnormal clonidine stimulation test.

Presenting Complaint

At 7 years of age, patient presented with chronic headaches.

History

CT-brain revealed acute hydrocephalus. Endoscopic 3rd ventriculostomy was done. MRI revealed a lesion posterior to mid-brain and pons as well as a hyper-intensity on T1 with contrast. A glioma was queried as the cause.

Patient recently was identified to have decreased growth velocity (4cm / year) in the preceding 6 months and was also admitted for a clonidine stimulation test. Patient was primed for 2 days.

Examination

N/A

Laboratory Investigations

Histology

GROSS DESCRIPTION: Specimen labelled Sub-occipital tumour consists of 5 fragments of tissue measuring 4-5mm each.

HISTOLOGY: Sections of the brain biopsies show sheets of bipolar neoplastic cells with piloid cytoplasmic processes. Scattered pleomorphic tumour cells, microcystic areas and areas resembling oligodendrocytes are present. Rosenthal fibres and eosinophilic granular bodies are not a prominent feature. There is no vascular proliferation or tumour necrosis.

Comment: See frozen section below.

PARAFFIN SECTIONS: Sections of the brain tumour show piloid areas (cells with hair-like processes, gemistocytic cells and focal Rosenthal fibres. Mild nuclear atypia is noted. There are no mitotic figures, vascular proliferation or areas of necrosis. Scattered entrapped neurons are noted.

IMMUNNOHISTOCHEMISTRY: Neurofilament: Highlight background neural processes

GFAP: Diffusely positive p53: Weak staining in less than 10% of the tumour cells, interpreted as negative.

Ki67 proliferation index: < 4%

CONCLUSION: Brain, biopsy: Juvenile pilocytic astrocytoma.

Other Investigations

Clonidine stimulation testing

Final Diagnosis

Since the hGH concentration did not rise to above 10 ug/L, the diagnosis is likely that of growth hormone deficiency, or hyposomatotropism.

Take Home Message

Provocative Testing for Growth Hormone deficiency (GHD)

The following provocative (dynamic) tests are available for hGH deficiency:

• Insulin tolerance test
• Clonidine stimulation test
• Levodopa-propranolol HCl test
• Arginine HCl test
• Glucagon test

Provocative GH testing is criticized for several reasons, including the following:

• None of the tests reproduces the physiologic secretory pattern of GH because they involve the use of pharmacologic stimuli to indirectly assess physiologic GH production.
• Individual clinicians assign what are essentially arbitrary definitions for subnormal responses (ie. cutoffs for peak serum GH values)
• The reproducibility of provocative tests and GH RIAs is limited. Many pediatric endocrinologists apply other clinical criteria (eg, growth velocity Z score below -2) and do not perform provocative GH tests to diagnose GHD.

Despite limitations, provocative GH tests remain helpful ways to measure GH reserve. Pediatric endocrinologists use physiologic stimuli (eg, strenuous exercise, fasting, deep sleep) and pharmacologic stimuli (eg, clonidine, levodopa-propranolol, glucagon, arginine, insulin) to provoke GH secretion. The tests must be performed after overnight fasting, firstly ensuring that the children are euthytoid.

To improve diagnostic sensitivity and specificity, at least 2 provocative tests are performed. Immediately before and during the earliest phases of puberty, GH production is often indistinguishable in unaffected children and in children with GHD. Serum GH concentrations typically rise during puberty. Many investigators suggest that children approaching puberty should be given gonadal steroids to prime the growth hormone-releasing hormone (GHRH)-GH axis before testing.

Most clinicians use a peak serum GH concentration of more than 10 ng/mL (30 IU) to exclude GHD in children. Specific provocative tests are described below.

Insulin tolerance test

Insulin-induced hypoglycemia is the most potent stimulus for GH secretion and the most dangerous tool for provocative GH testing in patients who may have GH deficiency. Insulin tolerance testing takes advantage of the hormonal counterregulatory response to hypoglycemia. In patients without GHD, plasma concentrations of glucagon, epinephrine, norepinephrine, cortisol, corticotropin, and GH are elevated in response to acute hypoglycemia.

To perform the test, patients fast for 8 hours. Then, lispro insulin 0.1 U/kg of body weight is administered rapidly as an intravenous bolus. Serial blood samples are subsequently obtained to measure GH, cortisol, and glucose concentrations at 0, 15, 30, 60, 75, 90, and 120 minutes. With each sample, the blood glucose level is simultaneously determined by using a bedside glucometer to document an appropriate reduction and to ensure safety. Performance of the test is considered adequate when the blood glucose level decreases below 50% of its baseline value.

Adverse effects expected during the procedure include symptoms secondary to hypoglycemia, such as lethargy, shaking, confusion, headache, abdominal pain, nausea, vomiting, syncope, and seizure activity. The test must be performed under the watchful eye of the physician who can begin prompt resuscitation with glucose and/or glucagon as soon as the diagnostic samples have been obtained. To date, the insulin tolerance test is the only provocative test associated with fatalities; therefore, personnel must be trained and conduct the test judiciously.

Clonidine stimulation test

Clonidine acts centrally to stimulate alpha-adrenergic receptors, which are involved in regulating GH release. Serum GH levels are obtained at baseline and at 60 minutes and 90 minutes after the oral administration of clonidine 0.1 mg/kg. Clonidine may induce hypotension during the test. Therefore, warn parents that they may experience lethargy and/or depression for 24 hours after clonidine is administered.

Levodopa-propranolol HCl test

Levodopa is a dopamine receptor agonist. Dopamine is involved in the stimulation of GH secretion. In the converse, beta-adrenergic control negatively regulates GH release.

Propranolol is a beta-blocker used to hinder inhibitory input affecting GH release, while levodopa simultaneously stimulates GH release by means of the dopaminergic pathway. Propranolol 0.75-1 mg/kg is orally administered before levodopa. The dosage of levodopa for levodopa-propranolol HCl testing varies with weight, so that children weighing less than 15 kg receive 125 mg, children weighing 10-30 kg receive 250 mg, and children weighing more than 30 kg receive 500 mg.

Blood samples for GH testing are drawn at 0, 60, and 90 minutes after the administration of levodopa. Adverse effects include nausea and, in rare cases, emesis. In addition, the patient’s heart rate may decrease because of the use of propranolol. Closely monitor his or her vital signs, and ensure that appropriate resuscitative measures are available.

Arginine HCl test

Arginine appears to exert a direct depolarizing action on somatropic neurons, increasing GH secretion. After an overnight fast, patients are given 10% arginine HCl in 0.9% NaCl 0.5 g/kg (not to exceed 30 g) as a constant intravenous infusion over 30 minutes. Blood samples for GH testing are obtained at 0, 15, 30, 45, and 60 minutes after the infusion of arginine is begun. Arginine has historically been used as a primer before insulin is administered during insulin tolerance testing.

Glucagon test

Glucagon increases peripheral glucose concentrations by means of glycogenolysis and gluconeogenesis. Because glucagon is rapidly metabolized, an abrupt reduction in serum glucose concentration ensues and triggers the release of counterregulatory hormones.

After fasting overnight, patients receive an intramuscular injection of glucagon 0.03 mg/kg (not to exceed 1 mg). Some clinicians advocate the concomitant use of propranolol to inhibit the catecholaminergic response to hypoglycemia. Serum GH concentrations are determined at 0, 30, 60, 90, 120, 150, and 180 minutes after glucagon administration. Nausea and, occasionally, emesis may occur.

Growth hormone reference intervals provided should be seen as a guideline only. Specifically, reference intervals or random growth hormone levels are not useful for diagnosing growth hormone deficiency.
For this, stimulation tests are required. A recent paper established clinical evidence-based cut-off limits for
diagnosing growth hormone deficiency in children and adolescents following growth hormone stimulation tests, by various commercially available immunoassays, compared to isotope dilution mass spectrometry (ID-MS).
Reference: Clinical evidence-based cutoff limits for GH stimulation tests in children with a backup of results with reference to mass spectrometry, Wagner et al, European Journal of Endocrinology (2014)171, 389-397

TrakCare Autocomment

Abnormal Result

Increased CSF IgG / albumin Index

Decreased CSF Glucose

Presenting Complaint

At this admission, she presented to Groote Schuur Hospital with a history of parasthesias and lower limb weakness. Her symptoms have had a rapid progression to completed paraplegia, with loss of sphincter control over three days. She does not report altered sensorium or alteration in her sleep wake cycle.

History

The patient had two prior episodes of TB and Systemic Lupus Erythematosis, which was diagnosed in 2017. Her presenting symptoms was malar rash, arthralgia, and having positive serological markers.

She has subsequently been diagnosed with class-II nephritis and autoimmune haemolytic anaemia. She was on maintenance immunosuppression regimen of prednisone and azathioprine between 2017-19.

She has had two prior episodes of TB in 2014 and 2018.

She was diagnosed with optic neuritis in October 2019, after she developed visual loss in her left eye with no perception of light. Retrospectively she reports a prior episode of unilateral visual loss in her right eye in February 2019, consistent with an episode of ON. On the basis of her ON she had her immunosuppression escalated to monthly pulses of cyclophosphamide, from November 2019 until march 2020.

She has now developed an acute severe inflammatory longitudinally extensive myelitis, as result of seropositive Neuromyelitis Optica Spectrum Disorder (NMOSD).

Neuromyelitis Optica Spectrum Disorder, or NMOSD, is a rare, lifelong and debilitating autoimmune disease of the central nervous system (CNS), characterized by inflammation in the optic nerve and spinal cord.

Examination

Higher function examination was normal. Cranial nerve examination revealed bilaterally dilated pupils with sluggish responses. Relative afferent pupillary defect (RAPD) was noted in the left eye. Visual acquity is reduced in both eyes, with the left being more severely affected than the right. Fundoscopy showed bilateral disc pallor, no active papillitis. She had no further cranial nerve or bulbar signs. Upper limb motor examination showed paratonia in the right upper limb. The left had mild loss of dexterity and power in her left hand. Reflexes were symmetrical. Power in her lower limbs was reduced with 0/5 power, she would have intermittent involuntary movements to tactile stimuli. She was hypertonic with a triple response. She has a T4 sensory level to light touch and pin prick and has marked joint position sensation loss in both her legs.

The patient was admitted for evaluation of her Myelitis. She was started on high dose methylprednisolone and after 3 days of no response she was started on Plasma exchange. Her spastic paraplegia remains unchanged. She has received pressure care, physiotherapy, OT and bowel regimen in the ward. Given her young age, and aggressive disease (developing an severe acute inflammatory myelopathy while on monthly cyclophosphamide pulse therapy), Department of Internal Medicine hopes she can undergo induction therapy with rituximab (375mg/m2) for her seropositive NMOSD in the hopes of avoiding progressive disability and recovering some voluntary function of her limbs.

Laboratory Investigations

CSF Glucose

CSF glucose was 1.4 mmol/L. This is low. It is unfortunately not known what the plasma glucose was at that time, as it wasn’t sent with a plasma glucose sample at the same time, as one would have hoped. If one assumes the plasma glucose have been normal (3.5-5.5mmol/L) and that CSF glucose’s normal range is 0.6-0.8 x plasma glucose, then this value (0.4 – 0.25 that of serum) is significantly low.

Low CSF glucose is indicative of:

• bacterial meningitis,
• mycobacterial infection (TB meningitis),
• mycoplasmal infection and
• fungal infection in the CSF.
• Leucocytes may also decrease CSF glucose – thus it may indicate a disease process rather than only bacterial infection.

CSF Protein

CSF protein of 2.47 g/L (0.15 – 0.45 g/L) is significantly raised. This is indicative of increased permeability of the blood-CSF barrier, most commonly due to inflammatory conditions, but can also be elevated due to:

• Decreased spinal CSF flow above the sampling level due to:
  • abscess
  • tumor (see also Froin Syndrome)
  • collapsed intervertebral disc
    • These allow for increased equilibration of fluid between CSF and plasma.
• Immune response within the CSF (intrathecal synthesis of IgG)
• Destruction of brain tissue releasing proteins directly into CSF.
• Neonates (term and pre-term) have a higher reference range – up to 1.7 g/L.

CSF Albumin

CSF albumin of 1271 mg/L (100 – 300) is elevated. This value is increased significantly, likely indicating an increase in the permeability of the blood-brain-barrier (severe impairment).

CSF Albumin Index

The CSF Albumin Index was 31.8 mg/g (<9).

CSF albumin is best interpreted in conjunction with the serum albumin. CSF albumin is corrected for leaking of albumin from the serum (thus incorporating the serum albumin concentration). This is called the CSF albumin index (mg/g) = CSF albumin(mg/L) / serum albumin (g/L)

• <9: intact blood-CSF barrier
• 9-14: slight impairment
• 14-30: moderate impairment
• >30: severe impairment

CSF IgG / Albumin Index

CSF IgG index = 0.86

IgG should under normal situations be almost totally excluded from CSF (blood:CSF ratio of >/= 500:1). Intrathecal production of IgG follows infiltration of the space by B-lymphocytes in demyelinating disorders.

Increase in CSF IgG can however be due to increased permeability form the plasma (dysfunctional barrier), increased plasma IgG concentration or intrathecal production. To correct for the former two, plasma IgG and serum albumin is used in an equation to get the above named index, which tries to isolate CSF IgG for the portion which was produced intrathecally.

The formula is: IgG index= (CSF IgG / Serum IgG) x (Serum Albumin / CSF Albumin). Units of measurement should be similar, at least for serum and CSF respectively.

Reference interval for CSF IgG index is 0.3 – 0.7. Ratios greater than 0.7 indicate increased intrathecal synthesis, as seen in more than 80% of cases of Multiple Sclerosis (MS) and other demyelinating disorders. False elevations occur in traumatic tap and SAH. CSF IgG index is more reliable when the CSF Albumin Index is normal (i.e. when the barrier function is not decreased, but obviously this is rarely the case, as inflammation leads to decreased barrier function).

CSF IgG synthesis rate

153 mg/day

The rate of intrathecal IgG synthesis can be estimated by the empirically derived Tourtellotte’s formula and a synthesis
rate of greater than 8 mg/day is found in most cases of multiple sclerosis. It is a more complex formula, with several constants and probably provides no additional information than the IgG index. (Ref: Tietz 6th Ed.)

Other Investigations

Investigations to this point include the following:

1. MRI brain and spine shows Longitudinal extensive transverse myelitis from the cervical to the lumbar level.

2. FBC and CEU was normal – Neutrophils were normal, lymphocyte count was 0.81.

3. Serologies were ANA, AdsDNA, AntiSm, Anti-Ro Positive. C3 and C4 were normal.

4. HIV, HBV, HCV serology was normal.

5. Serum AQP4 serology positive with a titre of 10

6. CSF: Protein-2.47, Gluc-1.4,P8, L37 R0, IgG index0.87, CLAT, GXP, Culture negative.

7. CD19: 250

8. CXR showed bilateral reticular nodular infiltrate and features of post of TB bronchiectasis

Final Diagnosis

Neuromyelitis Optica Spectrum Disorder

Take Home Message

CSF Glucose

Recovery from meningitis results in recovery of glucose before CSF cell counts and Total Protein.

CSF glucose is typically normal in CSF viral infections, exceptions may include: Meningoencephalitis with Mumps, EV, HSV, HZV.

Leucocytes may also decrease CSF glucose – thus it may indicate a disease process rather than only bacterial infection.

Ratio of CSF:serum glucose has limited utility in neonates and in pts. with hyperglycemia.

CSF glucose <1.0 mmol/L is highly indicative of bacterial meningitis.

If intellectual disability, seizures and motor impairment is present along with low ratio, think of GLUT-1 transporter deficiency – then measure CSF lactate.

CSF Chemistry

As described above

Correcting total protein for Traumatic tap

The presence of CSF bleeding results in approximately 0.010 g of protein / L per 10^6 RBCs / L

Abnormal Result

Total Vitamin D of 27.1 nmol/L on 18 March 2020.

Total Vitamin D of 65.4 nmol/L on 01 April 2020.

Presenting Complaint

Patient had a low impact femur fracture on 18 March 2020 :

History

Patient is known with:

• previous deep venous thrombosis in 2018, on Warfarin therapy
• ?Epilepsy, patient is on carbamazepine, for which the Endocrinology specialists were of opinion that it may have been the cause of the low Vitamin D level.

Examination

Laboratory Investigations

Other Investigations

Final Diagnosis

Vitamin D deficiency likely due to carbamazepine therapy.

Take Home Message

I was not aware that patients on carbamazepine (or other enzyme inducing agents) have lower Vitamin D levels, and it became evident after a quick literature search that it was in fact the case, see the abstract of the article below, also see another article written by a colleague of mine, Jusine Cole, on the Vitamin D controversies.

Also, I have learned that although “total Vitamin D”, as the assay is named in our immunoassay package insert and on TrakCare LIS, has to do with the total portion with regards to protein binding (to Vitamin D binding protein) and not so much to the fact that calcitriol and calcidiol is measured.

It is however evident that, since the cross-reaction in the immuno-assay is quite pronounced with the various forms of Vitamin D, that total indeed, might be an accurate description. In reality, the assay is however called the Total 25-hydroxy Vitamin D.

Vitamin D levels and bone turnover in epilepsy patients taking carbamazepine or oxcarbazepine.

Abstract

PURPOSE:

Evidence suggests that enzyme-inducing antiepileptic drugs (AEDs) may decrease serum 25-hydroxyvitamin D (25-OHD) levels and increase bone turnover. We sought to determine whether these are affected by treatment with carbamazepine (CBZ) or oxcarbazepine (OXC).

METHODS:

We measured serum levels of 25-OHD, parathyroid hormone (PTH), osteocalcin (OCLN), bone alkaline phosphatase (BAP), and urinary N-telopeptides of type I collagen cross-links (NTX) in normal controls (n=24) and in epilepsy patients taking CBZ (n=21) or OXC (n=24) in monotherapy. CBZ patients were subsequently switched overnight to OXC monotherapy, and after 6 weeks, the tests were repeated.

RESULTS:

25-OHD levels were lower in each drug-treated group (OXC, 19.4+/-2.3 pg/ml; CBZ, 20.4+/-2.4) than in the controls (27.5+/-2.8) (ANOVA, p=0.052). This difference was significant for the OXC group (p<0.05). PTH, BAP, and NTX did not differ significantly among groups. OCLN levels were somewhat elevated in the OXC group (2.79+/-0.47 ng/ml) and more clearly and significantly elevated in the CBZ group (3.63+/-0.36) compared with controls (2.38+/- 0.41) (p=0.053). Because the data were very similar between OXC and CBZ groups, they were combined to increase statistical power. The combined drug-treatment group had significantly higher BAP (p=0.02) and lower 25-OHD (p=0.015) than did controls. The latter remained significant even after accounting for the confounding effects of age on 25-OHD levels (p<0.05). No significant differences were found after CBZ patients were switched to OXC.

CONCLUSIONS:

Epilepsy patients taking OXC or CBZ have significantly lower 25-OHD than do normal controls, with a pattern of changes in other bone biomarkers suggestive of secondary hyperparathyroidism. It may be prudent for patients taking CBZ or OXC to be prescribed 25-OHD replacement.

Abnormal Result

CSF Glucose: <0.1 mmol/L

CSF Protein: <0.01 mmol/L

Both of the above results have already been rerun on the same analyzer with the same result.

Presenting Complaint

It was then suspected in the lab that the results might have been obtained from a urine (or other fluid) sample rather, which might accidentally been registered / recorded as CSF.

An MC&S (Microscopy, Culture and Sensitivity testing) was also requested on an aliquot of the sample.

History

The above result was obtained from a patient known with hydrocephalus. No other information was available at the time of analysis.

Examination

N/A

Laboratory Investigations

As above abnormal results.

Other Investigations

Microscopy: No leucocytes; No bacteria; India Ink stain was pending.

The following possibilities were thought of:

• Pre-analytical factors:
  • A urine sample was sent and incorrectly registered as CSF
    • A possibility to quickly exclude this was to determine the creatinine on the sample if urine, then the creatinine will measure in the thousands (umol/L) or in the mmol/L range.
    • One could also do CSF identification by CSF electrophoresis for the presence of beta-2 transferrin or beta trace protein identification, but this is expensive and laborious.
  • Bacteria metabolised all the glucose, and for some reason the protein did not go up in the patient, although the protein <0.01g/L is extremely low, making the possibility of this scenario unlikely.
• Analytical factors:
  • Bubble aspiration, although the rerun gave a similar result.
  • Interferents: none could be thought of in this scenario.
• Post-analytical:
  • The result was obtained directly from the analyzer’s user interface, hence making transcription / translation errors unlikely.

Final Diagnosis

The clinician, a neurosurgery doctor, was phoned to enquire about the history, and it was indeed a sample from the cerebro-spinal space, but upon questioning the Neurosurgery doctor, it became known that:

• This sample was obtained intra-operatively during placement of a ventriculo-abdominal shunt for hydrocephalus
• During the procedure, the ventricles have been flushed with Normal Saline, which explains the low CSF protein and the low Glucose.

Take Home Messages

The history from the clinician was confirmation that this sample was indeed from the cerebrospinal space, although not representative of the cerebrospinal space, as it was taken during a procedure, hence likely pure saline was measured.

Adequate history from the clinician is most often the most useful information that can be obtained, especially when severely abnormal results are obtained.

When results do not make sense, one should not authorize them without discussing with the clinician first.

The neurosurgery doctor agreed that to request the chemistry on this sample was not indicated and they actually only wanted the Microbiology investigations as proof of some sort that an infection was not present, for which exact indication the Microbiology team might comment on.

Results was thus not authorized, but rather cancelled with a comment, stating that sampling was not representative of the physiological CSF.

Abnormal Result

Hypokalemia in a 86 year old lady.

Presenting Complaint

Muscle weakness for the preceding 3 weeks.

History

Known with hypertension, dyslipidaemia and chronic kidney disease.

Presented at a private practice with symptomatic hypokalemia. Patient gave a history of 3 weeks body weakness and dizziness and said she hasn’t been eating well for the prior three weeks.

No other symptoms. No previous medical admissions or procedures.

The patient has just had persistent hypokalemia in hospital despite supplementation. The consultant in Internal Medicine attributed her persistent CMP disturbances to refeeding type syndrome.

In the hospital admission the patient was receiving:

• Simvastatin 20mg dly PO
• Calcium carbonate (1g elemental Calcium) q8h PO
• SlowMag 2 tabs daily PO
• Metoclopramide 10mg q8h PO

Examination

Not available

Laboratory Investigations

Other Investigations

Final Diagnosis

Take Home Message

A study by Elisaf, M & Siamopoulos, KC (Postgrad Med J 1995; 71: 211-212), clearly showed that in patients with hypokalaemia of extrarenal origin FEK is less than 6.5%, i.e. a FEK more than 6.5% is indicative of inappropriate potassium loss. FEK was more than 9% in all patients with hypokalaemia of renal origin.
The study concluded that in hypokalaemic patients with normal renal function, FEK is a useful tool in the diagnostic approach of hypokalaemia.

Limitations: A random urine sample was used to determine these values. Ideally a 24 hour urine collection should be used and theoretically should provide a more accurate estimation of tubular function. The fractional excretion of potassium is however inherently normalized to creatinine.
No clinical info / medication history was supplied. The most likely cause of inappropriate potassium loss in the urine is medication (iatrogenic) like diuretics and some antihypertensives. Ideally, if a pathogenic, rather than iatrogenic cause of potassium loss is suspected, the patient needs to be free of potassium supplementation and all medication which could influence tubular function must be stopped before analysis of the renal tubular electrolyte handling.

Abnormal Result

Estrogen 4823 pmol/L in a patient with in vitro vertillization.

Presenting Complaint

The Doctor called, querying if this might be a possible high dose hook effect. They expected a much higher result with this particular patient.

I explained that this is a competitive immunoassay and that high dose hook effect is most likely observed rather with sandwich immunoassays.

History

This patient was undergoing IVF for multiple pregnancy – higher value anticipated (10000 – 12000 pmol/L)

Examination

N/A

Laboratory Investigations

1 in 10 dilution made, result of the rerun was 415 x 10 = 4150 pmol/L (-14% difference).

Other Investigations

Final Diagnosis

The estradiol was indeed likely close to a true result, even though queried by the clinician.

This was confirmed by the duplicate result when running this sample in dilution. The -14% difference from the original result can likely be explained by imprecision from:

• Pipetting error when doing the manual dilution
• Imprecision of the analyser
• Matrix effects when using the universal diluent from the analyser

Take Home Messages

Competitive immunoassays are NOT prone to high dose hook effect, due to the inherent characteristics of the assay.

It is however known that measurement of estradiol at the levels required for IVF is not in the linear range of the assay and that there are likely to be imprecision as noted by the points above.

The measuring range as quoted by the package insert for our Roche Cobas 6000 E2 assay is 18.4 – 11010 pmol/L (LOD to max of master curve). It can however be reported up to 110100 pmol/L for 10-fold diluted samples.

It is however a pity that the reading off the standard curve (signal) cannot be seen on the analyzer’s firmware, as can be seen with routine chemistry analytes eg. liver enzymes etc.

An interesting article which I’ve also forwarded to the doctor is added below.

Download File

Abnormal Result

Oral glucose tolerance test with the Sodium Fluoride (NaF) tubes registered by the lab 20 hours after being sampled.

Collection date: 07h22 05/03/2020

Received date: 18h44, 05/03/2020

Registered date: 03h26, 06/03/2020

Please note that samples in our lab are being centrifuged after being registered.

Presenting Complaint

My thoughts were, that if the sample isn’t centrifuged in a timely manner, metabolism would still happen, albeit at a slower rate. I also thought that metabolism (glycolysis) would continue if left for a long period (>8 hours) uncentrifuged.

Would you argue the result as given below at “Laboratory Investigations” is reliable, given the following info?

1. Stability of glucose in whole blood in NaF tubes?
2. Could this be a false normal result?

The stability spreadsheet as summarized by our lab did not have the stability info for glucose in whole blood:

History

Patient is most likely pregnant (being from an antenatal clinic) and this is then a screening test for gestational diabetes.

Examination

N/A

Laboratory Investigations

Other Investigations

Literature search on Google Scholar yielded the following interesting article:

Effectiveness of sodium fluoride as a preservative of glucose in blood.

A Y Chan, R Swaminathan, C S CockramClinical Chemistry, Volume 35, Issue 2, 1 February 1989, Pages 315–317, https://doi.org/10.1093/clinchem/35.2.315 Published: 01 February 1989

Abstract

How effective is sodium fluoride as a preservative of blood glucose? We compared changes in glucose concentration in fluoride-treated blood specimens with those of heparin-treated specimens. The former declined rapidly during the first hour; thereafter the rate of decrease was slower, and after 4 h the glucose concentration in the blood samples remained stable for up to three days. In contrast, the glucose concentration in the heparin-containing samples declined continuously. During the first hour, however, the rates of decline in the two types of samples were similar. Evidently sodium fluoride takes effect slowly but effectively in preserving glucose in blood for at least three days. Its use, however, is unnecessary if the concentration of glucose is to be measured within the first hour after sampling.

Final Diagnosis

This is likely a true result, meaning the patient is normal and does not have impaired glucose tolerance, nor diabetes.

Take Home Message

Blood glucose is stable for 3 days in plasma from NaF tubes, whether being centrifuged in a timely manner or not.

The stability of glucose in specimens is affected by storage temperature, bacterial contamination, and glycolysis. Plasma or serum samples without preservative (NaF) should be separated from cells or clot within half an hour of being drawn. When blood is permitted to clot and to stand uncentrifuged at room temperature, the average decrease in serum glucose is ~7% per hour (0.28 – 0.56 mmol/L/hour), as a result of glycolysis. Glycolysis can be inhibited by collecting the specimen influoride tubes (1).

(1) Sacks DB. Carbohydrates. In: Tietz NW, ed. Fundamentals of Clincal Chemistry. 4th ed. Philadephia: WB Saunders 1996;351-374.

Abnormal Result

Presenting Complaint

Upon signing blood results out, a creatinine result was measured by the analyzer as <0.1 mmol/L on a urine sample.

History

Clinical History was not available, but in my personal experience at the time, I haven’t seen such a low urine creatinine yet.

The possibilities in my mind, was that this was either a serum sample and could perhaps be incorrectly sent / registered from the Porphyria lab as serum, hence the result being < 0.1 mmol/L (<100umol/L if translated into serum reporting units).

Examination

The sample smelt and looked like urine. According to our new registrar, Mrs. Mariam Mahomed, it also tasted like urine. I was personally not capable of this task, so we decided to rerun the sample.

Mrs. Bilqees Jacobs, our technologist on the bench this day was of opinion that when such a low result is seen, it is usually due to a bubble aspirated by the analyzer’s sampling probe.

Laboratory Investigations

The rerun of the sample as a urine creatinine gave the result as 1.9 mmol/L, more in keeping with a true urine creatinine result.

Other Investigations

None was necessary, but should the result have been < 0.1 mmol/L on the rerun, we would then have run the sample as a serum on the analyzer to more accurately quantify the value.

Final Diagnosis

Possibly, a bubble was aspirated by the analyzer’s sampling probe and hence it did not pipette enough sample into the reagent well, or likely not pipette any sample therein.

Take Home Messages

Try to avoid bubbles in samples.

This brings me to the point: whenever a ” lower than detection limit” is seen, think of the cause:

“Tiny Bubbles!”

Abnormal Result

Upon signing out blood results:

Calcium = 1.41 mmol/L – Critically low Calcium result

Magnesium = 0.37 mmol/L – Critically low Magnesium

Presenting Complaint

Loss of breath initially accompanied by weight loss.

Upon admission to the ward, patient was slightly delirious, but still able to walk and talk.

History

Patient with metastatic lung cancer and accompanying hypercalcemia, a week prior to the results as at present.

Doctor has given IV Bisphosphonate after the hypercalcemia was noted a week prior (Calcium = 4.23 mmol/L; Alb = 21 g/L; Corrected Ca = 4.61 mmol/L)

Examination

Extensive Crepitations over all the right lung fields.

Laboratory Investigations

Other Investigations

Final Diagnosis

Invasive lung CA with “hungry bones” after IV Zolendronic Acid

Vitamin D deficiency, preventing Calcium absorbtion after the Zolendronic acid started its action of inhibiting bone resorption.

Take Home Messages

CA causes hypercalcemia

Bisphosphonates inhibits bone resorption. Because 99.95% of Ca in the body resides in bone, the effect in serum (the remaining 0.05% of total body Ca) can be significant.

All bisphosphonate drugs share a common phosphorus-carbon-phosphorus “backbone”:

They differ in the R-groups as above. It binds to calcium hydroxyapatite in bone.

Of the dose infused / absorbed, 50% is excreted unchanged by the kidney, the rest binds to bone tissue, where its elimination half life can apparently be up to 10 years! (UW Courses Web Server- https://courses.washington.edu/bonephys/opbis.html )

Because a bisphosphonate group mimics the structure of pyrophosphate, it can inhibit activation of enzymes that utilize pyrophosphate.

Magnesium follows Calcium levels, but Mg deficiency itself can also cause hypocalcemia.

Abnormal Result

Urine toxicology screening / Urine organic acid analysis

Presenting Complaint

ESKOM load shedding resulted in failure of the power supply (including the emergency power generator) at a part of the hospital which included the routine toxicology screening lab at the Department of Pharmacology.

Two patient samples arrived at Red Cross Hospital IMD lab one Wednesday morning, brought personally by the Lab Manager of the Pharmacology Toxicology Department, requesting help to identify the cause of poisoning in two of six patients with alleged poisoning. At the time, the patients were both on ventilatory support in ICU due to depressed level of consciousness.

The repeated electricity power failures over a weekend, resulted in Toxicology’s state-of-the art tripple-quad tandem qTOF mass spectrometer to fail.

History

Six individuals were given a drink by a taxi driver in Landsdowne, Cape Town, on that Monday morning, all who collapsed few minutes later in the streets. Paramedics were on the scene soon and all affected patients were rushed to respective hospitals. See more on the Daily Voice.

Examination

The two individuals from whom we obtained urine for analysis, both had similar signs:

GCS was apparently 4/15, requiring intubation and ventilation.

Pupils were not miotic nor midriatic and signs were not in keeping with organophosphate poisoning.

Laboratory Investigations

No other laboratory investigations were available at the time. In retrospect, one of the patient’s results are available and serum electrolytes, liver enzymes, liver function and renal function tests were within normal range.

Other Investigations

Urine organic acid analysis by GC-MS were done by us that day and we could identify Gamma-hydroxybutyrate (GHB) with an accurate hit to a known library adequately. We also ran a control urine sample of myself to prove the absence thereof as a negative control.

In the chromatograms below, note the height of the base peak, Pentadecanoic Acid (PDA) in relation to the peaks eluting at 11.45 minutes, 4-OH-butyrate, the analyte of interest. It can be seen that it is present in both the patient’s samples and is absent in the control sample.

Final Diagnosis

It was confirmed that both cases as analysed by us was in keeping with intoxication with 4-OH-butyrate (gammahydroxybutyrate; GHB).

Take Home Messages

We concluded that cases of gammahydroxybutyrate intoxication can be confirmed by urine GC-MS analysis at our IMD chemistry lab at Red Cross Children’s Hospital.

Gammahydroxybutyrate (GHB) has appeared to be quite a common (and perhaps re-emerging) drug in the Cape Town area. It can relatively easily be manufactured without sophisticated equipment and due to its ease of manufacture is not extensively made / traded by illicit drug traders.

GHB (an agonist of certain GABA receptors) can be manufactured relatively easily by addition of two ingredients, a prodrug gammabutyrolactone (GBL; a commercially available product) and an alkali hydroxide, such as sodium hydroxide to form GHB salt.

Other trade names of GHB include “Liquid Ecstasy”, “lollipops”, “Liquid X” or “Liquid E”.

See below a copy of the article as it appeared in the Daily Voice, a Cape Tonian newspaper: